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as a PDF - Forschungszentrum Borstel
Jahresbericht 2007-2008
Forschungszentrum Borstel – Leibniz-Zentrum
für Medizin und Biowissenschaften
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Leibniz-Zentrum für Medizin und Biowissenschaften
Parkallee 1-40
23845 Borstel
Tel. +49 (0)4537.188 0
E-mail. [email protected]
www.fz-borstel.de
Impressum/Imprint
Herausgeber/Editorial Board
Prof. Dr. Dr. Silvia Bulfone-Paus
Prof. Dr. P. Peter Zabel
Prof. Dr. U. E. Schaible
Koordination/Coordination
Dr. Bettina C. Brand
Design/Layout
Stil.3 Designbüro AGD
www.stil-punkt-3.de
Hamburg
Jahresbericht 2007-2008
Forschungszentrum Borstel – Leibniz-Zentrum
für Medizin und Biowissenschaften
Wissenschaftlicher Jahresbericht 2007-2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Verstärkung fuer die Wissenschaft (von li. nach re.)
S. Ehlers, Professor für Molekulare Entzündungsmedizin, Kiel;
Ulrich E. Schaible, Direktor am FZB & Professur für Biochemische Mikrobiologie, Lübeck;
Heinz Fehrenbach, Professur für Experimentelle Pneumologie, Lübeck.
Strengthening science (left to right):
S. Ehlers, Chair, Molecular Inflammation Medicine, Kiel;
Ulrich E. Schaible, Director at the Research Center Borstel & Chair, Biochemical Microbiology, Lübeck;
Heinz Fehrenbach, Chair, Experimental Pneumology, Lübeck.
Inhalt/Content
Vorwort/Preface
. . . . . . . . . . . . . . . . . . . 6
Abteilung Klinische Medizin
Organe des FZB/Boards of the FZB
Stiftungsversammlung . . .
Kuratorium . . . . . . . . . .
Direktorium . . . . . . . . . .
Kollegium . . . . . . . . . . .
Wissenschaftlicher Beirat .
Betriebsrat . . . . . . . . . .
Zentrale Verwaltung . . . .
Organigramm . . . . . . . .
Department of Clinical Medicine
. . . . . 130
. . . . . . . . . . . . . . 10
Zusammenfassung/Summary . . . . . . . . . . . 132
. . . . . . . . . . . . . . 10
Research Reports
. . . . . . . . . . . . . . 10
Division of Clinical Immunpharmacology . . . . . 143
. . . . . . . . . . . . . . 11
Division of Clinical and Experimental Pathology . . 149
. . . . . . . . . . . . . . 11
Division of Mycobacteriology and National
. . . . . . . . . . . . . . 11
Reference Center (NRC) for Mycobacteria . . . . 157
. . . . . . . . . . . . . . 11
Division of Molecular Mycobacteriology . . . . . . 164
. . . . . . . . . . . . . . 12
Division of Molecular and Clinical Allergology . . 175
Division of Cellular Allergology . . . . . . . . . . . 180
Division of Mucosaimmunology . . . . . . . . . . . 184
Division of Clinical Infectious
Abteilung für Immunchemie
und Biochemische Mikrobiologie
Department of Immunochemistry
and Biochemical Microbiology
Diseases and Clinical Studies . . . . . . . . . . . . 191
Publikationen/Publications
. . . . . . . . 14
Peer reviewed papers . . . . . . . . . . . . . . . . 204
Zusammenfassung/Summary . . . . . . . . . . . . . 16
Research Reports
Book chapters and reviews . . . . . . . . . . . . . 224
Division of Medical and Biochemical Microbiology . . 32
Division of Structural Biochemistry . . . . . . . . . . 36
Ernennungen/Appointments
Division of Immunochemistry . . . . . . . . . . . . . 45
Auszeichnungen/Awards
Division of Biophysics . . . . . . . . . . . . . . . . . 52
Preise . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Jennifer Debarry,
best PhD
graduation 2007
Emmy-Noether Junior Research Group
Ehrungen . . . . . . . . . . . . . . . . . . . . . . . . . 232
Immunobiophysics . . . . . . . . . . . . . . . . . . . 59
Division of Molecular Infection Biology . . . . . . . 62
Berufungen . . . . . . . . . . . . . . . . . . . . . . . . 232
Junior Research Group Molecular
Ernennungen. . . . . . . . . . . . . . . . . . . . . . . 232
Infection Biology . . . . . . . . . . . . . . . . . . . . 69
Research Group of Cellular Pneumology . . . . . . 74
Wissenschaftliche Veranstaltungen
Colloquia and Symposia
Abteilung Immunologie und Zellbiologie
Department of Immunology
and Cell Biology . . . . . . . . . .
Zentrumsseminare . . . . . . . . . . . . . . . . . . . 236
Weiterbildungsseminare der Klinik . . . . . . . . 242
Symposien und Workshops . . . . . . . . . . . . . 244
. . . . . . . . . 78
Zusammenfassung/Summary . . . . . . . . . . . . . 80
Research Reports
Division of Immunbiology . . . . . . . . . . . . . . . 88
Lehre/Teaching .
. . . . . . . . . . . . . . . . . . . 250
Ausbildung/Training .
. . . . . . . . . . . . . . . 256
Division of Cellular Immunology . . . . . . . . . . . 92
Division of Molecular Immunology . . . . . . . . . 97
Division of Immune Cell-Analytics . . . . . . . . . . 99
Chronik/Chronicle
. . . . . . . . . . . . . . . . . . 260
Division of Biological Chemistry . . . . . . . . . . 102
Division of Tumor Biology . . . . . . . . . . . . . . 106
Neue Entwicklungen
Division of Veterinary Infectiology
New Developments .
. . . . . . . . . . . . . . . . 266
and Immunology . . . . . . . . . . . . . . . . . . . 109
Division of Biochemical Immunology . . . . . . . . 119
Haushaltsplan/Budget .
. . . . . . . . . . . . . 273
Division of Innate Immunity . . . . . . . . . . . . . 125
Personenregister/Register of Persons
. . . 274
5
Jennifer Debarry,
Promotionspreis
2007
Vorwort
Preface
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Vorwort
Preface
Das Forschungszentrum Borstel (FZB), Leibniz-Zen-
The Research Center Borstel (RCB), Leibniz Center
trum für Medizin und Biowissenschaften, sieht sei-
for Medicine and Biosciences, sees its scientific
ne gesellschaftspolitisch und gesamtstaatlich be-
mission in the field of pneumology with the main
deutsame wissenschaftliche Mission in der
emphases on infectious diseases, allergy, and in-
grundlagen-, krankheits- und patientenorientier-
flammation.
ten Forschung auf dem Gebiet der Pneumologie
mit den Schwerpunkten Infektiologie, Allergologie und Entzündung.
The members of Borstel may look back upon a
number of impressive and pioneering events during 2007 and 2008. During this time of financial cri-
In den Jahren 2007 und 2008 können die Borste-
sis, we may look back upon two years during which
ler auf eine Reihe prägender und wegweisender
the RCB turned out to be a very respectable motor
Ereignisse zurückblicken. Der sich abzeichnenden
of scientific excellence in the North.
Finanzkrise trotzend, erwies sich das Forschungszentrum Borstel auch in den vergangenen beiden
Reopening of the Manor
House as a science and
communication center
Jahren alles andere als krisengeschüttelt. Wir
One of the emblems of the Center is our manor
können auf zwei Jahre zurückblicken, in denen
house, which could finally be reopened in June
sich das FZB als grundsolider Motor wissen-
2007 as a science and communication centre after
schaftlicher Exzellenz im Norden erwiesen hat.
five years of renovation and restauration activities.
Since then it has anew become the Center within
the Center.
Dreh- und Angelpunkt des Zentrums ist unser Herrenhaus, das nach fünf-jährigen Renovierungs-
Zentrum im Zentrum:
das wiedereröffnete
Herrenhaus
und Restaurierungsarbeiten im Juni 2007 als Wis-
Although financial conditions have become more
sens -und Kommunikationszentrum endlich wie-
difficult on the whole, the acquisition of non-public
dereröffnet wurde. Seitdem ist es wieder Zentrum
funds could once more be increased. This applies
im Zentrum.
also to research funds which are especially significant for a gain in prestige and could be acquired
from the EU, the BMBF, and the DFG. During the
Die Drittmitteleinwerbung konnte trotz der hierfür
last years, the RCB was again a spearhead of cre-
insgesamt härter gewordenen Bedingungen er-
ative biomedical research in the North and ac-
neut gesteigert werden. Dies gilt auch für beson-
tively participated in new initiatives for research
ders prestigereiche Forschungsmittel, die von der
cooperations such as Transregios and Collabora-
EU, dem BMBF und der DFG eingeworben werden
tive Research Centers.
konnten. So war in den vergangenen Jahren das
FZB wieder eine Speerspitze kreativer biomedizinischer Forschung im Norden und beteiligte sich
As part of the Pact for Innovation and Research the
aktiv an neuen Initiativen für Forschungsverbünde
RCB was again successful in gaining funds from
wie Transregios und Sonderforschungsbereichen.
the Leibniz-Association. Because of this success,
the financing for the group “Barrier Integrity“, a cooperation project together with the Christian-Al-
Auch die Antragstellung im „Pakt für Forschung
6
brechts-University of Kiel, was guaranteed. In col-
Wissenschaftlicher Jahresbericht 2007-2008
Vorwort
Preface
und Innovation“ der Leibniz-Gemeinschaft war
laboration with the “Leibniz-Center for Infection
zum wiederholten Male erfolgreich. Zum einen
Research “, i.e the Bernhard-Nocht-Institute and the
hat das die Finanzierung für die Gruppe „Barrie-
Heinrich-Pette-Institute (both in Hamburg), funds
re-Integrität“, ein gemeinsames Kooperationspro-
for a graduate school were raised. The “Leibniz
jekt mit der Christian-Albrechts Universität Kiel, si-
Graduate School – Model Systems for Infectious
chern können. Zum anderen konnte mit den
Diseases“ will start its training program in spring
Partnern des „Leibniz-Zentrum für Infektionsfor-
2009.
Leibniz Graduate School
schung“, dem Bernhard-Nocht-Institut und dem
Heinrich-Pette-Institut (beide Hamburg) Gelder für
eine Graduiertenschule eingeworben werden. Die
Communication and transfer of research results
„Leibniz-Graduate School – Modellssystem für In-
play an especially important role in life of the Re-
fektionskrankheiten“ wird im Frühjahr 2009 ihr an-
search Center. National and international meet-
spruchsvolles Ausbildungsprogramm starten.
ings were organised on the part of the Center within the framework of several scientific events in
Die Kommunikation und der Transfer von For-
2007 and 2008. The “Second Joint German Allergy
Congress” and the “14th European Carbohydrate
schungsergebnissen haben einen besonders ho-
Symposium” (2007) as well as the “49th Annual
hen Stellenwert im Zentrum. Dafür wurden in 2007
Congress of the German Society for Pneumology
und 2008 seitens des Zentrums im Rahmen zahl-
and Ventilation Medicine” and the “Symposium of
reicher wissenschaftlicher Veranstaltungen natio-
the European Respiratory Society, State of the Art
nale und internationale Diskussionsforen organi-
in Tuberculosis” in 2008 come to mind .
siert. Besonders hervorzuheben sind der „2.
Gemeinsamen Deutschen Allergie-Kongress“ und
das „14th European Carbohydrate Symposium“
Another new development is the new “Manor
(2007), sowie der „49. Kongress der Deutschen
House Lecture” of the Excellence Cluster “Inflam-
Gesellschaft für Pneumologie und Beatmungsme-
mation at Interfaces”, in which outstanding scien-
dizin e.V.“ und das „Symposium der European Re-
tists from all over the world are invited to present
spiratory Society, State of the Art in Tuberculosis“
their scientific knowledge and their expertise in the
in 2008.
field of inflammation.
Ein weiteres Aushängeschild ist die neue „Manor
Scientists at the RCB have appeared in highest-
House Lecture“ des Exzellenzclusters „Entzündung
ranking journals such as Nature Medicine, Nature
an Grenzflächen“, die einmal im Monat heraus-
Immunology, Journal of Experimental Medicine,
ragende Kolleginnen und Kollegen aus aller Welt
New England Journal of Medicine demonstrating
einlädt, ihre wissenschaftliche Erkenntnisse und
the scientific quality of the RCB. Scientific success
ihre Vision der Forschungsentwicklung im Bereich
is also shown by scientific prizes, awards and hon-
Entzündung darzustellen.
ours to people working at the RCB.
Zur wissenschaftlichen Bilanz des FZB trägt auch
In 2008 Ulrich Schaible succeeded Ernst Rietschel
die beeindruckende Publikationsleistung der Mit-
as head of the department “Immunochemistry and
arbeiterinnen und Mitarbeiter des FZB in höch-
Biochemical Microbiology”. Meanwhile he has re-
strangigen Journalen wie Nature Medicine, Na-
named it into “Molecular Infection Biolology”, re-
Wissenschaftlicher Jahresbericht 2007-2008
7
Manor House Lecture
Vorwort
Preface
ture Immunology, J Exp. Medicine, New England
structured and extended it and is on his way to lit-
J. of Medicine bei. Wissenschaftlicher Erfolg zeigt
erally become a “Borsteler”.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
sich aber auch in der Vergabe wissenschaftlicher
Preise, Auszeichnungen und Ehrungen an FZB-Mitarbeiterinnen und -Mitarbeiter.
In the summer of 2008 Heinz Fehrenbach took up
his work as the new professor for Experimental
Pneumology (Lübeck). From now on, the Experi-
Ulrich Schaible ist neuer
Direktor der Abtl.
Molekulare Infektiologie
Das wichtigste Borsteler Ereignis des Jahres 2008
mental Pneumology will be part of the department
war der Amtsantritt von Ulrich Schaible als Nach-
of Clinical Medicine as an independent section
folger von Ernst Rietschel im August. Inzwischen
which comprises several groups.
hat er seine Abteilung „Immunchemie und Biochemische Mikrobiologie“ umbenannt in „Molekulare Infektiologie“, umstrukturiert und erweitert
PD Dr. Thomas Gutsmann, a colleague in the divi-
und ist im besten Sinne dabei, ein Borsteler zu
sion of Biophysics for many years, received a call
werden.
for the W3 professorship for Nanobiomechanics at
the University of Ulm, for which we warmly congratulate him.
Heinz Fehrenbach leitet
den Bereich 'Experimentelle Pneumologie'
Im Sommer 2008 hat auch Heinz Fehrenbach seine Tätigkeit als neuer Professor für Experimentelle Pneumologie (Lübeck) aufgenommen. Die Ex-
In 2007, a Medical Treatment Center (MVZ) was
perimentelle Pneumologie wird zukünftig als
started at the Medical Clinic in Borstel after long
eigenständiger Bereich in der Abteilung Klinische
negotiations, which organisationally unites the
Medizin stehen und mehrere Gruppen umfassen.
Pathology (E. Vollmer) and Bronchopulmonary
Medicine (U. Greinert) and therefore guarantees
out patient care in Borstel. This new structure is
PD Dr. Thomas Gutsmann, langjähriger Mitarbei-
able to ensure the execution of the multiple clini-
ter in der Biophysik, erhielt einen Ruf auf eine W3-
cal studies at the Medical Clinic in Borstel togeth-
Professur für Nanobiomechanik an der Univer-
er with the Clinical Study Center (head: C. Lange)
sität Ulm, zu dem wir ihm herzlichst gratulieren.
existing since 2005.
Im Jahr 2007 konnte an der Medizinischen Klinik
Our sincere thanks for the support and solidarity
Borstel nach langen Verhandlungen endlich ein
goes to the trustee of the RCB, Secretary of State
Medizinisches Versorgungszentrum (MVZ) ge-
de Jager. We are also especially obliged to the
gründet werden, das die Praxen für Pathologie
representative trustee, Senior Government Official
(E. Vollmer) und Lungen- und Bronchialheilkunde
PD Dr. L. Schaade (Federal Ministry for Health,
(U. Greinert) organisatorisch zusammenführt und
Bonn), the head of the administrative district of
eine Sicherung der ambulanten Krankenversor-
Bad Segeberg, G. Gorrisen, the chairman of the
gung in Borstel bedeutet. Diese neue Versor-
Scientific Advisory Board, Prof. Dr. Seeger, Gießen,
gungsstruktur kann zusammen mit dem seit 2005
his representative Prof. Dr. B. Wollenberg, Lübeck,
bestehenden Klinischen Studienzentrum (Leiter:
the chairman of the Foundation Board, Senior Gov-
C. Lange) an der Medizinischen Klinik Borstel die
ernment Official M. Wagner (Schleswig-Holstein
Durchführung der vielfältigen klinischen Studien
Ministry of Science, Economy and Traffic), the rep-
gewährleisten.
resentative chairman of the Foundation Board, As-
8
Wissenschaftlicher Jahresbericht 2007-2008
Vorwort
Preface
Herzlicher Dank für die zuverlässige Unterstüt-
sistant Secretary Franz J. Bindert (Federal Ministry
zung und Solidarität, ohne die wir in Borstel nicht
for Health, Bonn) as well as the Circle of Friends
arbeiten könnten, gilt Herrn Staatssekretär de Ja-
of the Research Center.
ger, dem Kurator des FZB. Unser besonderer
Dank für vielfältige Unterstützungen und freundschaftliche Verbundenheit gilt auch dem stellver-
The Board of Directors is most obliged to all sci-
tretenden Kurator, Herrn Oberregierungsrat PD
entists, doctors, members of the clinical staff, grad-
Dr. L. Schaade (Bundesministerium für Gesund-
uate students and laboratory assistants, adminis-
heit, Bonn), dem Landrat des Kreises Segeberg,
trative officials, the technical staff and all other
Herrn G. Gorrissen, dem Vorsitzenden des
Borstel colleagues, who actually made the above
Wissenschaftlichen Beirates, Prof. Dr. Seeger,
mentioned successes possible.
Giessen, dessen Stellvertreterin Prof. Dr. B. Wollenberg, Lübeck, dem Vorsitzenden der Stiftungs-
Silvia Bulfone-Paus
versammlung, Herrn Ministerialrat M. Wagner
Peter Zabel
(Ministerium für Wissenschaft, Wirtschaft und Ver-
Ulrich Schaible
kehr des Landes Schleswig-Holstein) und dem
stellvertretenden Vorsitzenden, Ministerialdirigent Franz J. Bindert (Bundesministerium für Gesundheit, Bonn), sowie dem Freundeskreis des
Forschungszentrums.
Es ist dem Direktorium ein ganz besonderes
Anliegen, an dieser Stelle allen Wissenschaftlerinnen und Wissenschaftlern, Ärzten, Krankenpflegemitarbeitern, Doktoranden und Laborassistenten, Verwaltungsangestellten, Mitarbeitern des technischen Dienstes und allen anderen
Borsteler Mitarbeitern und Mitarbeiterinnen, welche die oben genannten Erfolge erst ermöglicht
haben, unsere Hochachtung und unseren Dank
auszusprechen.
Silvia Bulfone-Paus
Peter Zabel
Ulrich Schaible
Wissenschaftlicher Jahresbericht 2007-2008
9
Organe des FZB
Boards of the FZB
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Die Organe der Stiftung
Forschungszentrum Borstel
Stiftungsversammlung/
Foundation Board
Kuratorium/
Board of Curators
Ministerialrat Michael Wagner
Staatssekretär Jost de Jager
Ministerium für Wissenschaft, Wirtschaft und Verkehr
Ministerium für Wissenschaft, Wirtschaft und Verkehr
des Landes Schleswig-Holstein, Kiel
Schleswig-Holstein (Vorsitzender)
(Vorsitzender)
oder
Oberregierungsrat PD Dr. Lars Schaade
Oberregierungsrat Klaus-Eckhard von Unruh
Bundesministerium für Gesundheit
Ministerium für Wissenschaft, Wirtschaft und Verkehr
(stv. Vorsitzender)
des Landes Schleswig-Holstein, Kiel
Referatsleiterin Maria Becker
Bundesministerium für Gesundheit
Ministerialdirigent Franz J. Bindert
Infektions- und Gesundheitsschutz
Bundesministerium für Gesundheit, Bonn
Mathias Diener-Sonnenberg
(stv. Vorsitzender)
Finanzministerium, Land Schleswig-Holstein
oder
Referatsleiterin Hanna Fangohr
Dr. Renée Buck
Außeruniversitäre Forschungseinrichtungen
Ministerium für Soziales, Gesundheit, Familie,
Behörde für Wissenschaft und Forschung
Jugend und Senioren, Schleswig-Holstein
Hochschulamt, Freie und Hansestadt Hamburg
Landrätin Jutta Hartwieg
Kreis Segeberg
Landrätin Jutta Hartwieg
Kreis Segeberg
Prof. Dr. Werner Seeger
Abteilungsdirektor Dr. Ulrich Reineke
Direktor der Klinik Innere Medizin, Justus von
Deutsche Rentenversicherung, Berlin
Liebig Universität Gießen
oder
Verwaltungsoberrat Uwe Wilhelm
Prof. Dr. Barbara Wollenberg
Deutsche Rentenversicherung, Berlin
Direktorin der Klinik für Hals-, Nasen- und Ohrenheilkunde, Universität zu Lübeck
Dr. Ingrid Künzler
Direktorium/Scientific Directors
Deutsche Rentenversicherung Nord, Lübeck
Prof. Dr. med. Dr. rer. nat. Silvia Bulfone-Paus
Abteilungsdirektor Helmut Josten
(Geschäftsführende Direktorin)
Deutsche Rentenversicherung Nord
Direktorin der Abteilung ‚Immunologie und Zell-
Freie und Hansestadt Hamburg
biologie’
Landesverwaltungsdirektor Peter-Oliver Weber
Prof. Dr. rer. nat. Ulrich E. Schaible
Deutsche Rentenversicherung Oldenburg-Bremen
Direktor der Abteilung ‚Immunchemie und Biochemische Mikrobiologie’
10
Wissenschaftlicher Jahresbericht 2007-2008
Organe des FZB
Boards of the FZB
Prof. Dr. med. Peter Zabel
Prof. Dr. Thomas Hünig
Direktor der Abteilung ‚Klinische Medizin’
Institutsvorstand des Institutes für Virologie und
Ärztlicher Direktor der Medizinischen Klinik Bor-
Immunologie,
stel und der Medizinischen Klinik III, Universitäts-
Bayerische Julius-Maximilians-Universität, Würzburg
klinikum Schleswig-Holstein/Campus Lübeck
Prof. Dr. Erika Jensen-Jarolim
Kollegium/Scientific Council
Head of the Department of Pathophysiology,
Prof. Dr. med. Dr. rer. nat. Silvia Bulfone-Paus
Center for Physiology and Pathophysiology,
(Geschäftsführende Direktorin)
Medical University of Vienna
Direktorin der Abteilung ‚Immunologie und Zellbiologie’
Prof. Dr. Thisbe Lindhorst
Direktorin des Institutes für Organische Chemie,
Prof. Dr. rer. nat. Ulrich E. Schaible
Christian-Albrechts-Universität zu Kiel
Direktor der Abteilung ‚Immunchemie und Biochemische Mikrobiologie’
Prof. Dr. Klaus Dieter Pfeffer
Direktor des Institutes für Medizinische Mikrobio-
Prof. Dr. med. Peter Zabel
logie und Krankenhaushygiene,
Direktor der Abteilung ‚Klinische Medizin’
Universität Düsseldorf
Ärztlicher Direktor der Medizinischen Klinik Borstel und
Betriebsrat/Working Council
der Medizinischen Klinik III, Universitätsklinikum
Heiko Käßner (Vorsitzender)
Schleswig-Holstein/Campus Lübeck
Manfred Richter (stv. Vorsitzender)
Prof. Dr. rer. nat. Heinz Fehrenbach
Zentrale Verwaltung/
Administration
Bereichsleiter ‚Experimentelle Pneumologie’
Susann Schrader
Prof. Dr. med. Stefan Ehlers
Verwaltungsdirektorin
Bereichsleiter ‚Mikrobielle Entzündungsforschung’
Sybille Lenkeit
Wissenschaftlicher Beirat/
Scientific Advisory Board
Haushalt-, Kassen- und Rechnungswesen
Prof. Dr. Werner Seeger
Bernd Poetzing
Direktor der Klinik für Innere Medizin II,
Finanz- und Patientenwesent
Justus von Liebig Universität Gießen
(Vorsitzender)
Karl-Heinz Lemm
Personalwesen
Prof. Dr. Barbara Wollenberg
Direktorin der Klinik für Hals-, Nasen- und Ohren-
Heinz-Jürgen Wefel
heilkunde,
Wirtschaftsdienst
Universität zu Lübeck (stv. Vorsitzende)
Dirk Grünes
Prof. Dr. Christoph Dehio
Technischer Dienst
Infection Biology, Biozentrum, Universität Basel
Wissenschaftlicher Jahresbericht 2007-2008
11
Organigramm des Forschungszentrums Borstel
Structural Plan of the Research Center Borstel
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Headline
Subline
deutsch
Fo r s c h u n g
szentrum
Bo r s t e l
Headline
Direktorium (Vorstand)
Subline englisch
Prof. Dr. Dr. S. Bulfone-Paus (geschäftsführende Direktorin)
Prof. Dr. P. Zabel
Prof. Dr. U. E. Schaible
Zentrale Verwaltung
Verwaltungsdirektorin
S. Schrader
Tel -220
Fax -721
Abteilung Immunchemie und
Biochemische Mikrobiologie
Prof. Dr. rer. nat. Ulrich E. Schaible
Tel -600
Fax -403
Abteilung Immunologie
und Zellbiologie
Prof. Dr. med. Dr. rer. nat. S. Bulfone-Paus
Tel -200
Fax -403
Medizinische Klinik
Abteilung Klinische Medizin
Prof. Dr. med. P. Zabel
zugleich Ärztlicher Direktor der Medizinischen Klinik
Tel -300
Fax -603
Tel -370
Oberärzte
PD Dr. C. Lange
Dr. U. Greinert
Dr. E. Müller
PD Dr. H.-P. Hauber
Ambulanzen
Umweltmedizin
Pneumologie
Päd. Allergologie
Fax -313
Tel -332
Tel -384
Tel -323
Tel -371
Dr. E. Müller
Prof. Dr. P. Zabel
Dr. G. Berger
Tel -351
Tel -351
Tel -351
Zentrale Einrichtungen
12
Wissenschaftlicher Jahresbericht 2007-2008
Organigramm des Forschungszentrums Borstel
Structural Plan of the Research Center Borstel
Telefon:
Zentrale
0 45 37-1 88 - 0
Durchwahl 0 45 37-1 88 - ...
Wissenschaftsreferat
Dr. B. Brand
Tel -439
Fax -403
Tel = Telefon-Durchwahl
Fax = Telefax-Durchwahl
LG = Laborgruppe
AG = Arbeitsgruppe
EU-Forschungsreferat
Dr. K. Loetzer
Tel -309
Fax -403
Haushalts-, Kassen- und
Rechnungswesen
Finanz- und Patientenwesen
Wirtschaftsdienst
S. Lenkeit
Tel -271
B. Poetzing
Tel -338
H.-J. Wefel
Tel -278
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Personalwesen
K.-H. Lemm
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Technischer Dienst
Fax -626
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Tel -277
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LG Immunchemie
LG Medizinische und Biochemische Mikrobiologie
LG Strukturbiochemie
Prof. Dr. U. Zähringer
Tel -462
Prof. Dr. H. Brade
Tel -474
Prof. Dr. O. Holst
Tel -472
Fax -745
LG Molekulare Infektiologie
LG Biophysik
Prof. Dr. S. Ehlers
Tel -481
PD Dr. T. Gutsmann
Tel -291
Fax -686
Juniorprofessur für
Molekulare Infektiologie
Emmy-Noether-NG
Immunbiophysik
Prof. Dr. C. Hölscher
Tel -586
PD Dr. A. Schromm
Tel -296
Fax -775
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AG Zelluläre Pneumologie
Fax -632
PD Dr. C. Stamme
Tel -581
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LG Immunbiologie
LG Zell. Immunologie
Prof. Dr. Dr. S. Bulfone-Paus
Tel -200
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Prof. Dr. A. J. Ulmer
Tel -448
Immunzell-Analytik
LG Biologische Chemie
LG Tumorbiologie
Dr. M. Ernst
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Tel -424
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Fax -435
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Tel -585
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LG Vet.-Infektiologie
und -Immunologie
LG Biochem. Immunologie
LG Angeborene Immunität
Prof. Dr. J. Ahmed
Tel -428
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Tel -451
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Tel -420
Fax -627
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LG Klin. u. Experim. Pathologie
LG Mykobakteriologie u.
Nat. Referenzzentrum
LG Molekulare und Klinische
Allergologie
Prof. Dr. Dr. E.Vollmer
Tel -250 o. -278
Dr. S. Rüsch-Gerdes
OA Dr. U. Greinert
Dr. W.-M. Becker
Dr. G. Berger
Fax -229
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LG Zelluläre Allergologie
AG Molekulare Mykobakteriologie
LG Klin. Immunpharmakologie
PD Dr. H. Haas
Tel -440
PD Dr. S. Niemann
Tel -762
Prof. Dr. P. Zabel
Tel -300
Fax -608
Fax -311
LG Klinische Infektiologie u.
Studienzentrum
LG Mukosaimmunologie
PD Dr. C. Lange
Tel -332
PD Dr. A. Frey
Tel -562
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Tierhaltung
Laborantenausbildung
Bibliothek und Dokumentation
Dr. I. Monath
Tel -742
Dr. S. Zähringer
Tel -381
PD Dr. N. Reiling
C. Engler, K. Geschuhn Tel -276/Fax -260
Fax -403
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EDV-Zentrale
Dr. K. Visser
Tel -605
Wissenschaftlicher Jahresbericht 2007-2008
Fax -745
13
Abteilung Immunchemie und Biochemische Mikrobiologie
Fo r s c h u n g s z e n t r u m
Bo r s t e l
14
Department of Immunochemistry and
Biochemical Microbiology
Wissenschaftlicher Jahresbericht 2007-2008
Abteilung Immunchemie und Biochemische Mikrobiologie
Department of Immunochemistry and
Biochemical Microbiology
Abteilung Immunchemie und
Biochemische Mikrobiologie
Department of Immunochemistry
and Biochemical Microbiology
Wissenschaftlicher Jahresbericht 2007 -2008
15
Zusammenfassung
Summary
Fo r s c h u n g s z e n t r u m
Bo r s t e l
new: Department
for Molecular
Infection Biology
neu: Abteilung für Molekulare Infektiologie
Abteilung für
Immunchemie
und Biochemische
Mikrobiologie
Department of
Immunochemistry
and Biochemical
Microbiology
Leiter
Head
Prof. Dr. rer. nat. Ulrich E. Schaible
Prof. Dr. rer. nat. Ulrich E. Schaible
Sekretariat
Secretary
Maike Kohlmorgen
Maike Kohlmorgen
Für die Abteilung Immunchemie und Biochemi-
The Department of Immunochemistry and Biochem-
sche
Jahre
ical Microbiology went through a transitional phase
2007/2008 eine Phase des Umbruchs. Professor
in 2007/2008. Professor Dr. Dr. Ernst Th. Rietschel,
Dr. Dr. Ernst Th. Rietschel, der die Abteilung (und
who directed the department (and as acting direc-
als Geschäftsführender Direktor immer wieder
tor the Research Center during several periods) su-
auch das Forschungszentrum) in beeindrucken-
perbly through many challenging times and estab-
der Weise durch oft schwierige Zeiten führte und
lished its excellent international standing, retired
ihr hervorragendes internationales Ansehen mit-
but took over the even greater task to lead the Leib-
begründete, wurde emeritiert, übernahm aber
niz Association as its President. As Professor Ri-
die ungleich größere Aufgabe des Präsidenten
etschel’s successor in 2008, I took over the fasci-
der Leibniz-Gemeinschaft. Als Nachfolger von
nating assignment to lead this outstanding
Prof. Rietschel übernahm ich 2008 die faszinie-
Department to future challenges. The change in di-
Mikrobiologie
bedeuteten
die
rende Aufgabe, diese hervorragende Abteilung
rectorship went along with a new name, Depart-
zu neuen Aufgaben zu führen. Die Übernahme
ment of Molecular Infection Biology (MIB), to ac-
dieser Position ging einher mit einer Umbenen-
commodate the interdisciplinary and holistic
nung der Abteilung in Abteilung für Molekulare
orientation study infectious diseases.
Infektiologie (MI), um die interdisziplinäre und
ganzheitliche Orientierung der Abteilung zu unterstreichen.
MIB combines research on the many facets of bacterial infections to study them in their entirety. The
research agenda covers from basic molecular anal-
MI kombiniert Forschung an den unterschiedlich-
ysis of bacterial structures and their genetic basis,
sten Facetten bakterieller Infektionen, um diese in
studies on pathogen and host interactions and the
ihrer Ganzheit zu erforschen. Das Forschungspro-
resulting immune responses as well as translation-
gramm der MI deckt verschiedene Bereiche von
al approaches in collaboration with the Clinical De-
der molekularen Analyse bakterieller Strukturen
partment. The prime foci of MIB are on pulmonary
und ihrer genetischen Grundlagen, Studien zu Er-
pathogens, in particular those, which cause Tuber-
reger-Wirt-Wechselwirkungen und den daraus re-
culosis and Cystic Fibrosis, but also on Chlamydia
sultierenden Immunantworten bis hin zu transla-
and other Gram-negative ones including environ-
tionalen Ansätzen in Zusammenarbeit mit der
mental bacteria and their influence on lung immu-
Abteilung Klinische Medizin ab. Den Hauptfokus
nity such as allergic reactions. Tuberculosis research
16
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
der Abteilung bilden Krankheitserreger der Lun-
comprises
studies
ge, insbesondere diejenigen, die Tuberkulose
cell/pathogen interactions and anti-tuberculosis im-
und Zystische Fibrose verursachen, aber auch
munity.
Chlamydien und andere Gram-negative Erreger,
pathogenicity - primarily in infections with Gram-
wie auch Umweltkeime und deren Einfluss auf die
negative bacteria - are studied with respect to their
Immunität der Lunge, wie beispielsweise auf all-
structural, genetic and functional characteristics
ergische Reaktionen. Die Tuberkuloseforschung
with a focus on immune modulation by bacterial
betreibt Studien zur Genetik des Wirtes, zu Wirts-
compounds and membrane biophysics of receptor-
Zell-Erreger-Interaktionen und der Immunität ge-
ligand interactions.
Factors
on
host
determining
genetics,
virulence
host
and
gen die Tuberkelbazillen. Faktoren, die die Virulenz und Pathogenität bestimmen – insbesondere
bei Infektionen mit Gram-negativen Bakterien –
MIB assembles a broad range of state of the art tech-
werden bezüglich ihrer strukturellen, genetischen
niques as well as the expertise and interdisciplinary
und funktionellen Charakteristika untersucht, wo-
approach of MIB’s excellent scientific staff. This al-
bei der Schwerpunkt auf der Immunmodulation
lows for a detailed but global analysis of a pathogen
durch bakterielle Moleküle und die Membran-
and the disease it causes, i.e. the infectious process
biophysik
in its entirety from the characterization of individual
von
Rezeptor-Ligand-Interaktionen
liegt.
bacterial molecules and their interactions with host
cell structures to their effect on the host and the resulting defence responses. Nuclear magnet reso-
MI vereinigt ein breites Spektrum an hochmoder-
nance (NMR) and mass spectroscopy (MS) are used
nen Techniken mit der Expertise und interdiszi-
for structural analysis of bacterial compounds. Atom-
plinären Ausrichtung seiner exzellenten wissen-
ic force microscopy (AFM) and micro calorimetry al-
schaftlichen Mitarbeiterinnen und Mitarbeiter.
low assessment of molecular interactions between
Das erlaubt eine detaillierte und umfassende
pathogen and host molecules. Using advanced life
Analyse eines Krankheitserregers und der durch
cell imaging and other microscopy techniques com-
ihn ausgelösten Krankheit, z.B. den Prozess einer
bined with experimental tissue culture, in vitro organ
Infektion in seiner Ganzheit von der Charakteri-
and animal models the host-pathogen interplay can
sierung eines einzelnen bakteriellen Moleküls
be dissected. Our large collection of gene knock-out
und seiner Interaktion mit Wirtszell-Strukturen bis
and transgenic mouse models paired with safety lev-
hin zu seinem Effekt auf den Wirt und die daraus
el 3 labs for work with tubercle bacilli (Mycobacteri-
resultierenden Abwehrmechanismen. Kernspin-
um tuberculosis) provides the great opportunity to
Magnet-Resonanz (Nuclear Magnet Resonance;
study the host response to tuberculosis, one of the
NMR) und Massenspektroskopie (MS) werden für
most important human infections world-wide. Finally,
die strukturelle Analyse von bakteriellen Be-
in collaboration with other groups within the Clinical
standteilen genutzt. Atomare Kraftmikroskopie
and Immunology/Allergy departments of the Re-
(Atomic Force Microscopy; AFM) und Mikrokalori-
search Center Borstel, we direct our research to-
metrie erlauben die Bestimmung molekularer In-
wards translational approaches. The allocation of
teraktionen zwischen Krankheitserregern und
novel findings and inventions to benefit medical
Wirtsmolekülen. Durch die Verwendung von Mi-
progress is facilitated by their translation into patents
kroskopie an lebenden Zellen (Advanced Life
and clinical applicability, by which we hope to con-
Cell Imaging) und anderer mikroskopischer und
tribute to the development of novel diagnostics and
bildgebender Verfahren in Kombination mit ex-
therapeutics including anti-tuberculosis and anti-al-
perimentellen Gewebekulturen können in vitro
lergy drugs.
Wissenschaftlicher Jahresbericht 2007-2008
17
NMR, MS, AFM,
micro calorimetry,
life cell imaging
NMR, MS, AFM,
Mikrokalorimetrie,
Life Cell Imaging
Zusammenfassung
Summary
Organ- und Tiermodelle der Wirts-Erreger-Wech-
At the intersection between infection and allergy re-
selwirkung analysiert werden. Unsere große
search, the Division of Structural Biochemistry lead
Sammlung von Gen Knock-Out und transgenen
by Prof. Dr. Otto Holst, unfolded structures of bac-
Mausmodellen und das Vorhandensein von La-
terial origin which are protective against allergic re-
boratorien der Sicherheitsstufe 3 für die Arbeit mit
actions. Several epidemiological studies revealed
Tuberkulosebakterien (Mycobacterium tuberculo-
an allergy protective effect of farm life in early child-
sis) bieten die großartige Möglichkeit, die Tuber-
hood. Children grown up on traditional farms in the
kulose, eine der wichtigsten Infektionskrankheiten
south of Germany presented with significantly re-
des Menschen weltweit, genauer zu untersuchen.
duced occurrence of asthmatic symptoms, hay fever
Schließlich arbeiten wir zusammen mit anderen
and atopic sensitization in comparison to other chil-
Gruppen
immunologi-
dren living in the same area. The division’s earlier
schen/allergologischen Bereiche am Forschungs-
studies provided insight to the protective effect of
zentrum Borstel an translationalen Forschungs-
cowshed dusts. Recently, Otto Holst and his col-
ansätzen. Wir hoffen, zur Entwicklung neuer
leagues analysed the microbial flora of protective
Diagnostika und Therapeutika (u. a. auch Medi-
cowshed dusts including the most prominent bac-
kamente gegen Tuberkulose und Allergien) bei-
terial spores. Of a number of bacterial species iden-
tragen zu können, auch indem wir neue Ent-
tified in cowsheds, two were selected, isolated, and
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Acinetobacter lwoffii &
Lactococcus lactis:
reduce allergic reactions
der
klinischen
und
deckungen und Erfindungen durch Patente
characterized, namely Acinetobacter lwoffii F78
klinisch anwendbar machen.
and Lactococcus lactis G121. The isolates were investigated with regard to their activation through
pattern recognition receptors and subsequent in-
Acinetobacter lwoffii &
Lactococcus lactis: Verringerung der allergischen Reaktion
An der Schnittstelle zwischen Infektion und Aller-
duction of human monocyte-derived dendritic cells
gie hat die von Prof. Dr. Otto Holst geleitete For-
maturation, inflammatory cytokines, T-helper cell 1
schergruppe Strukturbiochemie Strukturen bakteriellen Ursprungs entschlüsselt, die gegen
(TH1) polarizing Notch ligand expression and their
effect on the allergic phenotype. It was found that
allergische Reaktionen schützen. Verschiedene
both bacterial isolates were capable to reduce al-
epidemiologische Studien zeigten, dass ein Le-
lergic reactions in mice and induce a T-helper 1 cell
ben auf einem Bauernhof in früher Kindheit ge-
polarizing program in dendritic cells. These data
gen Allergien schützt. Kinder, die auf traditionel-
strongly support the hygiene hypothesis, i.e. that an
len Bauernhöfen in Süddeutschland aufwachsen,
environment rich in microbiological structures such
entwickeln deutlich seltener asthmatische Sym-
as the farming environment can protect from aller-
ptome, Heuschnupfen oder atopische Erkrankun-
gies. Furthermore, it was found that bacterial spores
gen im Vergleich zu anderen Kindern, die in der
in contrast to vegetative bacteria stimulated a TH1
gleichen Gegend leben. Frühere Untersuchungen
biasing cytokine profile. Application of Bacillus
dieser Forschergruppe erlaubten Einblicke in den
licheniformis spores prevented allergy-associated
Schutzeffekt von Stäuben aus Kuhställen. Kürzlich
eosinophilia and goblet cell hyperplasia in mice.
analysierten Otto Holst und seine Kollegen die mi-
This suggests that bacterial spores from a farming
krobielle Flora von schützenden Stäuben aus Kuh-
environment can suppress development of allergic
ställen inklusive der häufigsten Bakteriensporen.
asthma focusing the hygiene hypothesis to the func-
Aus einer Vielzahl von Bakterien, die in Kuhstäl-
tion of bacterial spores in allergy protection.
len gefunden wurden, wurden zwei ausgewählt,
isoliert und charakterisiert, nämlich Acinetobac-
ter lwoffii F78 und Lactococcus lactis G121. Die
The historically strong engagement of the Research
Isolate wurden auf ihren Effekt hin untersucht, den
Center in analysing the biological properties and
18
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
sie auf Strukturerkennungsrezeptoren haben und
structural entities of the main cell wall glycolipid of
auf in der Folge ablaufende Aktivierungssignale:
Gram-negative bacteria, lipopolysaccharide (LPS)
Reifung menschlicher dendritischer Zellen, Aus-
was still a main topic in 2007/2008. The Division of
schüttung entzündlicher Zytokine, Polarsierung
Structural Biochemistry found that the hexasaccha-
hin zu T-Helfer 1 Zellen durch Notch-Liganden Pro-
ride of the outer core (OC) region of Yersinia ente-
duktion und Effekt auf allergische Reaktionen. Es
rocolitica serotype O:3 is essential for the integrity
wurde festgestellt, dass beide bakteriellen Isola-
of the outer membrane and virulence during early
te in der Lage waren, allergische Reaktion in
phases of infection. This hexasaccharide is most
Mäusen zu verringern und dass sie ein T-Helfer 1
likely involved in Yersinia’s resistance against
(TH1) Zellpolarisierungsprogramm in dendriti-
cationic antimicrobial peptides (AMP). It was found
schen Zellen anregen. Diese Daten stützen nach-
that the proximal sugar residue of the OC hexas-
haltig die Hygienehypothese, also, dass eine Um-
accharide is a 2-acetamido-2,6-dideoxy-D-xylo-hex-
gebung,
4-ulopyranose but not a 2-acetamido-2,6-dideoxy-D-
die
reich
an
mikrobiologischen
(D-FucpNAc,
N-acetyl-D-fuco-
Strukturen ist, wie z.B. ein Bauernhof, vor Allergi-
galactopyranose
en schützen kann. Weiterhin wurde herausgefun-
samine) as previously published. It was also demon-
den, dass Bakteriensporen im Gegensatz zu ve-
strated that WbcP is the biosynthetic enzyme con-
getativen
förderndes
verting UDP-2-acetamido-2-deoxy-D-glucopyranose
Zytokinprofil auslösen. Die Anwendung von Spo-
(UDP-D-GlcpNAc, UDP-N-acetyl-D-glucosamine) to
ren des Bacillus licheniformis verhinderte die mit
UDP-2-acetamido-2,6-dideoxy-D-xylo-hex-4-ulopyra-
Allergien assoziierte Eosinophilie und Hyperpla-
nose. Furthermore, evidence was provided that the
sie der Goblet Zellen in Mäusen. Dies lässt ver-
keto/diol-character of the sugar residue is crucial for
muten, dass Bakteriensporen von Bauernhöfen
the biological role of OC, i.e. recognition by bacte-
die Entwicklung von allergischem Asthma unter-
riophage, enterocoliticin and specific monoclonal
drücken können, was das Augenmerk der Hygie-
antibodies.
Bakterien
ein
TH1
Hexasaccharide may be
involved in Yersinia resistance against antimicrobial peptides
nehypothese auf die Funktion von Bakteriensporen im Schutz gegen Allergien lenkt.
The Division of Biophysics headed by PD Dr. Thomas
Gutsmann established a system to analyse the inDas historisch gewachsene große Engagement
teraction between AMP and other host proteins and
des Forschungszentrums Borstel bei der Analyse
model membranes. Atomic force microscopy was
der biologischen Eigenschaften und strukturellen
used to define the localization of the LPS binding
Eigenheiten des Hauptzellwandglykolipids Gram-
protein (LBP), essential for LPS-signalling, to choles-
negativer Bakterien, Lipopolysaccharid (LPS),
terol-rich domains in reconstituted lipid membranes.
war auch in den Jahren 2007/2008 ein Hauptthe-
Through the collaborative research centre SFB 617
ma der MI. Die Forschergruppe Strukturbioche-
and the Fond for Innovation of Schleswig-Holstein,
mie fand heraus, dass das Hexasaccharid der
a novel biosensor and fluorescence spectrometry
äußeren Kernregion von Yersinia enterocolitica
system, respectively, became available in order to
Serotyp O:3 unerlässlich ist für die Integrität der
study receptor-ligand binding and membrane in-
äußeren Membran und die Virulenz während der
teractions with tremendously improved sensitivity
frühen Phase der Infektion. Dieses Hexasaccharid
and resolution. These techniques allowed studies
ist wahrscheinlich an der Resistenz von Yersinia
on the microbicidal effectiveness of AMP against a
gegen kationische antimikrobielle Peptide (AMP)
number of bacterial species including mycobacte-
beteiligt. Der proximale Zucker der äußeren Kern-
ria and the underlying molecular mechanism. It
region des Hexasaccharids ist ein 2-Acetamido-
should be mentioned that Arenicin-1, an AMP de-
Wissenschaftlicher Jahresbericht 2007-2008
19
Resistenz von Yersinia
gegen kationische antimikrobielle Peptide
Zusammenfassung
Summary
2,6-Dideoxy-D-xylo-hex-4-Ulopyranose und nicht
rived from the marine sand worm Arenicola areni-
ein 2-Acetamido-2,6-Dideoxy-D-Galactopyranose
cola, is of special therapeutic interest due to its re-
(D-FucpNAc, N-acetyl-D-Fucosamine) wie ursprüng-
quirements in temperature and salt concentrations.
lich beschrieben. WbcP ist das biosynthetisch re-
In cooperation with the novel Division of Cellular Mi-
levante Enzym, das UDP-2-Acetamido-2-deoxy-D-
crobiology (Prof. Dr. Ulrich Schaible), a simple re-
Glucopyranose (UDP-D-GlcpNAc, UDP-N-acetyl-
constituted membrane model of the outer mem-
D-glucosamine) in UDP-2-Acetamido-2,6- dideoxy-
brane of the tubercle bacillus was established,
D-xylo-hex-4-Ulopyranose umwandelt. Es konnte
which demonstrated that AMP effectiveness was
auch gezeigt werden, dass der Keton/Diol-Cha-
hampered by mycobacterial cell wall lipids. This ex-
rakter des Zuckeranteils für die biologische Rolle
plains the surprisingly low anti-mycobacterial activ-
der äußeren Kernregion, z.B. für die Erkennung
ity of such AMP. The Division of Biophysics works in
durch Bakteriophagen, Enterocoliticin und spezi-
close association with the Emmy-Noether-Junior Re-
fische monoklonale Antikörper, wichtig ist.
search Group of Immunobiophysics headed by PD
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Dr. Andra Schromm. This group characterized the biological activity of bacterial lipopeptides in both vir-
The concept of 'endotoxic
conformation' may be
also applicable to
lipopeptides
Die von PD Dr. Thomas Gutsmann geleitete For-
ulence and immune stimulation. Physicochemical
schergruppe Biophysik hat ein System etabliert,
and biological characterisation of synthetic
mit dem die Wechselwirkung zwischen AMP und
lipopeptides modelled by bacterial blue prints re-
anderen Wirtsproteinen sowie Modellmembra-
vealed that the concept of ‘endotoxic conformation’,
nen analysiert werden kann. Atomic Force Mikro-
i.e. the physicochemical information of the
skopie wurde benutzt, um mit Hilfe von rekonsti-
molecules is also applicable to lipopeptides. This
tuierten Lipidmembranen die Lokalisation des
concept, which has originally been developed for
LPS-bindenden Proteins (LBP), welches essentiell
lipopolysaccharides, constitutes a general principle
für die Zellaktivierung durch LPS ist, Cholesterol-
for the molecular basis of cell activation by am-
reichen Bereichen zuzuordnen. Mit Mitteln aus
phiphilic molecules. Collaborative studies together
dem Sonderforschungsbereich 617 und dem In-
with the group of PD Dr. Cordula Stamme (Cellular
novationsfond Schleswig-Holstein konnten ein
Pneumology) revealed mechanisms of lung-specific
neues Biosensorsystem und ein Fluoreszenz Spek-
immune regulation as executed by pulmonary C-
trometrie System angeschafft werden, mit dem
type lectins.
Rezeptor-Ligand-Bindungen und Membran-Wechselwirkungen mit enorm verbesserter Sensitivität
und Auflösung untersucht werden können. Diese
Also highly active in LPS research, the Division of Im-
Techniken erlauben Untersuchungen der anti-bak-
munochemistry headed by Prof. Dr. Ulrich Zähringer
teriellen Aktivität von AMP gegen eine Anzahl
pioneered the usage of nuclear magnetic reso-
von Bakterienspezies, u.a. auch Mykobakterien,
nance (NMR) to analyse the structures and dynam-
und der zugrunde liegenden molekularen Me-
ics of endotoxins, the molecular principle of sepsis
chanismen. Es sollte nicht unerwähnt bleiben,
resulting from exaggerated innate immune re-
dass Arenicin-1, ein aus dem Wattwurm Arenico-
sponses to bacterial endotoxins. Sepsis remains a
la arenicola gewonnenes AMP, aufgrund seiner
global health threat with high risk of morbidity and
Anforderungen an Temperatur und Salzgehalt
mortality. LPS of Gram-negative bacteria is the pro-
von besonderem therapeutischem Interesse ist. In
totypic inducer of sepsis based on its activation of
Zusammenarbeit mit der neuen Forschergruppe
haematopoietic immune cells through the Toll-like
Zelluläre Mikrobiologie wurde ein einfaches re-
receptor (TLR) 4/MD-2 complex and subsequent re-
konstituiertes Membranmodell der äußeren Mem-
lease of pro- and anti-inflammatory mediators.
20
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
bran des Tuberkelbazillus etabliert, welches zeig-
However, the molecular interaction between the LPS
te, dass die Effektivität bestimmter AMPs durch
and the TLR4/MD-2 complex is still undefined on a
mykobakterielle Zellwandlipide gehemmt wurde.
structure-function relationship. Gaining insight into
Dies erklärt die überraschend niedrige antimyko-
this interaction will hopefully improve therapeutic
bakterielle Aktivität solcher AMPs. Die Forscher-
regimens against sepsis. In cooperation with the
gruppe Biophysik arbeitet eng mit der von PD Dr.
group of Stephan Grzesiek, NMR Structural Biology,
Andra Schromm geleiteten Emmy-Noether-Nach-
Biozentrum Basel, Switzerland, the Division of Im-
wuchsgruppe Immunbiophysik zusammen. Diese
munochemistry successfully developed a novel nu-
Gruppe hat die biologische Aktivität von bakteri-
clear magnetic resonance (NMR) approach to de-
ellen Lipopeptiden sowohl in der Virulenz wie
auch in der Immunstimulation charakterisiert. Die
termine structure and dynamics of LPS in aqueous
solution. This was achieved by uniformly 13C,15N-la-
physikochemische und biologische Charakterisie-
belling of LPS in genetically well-defined Es-
rung von synthetischen Lipopeptiden, die den
cherichia coli strains and keeping isolated LPS in so-
bakteriellen Vorlagen nachgebaut wurden, ha-
lution by di-hexanoyl-phosphatitdylcholine (DHPC)
ben gezeigt, dass das Konzept der endotoxi-
as a mimetic of the bacterial membrane. These LPS
schen Konformation, also die physikochemische
preparations showed well-resolved NMR spectra,
Information der Endotoxinmoleküle (LPS), auch
which made LPS amenable to analysis by modern
auf Lipopeptide übertragbar ist. Dieses Konzept,
heteronuclear NMR methods for the first time to
welches ursprünglich für LPS entwickelt wurde,
study conformation and dynamics of LPS as a key-
stellt damit ein allgemeines Prinzip für die mole-
event in LPS-TLR4/MD-2 interaction. Moreover, this
kulare Basis der Zellaktivierung durch amphiphi-
NMR approach proved to be applicable to analyse
le Moleküle dar. Studien, die in Zusammenarbeit
a broad panel of complex LPS and lipid A structures
mit der Gruppe von PD Dr. Cordula Stamme (Zel-
under natural conditions resembling the bacterial
luläre Pneumologie) durchgeführt wurden, deck-
membrane. Within the Division of Immunochemistry,
ten Mechanismen Lungen-spezifischer Immunre-
the group of PD Dr. Buko Lindner, specialized in dif-
gulation auf, die von pulmonaren C-Typ Lektinen
ferent methods of mass spectrometry, used capil-
ausgeführt wurden.
lary electrophoresis coupled to Fourier-transform
ion cyclotron resonance mass spectrometry to separate and analyse complex mixtures of glycolipids.
Auch die von Prof. Dr. Ulrich Zähringer geleitete
Native glycolipid preparations (glycosphingolipid,
Forschergruppe Immunchemie ist sehr aktiv in der
lipopolysaccharides) are inherently highly hetero-
LPS-Forschung. Sie war wegbereitend für die Nut-
geneous with respect to numbers and types of fat-
zung von NMR für die Analyse der Struktur der
ty acids, oligosaccharides and non-stoichiometric
Endotoxine (LPS) und ihrer Dynamik. Endotoxine
substituents (P, P-Etn, and Ara4N), which hamper
stellen das molekulare Prinzip der Sepsis dar, die
their structural characterisation. In the past, chro-
aus der überbordenden angeborenen Immun-
matographic separation of highly amphiphilic com-
antwort gegen bakterielle Endotoxine entsteht.
pounds by HPLC or TLC for subsequent chemical
Sepsis bleibt eine globale Gesundheitsbedro-
analyses failed. To overcome these problems, cap-
hung mit einem hohen Krankheits- und Sterblich-
illary electrophoresis (CE) was linked online to a
keitsrisiko. LPS von Gram-negativen Bakterien ist
Fourier-transform ion cyclotron resonance mass
der prototypische Verursacher einer Sepsis, da-
spectrometer (FT-ICR MS) using a sheath liquid in-
durch, dass es haematopoetische Immunzellen
terface. This novel technique allowed for the first
über den Toll-like Rezeptor (TLR) 4/MD-2 Komplex
time separation and online mass analyses of com-
aktiviert und dadurch zur Freisetzung von pro-
ponents within non-derivatized R-Form LPS and lipid
Wissenschaftlicher Jahresbericht 2007-2008
21
Physikochemische Information des LPS auch auf
Lipopeptide übertragbar
Zusammenfassung
Summary
und anti-inflammatorischen Mediatoren führt. Al-
A as well as more complex mixtures with detection
lerdings sind die Struktur-Funktion-Zusammen-
limits in the low fmol range.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
hänge der molekularen Interaktion zwischen LPS
und TLR4/MD-2 noch immer unverstanden. Die
NMR Methode zur Bestimmung der Struktur
und der Dynamik von
LPS in wässriger Lösung
Aufdeckung dieser Zusammenhänge wird hof-
The Division of Medical and Biochemical Microbi-
fentlich die therapeutischen Möglichkeiten gegen
ology headed by Prof. Dr. Helmut Brade generated
Sepsis verbessern. In Kooperation mit der Grup-
and characterized antibodies against LPS of Gram-
pe von Stephan Grzesiek, NMR Structural Biology,
negative bacteria. The aim was to get hold of anti-
Biozentrum Basel, Schweiz, entwickelte die For-
bodies which can neutralize LPS of sepsis agents
schergruppe Immunchemie erfolgreich eine neue
such as Escherichia coli. As previous approaches us-
NMR-Methode, mit der Struktur und Dynamik von
ing neoglycoconjugates for immunization failed, a
LPS in wässriger Lösung bestimmt werden kön-
novel strategy through vaccination with anti-idio-
nen. Dies wurde dadurch erreicht, dass LPS in ge-
typic antibodies was successful in inducing LPS-neu-
netisch gut definierten Escherichia coli Stämmen
einheitlich mit 13C,15N gekennzeichnet wurden.
tralizing antibodies. The single chain technique is
Isoliertes LPS wurde dann durch Di-Hexanoyl-
which represent as putative therapeutics against
Phosphatitdylcholine (DHPC) quasi zur Nachah-
sepsis. Of special interest was the LPS of intracellu-
mung der bakteriellen Membran in Lösung ge-
lar Chlamydia pathogens. The crystal structure of
halten. Diese LPS-Präparationen zeigten gut
antibodies specific for Chlamydia LPS complexed
aufgelöste NMR-Spektren, die es zum ersten Mal
with natural oligosaccharides lead to the rational
ermöglichten, LPS mit modernen heteronuklearen
design of a synthetic neoglycoconjugate. This con-
Methoden zu analysieren, um so Konformation
jugate was used to generate specific antibodies
und Dynamik des LPS als Schlüsselereignis in der
against Chlamydophila psittaci but not other
LPS-TLR4/MD-2 Interaktion zu untersuchen. Außer-
Chlamydia species, which proved to be a useful tool
dem zeigte sich, dass diese NMR-Methode auf
to specifically diagnose C. psittaci. Successful co-
die Analyse eines breiten Spektrums komplexer
crystallization of antibodies and Chlamydia-LPS- de-
LPS und Lipid A Strukturen unter natürlichen Be-
rived oligosaccharides revealed that specificities to
dingungen ähnlich der bakteriellen Membran an-
3-Desoxy-d-manno- oct-2-ulosonacid (Kdo) are germ
wendbar ist. Innerhalb der Forschergruppe Im-
line determined. Studies using synthetic oligosac-
munchemie hat sich die Gruppe von PD Dr. Buko
charide ligands therefore revealed that KDO-spe-
Lindner auf verschiedene Methoden der Mas-
cific antibodies expose a high flexibility with re-
senspektrometrie spezialisiert. Die Gruppe ent-
spect to their species specificity, which is due to low
wickelte eine Kapillarelektrophorese Methode in
affine cross reactivity allowing affinity maturation
Kombination mit Fourier-Transform Ion Cyclotron
and specification upon antigen encounter.
currently used to generate recombinant antibodies,
Resonanz Massenspektrometrie, um komplexe
Mixturen von Glykolipiden trennen und analysie-
Phage display to identify
a novel single chain antibody specific for Man6P
on glycoproteins
ren zu können. Natürliche Glykolipid-Präparatio-
Within the Division of Medical and Biochemical Mi-
nen (Glykosphingolipide, Lipopolysaccharide)
crobiology, PD Dr. Sven Müller-Loennies in cooper-
sind grundsätzlich höchst heterogen bezüglich
ation with Prof. Dr. Thomas Braulke, University Clin-
Anzahl und Art von Fettsäuren, Oligosacchariden
ics Hamburg-Eppendorf, employed phage display
und nicht-stoichiometrischen Bestandteilen (P, P-
to identify a novel single chain antibody specific for
Etn, und Ara4N), welche die strukturelle Charak-
mannose-6-phosphate (Man6P) on glycoproteins.
terisierung behindern. In der Vergangenheit
As a proof-of-principle, this antibody fragment can
scheiterte die chromatographische Auftrennung
be used to diagnose certain lysosomal storage dis-
22
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
von hoch amphiphilen Bestandteilen durch HPLC
eases and to purify Man6P-carrying proteins to im-
oder TLC für die anschließende chemische Ana-
prove enzyme supplementation therapy which will
lyse. Um dieses Problem zu umgehen, wurde die
gain lead to translational approaches.
Kapillarelektrophorese (KE) online mit einem Fourier-Transform Ion Cykolotron Resonanz Massenspektrometer (FT-ICR MS) verbunden, wofür ein
Beneath structural analyses of Gram-negative cell
Sheath-Liquid-Interface genutzt wird. Diese neue
wall glycolipids, the prime focus of MIB in
Technik erlaubt zum ersten Mal sowohl die Tren-
2007/2008 laid in tuberculosis research. In the Divi-
nung und Online-Massenanalyse von Bestandtei-
sion of Molecular Infection Biology (Prof. Dr. Stefan
len im nicht-derivatisierten R-Form LPS und im Li-
Ehlers), the model of aerosol tuberculosis in mice
pid A wie auch von komplexeren Mixturen mit
was employed to define host- and pathogen-de-
Nachweisgrenzen im niedrigen fmol Bereich.
pendent determinants of tuberculosis pathogenesis
with a focus on innate receptors. Using a humanized
mouse model to investigate the role of the C-type
Die von Prof. Dr. Helmut Brade geleitete For-
lectin DC-SIGN, novel data suggest that instead of
schergruppe Medizinische und Biochemische
favouring the immune evasion of mycobacteria, hu-
Mikrobiologie hat Antikörper gegen LPS von
man DC-SIGN may have evolved as a pathogen re-
Gram-negativen Bakterien entwickelt und cha-
ceptor promoting protection by limiting TB-induced
rakterisiert. Das Ziel war, einen Antikörper zu er-
pathology. Focusing on the relative role of Toll-like
halten, der LPS von Sepsiskeimen wie Escherichia
receptors and the common adapter molecule
coli neutralisieren kann. Erste Versuche mit Neo-
MyD88, recent work demonstrates that Toll-like re-
glykokonjugaten als Immunisierung schlugen fehl.
ceptors are not essential for effective immunity to M.
Hingegen war eine neue Strategie der Vakzinati-
tuberculosis in mice. However, MyD88 is essential
on mit anti-idiotypischen Antikörpern erfolgreich
for protective host defense in tuberculosis, which is
und induzierte LPS-neutralisierende Antikörper.
however not due to signal transduction from TLR 2,
Die Einzelkette - (Single-Chain) Technik wird der-
4 or 9. Of interest, adaptive immunity to M. tuber-
zeit genutzt, um rekombinante Antikörper zu ge-
culosis is largely TLR/MyD88-independent. Studies
nerieren, die möglicherweise für die Therapie ge-
using M. tuberculosis gene deletion mutants re-
gen Sepsis eingesetzt werden könnten. Von
vealed that inflammasome activation and IL-1beta
besonderem Interesse war das LPS der intrazel-
processing depends on a putative Zn(2+) metallo-
lulären Bakterien aus der Chlamydien Familie.
protease. Although nitrate reductase and arginine
Die Kristallstruktur von Antikörpern, die charakte-
deaminase
ristisch für Chlamydien-LPS sind, zusammen mit
metabolism of M. tuberculosis, mutants deficient in
natürlichen Oligosacchariden führte zum Design
these enzymes were not defective in persistence.
eines synthetischen Neoglykokonjugates. Dieses
This prompted a thorough investigation of the phys-
Konjugat wurde benutzt, um spezielle Antikörper
iological status of granulomatous lesions in animal
gegen Chlamydophila psittaci, aber nicht gegen
models. These studies revealed that tuberculosis le-
andere Chlamydien-Spezies, herzustellen, was
sions in the murine lung were not hypoxic, which
sich als nützliches Werkzeug bei der Diagnose
was however in sharp contrast to necrotizing gran-
von C. psittaci erwies. Die erfolgreiche Ko-Kristal-
ulomas in guinea pigs showing a hypoxic environ-
lisation von Antikörpern und Oligosacchariden
ment. In contrast to M. tuberculosis, the opportunist
are
important
for
anaerobic
aus dem Chlamydien-LPS ließ erkennen, dass die
M. avium induces necrotizing granulomas in mice.
Antikörperspezifität gegen 3-Desoxy-d-manno-oct-
These lesions, which develop in an IFN-γ and IRF-1-
2-Ulosonsäure (Kdo) von den Keimzellen deter-
dependent manner, were also hypoxic. To further
Wissenschaftlicher Jahresbericht 2007-2008
23
DC-SIGN may have
evolved as a pathogen
receptor
Induktion von
LPS neutralisierenden
Antikörpern
Zusammenfassung
Summary
miniert ist. In Untersuchungen, bei denen synthe-
address the microenvironment in which mycobac-
tische Oligosaccharide verwendet wurden, zeig-
teria reside in vivo, a new technique was estab-
te sich, dass Kdo- spezifische Antikörper eine ho-
lished to isolate mycobacteria-containing phago-
he Flexibilität bezüglich ihrer Spezies- Spezifität
somes from primary macrophages based on
zeigten, was durch eine niedrige affine Kreuzre-
immunomagnetic labelling.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
aktivität verursacht wird. Diese erfahren allerdings nach Antigenerkennung eine mit der AffiThe Divison of Infection Biology (Junior Professor)
nitätsreifung einhergehenden Spezifizierung.
lead by Prof. Dr. Christoph Hölscher focused on the
cytokine milieu essential for protective immunity in tuInnerhalb der Forschergruppe Medizinische und
berculosis. This group revealed that alternative
Biochemische Mikrobiologie hat PD Dr. Sven Mül-
macrophage activation is associated with reactiva-
ler-Loennies in Kooperation mit Prof. Dr. Thomas
tion and lung pathology in tuberculosis. Reactivation
Braulke, Universitätsklinikum Hamburg-Eppen-
and granuloma necrosis are pivotal events of post-
dorf, Phage Display eingesetzt, um einen neuen
primary tuberculosis. In patients with pulmonary tu-
Single Chain Antikörper zu herzustellen, der für
berculosis from Ghana, a structural variant of the in-
Mannose-6-Phosphat- (Man6P) auf Glycoprotei-
terleukin (IL)-4 receptor-alpha (Rα) was found to be
nen spezifisch ist. Dieses Antikörperfragment
associated with an increased transcription of the
kann genutzt werden, um bestimmte lysosomale
gene and increased cavity size indicating a promi-
Speicherkrankheiten zu diagnostizieren und um
nent role of the IL-4Rα in human granuloma necrosis.
Man6P-tragende Proteine zur Verbesserung der
Detailed analysis in mice overexpressing IL-4Rα-me-
Enzym-Ergänzungs-Therapie zu reinigen. Diese Er-
diated signals revealed that alternative macrophage
gebnisse zeigen interessante Ansätze für die
activation is associated with M. tuberculosis re-
translationale Forschung auf.
crudescence and granuloma necrosis independent
of overt effects on anti-mycobacterial T cell immunity. These studies demonstrated the contribution of IL-
Therapeutic interference
with IL-17-dependent
pathways holds the risk
of reactivating latent
tuberculosis
Neben der strukturellen Analyse Gram-negativer
4Rα-dependent mechanisms to the pathogenesis of
Zellwandglykolipide lag der Schwerpunkt der Ab-
post-primary tuberculosis. In recent years, the cy-
teilung Molekulare Infektiologie in den Jahren
tokine IL-17 attracted the attention of tuberculosis re-
2007/2008 in der Tuberkuloseforschung. Die For-
search. The Group of Christoph Hölscher could
schergruppe Molekulare Infektionsbiologie (Prof.
demonstrate that IL-17 is essential to maintain pro-
Dr. Stefan Ehlers) nutzt insbesondere das Maus-
tective immunity during M. tuberculosis infection. A
modell der Inhalationstuberkulose, um Wirts- und
variety of autoimmune disorders have been shown to
Erreger abhängige Determinanten der Krankheit-
depend on IL-17-producing T helper (TH) 17 cells.
sentwicklung zu definieren. Untersuchungen an
Therapeutic blockade of TH17 development may pro-
für das menschliche C-Typ Lectin DC-SIGN trans-
vide a novel approach to avoid adverse conse-
genen Mäusen lassen vermuten, dass dieser Re-
quences of anti-inflammatory strategies such as re-
zeptor vor allem zum Schutz vor der infektions-
activation of latent tuberculosis as a result of
vermittelten Gewebepathologie beiträgt. Bei der
gezielten Analyse einzelner Toll-like Rezeptoren
anti-autoimmune therapies. Their findings, however,
revealed that IL-17 is essential for maintaining CD4+
(TLR) und deren gemeinsamen Signal-Adapter-
TH1 cell-dependent protection during the chronic
moleküls MyD88 fiel auf, dass TLRs und MyD88 für
stage of tuberculosis. In conclusion, therapeutic in-
die Entwicklung der anti-tuberkulösen T-Zell-Im-
terference with IL-17-dependent pathways holds the
munität nicht essentiell sind. Hingegen ist intaktes
risk of reactivating latent tuberculosis.
24
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
MyD88 unabdingbar für den antibakteriellen
The new division of Cellular Microbiology headed
Schutz, jedoch nicht aufgrund seiner Funktion als
by Prof. Dr. Ulrich Schaible is also primarily focusing
Adaptermolekül für TLRs. Gezielte Deletionsmu-
on tuberculosis research. The main interests are the
tanten in M. tuberculosis haben gezeigt, dass die
role of micronutrients and innate immune players in
Aktivierung des Inflammasoms und die IL-1beta-
host defence against M. tuberculosis and the intra-
Prozessierung durch eine Zn(2+) Metalloprotease
cellular survival strategies of tubercle bacilli. Recent
des Erregers moduliert werden. Obwohl die Akti-
studies of this group revealed the importance of
vität der Nitratreduktase und der Arginin-Deami-
iron for exaggeration of tuberculosis and lead to the
nase für den anaeroben Metabolismus von M.
generation of novel iron sensors to measure cellu-
tuberculosis wichtig sind, war die Persistenz-
lar iron status in collaboration with Prof Dr. Robert
fähigkeit von Mutanten, in denen diese Enzyme
Hider, Kings College London. Innate immune play-
inaktiviert waren, im Mausmodell nicht einge-
ers such as IL-18 and neutrophils seem to contribute
schränkt. Dieser Befund war der Auslöser für um-
to protective host defence in a yet underappreciat-
fangreiche vergleichende Untersuchungen zur
ed manner. Moreover, apoptotic cell death has
Physiologie des Granuloms in Tiermodellen: Hier-
been demonstrated to facilitate cross-presentation
bei erwiesen sich TB-Läsionen in der Maus als
of mycobacterial antigens to properly prime pro-
nicht schwer hypoxisch, während nekrotisierende
tective T cell responses, which will be of importance
Granulome in Meerschweinchen quasi anaerob
for rational vaccine design against tuberculosis. Fi-
waren. Die experimentelle Infektion mit M. avium
nally, the mycobacterial cell wall glycolipid, tre-
induziert auch in Mäusen nekrotisierende Granu-
halose dimycolate, was identified as a key factor in
lome - diese Läsionen, die sich abhängig von IFN-
inhibiting phagosome maturation by M. tuberculo-
gamma und IRF-1 entwickeln, erwiesen sich eben-
sis, a hallmark of mycobacterial virulence. Howev-
falls als hypoxisch. Um das Mikromilieu, in dem
er,
Mykobakterien intrazellulär leben, näher zu cha-
macrophages is able to chemically inactivate this
rakterisieren, wurde daraufhin eine neue Metho-
virulence factor. These studies reveal a complex in-
de entwickelt, die die Isolierung Mykobakterien-
terplay between the tubercle bacillus and the host,
haltiger Phagosome aus primären Makrophagen
which ultimately determine either latency or dis-
mittels eines immunmagnetischen Verfahrens er-
ease progression.
nitric
oxide
generated
by
activated
laubt.
The year 2007 was marked by the successful grantDie Forschergruppe Juniorprofessur Molekulare
ing of the initiative for a Cluster of excellence “In-
Infektionsbiologie unter der Leitung von Prof. Dr.
flammation at Interfaces” between the universities
Christoph Hölscher beschäftigt sich mit der Zyto-
Kiel, Lübeck and the Research Centre Borstel. The
kin-vermittelten Interaktion verschiedener Immun-
cluster bundles research on the various aspect of in-
zellen des unspezifischen und spezifischen Im-
flammation in human diseases caused by infec-
munsystems während der Infektion mit M.
tions, allergies and autoimmunity in all three loca-
tuberculosis. Das Labor fand heraus, dass eine
tions. MIB with its expertise in infection research is
durch den Interleukin (IL)-4 Rezeptor-alpha (Ra)
an integral part of the Cluster providing structural
vermittelte alternative Aktivierung von Makro-
analyses and infection models. As a consequence
phagen mit der Reaktivierung und der für die
of the establishment of the cluster, Prof. Dr. Stefan
post-primäre Tuberkulose typischen Pathologie
Ehlers accepted the new Chair of Molecular In-
assoziiert ist. In Patienten mit einer pulmonalen
flammation Medicine at Kiel University. In the fol-
Tuberkulose aus Ghana wurde eine strukturelle
lowing, the novel section of Microbial Inflammation
Wissenschaftlicher Jahresbericht 2007-2008
25
Isolierung Mykobakterien-haltiger Phagosome
mittels eines immunmagnetischen Verfahrems
Zusammenfassung
Summary
Variante des IL-4Ra gefunden, welche zu einer er-
Research headed by Prof. Dr. Stefan Ehlers was es-
höhten Signalweiterleitung führt und mit einer ver-
tablished within MIB and now encompasses three
stärkten Pathologie verbunden ist. Somit scheint
independent divisions: Microbial Interface Biology
die IL-4Ra Untereinheit von großer Bedeutung für
headed by PD Dr. Norbert Reiling, Infection Im-
die Ausbildung zentral nekrotisierender Granulo-
munology headed by Prof. Dr. Christoph Hölscher
me zu sein. Detaillierte funktionelle Analysen in
and Molecular Inflammation Medicine headed by
Mäusen,
Prof. Dr. Stefan Ehlers.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
welche
IL-4Ra-vermittelte
Signale
überexprimieren, zeigten, dass eine alternative
Makrophagen Aktivierung involviert ist in die
Therapy of infectious
diseases with a special
focus on bacterial sepsis
Reaktivierung und Granulomnekrose einer M. tu-
However, further numerous grants were awarded to
berculosis Infektion ohne die anti-mykobakteriel-
the researchers within MIB. The final period of fund-
le T-Zell-vermittelte Immunantwort zu beeinträch-
ing was granted to the Emmy-Noether Junior Re-
tigen.
eine
search Group Immunobiophysics headed by PD Dr.
bedeutsame Beteiligung von IL-4Ra-vermittelten
Andra Schromm. The excellent research output of
Mechanismen an der Pathogenese der post-
this group lead to the decision to continue Im-
primären Tuberkulose. In den vergangenen Jah-
munobiophysics as an inherent part of MIB. The fi-
ren zog das Zytokin IL-17 die Aufmerksamkeit der
nal period of the collaborative research centre (SFB
Tuberkuloseforschung auf sich. Die Forschergrup-
470) “Glycostructures in Biosystems” ending in 2009,
pe um Christoph Hölscher konnte belegen, dass
was highly successful and a novel initiative is under
IL-17 für den Immunschutz während einer Tuber-
way to hopefully continue this important research in
kulose-Infektion notwendig ist. Da die Pathoge-
a wider collaborative manner. Prof. Dr. Branden-
nese zahlreicher autoimmuner und chronisch
burg and PD Dr. Thomas Gutsmann were awarded
entzündlicher Erkrankungen von IL-17-produzie-
a grant by the support program of the German Min-
renden T-Helfer (TH)17 Zellen abhängt, wird die
istry of Education and Research (BMBF) “Innovative
Inhibierung einer TH17 Entwicklung als neuartige
molecular and cellular therapeutic approaches”.
anti-entzündliche Therapie-Strategie diskutiert.
The project “Therapy of infectious diseases with a
Die Forschergruppe fand nun allerdings heraus,
special focus on bacterial sepsis”, a cooperative en-
dass IL-17 essentiell ist für die Aufrechterhaltung
des CD4+ TH1 Zell-vermittelten Schutzes während
deavour with Prof. Dr. M. Hornef (Medizinische
der chronischen Phase einer Tuberkulose. Daher
(Rheinisch-Westfälische Technische Hochschule
birgt eine Neutralisierung von IL-17-abhängigen
Aachen), will combine basic research and preclini-
Mechanismen im Rahmen einer anti-entzündli-
cal studies representing a fine example of transla-
chen Therapie das Risiko der Reaktivierung einer
tional research in the Department of MIB. Prof. Dr.
latenten Tuberkulose.
Otto Holst and PD Dr. Buko Lindner were also suc-
Diese
Studien
belegen
also
Hochschule Hannover) and Dr. T. Schürholz
cessfully involved in the collaborative research centre SFB-TR 22 between the universities of Marburg,
Die neue Forschergruppe Zelluläre Mikrobiolo-
Munich and Borstel.
gie unter der Leitung von Prof. Dr. Ulrich Schaible
richtet ihr Augenmerk ebenfalls auf die Tuberkuloseforschung. Das Hauptinteresse gilt der Rolle
In November 2007, scientists from the Universities of
bestimmter Nährstoffe und angeborener Be-
Kiel, Lübeck, Hamburg, and Bremen, as well from
standteile des Immunsystems in der Abwehr des
the GKSS, the EMBL (DESY, Hamburg), and the Re-
Wirtes gegen M. tuberculosis und den intrazel-
search Centre Borstel came together to discuss
lulären Überlebensstrategien der Tuberkulose-
membrane biophysics and structural biology in the
26
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
bakterien. Jüngere Studien der Gruppe zeigten,
first Meeting of Northern Biophysicists, which was
dass Eisen eine wichtige Rolle bei der Verschlim-
held at the Manor House in Borstel. This novel con-
merung der Tuberkulose spielt, und führten zu der
vention will hopefully develop into a platform for
Entwicklung von neuen Eisensensoren für die Mes-
biophysicists with Borstel in its centre. In September
sung des zellulären Eisenstatus (Kooperation mit
2007, the meeting „Scanning Probe Microscopy and
Prof Dr. Robert Hider, Kings College London). An-
Organic Materials XVI“ was organized by the Divi-
geborene Bestandteile des Immunsystems wie IL-
sion of Biophysics. Through such endeavours, MIB
18 und Neutrophile scheinen in einer bisher un-
will become a centre of excellence for infection-fo-
terschätzten Weise zur protektiven Wirtsabwehr
cused biophysics. In 2008, PD Dr. Thomas Gutsmann
gegen Tuberkulose beizutragen. Die Gruppe fand
was appointed the new leader of the division of Bio-
auch, dass der programmierte Zelltod die Kreuz-
physics. He was offered a professorship at the Uni-
präsentation mykobakterieller Antigene ermög-
versity Ulm, which we were successfully in compet-
licht und bei der Auslösung einer schützenden T-
ing with and we are happy to keep Dr. Gutsmann
Zellantwort eine Rolle spielt. Diese Ergebnisse
as leader of the Division of Biophysics. Strategical-
sind für das rationale Impfstoffdesign gegen die
ly, this Division will synergize with the group of Im-
Tuberkulose von Bedeutung. Schließlich wurde
munobiophysics headed by PD Dr. Andra Schromm
das mykobakterielle Zellwandglykolipid Trehalo-
in order to strengthen focus in biophysics at the
se-Dimycolat als Schlüsselfaktor bei der Hem-
Borstel Research Center.
mung der Phagosomreifung, eines zentralen Virulenzmechanismus des Tuberkuloseerregers,
durch M. tuberculosis identifiziert. Allerdings ist
In 2008, Prof. Dr. Ulrich E. Schaible accepted the
Stickoxid, welches aktivierte Makrophagen pro-
directorship of the Department of MIB and as
duzieren, in der Lage, diesen Virulenzfaktor che-
head of the division of Cellular Microbiology. This
misch zu deaktivieren. Diese Studien enthüllen
recruitment will further strengthen tuberculosis re-
ein komplexes Zusammenspiel zwischen dem Tu-
search within MIB and at the Borstel Research
berkulosebazillus und seinem Wirt, was letztend-
Centre. Part of this group is still placed for anoth-
lich über Latenz- oder Krankheitsentwicklung ent-
er two years at the London School of Hygiene and
scheidet.
Tropical Medicine, UK and strong links will be built
between both institutions to facilitate research initiatives at the EU level and interchange expertises
Das Jahr 2007 war durch die erfolgreiche Bewilli-
from both partners, namely epidemiology and
gung des Exzellenzclusters “Inflammation at In-
structural biology.
terfaces”, einer gemeinsamen Initiative der
Universitäten Kiel und Lübeck und des Forschungszentrums Borstel, geprägt. Der Cluster
The excellence of the present member groups of
bündelt die Forschungsaktivitäten aller drei Stan-
MIB forms the basis for the future development of
dorte in verschiedenen Bereichen der Entzün-
the department. New challenges of transition lay
dungserkrankungen, die durch Infektionen, Aller-
ahead of us. Outstanding and valued colleagues
gien und Autoimmunitätserkrankungen verursacht
will leave the department in the years to come and
werden. Die Abteilung Molekulare Infektiologie
adequate successors need to be recruited. This re-
mit ihrer Expertise auf dem Gebiet der Infekti-
quires maintaining state of the art technologies
onsforschung ist integraler Bestandteil des Clu-
within MIB. Continuing our excellence in structural
sters und steuert Strukturanalysen und Infektions-
biology requires upgrading the accoutrement in
modelle bei. Im Rahmen der Etablierung des
NMR and MS to remain technically up to date and
Wissenschaftlicher Jahresbericht 2007-2008
27
Goal: Center of
Excellence for infectionfocused biophysics
Apoptose ermöglicht
Kreuzpräsentation von
Antigenen: Induktion
einer schützenden
T-Zellantwort.
Zusammenfassung
Summary
Clusters wurde Prof. Dr. Stefan Ehlers auf die Pro-
internationally competitive with respect to sensitivi-
fessur Molekulare Entzündungsmedizin an der
ty and resolution. The alarming menace of arising
Universität Kiel berufen. Zugleich wurde inner-
strains of the tubercle bacillus resistant to two and
halb der Abteilung Molekulare Infektiologie der
more antibiotics will require above category 3 safe-
Bereich Mikrobielle Entzündungsforschung eta-
ty levels in order to study such clinical isolates safe-
bliert, der von Prof. Dr. Stefan Ehlers geleitet wird
ly. Novel imaging technologies combined with ge-
und zur Zeit drei Forschergruppen umfasst: Mi-
netically tagged bacterial strains will provide the
krobielle Grenzflächenbiologie unter der Leitung
opportunity to study the infectious process in real
von PD Dr. Norbert Reiling, Infektionsimmunolo-
time in life animal models, an approach that is es-
gie unter der Leitung von Prof. Dr. Christoph Höl-
pecially challenging with respect to research in tu-
scher und Molekulare Entzündungsmedizin unter
berculosis. A future task will also be the recruitment
der Leitung von Prof. Dr. Stefan Ehlers.
of patient cohorts to translate our basic research in-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Neu: Bereich Mikrobielle
Entzündungsforschung
to clinical studies. Thereby, we hope to contribute to
novel diagnostics and therapeutics including anti-tuNeben dem Exzellenzcluster gab es noch weitere
berculosis and anti-allergy drugs. These future tasks
Bewilligungen von Forschungsmitteln für die Wis-
will allow MIB to tackle the great challenges in in-
senschaftlerinnen und Wissenschaftler der Abtei-
fection research and human health.
lung. Die letzte Förderperiode wurde für die Emmy-Noether-Nachwuchsgruppe Immunbiophysik,
die von PD Dr. Andra Schromm geleitet wird, bewilligt. Die exzellenten Forschungsergebnisse dieser Gruppe führten zu der Entscheidung, die Immunbiophysik als inhärenten Teil der Abteilung zu
erhalten. Die letzte Förderperiode des sehr erfolgreichen SFB 470 “Glykostrukturen in Biosystemen
– Darstellung und Wirkung” endet 2009. Zur Zeit
gibt es eine neue Initiative, die hoffentlich diese
wichtige Forschung in einem breiteren kooperativen Rahmen fortsetzen wird. Prof. Dr. Klaus Brandenburg und PD Dr. Thomas Gutsmann erhielten
die Bewilligung für das Projekt “Therapie von Infektionskrankheiten mit speziellem Bezug auf die
bakterielle Sepsis“ im Förderprogramm des Bundesministeriums für Bildung und Forschung
(BMBF)
‚Innovative
Therapieverfahren
auf
molekularer und zellulärer Basis’. Das Projekt, eine Kooperation mit Prof. Dr. M. Hornef (Medizinische Hochschule Hannover) und Dr. T. Schürholz
(Rheinisch-Westfälische Technische Hochschule
Aachen), wird Grundlagenforschung und präklinische Studien kombinieren, was ein wunderbares Beispiel für die fachübergreifende Forschung
der Abteilung Molekulare Infektiologie darstellt.
Prof. Dr. Otto Holst und PD Dr. Buko Lindner wa-
28
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
ren ebenfalls innerhalb des SFB-TR 22 erfolgreich,
einer gemeinsamen Initiative der Universitäten
Marburg und München und des Forschungszentrums Borstel.
Im November 2007 diskutierten Wissenschaftler
der Universitäten Kiel, Lübeck, Hamburg und Bremen gemeinsam mit Vertretern des GKSS, des EMBL (DESY, Hamburg) und des Forschungszentrums
Borstel Themen der Membranbiophysik und der
Strukturbiologie während des ersten Treffens der
Norddeutschen Biophysiker im Herrenhaus des
Forschungszentrums Borstel. Diese neue Zusammenkunft wird sich hoffentlich zu einer Plattform
für Biophysiker entwickeln, in deren Zentrum Borstel steht. Im September 2007 organisierte die Forschergruppe Biophysik den Kongress „Scanning
Probe Microscopy and Organic Materials XVI“.
Mit Hilfe solcher Veranstaltungen wird sich die Abteilung Molekulare Infektiologie zu einem Exzellenzzentrum für Infektions-orientierte Biophysik
entwickeln. 2008 wurde PD Dr. Thomas Gutsmann
Thomas Gutsmann
leitet die Biophysik
zum Gruppenleiter der Forschergruppe Biophysik
ernannt. Ihm wurde eine Professur an der Universität Ulm angeboten. Glücklicherweise konnten
wir Dr. Gutsmann jedoch als Leiter der Biophysik
in Borstel halten. Strategisch wird diese Forschergruppe mit der Gruppe Immunbiophysik, die von
PD Dr. Andra Schromm geleitet wird, zusammenarbeiten, um den Schwerpunkt Biophysik am Forschungszentrum weiter zu stärken.
2008 übernahm Prof. Dr. Ulrich E. Schaible die Position des Direktors der Abteilung Molekulare Infektiologie und als Leiter den Bereich Mikrobiologie und Infektiologie und die Forschergruppe
Zelluläre Mikrobiologie. Diese Berufung wird die
Tuberkuloseforschung in der Abteilung und am
Forschungszentrum Borstel weiter stärken. Ein Teil
seiner Gruppe wird für die nächsten zwei Jahre
weiterhin an der London School of Hygiene and
Tropical Medicine angesiedelt sein. Dies wird da-
Wissenschaftlicher Jahresbericht 2007-2008
29
Zusammenfassung
Summary
Fo r s c h u n g s z e n t r u m
Bo r s t e l
zu beitragen, eine enge Verbindung zwischen
den Institutionen zu etablieren, um Forschungsinitiativen auf der EU-Ebene und den Austausch
von Fachwissen zwischen den Partner, insbesondere in den Bereichen Epidemiologie und Strukturbiologie, zu erleichtern.
Die Exzellenz der Gruppen innerhalb der Abteilung Molekulare Infektiologie bildet die Basis für
die künftigen Entwicklungen der Abteilung. Neue
Herausforderungen liegen vor uns. Herausragende und geschätzte Kollegen werden in den kommenden Jahren die Abteilung verlassen und würdige Nachfolger müssen rekrutiert werden. Dies
erfordert die Etablierung von hochmodernen
Technologien in der Abteilung. Für die Fortführung unserer exzellenten Arbeit in der Strukturbiologie müssen NMR und MS in Hinblick auf
Sensitivität und Auflösung modernisiert werden,
um technisch auf dem neuesten Stand zu bleiben
und international kompetitiv sein zu können. Die
Höhere Sicherheitssufen
für die Untersuchung
multiresistenter,
klinischer Isolate in der
Tuberkuloseforschung
alarmierende Bedrohung durch neue, gegen
zwei und mehr Antibiotika resistente Stämme des
Tuberkelbazillus wird höhere Sicherheitsstufen als
die Stufe 3 erfordern, um solche klinischen Isolate sicher untersuchen zu können. Neue bildgebende Technologien in Kombination mit genetisch markierten Bakterienstämmen werden die
Möglichkeit bieten, den infektiösen Prozess in
Echtzeit in lebenden Tiermodellen zu untersuchen,
eine Vorgehensweise, die mit Blick auf die
Tuberkuloseforschung eine besondere Herausforderung darstellt. Eine weitere Aufgabe in der Zukunft wird die Rekrutierung von Patientenkollektiven sein, um unsere Grundlagenforschung in
klinischen Studien umzusetzen. Dabei hoffen wir,
zu der Entwicklung neuer Diagnoseverfahren und
Therapien insbesondere gegen Tuberkulose und
Allergien beitragen zu können. Diese zukünftigen
Aufgaben werden es der Abteilung erlauben,
sich, in unserer Verantwortung für die Gesundheit
der Menschheit, den großen Herausforderungen
in der Infektionsforschung zu stellen.
30
Wissenschaftlicher Jahresbericht 2007-2008
Division of Medical and Biochemical Microbiology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Medical and Biochemical
Microbiology
Head
(mAb) S69-4 which was able to differentiate C.
Prof. Dr. Helmut Brade
psittaci from C. pecorum, C. pneumoniae and C. tra-
Principal Investigators
infected cell monolayers and by ELISA. The epitope
Dr. Lore Brade
specificity of mAb S69-4 was determined by binding
PD Dr. Sven Müller-Loennies
and inhibition assays using bacteria, LPS, and
chomatis in double labelling experiments of
natural or synthetic Kdo oligosaccharides as free
Staff Scientists and Postdoctoral Fellows
ligands or conjugated to bovine serum albumin. The
Prof. Satish Raina
mAb bound preferentially Kdo(2→8)[Kdo(2→4)]
Prof. Graciana Klein
Kdo(2→4)Kdo with a KD of 10 µM as determined by
Dr. Elder Pupo (recipient of an Alexander von Hum-
surface plasmon resonance for the monovalent in-
boldt grant)
teraction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2→4)Kdo(2→4)Kdo
Graduate and Diploma Students
but not with Kdo(2→8)Kdo(2→4)Kdo, Kdo disac-
Dipl. Biol. Sandra Gerstenbruch
charides in 2→4- or 2→8-linkage or with Kdo
Dipl. Biol. Lena Heinbockel
monosaccharide. MAb S69-4 was able to detect LPS
on thin layer chromatography plates in amounts of
Technicians
less than 10 ng by immuno-staining. Due to the high
Ute Agge
sensitivity achieved in this assay, the antibody also
Nadine Harmel
detected in-vitro products of cloned Kdo-transferas-
Dagmar Meyer
es of Chlamydia. The antibody can therefore be
Christine Schneider
used in medical and veterinarian diagnostics, gen-
Veronika Susott
eral microbiology, analytical biochemistry and stud-
Irina von Cube
ies of chlamydial LPS biosynthesis.
Research Reports
A monoclonal antibody against a carbohydrate
epitope in lipopolysaccharide differentiates
Chlamydophila psittaci from Chlamydophila
pecorum, Chlamydophila pneumoniae and
Chlamydia trachomatis
Müller-Loennies S, Gronow S, Brade L, Brade H.
Lipopolysaccharide (LPS) of Chlamydophila psittaci
but not of Chlamydophila pneumoniae or Chla-
mydia trachomatis contains a tetrasaccharide of
3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) of the
Fig. 1. Characterization of LPS from C. trachomatis and C. psittaci
using monoclonal antibodies. Isolated LPS of C. trachomatis (lane
1-3) or C. psittaci (lane 4-6) in amounts of 100 ng (lane 1 and 4), 50
ng (lane 2 and 5) or 20 ng (lane 3 and 6) were separated by TLC
and stained with mAb S67-27 recognizing a single Kdo residue, mAb
S25-23 recognizing aKdo-(2→8)-αKdo-(2→4)-Kdo trisaccharide or
mAb S69-4.
sequence Kdo(2→8)[Kdo(2→4)]Kdo(2→4)Kdo. Af-
Further contribution to the general understanding of
ter immunization with the synthetic neoglycoconju-
carbohydrate binding antibodies was obtained by a
gate antigen Kdo(2→8)[Kdo(2→4)]Kdo(2→4)Kdo-
comparison of the primary structure of mAb S69-4 to
BSA, we obtained the mouse monoclonal antibody
that of mAb S45-18 of which the crystal structure in
32
Wissenschaftlicher Jahresbericht 2007-2008
Division of Medical and Biochemical Microbiology
Research Reports
complex with its ligand has been elucidated recent-
nation of the diffraction data revealed that the crys-
ly [Nguyen, H. P., N. O. Seto, C. R. MacKenzie, L.
tal was likely to be twinned and that the correct
Brade, P. Kosma, H. Brade, and S. V. Evans. 2003.
space group was P212121. Both translational pseu-
Germline antibody recognition of distinct carbohy-
dosymmetry and pseudo-merohedral twinning were
drate epitopes. Nat. Struct. Biol. 10:1019-1025].
observed in one crystal of SAG506-01 and pseudomerohedral twinning was observed for a second
crystal. The final R factor for SAG506-01 after
refinement in P212121 was 20.5 %.
Fig. 2. Detection of chlamydial inclusions in tissue culture by double labelling with monoclonal antibody S5-10 and S69-4. Tissue culture monolayers were infected with C. psittaci, C. pneumoniae or
C. trachomatis, reacted with mAb S5-10 (IgG3) and S69-4 (IgG1)
and developed with isotype specific second antibodies conjugated to FITC or HRP, respectively.
Collaboration: Roger MacKenzie, Institute for Biological Sciences, National Research Council Ottawa, Canada; Paul Kosma, Department of Chemistry, University of Natural Resources and Applied
Life Sciences, Vienna, Austria.
Pseudo-symmetry and twinning in crystals of homologous antibody Fv fragments
Fig. 3. Amino-acid sequence of the variable regions of SAG173-04
and SAG506-01 (top) and positions in the crystal structures (bottom). There are a total of three amino-acid differences in the two
variable light-chain regions and four amino-acid differences in the
two heavy-chain variable regions; however, only one of these differences lies in the hypervariable regions that potentially contact
the antigen. The amino-acid sequence is numbered according to
the Kabat numbering scheme. CDRs are indicated by grey shading. The magenta spheres represent the locations of the aminoacid differences, with the light chain in yellow and the heavy chain
in white . A single molecule within the asymmetric unit of SAG50601 is shown alone for clarity.
Müller-Loennies S, Gerstenbruch S, Brade H.
A difference of seven conservative amino-acid substitutions between two single-chain antibodies
(scFvs) specific for chlamydial lipopolysaccharide
does not significantly affect their molecular structures or packing contacts, but dramatically affects
their crystallization. The structure of the variable
domain (Fv) of SAG173-04 was solved to 1.86 Å
resolution and an Rcryst of 18.9 % in space group
P212121. Crystals of the homologous SAG506-01
Collaboration: Stephen V. Evans, Cory L. Brooks,
diffracted to 1.95 Å resolution and appeared at first
Ryan J. Blackler, Department of Biochemistry and
to have Patterson symmetry I4/m or P4/mmm; how-
Microbiology, University of Victoria, Victoria, BC,
ever, no solution could be found in space groups
Canada; Paul Kosma, Department of Chemistry,
belonging to the former and refinement in the only
University of Natural Resources and Applied Life Sci-
solution corresponding to the latter (in space group
ences, Vienna, Austria.
P43212) stalled at Rfree = 30.0 %. Detailed exami-
Wissenschaftlicher Jahresbericht 2007-2008
33
Division of Medical and Biochemical Microbiology
Research Reports
Exploration of specificity in germline monoclonal
Collaboration: Stephen V. Evans, Cory L. Brooks, De-
antibody recognition of a range of natural and
partment of Biochemistry and Microbiology Univer-
synthetic epitopes
sity of Victoria, Canada; Paul Kosma, Department of
Müller-Loennies S, Brade L, Brade H.
Chemistry, University of Natural Resources and Ap-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
plied Life Sciences, Vienna, Austria; Roger MacKenTo explore the molecular basis of antigen recogni-
zie, Tomoko Hirama, Institute for Biological Sci-
tion by germline antibodies, we have determined to
ences, National Research Council Ottawa, Canada.
high resolution the structures of the near-germline
monoclonal antibody S25-2 in complex with seven
distinct carbohydrate antigens based on the bacte-
Recognition of heptoses and the inner core of
rial sugar 3-deoxy-α-D-manno-oct-2-ulosonic acid
bacterial lipopolysaccharides by surfactant pro-
(Kdo). In contrast to previous findings, the inherited
tein D
germline Kdo monosaccharide binding site is not re-
Müller-Loennies S, Brade H
stricted to this bacterial sugar but is able to accommodate an array of substitutions and chemical mod-
Lipopolysaccharides (LPS) of Gram-negative bacte-
ifications of Kdo, including naturally occurring
ria are important mediators of bacterial virulence
antigens containing the related monosaccharide D-
that can elicit potent endotoxic effects. Surfactant
glycero-α-D-talo-oct-2-ulosonic acid as well as non-
protein D (SP-D) shows specific interactions with
terminal Kdo residues. However, we show by surface
LPS, both in vitro and in vivo. These interactions in-
plasmon resonance and ELISA how antibody S25-2
volve binding of the carbohydrate recognition do-
specificity is so dependent on the context in which
main (CRD) to LPS oligosaccharides (OS); however,
the antigen is presented that a free disaccharide dis-
little is known about the mechanisms of LPS recog-
plays strong binding while the same lipid-A-bound
nition. Recombinant neck+CRDs (NCRDs) provide
disaccharide does not bind. These structures pro-
an opportunity to directly correlate binding interac-
vide insight into how inherited germline genes code
tions with a crystallographic analysis of the binding
for immunoglobulins of limited flexibility that are ca-
mechanism. In these studies, we examined the in-
pable of binding a range of epitopes from which
teractions of wild-type and mutant trimeric NCRDs
affinity-matured antibodies are generated.
with rough LPS (R-LPS). Although rat NCRDs bound
more efficiently than human NCRDs to Escherichia
coli J-5 LPS, both proteins exhibited efficient binding
to solid-phase Rd2-LPS and to Rd2-LPS aggregates
presented in the solution phase. Involvement of
residues flanking calcium at the sugar binding site
was demonstrated by reciprocal exchange of lysine
and arginine at position 343 of rat and human
CRDs. The lectin activity of hNCRDs was inhibited by
specific heptoses, including L-glycero-α-D-mannoheptose (L,D-heptose), but not by 3-deoxy-α-D-mannooct- 2-ulosonic acid (Kdo). Crystallographic analysis
Fig. 4. Synthetic Kdo analogues crystallized with monoclonal antibody S25-2. The oligosaccharides (A) Kdo(2→4)Kdo(2→4)Kdo; (B)
Ko; (C) Ko(2→4)Kdo; (D) 7-epi-Kdo; (E) 3,4-dehydro-3,4,5-trideoxyKdo(2→8)Kdo; (F) 5-deoxy-4-epi-2,3-dehydro-Kdo(4→8)Kdo; (G)
Kdo(2→4)KdoC1red represent natural partial structures of bacterial LPS as well as artificial analogues of Kdo.
of the hNCRD demonstrated a novel binding orientation for L,D-heptose, involving the hydroxyl groups
of the side chain. Similar binding was observed for
a synthetic α1→3-linked heptose disaccharide corresponding to heptoses I and II of the inner core
34
Wissenschaftlicher Jahresbericht 2007-2008
Division of Medical and Biochemical Microbiology
region in many LPS. 7-O-Carbamoyl-L,D-heptose
Research Reports
Theses
and D-glycero-α-D-manno-heptose were bound via
ring hydroxyl groups. Interactions with the side
Diploma
chain of inner core heptoses provide a potential
Lena Heinbockel
mechanism for the recognition of diverse types of
Struktur- und Wirkungsbeziehungen bei der Bindung
LPS by SP-D.
von Antikörpern an Lipopolysaccharide aus Salmo-
nella enterica
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2007.
Fig. 5. Crystallographic complexes of the wild-type human NCRD
with L,D- and D,D-heptose. (A) L,D-Heptose (red and green sticks)
binds to Ca1 (green) by the 6- and 7-OH groups of its side chain.
(B) D,D-Heptose (red and pink sticks) also interacts with Ca1 via
the sugar ring.
Fig. 6. Superimpositions of L,D- and D,D-heptose. (A) D,D-Heptose
(pink sticks) and L,D-heptose (green sticks) crystal complexes illustrated in panels A and D of Figure 7 were aligned. The side
chain OH groups of L,D-heptose show the same spatial distribution as the vicinal OH groups of the pyranose ring of D,D-heptose.
(B) D,D-Heptose (pink sticks) was reoriented and superimposed on
the L,D-heptose crystal structure (green sticks). The spatial distribution of the 6- and 7-OH groups of the side chain of D,D-heptose
does not allow coordination with Ca1.
Collaboration: Hua Wang, James Head, Tanya Cafarella, Barbara Seaton, Department Physiology
and Biophysics, Boston University School of
Medicine, Boston, USA; Sharmin Sheikh, Barbara
McDonald, Kelly Smith, Erika Crouch, Department of
Pathology and Immunology, Washington University
School of Medicine, St. Louis, USA; Paul Kosma, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Wissenschaftlicher Jahresbericht 2007-2008
35
Division of Structural Biochemistry
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Structural Biochemistry
Head
and providing sufficient amounts of LPS precursors
Prof. Dr. Otto Holst
to support OM biogenesis. We now report the identification of an arginine to cysteine substitution at
Principle Investigator
position 134 of the conserved IM protein YhjD in
Dr. Uwe Mamat
KPM22 that acts as a compensatory suppressor mutation of the lethal DKdo phenotype. Further, the
Staff Scientists and Postdoctoral Fellows
yhjD400 suppressor allele renders the LPS trans-
Dr. Katarzyna Duda
Dr. Nestor Gonzalez Roldan
porter MsbA dispensable for lipid IVA transmembrane trafficking. The independent derivation of a
Dr. Anna Hanuszkiewicz
series of nonconditional KPM22-like mutants from
Dr. Patricia Sanchez-Carballo
the Kdo-dependent parent strain TCM15 revealed a
Dr. Kay Vogel
second class of suppressor mutations localized to
MsbA. Proline to serine substitutions at either
Graduate Student
residue 18 or 50 of MsbA relieved the Kdo growth
Elena Ciliberti
dependence observed in the isogenic wild-type
strain.
Technicians
Rainer Bartels
Collaboration: Meredith TC, Department of Micro-
Helga Bartels
biology and Molecular Genetics, Harvard Medical
Petra Behrens
School, Boston, USA; Woodard RW, Kirchhoff P, Kühl
Sylvia Düpow
A, Department of Medicinal Chemistry, College of
Regina Engel
Pharmacy, University of Michigan, Ann Arbor, USA;
Volker Grote
Aggarwal P, Nanotechnology Characterization Lab-
Katharina Jakob
oratory, Advanced Technology Program, SAIC-Fred-
Gudrun Lehwark-Yvetot
erick, Inc., Frederick, USA; Lindner B, Division of Im-
Kerstin Viertmann
munochemistry, Research Center Borstel.
Research Reports
Supported in part by: Deutsche Forschungsgemein-
Single amino acid substitutions in either YhjD or
schaft (Ma 1408/2-1 and Li-448/4-1).
MsbA confer viability to 3-deoxy-D-manno-oct-2ulosonic acid-depleted Escherichia coli
Mamat U, Hanuszkiewicz A, Holst O.
Modification of lipopolysaccharide with colanic
acid (M-antigen) repeats in Escherichia coli
The Escherichia coli K-12 strain KPM22, defective in
Mamat U, Holst O.
synthesis of 3-deoxy-D-manno-oct-2-ulosonic acid
(Kdo), is viable with an outer membrane (OM) com-
Colanic acid (CA) or M-antigen is an exopolysac-
posed predominantly of lipid IVA, a precursor of li-
charide produced by many enterobacteria, includ-
popolysaccharide (LPS) biosynthesis that lacks any
ing the majority of Escherichia coli strains. Unlike
glycosylation. To sustain viability, the presence of a
other capsular polysaccharides which have a close
second-site suppressor was proposed for transport
association with the bacterial surface, CA forms a
of lipid IVA from the inner membrane (IM), thus relieving toxic side effects of lipid IVA accumulation
loosely associated saccharide mesh that coats the
36
bacteria, often within biofilms. We show that a high-
Wissenschaftlicher Jahresbericht 2007-2008
Division of Structural Biochemistry
Research Reports
ly mucoid strain of E. coli K-12 ligates CA repeats to
early-life contact with cowsheds, farm animals,
a significant proportion of lipopolysaccharide (LPS)
and/or consumption of products like raw milk. Also,
core acceptor molecules, forming the novel LPS gly-
it had been indicated that microorganisms might
coform we call MLPS. MLPS biosynthesis is dependent upon i) CA induction, ii) LPS core biosynthesis,
have an important effect on the development of
and iii) the O-antigen ligase WaaL. Compositional
microbial organisms, their products, or both might
analysis, mass spectrometry, and nuclear magnet-
induce or influence allergy-protective mechanisms.
ic resonance spectroscopy of a purified MLPS sam-
We sought to gain further insight into the potential
ple confirmed the presence of a CA repeat unit
allergy-protective properties of microbes isolated
identical in carbohydrate sequence, but differing at
from the farming environment. Of a number of bac-
multiple positions in anomeric configuration and
terial species identified in cowsheds of farms,
linkage, from published structures of extracellular
2 were selected, isolated, and characterized, name-
CA. The attachment point was identified as O-7 of
ly Acinetobacter lwoffii F78 and Lactococcus lactis
allergies, and thus the question arose of which farm
the L-glycero-D-manno-heptose of the outer LPS
G121. The isolates were investigated with regard to
core, the same position used for O-antigen ligation.
their activation of pattern-recognition receptors, the
When O-antigen biosynthesis was restored in the
maturation of human monocyte-derived dendritic
K-12 background and grown under conditions meet-
cells, the upregulation of inflammatory cytokines,
ing the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its
the TH1-polarizing Notch ligand expression, and
proximal covalently linked cell surface polysaccha-
that both bacterial isolates were able to reduce al-
ride coat from O-antigen to CA in response to
lergic reactions in mice, to activate mammalian
certain environmental stimuli. The identification of
cells in vitro, and to induce a TH1-polarizing pro-
MLPS has implications for potential underlying
gram in dendritic cells. Our data strongly support
mechanisms coordinating the synthesis of various
the hygiene hypothesis, which states that an envi-
surface polysaccharides.
ronment rich in microbiologic structures, such as a
their influence on the allergic phenotype. It is shown
farming environment, might protect against the
Collaboration: Meredith TC, Woodard RW; Depart-
development of allergies.
ment of Medicinal Chemistry, College of Pharmacy,
University of Michigan, Ann Arbor, USA; Kaczynski Z,
Collaboration: Debarry J, Dickgreber N, Heine H,
Faculty of Chemistry, University of Gdansk, Gdansk,
Division of Innate Immunity, Research Center Bors-
Poland; Lindner B, Division of Immunochemistry, Re-
tel; Garn H, Blümer N, Renz H, Department of Clini-
search Center Borstel.
cal Chemistry and Molecular Diagnostics, Medical
Faculty, Philipps University, Marburg; von Mutius E,
Dr von Hauner Children’s Hospital, Ludwig-Maxim-
Acinetobacter lwoffii and Lactococcus lactis
ilians-University of Munich; Bufe A, Experimental
strains isolated from farm cowsheds possess
Pneumology, BGFA-XU18, Ruhr-University Bochum;
strong allergy-protective properties
Gatermann S, Institute for Hygiene and Microbiolo-
Hanuszkiewicz A, Holst O.
gy, Department of Medical Microbiology, Ruhr-University Bochum, Bochum.
Children who grow up in a farming environment
show lower levels of atopic sensitization, hay fever,
Supported in part by: Deutsche Forschungsgemein-
and asthma than children of the same age not liv-
schaft (SFB-TR22, Project A2).
ing in such an environment. A number of investigations provided good evidence that this is due to an
Wissenschaftlicher Jahresbericht 2007-2008
37
Division of Structural Biochemistry
Research Reports
Investigation of the chemical structure and bio-
non-virulent smooth-opaque (SmO) type. From both
logical activity of oligosaccharides isolated from
morphotypes we separated a non-acylated arabi-
rough-type Xanthomonas campestris pv. cam-
nomannan (AM) from an acylated polysaccharide
pestris B100 lipopolysaccharide
fraction by affinity chromatography, of which the
Kaczynski Z, Holst O.
AMs were structurally characterised. The AMs from
Fo r s c h u n g s z e n t r u m
Bo r s t e l
the virulent morphotype in contrast to that from the
The rough-type lipopolysaccharide (LPS) of the phy-
non-virulent form possessed a larger mannan chain
topathogenic bacterium Xanthomonas campestris
and a shorter arabinan chain. Incubation of murine
pv. campestris B100 was isolated utilizing the hot
bone marrow derived macrophages and human
phenol-water method and successively deacylated
dendritic cells showed that the acylated polysac-
by treatment with hydrazine and hot potassium
charide fractions were potent inducers of TNF-α,
hydroxide. Four compounds were separated by
IL-12 and IL-10, compared to non-acylated AMs
preparative high-performance anion-exchange
which only led to a marginal cytokine release. Fur-
chromatography and studied by sugar analysis and
by 1D and 2D homonuclear and heteronuclear 1H,
lated polysaccharide fractions as well as the non-
13C and 31P NMR spectroscopy as well as ESI FT-
acylated AMs were able to induce in vitro anti-tumor
MS. The two main products were a heptasaccharide
cytotoxicity of human peripheral blood mononucle-
ther in vitro experiments showed that both, the acy-
and a pentasaccharide of the structures α-D-Manp-
ar cells. Thus, morphotype specific structural differ-
(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→4)-α-D-Manp-
ences in capsular AMs of M. avium do not correlate
3 P -(1→5)-α-Kdo-(2→6)-β-D-Glc pN -4 P -(1→6)-α-D-
with biological activity, however, their acylation is a
GlcpN-1P (1) and β-D-Glcp-(1→4)-α-D-Manp-3P-
prerequisite for effective stimulation of murine
(1→5) -α- Kdo-(2→6)-β-D-GlcpN-4P-(1→6)-α-D-GlcpN-
macrophages and human dendritic cells.
1P (2), respectively. The products in smaller amounts
were a heptasaccharide and pentasaccharide pos-
Collaboration: Mittelstädt J, Brandau S, Division of
sessing the above structures plus a phosphate
Immunotherapy, Research Center Borstel; Reiling N,
group at C-4 of the Kdo residue (compounds 3 and
Division of Molecular Infectiology, Research Center
4). Both, heptasaccharide 1 and pentasaccharide 2
Borstel; Lindner B, Division of Immunochemistry, Re-
were able to induce an oxidative burst in cell cul-
search Center Borstel; Torrelles J, Brennan PJ, De-
tures of the non-host plant tobacco.
partment of Microbiology, Immunology and Pathology,
College
of
Veterinary
Medicine
and
Collaboration: Lindner B, Division of Immunochemi-
Biomedical Sciences, Colorado State University, Fort
stry, Research Center Borstel; Braun, S, Niehaus, K,
Collins, CO, USA.
Abt. 7, Proteom- und Metabolomforschung, Fakultät
für Biologie, Universität Bielefeld, Bielefeld.
Supported in part by: Deutsche Forschungsgemeinschaft (SFB 470, Project B1).
Capsular arabinomannans from Mycobacterium
avium with morphotype specific structural differ-
The structure of the O-specific polysaccharide of
ences but identical biological activity
the lipopolysaccharide from Pantoea agglomer-
Wittkowski M, Holst O.
ans strain FL1
Hanuszkiewicz A, Holst O.
The capsules of two colony-morphotypes of My-
cobacterium avium strain 2151 were investigated,
A neutral O-specific polysaccharide consisting of D-
i.e. of the virulent smooth-transparent (SmT) and the
rhamnose was obtained by mild acid hydrolysis of
38
Wissenschaftlicher Jahresbericht 2007-2008
Division of Structural Biochemistry
Research Reports
the lipopolysaccharide of the plant pathogenic bac-
tetrasaccharidic repeating unit containing a high
terium Pantoea agglomerans strain FL1, a common
number of acidic monosaccharides. It was hypoth-
epiphyte of many plant species, and associated
esized that the carboxylate groups might serve as
with Pseudomonas savastanoi pv. savastanoi in
a protective buffer for bacterium under the extreme
young and apparently intact olive knots. By means
life conditions. To provide insight into the relation-
of compositional and methylation analyses, and
ship and interactions between the environmental
NMR spectroscopy, the chemical repeating unit of
factors and microbial life, the core structure was al-
the polymer was identified as a linear tetrasaccha-
so characterized. The LPS was hydrolyzed under
ride of the structure
both mild acid and strong alkaline conditions. This
last treatment was the best one to
→2)-a-D-Rhap-(1→2)-β-D-Rhap-(1→3)-a-D-Rhap-(1→2)-a-D-Rhap-(1→
obtain the whole core backbone
and to gain information about the phosphates poCollaboration: Cimmino A, Evidente A, Dipartimen-
sition. Moreover, the strong alkaline treatment prod-
to di Scienze del Suolo, della Pianta, dell’ Ambien-
uct allowed us to identify the linkage between the
te e delle Produzioni Animali, Universitá Degli Studi
O-chain and the core structure. Two core oligosac-
di Napoli Federico II, I-80055 Portici (NA), Italy; Mar-
charides were found and their structures were de-
chi G, Surico G, Dipartimento di Biotecnologie Agra-
termined by FTICR MS and NMR spectroscopy. To
rie-Patologia Vegetale, Università di Firenze, I-50144
the best of our knowledge, this represents the first
Firenze, Italy.
description of the core structure of a lipopolysaccharide of an extremophile bacterium.
Structural characterization of the core region of
Collaboration: Pieretti G, Corsaro MM, Lanzetta R,
the lipopolysaccharide from the haloalkaliphilic
Parrilli M, Dipartimento di Chimica Organica e
Halomonas pantelleriensis: Identification of the
Biochimica, Università Federico II di Napoli, Napoli,
biological O-antigen repeating unit
Italy; Nicolaus B, Gambacorta A, Istituto di Chimica
Holst O.
Biomolecolare ICB-CNR, Pozzuoli, Italy; Lindner B, Division of Immunochemistry, Research Center Borstel.
Halomonas pantelleriensis is an extremophile, haloalkaliphilic microorganism that requires strictly aerobic conditions for growth. It is able to optimally
The structure of the O-specific polysaccharide
grow in media containing 3–15 % (w/v) total salt at
from the lipopolysaccharide of Pseudomonas sp.
a pH value between 9 and 10. To survive in these
OX1 cultivated in the presence of the Azo Dye Or-
harsh conditions the extremophiles have developed
ange II
several strategies that allow the microorganism to
Holst O.
thrive. These adaptative strategies probably concern the bacteria outer membrane, which is a bar-
The Gram-negative bacterium Pseudomonas sp.
rier regulating the exchange with the environment.
OX1, previously known as P. stutzeri OX1, is en-
In such a context, the lipopolysaccharides (LPSs),
dowed with a great metabolic versatility and is
which are among the major constituent of the Gram-
able to utilize a wide range of toxic organics as the
negative outer membrane, are thought to contribute
only source of carbon and energy for its growth. It
to restrictive membrane permeability properties.
has been recently observed that Pseudomonas sp.
Previous studies concerning the structure of the O-
OX1 can reduce azo dyes, ubiquitous pollutants ex-
chain repeating unit of the lipopolysaccharide from
hibiting resistance toward chemical and physical
this bacterium showed that it is constituted of a
degradation, with this azoreduction being a pro-
Wissenschaftlicher Jahresbericht 2007-2008
39
Division of Structural Biochemistry
Research Reports
cess able to generate enough energy to sustain
etamido-2,6-dideoxy-α- and β-D-glucopyranosides
Fo r s c h u n g s z e n t r u m
Bo r s t e l
bacterial survival. When such culture conditions oc-
and N-(5-hydroxy-6-methyl-4-oxo-4H-pyran-3-yl) acet-
cur, modifications in the primary structure of the O-
amide as side-products.
specific polysaccharide (OPS) within the lipoto
Collaboration: Borowski S, Michalik D, Reinke H, Vo-
remarkable changes both in the monosaccharide
gel C, University of Rostock, Institute of Chemistry,
composition and in the architecture of the repeat-
Rostock; Duda KA, Department of Microbiology, Uni-
ing unit, with respect to the polysaccharide pro-
versity of Silesia, Katowice, Poland.
polysaccharides
are
observed,
leading
duced under standard culture conditions. In the present paper, we present the complete structure of the
O-specific polysaccharide being
3)-β-D-QuiNAc4NBuOH(1→4)-α-D-GalNASer-(1→4)-α-D-ManNAcA-(1→4)-α-L-GulNAcA-(1
3
β-D-Glc-(1
This structure is profoundly different from the one
Activation of pro-matrix metalloproteinase-9 and
isolated from Pseudomonas sp. OX1 grown on rich
degradation of gelatin by the surface protease
medium.
PgtE of Salmonella enterica serovar Typhimurium.
Holst O.
Collaboration: Leone S, Lanzetta R, Scognamiglio R,
Alfieri F, Parrilli M, Molinaro A, Dipartimento di Chi-
Mammalian matrix metalloproteinases (MMPs) de-
mica Organica e Biochimica, Università degli Studi
grade collagen networks in extracellular matrices
di Napoli Federico II, Napoli, Italy; Izzo V, Di Dona-
by cleaving collagen and its denatured form
to A, Dipartimento di Biologia Strutturale e Funzio-
gelatin, and thus enhance migration of mammalian
nale, Università degli Studi di Napoli Federico II,
cells. The gastrointestinal pathogen Salmonella en-
Napoli, Italy.
terica survives and grows within host macrophages
and dendritic cells, and can disseminate in the host
by travelling within infected host cells. Here, we re-
αSynthesis of methyl 2-acetamido-2,6-dideoxy-α
port that S. enterica serovar Typhimurium activates
and β-D-xylo-hexopyranosid-4-ulose, a keto sugar
proMMP-9 (gelatinase B) secreted by human pri-
which misled the analytical chemists
mary macrophages, and degrades gelatin after
Hanuszkiewicz, Holst O.
growth within J774A.1 murine macrophage-like cells.
Both proMMP-9 activation and gelatin degradation
To understand the contradictory results on the struc-
were due to expression of the Salmonella surface
ture of the lipopolysaccharide isolated from a Yer-
protease PgtE. Following intraperitoneal infection in
sinia enterocolitica O:3, both anomers of methyl 2-
BALB/c mice, the amount of a pgtE deletion deriva-
acetamido-2,6-dideoxy-D-xylo-hexopyranosid-4-ulos
tive was nearly ten-fold lower in the livers and
e were prepared. The key steps of the synthetic
spleens of mice than the amount of wild-type S. en-
pathway were the selective acetylation of the
terica, suggesting that PgtE contributes to dissemi-
methyl 2-acetamido-2,6-dideoxy-α,β-D-glucopyrano-
nation of Salmonella in the host. PgtE belongs to the
sides, the oxidation of the 4-position to form the ke-
omptin family of bacterial beta-barrel transmem-
to-sugars, and deacetylation to provide the target
brane proteases. The ortholog of PgtE in Yersinia
compound. Surprisingly, the last step was accom-
pestis, Pla, which is central for bacterial virulence in
panied by a disproportionation to give methyl 2-ac-
plague, was poor in proMMP-9 activation and in
40
Wissenschaftlicher Jahresbericht 2007-2008
Division of Structural Biochemistry
Research Reports
gelatin degradation. To model the evolution of
ing infection with either strain. Aerosol challenge of
these activities in the omptin barrel, we performed
mice showed that wild type and Rv1500 mutant
a substitution analysis in Pla and genetically modi-
strains had identical growth rates in infected organs
fied it into a PgtE-like gelatinase. Our results indi-
over time. We verified mRNA expression of Rv1500
cate that PgtE and Pla have diverged in substrate
in H37Rv and conclude that Rv1500 must serve a re-
specificity, and suggest that Salmonella PgtE has
dundant role in viability and virulence of M. tuber-
evolved to functionally mimic mammalian MMPs.
culosis.
Collaboration: Ramu P, Lobo LA, Kukkonen M, Bjur
Collaboration: Malm S, Bange F-C, Department of
E, Suomalainen M, Raukola H, Miettinen M,
Medical Microbiology and Hospital Epidemiology,
Julkunen I, Rhen M, Korhonen TK, Lähteenmäki K,
Medical School Hannover, Hannover; Walter K,
General Microbiology, Department of Biological
Maass S, Pfau S, Ehlers S, Division of Molecular In-
and Environmental Sciences, Faculty of Biosciences,
fection Biology, Research Center Borstel, Borstel;
University of Helsinki, Helsinki, Finland.
Hübner G, Lindner B, Division of Immunochemistry,
Research Center Borstel, Borstel.
In vitro and in vivo characterization of a Mycobacterium tuberculosis mutant deficient in gly-
Animal shed Bacillus licheniformis spores possess
cosyltransferase Rv1500
allergy-protective as well as inflammatory prop-
Engel R, Holst O.
erties
Vogel K, Holst O.
In Mycobacterium marinum the homologue of
Rv1500 of Mycobacterium tuberculosis encodes a
Numerous epidemiological studies have demon-
glycosyltransferase. Initially, it was suggested that
strated an allergy-protective effect of farm life early
this gene is involved in the synthesis of phos-
in childhood. It has been hypothesized that environ-
phatidylinositol mannosides (PIM), generating
mental exposure to microbes may at least in part
Ac2PIM7 from Ac2PIM5. PIM and its related compounds lipomannan (LM) and lipoarabinomannan
contribute to this effect. Due to their small size and
(LAM) have been shown to modulate the host re-
of small children bacterial spores may be candi-
thereby their potential for deposition in lower airways
sponse to an infection with M. tuberculosis. Here,
dates for such allergy protective effects. To investi-
we generated a deletion mutant of Rv1500 in My-
gate immune responses elicited by exposure to Bacil-
cobacterium tuberculosis H37Rv, and analyzed the
lus spores in experimental settings. Animal shed and
mutant using a biochemical approach as well as in
mattress dusts were analyzed for bacteria and fungi
vitro and in vivo infection models. Inactivation of
by aerobic and anaerobic growth. Bacillus licheni-
Rv1500 did not lead to an altered expression pat-
formis 467, the most prominent microorganism found
tern of PIMs in M. tuberculosis H37Rv. We found
in these samples, was investigated with respect to
phophatidylinositol (PI), AcPIM2, Ac2PIM2, and
AcPIM6 in both strains, but were unable to detect
spore specific stimulation of pattern recognition re-
Ac2PIM7 or Ac2PIM5 either in the wild type or the
mutant strain. Uptake and growth of H37Rv and
ceptors, monocyte derived dendritic cells and TH-cell
polarization in vitro as well as to the prevention of
asthma development in a mouse model of allergic
Rv1500 mutant strains in murine bone marrow-de-
asthma. In vitro, Bacillus licheniformis spores acti-
rived macrophages was identical, and TNF
IL-12p40 production in mouse macrophages and
vated a TH1 cytokine expression profile. In vivo application of these spores resulted in less spore-spe-
dendritic cells was induced to similar levels follow-
cific but long lasting immune activation preventing
and
Wissenschaftlicher Jahresbericht 2007-2008
41
Division of Structural Biochemistry
Research Reports
eosinophilia and goblet cell hyperplasia, however
troscopy and mass spectrometry, the structure of the
they provoked an influx of neutrophils in lung tissue
rough lipopolysaccharide (LPS) isolated from Acine-
of asthmatic mice. Bacterial spores may contribute to
tobacter lwoffii F78 was elucidated. As a prominent
the allergy-protective properties of farming environ-
feature, the core region of this LPS contained the
ments, but their persistence in the lung causes on-
disaccharide α-Kdo-(2→8)-α-Kdo which so far has
going immune activation in mouse experiments.
been identified only in chlamydial LPS. In serologi-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
cal investigations, the anti-chlamydial LPS monoCollaboration: Blümer N, Garn H, Renz H, Depart-
clonal antibody S25-2 which is specific for the epi-
ment of Clinical Chemistry and Molecular Diagnos-
tope α-Kdo-(2→8)-α-Kdo reacted with A. lwoffii F78
tics, Medical Faculty, Philipps University, Marburg; Ko-
LPS. Thus, for the first time an LPS was identified out-
rthals M, Schwaiger K, Bauer J, Chair of Animal
side Chlamydiaceae which contains a Chlamydia-
Hygiene, Faculty of Life and Food Sciences, Technis-
specific LPS epitope in its core region.
che Universität München, Freising-Weihenstephan;
Mittel-
städt J, Brandau S, Division of
Immunotherapy,
Research
Center Borstel; Ege M, von Mutius E, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians-University of Munich,
Munich; Gatermann S, Institute
for Hygiene and Microbiology,
Department of Medical Microbiology,
Ruhr-University
Bochum, Bochum; Bufe A, Department
of
Pneumology,
Experimental
Ruhr-University
Bochum, Bochum; Goldmann T,
Division of Clinical and Experimental Pathology, Research
Center Borstel; Heine H, Division of Innate Immunity,
Fig. 7
Research Center Borstel.
Collaboration: Hübner G, Lindner B, Division of
Supported in part by: Deutsche Forschungsgemein-
Immunochemistry, Research Center Borstel; Vino-
schaft (SFB-TR22, Project A1).
gradov E, National Research Council, Institute for
Biological Sciences, Ottawa, Canada; Brade L,
Brade H, Division of Biochemical Microbiology,
Structural and immunochemical analyses of the
Research Center Borstel; Debarry J, Heine H, Divi-
lipopolysaccharide (LPS) from Acinetobacter
sion of Innate Immunity, Research Center Borstel.
lwoffii F78 – the first LPS outside Chlamydiaceae
carrying a Chlamydia-specific LPS epitope
Supported in part by: Deutsche Forschungsgemein-
Hanuszkiewicz A, Holst O.
schaft (SFB-TR22, Project A2).
´
By the means of chemical analyses, NMR spec-
42
Wissenschaftlicher Jahresbericht 2007-2008
Division of Structural Biochemistry
Research Reports
Modulation of dendritic cell function by cowshed
The structural analysis of the capsular polysac-
dust extract
charide from Acinetobacter lwoffii F78
Holst O.
Hanuszkiewicz A, Holst O.
We have shown previously that inhalation of
The capsular polysaccharide from Acinetobacter
cowshed dust extract (CDE) resulted in decreased
lwoffii F78 was isolated and purified, and its struc-
airway reactivity, eosinophilic inflammation and
ture was elucidated by the means of chemical anal-
sensitization in a mouse model of allergic asthma.
yses, NMR spectroscopy and mass spectrometry.
Our data suggested down regulation of allergic
The presence of a capsule on this bacterium was
immune response rather than activation of a Th1
confirmed by transmission electron microscopy ex-
response towards the model allergen. However, the
periments and utilization of anti-lipid A monoclonal
precise mechanism of allergy protection is not
antibody proved non-endotoxin origin of the isolat-
understood in detail until now. To get deeper in-
ed material. The structure represents a novel non-
sides into CDE induced immune modulation we
branched amino-polysaccharide, with high hetero-
have analysed effects of CDE on dendritic cell biol-
geneity of amino groups substituents:
ogy. Dendritic cells were generated from murine bone marrow
→3)-α-L-FucNAc-(1→3)-β-D-QuiNR14NR2-(1→4)-β-L-GlcNR33NR4A-(1→
(BMDC). Cells were stimulated
via the airways. Our results showed that cells were
where R1 represents 3-hydroxybutyric acid (3-HBA)
or N-acetylated alanine (AlaNAc); R2 - AlaNAc or 3-
not able to prime mice for allergic immune re-
HBA; R3 - acetyl or 3-HBA; and R4 - 3-HBA or acetyl.
with CDE and subsequently used to sensitize mice
sponse when they were treated with CDE two days
before pulsing with allergen whereas cells that
Collaboration: Kaczynski Z, Faculty of Chemistry,
were stimulated with CDE simultaneously to OVA
University of Gdansk, Gdansk, Poland; Lindner B, Di-
pulsing induced a fully developed allergic immune
vision of Immunochemistry, Research Center Borstel;
response. Surprisingly CDE treated cells that were
Goldmann T, Vollmer E, Division of Clinical and Ex-
not able to prime mice for allergic immune re-
perimental Pathology, Research Center Borstel; De-
sponse exhibit an activated phenotype with high ex-
barry J, Heine H, Division of Innate Immunity, Re-
pression of costimulatory surface molecule CD86.
search Center Borstel.
Moreover CDE treated cells transiently produced
high amounts of cytokines like IL-10, IL-12p70 and
Supported in part by the Deutsche Forschungsge-
TNF-0. Interestingly, blocking of autocrine produced
meinschaft (SFB-TR22, Project A2).
IL-10 in vitro partially restored allergy inducing capacity of CDE exposed cells. Thus we conclude that
prolonged contact to CDE reduces allergy inducing
Host and non-host plant response to the
capacity of dendritic cells. Furthermore we present
lipopolysaccaride
evidence that an autocrine IL-10 dependent mech-
solanacearum, a causal agent of a devastating
anism seems to be involved in down regulation of
disease of potato
dendritic cell function due to stimulation with CDE.
Ovchinnikova OG, Holst O.
Collaboration: Gorelik L, Kauth M, Gehlhar K, Bufe
Ralstonia solanacearum is one of the most devas-
A, Peters M, Department of Experimental Pneumol-
tating phytopatogenic bacteria, in particular its
ogy, Ruhr-University Bochum, Bochum.
race 3. This microorganism is the causative agent
isolated
from
Ralstonia
of destructive diseases of different agrarian crops
Wissenschaftlicher Jahresbericht 2007-2008
43
Division of Structural Biochemistry
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
including tomato and potato. An important aspect
of the interaction between this pathogen and the
host and non-host plants was its biochemical and
molecular basis. Thus, the lipopolysaccharides (LPS)
were extracted from the R. solanacearum cell wall,
purified, and the O-specific polysaccharide (OPS)
was isolated and chemically characterized by compositional analyses and NMR spectroscopy. The
OPS was constituted of two linear polymers of an
approximate ratio of 3:1, both of which were built
up from three rhamnose and one N-acetylglucosamine residues and differed only in the substitution of one rhamnose residue. Also, traces of
branched repeating units arising from nonstoichiometric xylosylation of the linear structure were present. The LPS inhibited the hypersensitivity reaction
(HR) in non-host tobacco plants and induced localized resistance in host potato plants, both of which
were pre-treated with the LPS before being inoculated with the pathogen.
Collaboration: Esposito N, Barone A, Evidente A, Dipartimento di Scienze del Suolo, della Pianta, dell’
Ambiente e delle Produzioni Animali, Universitá di
Napoli Federico II, Portici, Italy; Zoina A, Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Universitá di Napoli Federico II, Portici, Italy.
Theses
Dissertation
Hanuszkiewic, Anna
Isolation and structural as well as immunological
characterization of the cell wall components from
cowshed bacteria.
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2008.
Vogel, Kay
Microbial spores from farming environments and
their role in allergy protection.
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2008.
44
Wissenschaftlicher Jahresbericht 2007-2008
Division of Immunochemistry
Research Reports
Division of Immunochemistry
Head
Research Reports
Prof. Dr. Ulrich Zähringer
Structure and dynamics of 13C,15N-labeled lipid A
in a membrane mimetic
Principle Investigators
Zähringer U.
PD Dr. Buko Lindner
Dr. Christian Alexander
After successful setup of a new approach to investigate intact deep rough type 13C,15N-labelled LPS
Postdoctoral Fellows
(Re LPS) from a heptosyltransferase deficient mutant
Dr. Carola Funk (since July 2007)
(WBB01) of Escherichia coli K-12 conformational
Dr. Sandra Albrecht
analysis of LPS in water became accessible (Re-
Dr. Göran Hübner (since August 2008).
search Report 2006). This gives access to the anal-
Dr. Catharina Crone (till September 2007)
ysis of conformation and dynamics of LPS, which is
believed to be the key-event of understanding how
Technicians
LPS and TLR4/MD-2 molecules interact. This method
Nina Grohmann
is also useful not only for different LPS but also for
Heiko Käßner
lipid A structures, independent of its acylation pat-
Brigitte Kunz
tern (Fig UZ1). The results of these investigations
Helga Lüthje
have been published in Angew Chem Intl Ed
Hermann Moll
2008;47:9870-9874.
Ursula Schombel
Simone Thomsen
Birte Wegner
Guests and Trainees
Dr. Evelyna Zdorovenko, N.D. Zelinsky Institute,
Moscow, Russia
03-05/07, and 09/07- 10/07, supported by SFB470,
Project B4
Dr. Anna Kondakova, N.D. Zelinsky Institute of Organic Chemistry Moscow, Russia
10 - 11/07, and 10/08 - 11/08
Hrkac’ Stjepan and Ankatrin Stange, graduate students University Kiel
voluntary MS trainee-coarse (08/08)
Ruth Kläver, master course student, Molecular Life
Sciences, University of Münster
voluntary internship (09/08 - 10/08)
Jakob Hufschmidt
voluntary student’s internship (08/08)
Wissenschaftlicher Jahresbericht 2007-2008
45
Division of Immunochemistry
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
bacteria. For further clarification, we aimed to isolate and characterize a TLR2-activating lipoprotein
from Staphylococcus aureus as a representative
lipoprotein of Gram-positive bacteria. Since the
bacterial cell wall of S. aureus contains over 60
lipoproteins, we concentrated on lipoprotein sitC, a
staphylococcal iron transporter. This lipoprotein
was over-expressed by a S. aureus mutant, thus facilitating the isolation of the single lipoprotein. A
new approach to isolate sitC was enabled by another S. aureus mutant over-expressing the hisFig. 8. Structure of E. coli lipid A (left) and part of the 2D contour
plot (right) of a 1H,13C HSQC-spectrum (800MHz) of 13C,15N-labelled penta-acylated (red) and hexa-acylated (blue) lipid A from
LPS (F515 and WBB01) in 3 % DHPC-d15 at 310K.
tagged sitC. Isolation of the nearly pure lipoprotein
The well resolved carbon signals of the fatty acids
100-fold higher than that of sitC isolated by other
in the HSQC, together with complex 3D HCCH -TOC-
methods such as electroelution. The TLR2-depen-
SY experiments allowed the unequivocal assign-
dent activity of the isolated his-tagged lipoprotein,
ment of all signals, which is a prerequisite for fur-
determined using HEK293 cells was significant. Our
ther conformational analyses. NOESY spectra finally
studies demonstrated the recognition of a single
showed distinct differences in both lipid A’s, sug-
lipoprotein by TLR2 for the first time
was achieved by using Ni-NTA-chromatography. The
amount of sitC-his isolated by this method was over
gesting that the conformation of the lipid A-backbone has a significant impact on the reduced endotoxic activity of penta- vs. hexa-acylated lipid A.
Collaboration: Grzesiek S, Wang W, Sass HJ, Division of Structural Biology, Biozentrum, University of
Basel, Basel, Switzerland.
Isolation and structural characterization of TLR2 activating lipoproteins from Staphylococcus aureus
Funk C, Alexander C, Zähringer U.
Recognition so called pathogen-associated molecular patterns (PAMPs), by Toll-like receptors (TLRs)
initiates signal transduction pathways of the innate
immune system. As a specific structure-function-relationship between PAMP and TLR is well established for most of the TLRs the structure of TLR2-lig-
Fig. 9. TLR2-dependent activity of cell wall extracts from S. aureus
SA113 Dlgt (lacking lipoproteins) SA113W TpTX sitC-His (overexpressing His-tagged lipoprotein sitC), and the His-tagged lipoprotein sitcC, isolated by Ni-NTA chromatography.
ands published till date is under dispute. This
project aims at defining a clear structure-activity
Collaboration: Götz F, Anstett M, Stoll H, Depart-
based relationship for TLR2. Previous studies of our
ment of Microbial Genetics, University of Tübingen;
group indicated that lipoproteins are the major if
Heine H, Division of Innate Immunity, Research Cen-
not sole TLR2-activating molecules of Gram positive
ter Borstel.
46
Wissenschaftlicher Jahresbericht 2007-2008
Division of Immunochemistry
Research Reports
Supported in part by: Deutsche Forschungsgemein-
chemistry. Grzesiek S, Wang W, Division of Structural
schaft, Schwerpunktprogramm 1110.
Biology, Biozentrum, University of Basel, Basel,
Switzerland.
Study of the pseudomonas LPS core region and
Supported in part by: Deutsche Forschungsgemein-
its binding to a Pseudomonas-specific monoclon-
schaft, SFB 470 B4 and C1.
al antibody
Albrecht S, Zähringer U.
Peptidoglycan in innate immunity: structure-funcThe inner core of the lipopolysaccharide (LPS) of all
tion relationship
Pseudomonas species contains a unique carbohy-
Zähringer U, Lindner B.
drate structure which we identified to be a highly
phosphorylated 7-O-carbamoyl L-glycero-D-manno-
During the last year we successfully established two
heptopyranose (7-O-Cm-L,D-Hep). Recent studies on
different projects which are devoted to the isolation
clinical isolates from patients suffering from Pseu-
and structural characterization of well defined and
domonas infections showed a significant specificity
highly purified peptidoglycan (PG) and PG-frag-
of mAb S60-4-14 for Pseudomonas strains isolated
ments. Both projects aim at isolation and character-
from the lung of cystic fibrosis patients (Annual Re-
ization of biological active peptidoglycan fragments
port 2006). For a detailed characterization of the an-
which are known to act as specific ligands for the so
tibody-LPS interaction we pursued two approaches.
called Nod-like receptors Nod1 and Nod2 within the
On the one hand we isolated the mAb S60-4-14 from
integrated research network (IRN) of nod-like recep-
culture supernatants, prepared Fab-fragments by
tors within the cluster of excellence “Inflammation at
papain-cleavage and purified appropriate scFv
Interfaces”. First interesting results further support
from an extract of a scFv S60-4-14-overexpressing E.
our hypothesis that besides muramic acid also the
coli strain. The binding of scFv S60-4-14 was ana-
stem-peptide of PG but not the glycan part in S. au-
lyzed by Western Blot analysis and showed the
reus PG is indispensible for the expression of Nod-2
same substrate. On the other hand we grew
13C,15N-labeled as well as unlabeled oligosaccha-
activity. The other PG-project, which is also devoted
rides from P. aeruginosa PAO1- to DalgC. Native and
non-purified 13C,15N-LPS from P. aeruginosa PAO1-
of Staphylococcus aureus, is a collaborative project
DalgC as analyzed by NMR (800 MHz) was shown
schmidt, University of Greifswald. This project aims at
to express structural heterogeneity differing in acy-
the characterization of the glycan part of PG which
lation, phosphorylation and, as we found, potential-
is known to act as a ligand of Thrombospondin-1
ly conformational features which may have an im-
(TSP-1). Our current hypothesis is that only the glycan
pact on the epitope structural analysis. Based on the
and not the peptide part of the PG is involved in the
availability of mAb, Fab and scFv as well as LPS and
specific interaction of TSP-1, which we hypothesize to
its substructures we are now able to investigate the
act as a lectin involved and mediating staphylococ-
epitopes involved in the binding by methods such as
cal adherence during infection.
to the chemical analysis of PG and PG-subfragments
which we started together with Prof. S. Hammer-
STD-NMR and Surface Plasmon Resonance.
Collaboration: H. Heine, Division of Innate immuniCollaboration: Müller-Loennies S, Brade L, Brade H,
ty, P. Rosenstiel and A. Till, CAU.
Division of Medical and Biochemical Microbiology;
Supported in part by: Excellence Cluster “Inflam-
Goldmann T, Division of Clinical and Experimental
mation at Interfaces” (Lübeck, Borstel, Kiel) and
Pathology; Mamat U, Division of Structural Bio-
DFG (SH, UZ, BL).
Wissenschaftlicher Jahresbericht 2007-2008
47
Division of Immunochemistry
Research Reports
Application of capillary electrophoresis – Fourier-
With an optimized CE electrolyte based on water,
transform ion cyclotron resonance mass spec-
isopropyl alcohol, triethylamine and acetic acid at
trometry to the separation of R-form lipopolysac-
pH 8.9 and short uncoated fused silica capillaries
charide and lipid A species.
we could for the first time separate and online mass
Hübner G, Lindner B.
analyse the different components of underivatized
Fo r s c h u n g s z e n t r u m
Bo r s t e l
R-Form LPS and lipid A even of complex mixtures
Native isolates of LPS comprise a high inherent het-
(Fig. 10 bottom). Measurements with well defined
erogeneity with respect to the number and type of
LPS standards revealed detection limits of single
fatty acids and core oligosaccharides and of non-
constituents in the low fmol range. Comparison of
stoichiometric substituents (P, P-Etn, and Ara4N)
MS and CE-MS measurements under equal condi-
which hamper their structural characterisation. So
tions enabled the investigation of ion suppression
far, chromatographic separation (e.g. by HPLC or
effects within heterogeneous LPS samples.
TLC) of these highly amphiphilic compounds for subsequent chemical analyses failed. Thus, high reso-
Supported in part by: SFB-TR 22 Project Z01 and
lution mass spectrometry is the method of choice for
DFG (Li 448/4-1).
the detailed analysis of these compounds. However, ion suppression effects within the complex sample mixture during electrospray ionisation can influ-
LipID – a software tool for automated assignment
ence the detection sensitivity of individual
of lipids in mass spectra
compounds dramatically. To overcome these prob-
Hübner G, Lindner B.
lems we coupled a home-made capillary electrophoresis (CE) online to a Fourier-transform ion
Gangliosides and phospholipids determine the bio-
cyclotron resonance mass spectrometer (FT-ICR MS)
physical properties of membrane structures (e.g.
using a sheath liquid interface (Fig.10 top).
transmembrane permeability, formation of microdomains) and are involved in
many essential biological functions e.g. cell signaling or energy
storage. Furthermore, defects in
lipid metabolism may play an essential role in many diseases. The
analysis of lipids and especially
those of subcellular membrane
compartments (e.g. phagosomes,
lipid rafts) is challenging because
of the low amounts of material
available for analysis and the
wide molecular diversity of this
class of compounds and their relative insolubility in aqueous systems. Using multiple stage mass
spectrometry, ion species may be
Fig. 10. Details of the sheath liquid online coupling of the homemade CE to the high resolution Qe FT-ICR MS (top) and the extracted ion chromatogram with respective mass spectra of different species of an R-form LPS isolated from an Rb-mutant of
Salmonella enterica.
48
unambiguously classified. However, this procedure is
time consuming. Our approach for detailed lipid analysis is based on normal phase HPLC separation cou-
Wissenschaftlicher Jahresbericht 2007-2008
Division of Immunochemistry
Research Reports
pled online to high resolution MS analysis. Due to the
Structural studies of lipopolysaccharides of med-
high mass accuracy (> 2ppm) of the FT-ICR MS in-
ically important Gram-negative bacteria by high-
strument the lipids can be unequivocally be identified
resolution ESI ICR-FT mass spectrometry
taking into account the HPLC retention time and the
Kondakova A, Lindner B.
exact mass of a lipid species. For the evaluation of
the huge amount of experimental data a new soft-
LPS plays a role in the activation of the host immune
ware tool called lipID was developed, which supports
system and alterations in its chemical structure may
the identification of glycerophospholipids, glycosph-
significantly influence biological activity of the bac-
ingolipids and fatty acids in mass spectra. The user-
terial strains. In the recent years we have devel-
extendable software is a Microsoft (MS) Excel Add-In
oped an efficient approach to structural studies of
and is compatible with all versions of MS Excel. It pro-
core oligosaccharide and lipid A parts of LPS
cesses singly given mass-to-charge values as well as
molecule based on high-resolution mass spectrom-
mass lists provided by the mass spectrometer con-
etry, which allows us to overcome the intrinsic het-
sidering a broad range of user-defined options. The
erogeneity of the sample and provide important
mode of operation is demonstrated in Fig 11. show-
structural information even when only limited
ing the mass spectrum of the total phospholipid ex-
amount of material is available. We applied this ap-
tract from a Gram-negative bacterium and the results
proach for the studies of LPS isolated from the bac-
produced by the lipID program giving information
teria of Yersinia pestis (causative agent of plague),
concerning the lipid headgroup, the total chain
Yersinia pseudotuberculosis (cause of broad range
lengths and the degree of saturation of the fatty acids.
of acute and chronic gastrointestinal disorders and
evolutionary predecessor of Y. pestis,) and Shigel-
Supported in part by: SFB-TR 22 Project Z01.
la (causative agent of gastrointestinal diseases, including shigellosis). While the structure of LPS in Y.
pestis has been studied extensively over last years,
most of the work has been done on specifically constructed mutant strains and little is known about the
biological diversity of LPS structures in wild type
strains. We have investigated the structure of LPS in
five wild type strains and found it to be in agreement with the structures reported earlier, but the variety of core oligosaccharide glycoforms and lipid
A acylation patterns was higher, e.g. in LPS of Y.
pestis A-1249, which produce mostly triacylated
form of lipid A, instead of more common tetraacylated one, but is not able to incorporate a residue
of galactose in the outer core. The structures of LPS
in Y. pseudotuberculosis strains were found to be
similar to those in Y. pestis strains. Studies of
Fig. 11. Example demonstrating the capability of the lipid identification program lipID. 27 out of 46 ion peaks in the detail of a mass
spectrum from a lipid extract from an E. coli mutant could be assigned with lipID, using an expected experimental mass accuracy
of 3 ppm. Besides several PE (or PC) species, also PG and PS
species were identified. All assigned signals in the mass spectrum
are depicted with an arrow.
oligosaccharide and lipid A fractions isolated from
eight Y. pseudotuberculosis strains, representing
serovars 1 to 4, revealed the following structure of
core oligosaccharide, with minor portion of core
molecules including phosphoethanolamine residue
at the unknown position.
Wissenschaftlicher Jahresbericht 2007-2008
49
Division of Immunochemistry
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
LPS in a catalytic manner, thus enhancing the
immunoactivating properties of these endotoxin
preparations. In the second project aqueous extracts of paraffin-embedded non small cell lung
Fig. 12
cancer-tissues conserved by the HOPE (Hepes-gluSimilarly, the mass spectrometric screening of LPS
tamic acid buffer mediated organic solvent protec-
samples isolated from 25 Shigella strains has shown
tion effect) fixation technique have been shown for
them to be structurally similar of the LPS of closely-
the first time also to be generally applicable to 2-
related bacteria Escherichia coli, but further investi-
DE-based proteome analysis and a set of four se-
gations are necessary to define significant structural
lected protein spots has been identified including
differences.
the keratin group that represents a highly expressed
marker protein fraction of lung adenocarcinoma.
Collaboration: Knirel Y, ND Zelinski Institute of Organic Chemistry RAS, Moscow, Russia.
Collaborations: Otto A, Berlin Max-Delbrück Center
of Molecular Medicine, Berlin, Germany; Goldmann
Supported in part by: DFG (Li 448/4-1).
T, Kähler D, Division of Clinical and Experimental
Pathology; Ulmer AJ, Division of Cellular Immunology; Gorczynski RM, Departments of Surgery and Im-
Proteomic analysis of clinically-relevant tissue
munology, University Health Network, Toronto,
preparations based on two-dimensional gel elec-
Canada; Mach JP, Institute de Biochimie, University
trophoresis
de Lausanne, Lausanne, Switzerland; Rietschel ET,
Alexander C, Lindner B, Zähringer U.
Waelli T, Clinique La Prairie, Clarens-Montreux,
Switzerland.
The proteomes of multiple tissue-derived samples
were analyzed by the high resolution two-dimen-
Supported in part by: Clinique La Prairie, Clarens-
sional gel electrophoresis (2-DE) system WITAVISIONä (WITA GmbH, Teltow, Germany) and subse-
Montreux, Switzerland.
quent identification of selected protein spots by
mass fingerprinting of tryptic peptides in two ma-
Theses
jor projects: In the first project within the research
network funded by the swiss clinic: Clinique La
Dissertation
Prarie (CLP) two major immunoactivating chro-
Crone, Catharina
matographic fractions of a fetal sheep liver extract
Aufbau einer quantitativen massenspekrtometri-
have been characterized by 2-DE-based proteomics
schen Analytik zur Untersuchung der Lipidzusam-
and a total of 30 different proteins including the pro-
mensetzung zellulärer Membranen und ihre An-
inflammatory cytokines macrophage-migration in-
wendung auf Membranmikrodomänen.
hibitory factor (MIF) and interleukin 1b (IL-1b) could
Technisch-Naturwissenschaftliche Fakultät
be identified. Moreover, fetal hemoglobin (HbF)
Universität Lübeck, 2007.
and the corresponding heme-free globin a and g
(supervisor PD Dr. Buko Lindner)
chains were shown to display LPS-binding activities
and to promote the disaggregation of S- and R-type
Hübner, Göran
Aufbau und Kopplung einer Kapillarelektrophorese
50
Wissenschaftlicher Jahresbericht 2007-2008
Division of Immunochemistry
Research Reports
an ein hochauflösendes Massenspektrometer zur
Analyse amphiphiler Zellmembrankomponenten.
Mathematisch-Naturwissenschaftliche Fakultät
Christian-Albrechts-Universität zu Kiel, 2008.
(supervisor PD Dr. Buko Lindner)
Patents
Alexander C, Zähringer U, Division of Immunochemistry; Ulmer AJ, Division of Immunology and
Cell Biology, Research Center Borstel, and members of the Clinique La Prairie Research.
Sa, L-1528 Luxembourg (LU).
International (Non EU) extension of the following European patent: Compositions comprising Fetal
Hemoglobin and Bacterial Endotoxin and optionally Additional Fetal Liver Components. International
Publication Number: WO/2004/073728, International Application No.: PCT/EP2004/001553.
Wissenschaftlicher Jahresbericht 2007-2008
51
Division of Biophysics
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Biophysics
Head
thermore, the sepsis shock syndrome, which in many
PD Dr. Thomas Gutsmann
cases is a result of the fact that antibiotics may kill
bacteria, but do not inactivate bacterial virulence
Principle Investigators
factors such as endotoxins (lipopolysaccharides), is
Prof. Dr. Klaus Brandenburg
worldwide of increasing importance regarding the
very high death rate (40 to 50 %). Therefore, the de-
Staff Scientists and Postdoctoral Fellows
velopment of suitable antimicrobial drugs is ur-
PD Dr. Jörg Andrä
gently requested. In recent years, various groups focus on the use of antimicrobial peptides (AMP)
Graduate and Diploma Students
derived from natural, innate immunity proteins,
Arne Böhling (until July 2007)
which in vivo bind to the virulence factors. Such bind-
Gesa Helms (until February 2008)
ing proteins are, for example, lactoferrin, the Limu-
Ahmed Jaber (until March 2008)
lus anti-LPS-factor, and the family of saposin-like
Malte Hammer (until August 2008)
proteins (NK-lysins, granulysins), which all have par-
Nico Grimm (until Oktober 2008)
ticular binding domains for example for bacterial
Annemarie Brauser
endotoxin. Furthermore, an alternative approach to
Jörg Howe
combat infections is the use of cyclooxygenase and
Yani Kaconis
lipoxygenase inhibitors. From the reviewed litera-
Lingyi LI
ture the mechanisms of action of antiinfective compounds on bacteria, on pathogenicity factors of the
Technicians
bacteria, and on viruses are summarized. In the last
Gerold von Busse
years, considerable progresses have been made in
Sabine Dabelstein
the fight against infections, specially also to over-
Nina Hahlbrock
come bacterial resistance.
Christine Hamann
Collaboration: Garidel P, Universität Halle
Kerstin Stephan
Guests and Trainees
Dr. Satoshi Fukuoka
Mechanism of interaction of optimized Limulus-
Health Technology Research Center, National Insti-
derived cyclic peptides with endotoxins. Thermo-
tute of Advanced, Industrial Science and Technolo-
dynamical, biophysical, and microbiological
gy, Takamatsu
analysis
Andrä J, Howe J, Gutsmann T, Brandenburg K.
Research Reports
On the basis of formerly investigated peptides cor-
Novel antiinflammatory and antiinfective agents
responding to the endotoxin-binding domain from
Andrä J, Howe J, Gutsmann T, Brandenburg K.
the Limulus anti-lipopolysaccharide factor (LALF), a
protein from L. polyphemus, we have designed and
Despite the availability of antibiotics, infectious dis-
synthesized peptides of different lengths with the
eases become an even increasing threatening for
aim to get potential therapeutic agents against the
human health, in particular due to resistance de-
septic shock syndrom. For an understanding of the
velopment, for example in animal husbandry. Fur-
mechanisms of action, we performed a profound
52
Wissenschaftlicher Jahresbericht 2007-2008
Division of Biophysics
Research Reports
physico-chemical and biophysical analysis of the in-
ticular enterobacterial forms of LPS represent ex-
teraction of rough mutant lipopolysaccharide (LPS)
tremely strong activator molecules of the human im-
with these peptides by applying Fourier-transform
mune system causing a rapid induction of cytokine
infrared spectroscopy (FTIR), small-angle X-ray scat-
production in monocytes and macrophages. Vari-
tering (SAXS), calorimetric techniques (differential
ous mammalian blood proteins have been docu-
scanning calorimetry DSC and isothermal titration
mented to display LPS binding activities mediating
calorimetry ITC), and freeze-fracture transmission
normally decreasing effects in the biological activ-
electron microscopy (FFTEM). Also, the action of the
ity of LPS. In more recent studies, the essential sys-
peptides on bacteria of different origin in microbial
temic oxygen transportation protein hemoglobin
assays was investigated. Using FTIR and DSC, our
(Hb) has been shown to amplify LPS-induced cy-
data indicated a strong fluidization of the lipid A
tokine production on immune cells. The mechanism
acyl chains due to peptide binding, with a decrease
responsible for this effect is poorly understood.
of the endothermic melting enthalpy change of the
Here, we characterize the interaction of hemoglobin
acyl chains down to a complete disappearance in
with LPS by using biophysical methods. The data
the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range.
presented, revealing the changes of the type and
Via ITC, it was deduced that the binding is a clear-
size of supramolecular aggregates of LPS in the
ly exothermic process which runs into saturation al-
presence of Hb, allow a better understanding of the
so at a [LPS]:[peptide] = 1:0.5 to 1:2 molar ratio. The
hemoglobin-induced increase in bioactivity of LPS.
data obtained with SAXS indicated a drastic
change of the aggregate structures of LPS into a
Collaboration: Garidel P, Universität Halle, Roessle
multilamellar stack, which was visualized in electron
M European Molecular Biology Laboratory, Alexan-
micrographs as hundreds of lamellar layers. This
der C, Div. of Immunochemistry, Fournier K and
can be directly correlated with the inhibition of the
Mach J.P. Universite Lausanne, Waelli T. Clinique La
LPS-induced production of tumor-necrosis-factor-a in
Prairie, Montreux, Gorczynski R University of Toron-
human mononuclear cells, but not with the action of
to, Ulmer AJ, Div. of Immunobiology, Zähringer U,
the peptides on bacteria.
Div. of Immunochemistry, Rietschel E Th, LeibnizGemeinschaft, Berlin.
Collaboration: Garidel P, Universität Halle, Rössle
M, European Molecular Biology Laboratory, Ham-
Supported in part by: Clinique La Prairie, Switzer-
burg, Richter W, Universität Jena, Leiva-León J and
land.
Moriyon I, Universidad de Navarra, Pamplona, Bartels R, Div. of Immunochemistry.
Thermodynamic analysis of the lipopolysaccharide-dependent resistance of Gram-negative bacBiophysical characterization of the interaction of
teria against polymyxin B
endotoxins with hemoglobins
Howe J, Andrä J, Gutsmann T, Brandenburg K.
Howe J, Hammer M, Brandenburg K.
Cationic antimicrobial cationic peptides (CAMP)
Bacterial endotoxin (lipopolysaccharide, LPS) is the
have been found in recent years to play a decisive
major component of the outer leaflet of the outer
role in host’s defence against microbial infection,
membrane in Gram-negative bacteria. During se-
but are also pharmaceutically developed as new
vere infections, bacteria may reach the blood circuit
therapeutic tool, necessary in particular due to the
of humans, and endotoxins may be released from
occurrence of resistance of microbiologic popula-
the bacteria due to cell division or cell death. In par-
tions to antibiotics. The structural basis of this phe-
Wissenschaftlicher Jahresbericht 2007-2008
53
Division of Molecular Infection Biology
Research Reports
nomenon is only partially elucidated, and may com-
mic reaction in the liquid crystalline phase (Fig. 13).
prise quite different mechanism at the site of the
In contrast, for resistant LPS the binding enthalpy
bacterial cell membranes or in their cytoplasm.
change remains endothermic in both phases. As in-
Polymyxin B (PMB) is a well-studied representative
frared data show, these differences can be ex-
of a CAMP in particular against Gram-negative bac-
plained by sterical changes in the head group re-
teria to understand the interaction with the outer
gion of the respective LPS.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
membrane or isolated membrane compounds such
as lipopolysaccharide (LPS) and to define the mech-
Collaboration: Conde R, Iriarte M, and Moriyón I.,
anism by which the peptides kill bacteria or neu-
Universidad de Navarra, Pamplona, Garidel P, Uni-
tralize LPS. Since PMB-resistance of bacteria is a
versiät Halle, Koch MHJ European Molecular Biolo-
long-known phenomenon and is attributed to struc-
gy Laboratory, Hamburg.
tural changes in the LPS moiety of the respective
bacteria, we have performed a thermodynamic and
biophysical analysis of the mechanisms of various
Rationale for the design of shortened derivatives
LPS:PMB interactions in comparison to LPS from sen-
of the NK-lysin derived antimicrobial peptide NK-
sitive strains.
2 with improved activity against gram-negative
pathogens
Andrä J, Brandenburg K.
The peptide NK-2 is an effective antimicrobial agent
with low hemolytic and cytotoxic activities and is
thus a promising candidate for clinical applications.
It comprises the a-helical, cationic core region of
porcine NK-lysin a homolog of human granulysin
and of amoebapores of pathogenic amoeba. Here
we visualized the impact of NK-2 on E. coli by electron microscopy and used NK-2 as a template for sequence variations to improve the peptide stability
and activity and to gain insight into the structure/function relationships. We synthesized 18 new
peptides and tested their activities on 7 Gram-negative and one Gram-positive bacterial strains, human erythrocytes and HeLa cells. Although all peptides appeared unordered in buffer, those active
Fig. 13. Isothermal calorimetric titration of LPS (0.05 mM) from P.
mirabilis R45 (top) and S. minnesota R595 (bottom) with PMB (3
mM) at 37 °C. For this, the LPS dispersion in the calorimetric cell
was titratetd every 5 min with 3 ml of PMB. The increase in the feedback power indicates an endothermic, the decrease an exothermic process.
against bacteria adopted an a-helical conformation in membrane-mimetic environments like triflouroethanol and negatively charged phosphatidylglycerol (PG) liposomes that mimick the
cytoplasmic membrane of bacteria. This conforma-
In isothermal titration calorimetric experiments we
tion was not observed in the presence of liposomes
have found considerable differences of PMB bind-
consisting of zwitterionic phosphatidylcholine (PC)
ing to sensitive and resistant LPS, for sensitive LPS
typical for the human cell plasma membrane. The
the endothermic enthalpy change in the gel phase
interaction was paralleled by intercalation of these
of the hydrocarbon chains converts into an exother-
peptides into PG-liposomes as determined by FRET-
54
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Infection Biology
Research Reports
spectroscopy. A comparative analysis between biological activity and the calculated peptide parameters revealed that the decisive factor for a
broad spectrum activity is not the peptide overall
hydrophobicity or amphipathicity but the possession of a minimal positive net charge plus an highly amphipathic anchor point of only 7 amino acid
residues (2 helical turns).
Collaboration: Goldmann T, Clinical and Experimental Pathology, Bartels R, Immunochemistry, Research Center Borstel; Monreal D, Martinez de Tejada G, Sanchez Gomez S, Moriyon I, Universidad
de Navarra, Pamplona, Spain; Olak C, Brezesinski
B, Max-Planck-Institut für Kolloid- und Grenzflächenforschung, Potsdam.
Supported in part by: SFB617, A17.
Structure and mode of action of the antimicrobial
Andrä J, Gutsmann T.
Fig. 14. Effect on the morphology of bacteria upon arenicin treatment (I). AFM images of untretated E. coli WBB01 (A) and incubated with 10 µM arenicin (B) in LB medium at 37°C for 30 min. Images were taken in air in AC (tapping) mode.
The solution structure and the mode of action of
Collaboration: Goldmann T, Clinical and Experi-
arenicin isoform 1, an antimicrobial peptide with a
mental Pathology, Research Center Borstel;
unique 18-residue loop structure, from the lugworm
Jakovkin I, Hecht O, Grötzinger J, Institute of Bio-
Arenicola marina were elucidated here. Arenicin
chemistry; Leippe, M, Zoological Institute, Christian-
folds into a two-stranded anti-parallel b-sheet. It ex-
Albrechts-University, Kiel; Krasnosdembskaya AD,
hibits high antibacterial activity at 37°C and 4°C
St. Petersburg State University, St Petersburg, Russia.
peptide arenicin
against Gram-negative bacteria, including polymyxin
B resistant Proteus mirabilis. Bacterial killing occurs
Supported in part by: SFB617, A17.
within minutes and is accompanied by membrane
permeabilisation, membrane detachment, and release of cytoplasm. Interaction of arenicin with re-
Surface acoustic wave biosensor as a tool to
constituted membranes that mimic the lipopolysac-
study the interaction of antimicrobial peptides
charide-containing
with phospholipid and lipopolysaccharide mod-
outer
membrane
or
the
phospholipid-containing plasma membrane of Gram-
el membranes
negative bacteria exhibited no pronounced lipid
Andrä J, Böhling A, Gutsmann T.
specificity. Arenicin-induced current fluctuations in planar lipid bilayers correspond to the formation of short
Surface acoustic wave biosensors are a powerful
lived heterogeneously structured lesions. Our data
tool for the study of biomolecular interactions. The
strongly suggest that membrane interaction plays a
modulation of a surface-confined acoustic wave is
pivotal role in the antibacterial activity of arenicin.
utilized here for the analysis of surface binding.
Wissenschaftlicher Jahresbericht 2007-2008
55
Division of Molecular Infection Biology
Research Reports
Phase and amplitude of the wave correspond
Collaboration: Gronewold TMA, Perpeet M,
roughly to mass loading and viscoelastic properties
Schlecht U, Biosensor GmbH, Bonn.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
of the surface, respectively. We established a procedure
to
reconstitute
phospholipid
and
Supported in part by: SFB617, A17.
lipopolysaccharide bilayers on the surface of a
modified gold sensor chip to study the mode of action of membrane-active peptides. The procedure
Physicochemical and biological analysis of syn-
included the formation of a self-assembled mono-
thetic bacterial lipopeptides: Validity of the con-
layer of 11-mercaptoundecanol, covalent coupling
cept of ’endotoxic conformation’
of carboxymethyl-dextran, and subsequent coating
Howe J, Gutsmann T, Andrä J, Brandenburg K.
with a poly-L-lysine layer. The lipid coverage of the
surface is highly reproducible and homogenous as
Lipoproteins from the outer or cytoplasmic mem-
demonstrated in atomic force micrographs.
branes of Gram-positive and Gram-negative bacte-
Ethanol/triton treatment removed the lipids com-
ria have been recognized increasingly to be im-
pletely which provided the basis for continuous se-
portant regarding their biological function and
quences of independent experiments. The setup
activity. They are, beside endotoxins (lipopolysac-
was applied to investigate the binding of human
charide, LPS) as main amphiphilic component of the
cathelicidin-derived peptide LL32, as an example
outer membrane of Gram-negative bacteria, es-
for antimicrobial peptides, to immobilized phos-
tablished as potent stimulants of the human innate
phatidylserine membranes. The peptide-membrane
immune system and elicit a variety of proinflamma-
interaction results in a positive phase shift and an
tory immune responses. Investigations of synthetic
increase in amplitude, indicating a mass increase
lipopeptides corresponding to N-terminal partial
along with a loss in viscosity. This suggests that the
structures of bacterial lipoproteins defined the
bilayer becomes more rigid upon interaction with
chemical prerequisites for their biological activity,
LL32.
especially with respect to the number and length of
acyl chains and sequence of the peptide part. Here
we present experimental data on the biophysical
mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed, that the molecular
geometry of these molecules, i.e. the measured
molecular conformations and supramolecular aggregate structures and the preference for membrane intercalation allow an understanding of the
biological activities of the different lipopeptides.
This refers in particular to the agonistic or antagonistic activity, i.e., their ability to induce cytokines in
mononuclear cells or to block this activity, respectively. The analytical data show that our concept of
‘endotoxic conformation’, originally developed for
LPS, can be applied also to the investigated
lipopeptides, and suggest that the molecular mech-
Fig. 15. Schematics of biosensor and the functionalization of the
gold surface by a self-assembled monolayer (SAM), a layer of CMdextran, Poly-L-lysine, and a phospholipid membrane.
56
anisms of cell activation by amphiphilic molecules
are governed by a general principle.
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Infection Biology
Research Reports
Collaboration: Schromm AB Div. of Immunobio-
of an increase in LPS mediating endotoxicity due to
physics, Ulmer AJ Div. of Immunobiology, Wies-
the action of Hb.
müller KH, EMC Microcollections, Tübingen, Seyberth T, Jung G, Universität Tübingen, Rössle M and
Collaboration: Garidel P, Universität Halle, Roessle
Koch MHJ, European Molecular Biology Laboratory,
M European Molecular Biology Laboratory, Richter
Hamburg.
W Universität Jena, Christian Alexander Div. of Immunochemistry, Fournier k and Mach J.P. Universite
Lausanne, Waelli T. Clinique La Prairie, Montreux,
Structural investigations into the interaction of
Gorczynski R University of Toronto, Ulmer AJ Div. of
hemoglobin and part structures with bacterial en-
Immunobiology, Zähringer U Div. of Immunochem-
dotoxins
istry, Hartmann A Clinique La Prairie, Montreux, Ri-
Howe J, Brandenburg K.
etschel E Th, Leibniz-Gemeinschaft, Berlin.
The interaction of bacterial endotoxins (lipopolysac-
Supported in part by: Clinique La Prairie, Montreux.
charide, LPS) with the oxygen transport protein
hemoglobin has been previously investigated, but
a detailed understanding of the mechanisms in-
Theses
volved is still lacking. Here, a biophysical investigation into the endotoxin:hemoglobin interaction is
Dissertation
presented which comprises the use of various rough
Böhling, Arne
mutant LPS as well as free lipid A, and, in addition
Biophysikalische Untersuchungen an bakteriellen
to the complete hemoglobin from fetal sheep ex-
Modellmembranen bezüglich ihrer lateralen Orga-
tract also the partial structures a-chain and the
nisation und der Interaktion mit humanen
heme-free sample. The investigations comprise the
sinen
determination of the gel to liquid crystalline phase
Mathematisch-Naturwissenschaftliche Fakultät
behaviour of the acyl chains of LPS, the ultrastruc-
Christian-Albrechts-Universität zu Kiel, 2007.
-Defen-
ture (type of aggregate structure and morphology)
of the endotoxins, and the incorporation of the
Hammer, Malte
hemoglobins into artificial immune cell membranes
Struktur-Wirkungsbeziehungen
and into LPS. Our data allow to suggest a model of
branaktiven Peptiden und bakteriellen Lipopoly-
the interaction between Hb and LPS in that
sacchariden
hemoglobins do neither react strongly with the hy-
Mathematisch-Naturwissenschaftliche Fakultät
drophilic nor with the hydrophobic moiety of LPS,
Christian-Albrechts-Universität zu Kiel, 2007.
zwischen
mem-
but with the complete endotoxin. Hb is able to incorporate into LPS with the longitudinal direction
Diploma
parallel to the lipid A double-layer. Although this
Brauser, Annemarie
does not lead to a strong disturbance of the acyl
Rasterkraftmikroskopische Untersuchungen zur In-
chain packing, the change of the curvature leads to
teraktion zwischen Phospholipidmembranen und
a slightly conical molecular shape with a change of
LBP (Lipopolysaccharid-bindendes Protein)
the three-dimensional arrangement from unilamel-
Fachbereich Angewandte Naturwissenschaften
lar into cubic aggregates. Our previous results show
Fachhochschule Lübeck, 2007.
that cubic LPS structures exhibit strong endotoxic activity. The here observed property of Hb on the
Helms, Gesa
physical state of LPS may explain the observation
Schichtdickenmessungen an rekonstituierten fest-
Wissenschaftlicher Jahresbericht 2007-2008
57
Division of Molecular Infection Biology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
körperunterstützten Lipidmembranen
Mathematisch-Naturwissenschaftliche Fakultät
Christian-Albrechts-Universität zu Kiel, 2008.
Grimm, Nico
Aufbau und Programmierung einer Filmwaage zur
biophysikalischen Untersuchung von Lipdmonoschichten
Fachbereich Angewandte Naturwissenschaften
Fachhochschule Lübeck, 2008.
Jaber, Ahmed
Spektroskopische und kalorimetrische Untersuchungen von Lipid-Peptid-Interaktionen
Fachbereich Angewandte Naturwissenschaften
Fachhochschule Lübeck, 2008.
Patents
Andrä, J., Blondelle, S. E., Brandenburg, K., Deutsch,
G., Japelj, B., Jerala, R., Leiva Leon, J., Lohner, K.,
Majerle, A., Martinez de Tejada, G., Moriyon, I., Porro, M., Pristovsek, P., Zorko, M. and Zweytick, D.
(2008) Antimicrobial Peptides. World Intellectual
Property Organization (WO 2008/006125 A1)
Brandenburg, K. Novel antimicrobial peptides. European patent office, P1712 EP.
58
Wissenschaftlicher Jahresbericht 2007-2008
Emmy-Noether Junior Research Group Immunobiophysics
Research Reports
Emmy-Noether Junior Research Group
Immunobiophysics
Head
lipopeptides revealed, that the molecular geome-
PD Dr. Andra Schromm
try of these molecules, i.e. the molecular conformations and supramolecular aggregate structures de-
Graduate Student
termined
Susanne Keese (since April 2008)
measurements and the preference for membrane
from
small-angle
X-ray
diffraction
intercalation allow an understanding of the biologTechnicians
ical activities of the different lipopeptides. This
Sabrina Groth
refers in particular to the agonistic or antagonistic
Nina Hahlbrock (until April 2008)
activity, i.e., their ability to induce cytokines in
mononuclear cells or to block this activity, respec-
Trainees
tively (Fig. 16). The analytical data show that our
Kai Reichelt (Jan-März 2008)
concept of ‘endotoxic conformation’, originally de-
Thore Masekowitz (August-November 2008)
veloped for LPS, can be applied also to the investigated lipopeptides, and suggest that the molecular
mechanisms of cell activation by amphiphilic
Research Reports
molecules are governed by a general principle.
Physicochemical and biological characterisation of synthetic bacterial lipopeptides: Validity of the concept of ’endotoxic
conformation’
Schromm AB, Howe J*, Brandenburg K*.
Lipoproteins from the outer or cytoplasmic
membranes of Gram-positive and Gramnegative bacteria have been recognized increasingly to be important regarding their biological function and activity. They are,
beside endotoxins (lipopolysaccharide, LPS)
as main amphiphilic component of the outer
membrane of Gram-negative bacteria, established
as potent stimulants of the human innate immune
system and elicit a variety of proinflammatory im-
Fig. 16. Correlation between the molecular conformation of lipid A
from E.coli, synthetic lipid A precursor IVa (“compound 406”), and
synthetic lipopeptides determined from small-angle X-ray diffraction and biological activity in human macrophages.
mune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures
Collaboration: Howe J, Brandenburg K, *Divison of
of bacterial lipoproteins defined the chemical pre-
Biophysics; Ulmer AJ, Division of Cellular Immunol-
requisites for their biological activity, especially with
ogy; Wiesmüller K-H, Seyberth T, EMC microcollec-
respect to the number and length of acyl chains and
tions GmbH, Tübingen; Rössle M, European Molec-
sequence of the peptide part. We obtained exper-
ular Biology Laboratory c/o DESY, Hamburg.
imental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of se-
Supported in part by: This work was funded by DFG
lected
grants (SCHR 621/2-2, SCHR 621/2-3 and Ul 68/3-1),
synthetic
diacylated
and
triacylated
Wissenschaftlicher Jahresbericht 2007-2008
59
Emmy-Noether Junior Research Group Immunobiophysics
Research Reports
the European Commission (ANEPID), and the Fed-
that the lipopeptide represents an important stim-
eral Ministry of Education and Research (project
ulus of the innate immune response in the early
Biochance no. 0312662).
stages of infection.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Collaboration: Reiling N, Division of Microbial InSerum-dependent immune recognition of a syn-
terface Biology; Howe J, Brandenburg K, Division of
thetic lipopeptide mimetic of the 19-kD lipopro-
Biophysics; Wiesmüller KH, EMC microcollections,
tein from Mycobacterium tuberculosis
Tübingen; Roessle M, European Molecular Biology
Groth S, Schromm AB.
Laboratory c/o DESY, Hamburg.
Host defense against invading pathogens is me-
Supported in part by: This work was funded by DFG
diated by the combined action of the innate and
grants SCHR 621/2-2 and SCHR 621/2-3.
the adaptive immune system. The innate immune
response provides a critical first line defense
against Mycobacterium tuberculosis, an intracel-
Innate immune regulation by pulmonal collectins
lular pathogen that represents a major health
Stamme C*, Moulakakis C*, Keese S, Sender V*,
threat worldwide. The 19-kDa lipoprotein is a cell-
Schromm AB.
wall-associated antigen expressed in M. tubercu-
losis. A synthetic lipopeptide mimicking the lipid
Pulmonary diseases caused by Gram-negative
moiety of the 19-kDa lipoprotein from M. tubercu-
bacteria are the leading cause of mortality from in-
losis has recently been assigned an important role
fectious diseases. The difficulties associated with
in the induction of an antibacterial immune re-
effective treatment of these diseases include the
sponse in host macrophages. We have investigat-
emergence of antibiotic-resistant pathogens, in-
ed the biological activities and the biophysical
creasing numbers of elderly individuals und im-
mechanisms underlying cell activation by synthet-
munocompromised patients. An attractive target
ic 19-kDa M. tuberculosis-derived lipopeptide. In-
for improving clinical outcomes is the modulation
vestigation of the geometry of the lipopeptide (i.e.
of the host pulmonary immune response itself. Sur-
the molecular conformation and supramolecular
factant protein A is one of the primary secreted
aggregate structure) and the preference for mem-
components of lung innate immunity eliciting es-
brane intercalation provide an explanation for the
sential immunomodulatory functions on alveolar
biological activities of the mycobacterial lipopep-
macrophages which constitute the majority of resi-
tide. Surprisingly, we found that activation of hu-
dent immune cells of the lung. We investigate the
man macrophages to induce proinflammatory me-
physico-chemical basis and molecular mechanisms
diators (TNFα, IL-6, IL-8) in response to the
of surfactant protein-A dependent regulation of the
Mtb-lipopeptide is strongly enhanced in the ab-
pulmonary
sence of serum proteins suggesting a lung-specif-
lipopolysaccharide. SP-A stabilizes IκB-α, the pri-
ic adaptation of the immune recognition. This ob-
mary regulator of NF-κB, in alveolar macrophages
servation could be confirmed for the immune
both constitutively and in the presence of LPS. Us-
response of murine macrophages which showed a
ing alveolar macrophages and PBMC from IκB-α
strongly enhanced mediator release (TNFα) in the
knockout/IκB-β knockin mice, we could show that
absence of serum. Our data suggest that the
SP-A fails to inhibit LPS-induced TNF-α production
molecular mechanisms of immune recognition of
(Fig. 17) and p65 nuclear translocation, confirming
the lipopeptide derived from M. tuberculosis are
a critical role for IκB-α in SP-A-mediated LPS inhi-
tailored to the ambient conditions of the lung and
bition.
60
immune
response
to
bacterial
Wissenschaftlicher Jahresbericht 2007-2008
Emmy-Noether Junior Research Group Immunobiophysics
Research Reports
*Division of Cellular Pneumology; Leitges M, The
Biotechnolgy Centre of Oslo, University of Oslo.
Supported in part by: This work was funded by DFG
grants 609/1-3, 609/1-4, SCHR 621/2-2 and
SCHR621/3-1.
Fig. 17. SP-A fails to inhibit LPS-induced activation of IκB-α knockout/IκB-β knockin (AKBI) cells. Pooled PBMC from either three to
four AKBI (A) or three wt mice (B) were left untreated or treated
with 20-60 mg/ml SP-A (37°C, 1 h), and then exposed either to media, or to 0.1-100 ng/ml LPS. Cell-free supernatants were harvested after 4 h for the determination of TNF-α by ELISA. The data
shown are mean ± SE of three independent experiments. Statistical analysis was performed using a two-way ANOVA with a Bonferroni post-test. * p < 0.05, ** p < 0.01, † p < 0.001 (versus LPS-induced TNF-a release in the absence of SP-A).
We identifed atypical (a) protein kinase C ζ as a
pivotal upstream regulator of SP-A mediated IκBα/NF-κB pathway modulation deduced from blocking experiments and confirmed by using alveolar
macrophages from aPKC ζ deficient mice. SP-A
transiently triggers aPKCThr410/403 phosphorylation, aPKC kinase activity, and translocation in primary rat alveolar macrophages. Coimmunoprecipitation experiments reveal that SP-A induces
aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory
macrophage activation via IκB-α by SP-A critically
depends on PKCζ activity, and thus attribute a novel, stimulus-specific signaling function to PKCζ in SPA-modulated pulmonary immune response.
Collaboration: Moulakakis C, Sender V, Stamme C,
Wissenschaftlicher Jahresbericht 2007-2008
61
Division of Molecular Infection Biology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Molecular Infection Biology
Head
(ii) MyD88 is essential, but not because it transduces
Prof Dr. Stefan Ehlers
signals from TLRs, (iii) adaptive immunity to TB is
largely TLR/MyD88-independent (Fig. 18).
Principle Investigator
PD Dr. Norbert Reiling
Postdoctoral Fellows
Dr. Kerstin Walter
Dr. Evelin Grage-Griebenow (since 2/08)
Dr. Sahar Aly (since 11/08)
Dr. Jochen Behrends (since 4/08)
Bachelor, Diploma/Master and Graduate Students
Fig. 18 Schematic summary of mycobacterial proliferation in the
lungs of Mtb-infected mice deficient for various TLRs, MyD88, IL-1R
or IFN-γ.
Dagmar Schneider (until 9/08)
Some of the discrepancies may be resolved by co-
Jan Neumann
gent attribution of distinct immune functions to the in-
Kollja Schaale
dividual components of the TLR/MyD88 system. In
Christine Steinhäuser
mice, TLRs and MyD88 are fully dispensable in sensing Mtb infection and instructing T cell-mediated
Technicians
adaptive immunity, and while TLRs are also redun-
Svenja Kröger
dant during macrophage effector immunity, MyD88
Silvia Maass
is essential for efficient killing of mycobacteria. This
Stefanie Pfau
distinction should help to molecularly pinpoint the
Kathrin Seeger
MyD88-dependent, yet TLR-independent critical
Kerstin Kopp
mechanisms of macrophage activation involved in in-
Manfred Richter
tracellular growth restriction of M. tuberculosis. Disrupted IL-1R and/or IFN-gamma signaling pathways
Guests and Trainees
likely play a much more prominent role in explaining
Daniel Behme
the exquisite susceptibility of MyD88-deficient mice to
Department of Haematology/Oncology, Georg-Au-
TB than the function of MyD88 as a TLR adaptor.
gust-University, Göttingen
Collaboration: Hölscher C, Junior Professorship
Molecular Infection Biology, FZB.
Research Report
MyDths and un-TOLLed truths: sensor, instructive
Supported in part by: DFG grant SFB415-C7.
and effector immunity to
tuberculosis
LspA inactivation in Mycobacterium tuberculosis
Reiling N, Ehlers S.
results in attenuation without affecting phagoControversy exists concerning the role of Toll-like re-
some maturation arrest
ceptors and MyD88 in immunity to tuberculosis (TB).
Walter K, Keller C, Ehlers S.
Our data argue that (i) Toll-like receptors are not es-
The success of Mycobacterium tuberculosis de-
sential for an effective immune response against TB,
pends on its ability to survive within host
62
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Infection Biology
Research Reports
macrophages. Here, M. tuberculosis avoids the
Ac(2)PIM(5). Phosphatidylinositol mannoside and
acidic, hydrolytically competent environment of the
its related compounds lipomannan (LM) and
phagolysosome by arresting phagosome matura-
lipoarabinomannan (LAM) have been shown to
tion. Having shown previously that a M. tuberculo-
modulate the host response to an infection with M.
sis mutant deficient in lipoprotein signal peptidase
tuberculosis. Here, we generated a deletion mutant
(LspA) is strongly attenuated in vivo in a mouse
of Rv1500 in M. tuberculosis H37Rv, and analyzed
model of infection, we now studied putative mech-
the mutant using a biochemical approach as well
anisms involved in attenuation of the lspA: : aph mu-
as in vitro and in vivo infection models. Inactivation
tant at a cellular level. In this work we investigated
of Rv1500 did not lead to an altered expression pat-
the ability of the mutant to interfere with two host de-
tern of PIMs in M. tuberculosis H37Rv. We found
fence mechanisms, i.e. Toll-like receptor (TLR)2-de-
phosphatidylinositol
pendent immune response and phagosome matu-
Ac(2)PIM(2), and AcPIM(6) in both strains, but were
ration. While mycobacterial lipoproteins have been
unable to detect Ac(2)PIM(7) or Ac(2)PIM(5) either
reported to trigger a TLR2 signalling pathway criti-
in the wild type or the mutant strain. Uptake and
cal for innate immune responses, we found that
growth of H37Rv and Rv1500 mutant strains in
growth control of the lspA : : aph mutant was inde-
murine bone marrow-derived macrophages was
pendent of TLR2. In addition, the lspA : : aph mutant
identical, and TNFalpha and IL-12p40 production in
arrested phagosome maturation to an extent simi-
mouse macrophages and dendritic cells was in-
lar to that of the wild-type, as measured by lysoso-
duced to similar levels following infection with either
mal-associated membrane protein 1 (LAMP1) co-lo-
strain. Aerosol challenge of mice showed that wild
calization
These
type and Rv1500 mutant strains had identical
observations demonstrate severe attenuation even
growth rates in infected organs over time. We veri-
in the presence of arrested phagosome maturation,
fied mRNA expression of Rv1500 in H37Rv and con-
and point to a role for the early phagosome in
clude that Rv1500 must serve a redundant role in vi-
growth restriction of the M. tuberculosis lspA mutant.
ability and virulence of M. tuberculosis.
Collaboration: Rampini SK, Selchow P, Böttger EC,
Collaboration: Malm S, Bange FC, Department of
Sander P, Institut für Medizinische Mikrobiologie,
Medical Microbiology and Hospital Epidemiology,
Universität Zürich, Switzerland.
Medical School Hannover, Germany; Holst O, Engel
and
intraphagosomal
pH.
(PI),
PIM(2),
AcPIM(2),
R, Structural Biochemistry, FZB; Lindner B, ImmunoSupported in part by: Cluster of Excellence, Re-
chemistry, FZB.
search Area H.
Supported in part by: DFG grant SFB470-C9.
In vitro and in vivo characterization of a My-
cobacterium tuberculosis mutant deficient in gly-
Mycobacterium tuberculosis prevents inflamma-
cosyltransferase Rv1500.
some activation
Walter K, Ehlers S.
Walter K, Keller C, Ehlers S.
In Mycobacterium marinum, the homologue of
Mycobacterium tuberculosis (Mtb) parasitizes host
Rv1500 of M. tuberculosis encodes a glycosyltrans-
macrophages and subverts host innate and adap-
ferase. Initially, it was suggested that this gene is in-
tive immunity. Several cytokines elicited by Mtb are
volved in the synthesis of phosphatidylinositol man-
mediators of mycobacterial clearance or are in-
nosides (PIMs), generating Ac(2)PIM(7) from
volved in tuberculosis pathology. Surprisingly, inter-
Wissenschaftlicher Jahresbericht 2007-2008
63
Division of Molecular Infection Biology
Research Reports
leukin-1beta (IL-1beta), a major proinflammatory cy-
fected by the mutation, indicating a separate path-
tokine, has not been implicated in host-Mtb inter-
way for arginine degradation under aerobic condi-
actions. IL-1beta is activated by processing upon as-
tions. Following aerosol infection in mice, the
sembly of the inflammasome, a specialized
DeltaarcA mutant and wild-type strain of M. tuber-
inflammatory caspase-activating protein complex.
culosis multiplied and persisted in infected organs
Here, we show that Mtb prevents inflammasome ac-
in a similar fashion.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
tivation and IL-1beta processing. An Mtb gene,
zmp1, which encodes a putative Zn(2+) metallo-
Collaboration: Sürken M, Röhker C, Bange FC. De-
protease, is required for this process. Infection of
partment of Medical Microbiology and Hospital
macrophages with zmp1-deleted Mtb triggered ac-
Epidemiology, Medical School Hannover, Germany.
tivation of the inflammasome, resulting in increased
IL-1beta secretion, enhanced maturation of Mtb containing phagosomes, improved mycobacterial
Location of persisting mycobacteria in a guinea
clearance by macrophages, and lower bacterial
pig model of tuberculosis revealed by r207910
burden in the lungs of aerosol-infected mice. Thus,
Aly S, Ehlers S.
we uncovered a previously masked role for IL-1beta in the control of Mtb and a mycobacterial system
The lengthy chemotherapy of tuberculosis reflects
that prevents inflammasome and, therefore, IL-1be-
the ability of a small subpopulation of Mycobac-
ta activation.
terium tuberculosis bacteria to persist in infected individuals. To date, the exact location of these per-
Collaboration: Master SS, Davis AS, Timmins GS,
sisting bacteria is not known. Lung lesions in guinea
Deretic V. Department of Molecular Genetics and
pigs infected with M. tuberculosis have striking sim-
Microbiology, University of New Mexico Health Sci-
ilarities, such as necrosis, mineralization, and hy-
ences Center, Albuquerque, USA; Rampini SK,
poxia, to natural infections in humans. Guinea pigs
Springer B, Sander P, Institut für Medizinische Mikro-
develop necrotic primary lesions after aerosol in-
biologie, Universität Zürich, Switzerland.
fection that differ in their morphology compared to
secondary lesions resulting from hematogenous dis-
Supported in part by: the Cluster of Excellence, Re-
semination. In infected guinea pigs conventional
search Area H.
therapy for tuberculosis during 6 weeks reduced the
bacterial load by 1.7 logs in the lungs and, although
this completely reversed lung inflammation associ-
Anaerobic arginine metabolism of Mycobacteri-
ated with secondary lesions, the primary granulo-
um tuberculosis is mediated by arginine deimi-
mas remained largely unaffected. Treatment of an-
nase (arcA), but is not essential for chronic per-
imals with the experimental drug R207910 (TMC207)
sistence in an aerogenic mouse model of
for 6 weeks was highly effective with almost com-
infection
plete eradication of the bacteria throughout both
Walter K, Keller C, Ehlers S.
the primary and the secondary lesions. Most importantly, the few remnants of acid-fast bacilli re-
In many pathogens, degradation of arginine via the
maining after R207910 treatment were to be found
arginine deiminase pathway supports anaerobic
extracellular, in a microenvironment of residual pri-
metabolism. Here we show by deletion of Rv1001
mary lesion necrosis with incomplete dystrophic cal-
(arcA) in Mycobacterium tuberculosis that this gene
cification. This zone of the primary granuloma is hy-
functions as an arginine deiminase. Arginine
poxic and is morphologically similar to what has
metabolism in the presence of oxygen was not af-
been described for human lung lesions (Fig. 19).
64
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Infection Biology
Research Reports
These results show that this acellular rim may, there-
IRF-1 appeared to be directly involved in the differ-
fore, be a primary location of persisting bacilli with-
entiation of a type 1 immune response to my-
standing drug treatment.
cobacterial infection. In summary, IRF-1, rather than
being a mere transcription factor downstream of
IFN-gamma, may be a master regulator of mycobacteria-induced immunopathology.
Fig. 19. Hypoxic and necrotizing granuloma in the lung of a M. tuberculosis-infected guinea pig detected by pimonidazole immunohistochemistry.
Collaboration: Lenaerts AJ, Hoff D, Andries K,
Cantarero L, Orme IM, Basaraba RJ. Department of
Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, USA.
Mycobacteria-induced granuloma necrosis depends on IRF-1
Aly S, Reiling N, Ehlers S.
In a mouse model of mycobacteria-induced immunopathology, wildtype C57BL/6 (WT), IL-18-knockout (KO), and IFNalphabeta-receptor-KO mice developed
circumscript,
centrally
necrotizing
granulomatous lesions in response to aerosol infection with M. avium, whereas mice deficient in the
IFN-gamma-receptor, STAT-1 or IRF-1 did not exhibit granuloma necrosis. Comparative, microarray-
Fig. 20. Differential gene expression in the lungs of WT versus IRF1-KO mice infected with Mycobacerium avium TMC724. Graphic
representation of a cluster analysis including 528 genes regulated
more than 2-fold between WT and IRF-1 KO lungs infected with M.
avium. Each row in the heat map represents one probe set, the
columns stand for the conditions tested. A red square indicates
higher expression, a green square indicates lower expression relative to a row-wise mean (black). The different groups A-F comprise genes regulated in a similar manner.
based gene expression analysis in the lungs of infected WT and IRF-1-KO mice identified a set of
Collaboration: Mages J, Lang R, Institute for Medi-
genes whose differential regulation was closely as-
cal Microbiology and Immunology, Technical Uni-
sociated with granuloma necrosis, among them
versity of Munich; Kalinke U, Paul-Ehrlich-Institute,
cathepsin K, cystatin F and matrix metalloprotease
Langen; Decker T, Institute for Biochemistry, Univer-
10 (Fig. 20). Further microarray-based comparison
sity of Vienna, Austria.
of gene expression in the lungs of infected WT, IFN-
Supported in part by: DFG grant SFB367, C9 and
gamma-KO and IRF-1-KO mice revealed four distinct
BMBF grant NGFN-2.
clusters of genes with variable dependence on the
presence of IFN-gamma, IRF-1 or both. In particular,
Wissenschaftlicher Jahresbericht 2007-2008
65
Division of Molecular Infection Biology
Research Reports
Mice that overexpress CC chemokine ligand 2 in
Collaboration: Schreiber O, Steinwede K, Ding N,
their lungs show increased protective immunity to
Srivastava M, Maus R, Länger F, Prokein J, Welte T,
infection with Mycobacterium bovis bacilli Cal-
Gunn MD, Maus UA, Department of Pulmonary
mette-Guérin
Medicine, Medical School Hannover, Germany.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Ehlers S, Behrends J.
The acute phase of mycobacterial lung infection is
Decreased pathology and prolonged survival of
characterized by a nearly exponential outgrowth of
human DC-SIGN transgenic mice during my-
mycobacteria in the alveolar airspace and lung
cobacterial infection
parenchymal tissue, suggesting insufficient early
Reiling N, Fessler C, Walter K, Ehlers S.
protective immunity against mycobacterial challenge. In the current study, we tested the hypothe-
Dendritic cell (DC)-specific intercellular adhesion
sis that a CC chemokine ligand 2 (CCL2)-dependent
molecule-3 grabbing nonintegrin (DC-SIGN: CD209)
increased mononuclear phagocyte subset accumu-
is a C-type lectin that binds ICAM-2,3 and various
lation in distal airspaces would improve the lungs’
pathogens such as HIV, helicobacter, and my-
protective immunity to infection with Mycobacterium
cobacteria. It has been suggested that Mycobac-
bovis bacille Calmette-Guérin (BCG). Wild-type
terium tuberculosis, the causative agent of pul-
mice and CCL2-overexpressing mice that exhibited
monary tuberculosis, interacts with DC-SIGN to
increased pools of alveolar and lung mononuclear
evade the immune system. To directly analyze the
phagocytes-due to the lung-specific overexpression
role of human DC-SIGN during mycobacterial infec-
of human CCL2 in type-II alveolar epithelial cells-
tion, we generated conventional transgenic (tg)
were infected intratracheally with BCG and the de-
mice (termed “hSIGN”) using CD209 cDNA under
veloping lung inflammatory response was ana-
the control of the murine CD11c promoter. Upon my-
lyzed. CCL2-overexpressing mice demonstrated
cobacterial infection, DCs from hSIGN mice pro-
significantly decreased mycobacterial loads in the
duced significantly less IL-12p40 and no significant
bronchoalveolar space, lung parenchymal tissue,
differences were be observed in the secretion lev-
and spleen compared with wild-type mice, when
els of IL-10 relative to control DCs.
both groups of mice were infected with BCG. Moreover, in BCG-infected mice, later-developing, accelerated resolution of lung granuloma formation
was noted, particularly in CCL2-overexpressing
mice as compared with wild-type mice. In addition,
CCL2-overexpressing mice demonstrated an increased trafficking of mycobacteria-loaded dendritic cells towards lung-draining lymph nodes that
was found to coincide with increased mycobacterial loads in this compartment. The data of the current study suggest that CCL2-dependent amplification of endogenous host-defense programs in the
lung may improve the lungs’ protective immunity
Fig. 21. Survival of hSIGN mice after infection with 2000 CFU Mtb.
Ten mice per group, log-rank test ***, p < 0.001.
against mycobacterial infections. Experiments are
ongoing to determine the relative importance of
After high dose aerosol infection with the strain M.
these mechanisms in protection against airborne
tuberculosis H37Rv, hSIGN mice showed massive
M. tuberculosis infection.
accumulation of DC-SIGN(+) cells in infected lungs,
66
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Infection Biology
Research Reports
reduced tissue damage and prolonged survival
as demonstrated for IFN-alpha response to virus.
(Fig. 21). Based on our in vivo data, we propose that
Currently, these PDC and myeloid DC subsets are in-
instead of favoring the immune evasion of my-
vestigated for their immunoregulatory role in the
cobacteria, human DC-SIGN may have evolved as
early pathogenesis of Wegener`s granulomatosis
a pathogen receptor promoting protection by limit-
and experiments with human cells are extended to
ing tuberculosis-induced pathology.
in vivo and in vitro studies in mice. These studies are
part of the project “Molecular induction mecha-
Collaboration: Schaefer M, Stephani J, Taniuchi I,
nisms of danger receptor PAR-2 on DC” performed
Hatam F, Ruland J, Wagner H, Sparwasser T. Insti-
in the Clinical Research Group on the early patho-
tute for Medical Microbiology, Immunology, and Hy-
genesis of Wegener`s disease.
giene, Technische Universität München, Munich,
Germany; Fehrenbach H, Yildirim AO, Experimental
Collaboration: Löseke S, Kauth M, Gehlhar K, Bufe
Pneumology, FZB.
A,
Experimental
Pneumology,
Ruhr-University
Bochum; Zawatzky R, Department FO30, German
Supported in part by: DFG GRK288 A6, SFB 470 C9,
Cancer Research Centre, Heidelberg; Holle J, Cser-
and the German National Genome Research Net-
nok E, Dept. of Rheumatology, UK-SH Lübeck and
work (01GS0412).
Clinic for Rheumatology, Bad Bramstedt.
Anti-BDCA-4 (neuropilin-1) antibody can suppress
A novel immunomagnetic approach to isolate my-
virus induced IFN-alpha production of plasmacy-
cobacteria-containing phagosomes from primary
toid dendritic cells
macrophages
Grage-Griebenow E.
Steinhäuser C, Pott J, Ehlers S, Reiling N.
Plasmacytoid dendritic cells (PDC) in human blood
M. tuberculosis (Mtb) is able to persist within the
are the main source of virus-induced IFN-alpha.
host alveolar macrophage by arresting the matu-
They exhibit a lineage-negative phenotype but all
ration of its phagosome. The Mtb phagosome re-
express BDCA-4, which is homologous to the neu-
tains characteristics of an early endosome and
ronal receptor Neuropilin-1. Specific staining with
does not fuse with lysosomes. Little is known about
anti-BDCA-4 antibody is used for positive isolation
signalling events starting from these intracellular
of PDC from blood by magnetic cells sorting. We
compartments. In order to analyse these processes
demonstrated that these positively selected PDC
we established a new technique to isolate my-
showed reduced or completely abolished IFN-alpha
cobacteria containing phagosomes from primary
release compared to unstained PDC which were
macrophages based on the immunomagnetic la-
negatively selected by magnetic depletion of lin-
belling of mycobacteria. After biotinylation of the
eage-positive blood mononuclear cells. In addition,
mycobacterial surface bacteria were coupled to
treatment of these unstained PDC with anti-BDCA-4
magnetic streptavidin nanobeads. The stability of
mAb also resulted in at least 2-fold lower or com-
the labelling was monitored by flow cytometry. Elec-
pletely reduced virus-induced IFN-alpha production.
tron microscopy (TEM) demonstrated the integrity of
It is shown that the antibody does not simply affect
magnetically labelled mycobacteria within phago-
cell survival or block virus attachment. IFN-alpha re-
somes of murine macrophages (Fig. 22). The plas-
lease induced by non-viral CpG oligodeoxynu-
ma membrane of the macrophages was carefully
cleotides is also reduced. In conclusion, data sug-
disrupted by sonification leaving phagosomes in-
gest an immunoregulatory role for BDCA-4 on PDC
tact as visualized by TEM. The solution containing
Wissenschaftlicher Jahresbericht 2007-2008
67
Division of Molecular Infection Biology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
the disrupted cells was used for the isolation of
phagosomes in a high magnetic field. The protein
composition of mycobacterial phagosomes was
analysed by Western blot and revealed a differential distribution of proteins between the magnetic
phagosome fraction and the non-magnetic fraction.
These data indicate that the new phagosome isolation assay is a sophisticated method to examine
molecular events at the interface of the host cell
and the mycobacterial invader.
Fig. 22. Electron microscopy of a murine macrophage infected with
magnetically labeled M. avium SE01.
Collaboration: Vladimir Tchikov, Stefan Schütze, Institute of Immunology, University Hospital of
Schleswig-Holstein, Kiel, Germany; Karl-Heinz Wiesmüller, EMC microcollections GmbH, Tübingen, Germany.
Supported in part by: DFG grant Re1228/4-1 und
Sch733/7-1.
Theses
Master of Science
Kolja Schaale
Wnt5a induced signaling events in the context of inflammatory processes: studies in primary murine
macrophages
Masterstudiengang “Molecular Life Sciences”
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2008.
68
Wissenschaftlicher Jahresbericht 2007-2008
Junior Research Group Molecular Infection Biology
Research Reports
Junior Research Group Molecular
Infection Biology
Head
after 12 weeks of infection and dying significantly
Prof. Dr. Christoph Hölscher (Juniorprofessor)
earlier than controls. The differentiation, recruitment
and activation of TH1 cells as well as the induction
Staff Scientists and Postdoctoral Fellows
of IFN-gamma-dependent effector genes against
Jochen Behrends (from 05/08)
Mtb were not affected by macrophage-derived IL-
Hanna Erdmann (from 10/08)
10. However, microarray analysis of pulmonary
gene expression revealed patterns characteristic of
Graduate Students
alternative macrophage activation that were over-
Lisa Heitmann (from 02/08)
represented in Mtb-infected macIL-10-transgenic
Manuela Heßmann (until 03/08)
mice (Fig. 23a).
Kristian Holz (from 09/08)
Caroline Roßnagel (until 08/07)
Dominik Rückerl (until 11/07)
Jan Christian Sodenkamp
Technicians
Alexandra Hölscher
Insa Lenz (until 03/07)
Gabriele Röver (from 01/08)
Tanja Sonntag
Kerstin Traxel (from 01/08)
Johanna Volz (from 02/08)
Research Reports
Autocrine IL-10 induces hallmarks of alternative
activation in macrophages and suppresses antituberculosis effector mechanisms without compromising T cell immunity
Heitmann L, Hölscher C.
Elevated IL-10 has been implicated in reactivation
tuberculosis (TB). Since macrophages rather than T
cells were reported to be the major source of IL-10
in TB, we analyzed the consequences of a
macrophage-specific overexpression of IL-10 in
transgenic mice (macIL-10-transgenic) after aerosol
Fig. 23. After Mtb-infection, macIL-10tg mice develop alternatively
activated macrophages. FVB (open symbols) and macIL-10tg (solid symbols) mice were infected with 100 CFU Mtb via the aerosol
route. (a) Signs of alternative macrophage activation in the lung
transcriptome of macIL-10tg mice after Mtb infection. Gene-expression analysis was performed in lung homogenates from uninfected and mice infected for 25 and 42 days. Changes in expression of selected genes associated with alternative macrophage
activation during Mtb infection of FVB and macIL-10tg mice were
visualized using hierarchical clustering of z-score normalized average expression values. Similarity between experimental conditions and patterns of individual genes is indicated by dendrograms. Gene symbols are given (Mrc, macrophage mannose
receptor; Chi3l3, Ym1; Chi3l4, Ym2; Il4ra, IL-4 receptor-alpha; Retlna, Fizz1; Arg2, arginase-2; Arg1, arginase-1; Chi3l1, chitinase 3l1).
(b, c) Increased arginase-1 activity is concomitant with reduced
production of RNI. (b) Gene-expression of Arg1 was determined in
lung homogenates from uninfected and mice infected for 21, 49
and 89 days by quantitative real time RT-PCR based on expression
of Hprt. (c) During the course of infection, the RNI content in sera
was measured by the Griess reaction.
infection with Mycobacterium tuberculosis (Mtb).
MacIL-10-transgenic mice were more susceptible to
Importantly, arginase-1 gene expression and activ-
chronic Mtb infection than non-transgenic litter-
ity were strikingly enhanced in transgenic mice ac-
mates, exhibiting higher bacterial loads in the lung
companied by a reduced production of reactive ni-
Wissenschaftlicher Jahresbericht 2007-2008
69
Division of Biophysics
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Together,
with Mtb. Aerosol infection of IL-13tg mice resulted in
macrophage-derived IL-10 triggers aspects of alter-
a profound induction of arginases concomitant with
native macrophage activation and promotes Mtb
increased bacterial loads at chronic stages of the
recrudescence independent of overt effects on an-
disease very much resembling reactivation TB. Importantly, in IL-13tg mice Mtb infection resulted in tis-
trogen
intermediates
(Fig.
23b).
ti-TB T cell immunity.
sue pathology similar to GN in human TB. From our
Collaboration: Ehlers S, Molecular infection Biology,
data we conclude that IL-4Rα-dependent mecha-
Research Center Borstel; Schreiber T, Robert Koch
nisms are involved in the pathogenesis of post-pri-
Institute, Berlin, Germany; Lang R, Molecular
mary TB.
Medicine, University Hospital Erlangen, Germany;
Murray P, Department of Infectious Diseases and Im-
Collaboration: Ehlers S, Molecular infection Biology,
munology, St. Jude Children’s Research Hospital,
Research Center Borstel, Thye T, Meyer CG,
Memphis, TN USA.
Horstmann R, Department of Molecular Medicine,
Bernhard Nocht Institute for Tropical Medicine,
Supported in part by: Collaborative research grant
Hamburg, Germany; McKenzie ANJ, MRC Labora-
(“Host defence against infections”) from the Medical
tory of Molecular Biology, Cambridge, United King-
University of Lübeck; BMBF, Nation-wide Collabora-
dom; Brombacher F, International Centre for Ge-
tive Grant “Pulmonary Tuberculosis – host and
netic Engineering and Biotechnology, University of
pathogen determinants of resistance and disease
Cape Town, South Africa.
progression”, workpackage E “Animal Models”.
Supported in part by: BMBF, Nation-wide Collaborative Grant “Pulmonary Tuberculosis - host and
Granuloma necrosis in tuberculosis is mediated
pathogen determinants of resistance and disease
by interleukin-4 receptor-alpha-dependent mech-
progression”, workpackage E “Animal Models”.
anisms
Heitmann L, Hölscher C.
IL-17 maintains protective immunity during MyReactivation and granuloma necrosis (GN) are piv-
cobacterium tuberculosis infection
otal events of post-primary tuberculosis (TB). To date,
Rückerl D, Behrends J, Heßmann M, Hölscher C.
there is no clear evidence whether interleukin (IL)-4
and IL-13 are involved in the pathogenesis of the dis-
Because a variety of autoimmune disorders have
ease. Therefore, we genotyped polymorphisms in IL-
now been shown to depend on interleukin (IL)-17-
4, IL-13 and the IL-4 receptor alpha-chain (IL-4Rα) in
producing T helper (TH)17 cells, therapeutic block-
patients with pulmonary TB from Ghana. A structural
ade of TH17 development may provide a novel ap-
variant of the IL-4Rα, which has been shown to be as-
proach to avoid adverse consequences of
sociated with an increased transcription of the gene,
anti-inflammatory strategies such as reactivation of
was found to be significantly associated with in-
latent tuberculosis (TB). To evaluate the potential
creased cavity size indicating a prominent role of the
risk of interfering with IL-17-dependent inflammation,
IL-4Rα in human GN. In Mycobacterium tuberculosis
(Mtb)-infected mice, however, IL-4 and IL-13 are only
we analyzed the outcome of experimental TB in IL17-deficient (-/-) mice after infection with Mycobac-
moderately induced and necrotizing granulomas are
terium tuberculosis (Mtb). IL-17 was important for the
not apparent. To address the question how IL-4Rα-
induction of neutrophil chemokines after Mtb infec-
mediated mechanisms affect the outcome of murine
TB, IL-13-overexpressing (IL-13tg) mice were infected
tion, but was not involved in granuloma formation
70
and protection during the first three months of Mtb
Wissenschaftlicher Jahresbericht 2007-2008
Division of Biophysics
Research Reports
infection. Mtb-infected IL-17-/- mice efficiently gen-
IL-12/23p40 production in activated macrophages,
erated interferon-gamma (IFNγ)-producing T cells
we hypothesise that IL-27 modulates Th17 immune re-
and IFNγ-dependent effector responses. However,
IL-17-/- mice were not able to control mycobacterial
sponses
replication during the chronic phase of experimen-
pha gp130 receptor complex. To evaluate these IL-27-
tal TB and died significantly earlier than corre-
mediated
sponding wildtype mice. This breakdown of immune
protection in IL-17-/- mice was associated with a
macrophages, we analyzed inflammatory immune
drop in the frequency of IFNγ-producing CD4+ T
cells. Our findings reveal that IL-17 is essential for
maintaining CD4+ T cell-dependent protection dur-
by
regulating
pro-inflammatory
macrophage responses through its IL-27 receptor-al-
suppressive
effects
Mtb
specifically
in
in
-/macrophage/neutrophil-specific gp130-deficient ( )
LysMcregp130flox/flox mice. In vitro, IL-6- and IL-27-
responses
after
infection
ing chronic stages of TB. Hence, interfering with IL-
mediated signal transduction was affected in
macrophages from LysMcregp130flox/flox mice lead-
17-dependent pathways as an anti-inflammatory
ing to an uncontrolled production of IL-12/23p40 af-
therapeutic approach will possibly incur the danger
ter stimulation. Following infection with Mtb, LysMcregp130flox/flox mice produced elevated amounts
of reactivating latent TB.
of IL-6 when compared to infected LysMcre-negative
Collaboration: Ehlers S, Molecular infection Biology,
littermates. Accordingly, the differentiation of Th17
Research Center Borstel Müller U and Alber G, University of Leipzig, Germany; Iwakura Y, University of
cells was increased in macrophage/neutrophil-specific gp130-/- mice whereas Th1 immune responses
Tokyo, Japan.
were not affected. In summary, our results so far revealed that after Mtb infection Th17 differentiation is
Supported in part by: Cluster of Excellence “Inflam-
regulated on the level of macrophages/neutrophils
mation at Interfaces, IRN F “Cytokine signalling via
through gp130-mediated signals. Together, our study
gp 130”, project 4 “Infection driven inflammation
may expand the understanding of regulatory mech-
models”.
anism during Th17 differentiation implying IL-27 as a
negative feedback controller which limits the Th17driving production of IL-6 and IL-23 by macrophages.
gp130 on macrophages/neutrophils negatively
regulates TH17 development during experimental
Collaboration: Ehlers S, Molecular Infection Biology,
tuberculosis
Research Center Borstel Förster I, Institut für umwelt-
Sodenkamp JC, Hölscher C.
medizinische Forschung, Heinrich Heine Universität,
Düsseldorf, Germany; Müller W, University of Man-
Interleukin (IL)-6- and IL-23-driven T helper cell (Th)17
chester, Manchester, England, UK.
differentiation is associated with the development of
autoimmune and chronic inflammatory diseases. Af-
Supported in part by: Cluster of Excellence “Inflam-
ter infection with Mycobacterium tuberculosis (Mtb),
mation at Interfaces, IRN F “Cytokine signalling via
IL-17 deficiency results in a loss of immune protection
gp 130”, project 4 “Infection driven inflammation
exceptionally during chronic stages of the disease.
models”; DFG HO 2145/4-3 “Regulation of inflam-
However, an increased Th17 immune response in the
matory and protective macrophage immune re-
absence of IL-27-mediated regulatory mechanisms is
sponses through Stat3-dependent signaling events”;
concomitant with the development of excessive in-
SFB 415, project C10 “Cell-specific mechanisms me-
flammation and premature death. Hence, Th17 dif-
diated by IL-27/WSX-1 during inflammatory immune
ferentiation after Mtb infection has to be tightly reg-
responses”.
ulated. Because IL-27 is able to inhibit IL-6 and
Wissenschaftlicher Jahresbericht 2007-2008
71
Division of Biophysics
Research Reports
Adjuvanticity of a synthetic cord factor analog for
Here, we demonstrate that these glycolipids acti-
subunit Mycobacterium tuberculosis vaccination
vate macrophages and dendritic cells (DC) via
requires FcRγ-Syk-Card9-dependent innate im-
Syk-Card9-Bcl10-Malt1 signaling to induce a spe-
mune activation
cific innate activation program distinct from the re-
Hölscher C.
sponse to Toll-like receptor (TLR) ligands. APC ac-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
tivation by TDB and TDM was independent of the
Novel vaccination strategies against Mycobacteri-
C-type lectin receptor Dectin-1, but required the
um tuberculosis (Mtb) are urgently needed. The
ITAM-bearing adapter protein Fc receptor gamma-
use of recombinant Mtb antigens as subunit vac-
chain (FcRγ). In vivo, TDB and TDM adjuvant activ-
cines is a promising approach, but requires adju-
ity induced robust combined TH1 and TH17 T cell
vants that activate antigen presenting cells (APC)
responses to a Mtb subunit vaccine and protection
for elicitation of protective immunity. The my-
against Mtb challenge in a Card9-dependent man-
cobacterial cord factor, Trehalose-6,6-dimycolate
ner (Fig. 24). These data provide a molecular ba-
(TDM), and its synthetic analog Trehalose-6,6-
sis for the immunostimulatory activity of TDB and
dibehenate (TDB) are effective adjuvants in com-
TDM, and identify the Syk-Card9 pathway as a ra-
bination with Mtb subunit vaccine candidates in
tional target for vaccine delelopment against tu-
mice. However, it is unknown which signaling path-
berculosis.
ways they engage in APC and how these are coupled to the adaptive immune response.
Collaboration: Ruland J, Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Germany; Lang R, Molecular
Medicine, University Hospital Erlangen, Germany;
Agger EM, Adjuvant Research, Dept. of Infectious
Disease Immunology, Statens Serum Institute,
Copenhagen, Denmark.
Supported in part by: Cluster of Excellence “Inflammation at Interfaces, IRN F “Cytokine signalling via
gp 130”, project 4 “Infection driven inflammation
models”.
Theses
Dissertation
Rückerl, Dominik
Die Bedeutung IL-27- und IL-23-vermittelter MechaFig. 24. Induction of protective immunity to Mtb by TDB/TDM adjuvant requires Card9. Wildtype and Card9-/- mice were immunized
with ESAT6/AG85 in DDA or DDA-TDB liposomes and infected by
aerosol with 100 CFU Mtb. (a) Immunization with ESAT6/AG85 in
DDA-TDB liposomes fails to reduce the number of bacterial loads
in the lungs of Card9-/- mice. After 6 weeks, mice were sacrificed
and the bacterial load was determined in the lung. (b) Protection
after immunization with ESAT6/AG85 in DDA-TDB liposomes correlates with the Card9-dependent induction of TH17 rather than TH1
cells. Frequency of CD4 T cells producing IL-17 or IFNg from mediastinal lymph nodes 6 weeks after Mtb infection.
72
nismen für den Verlauf entzündlicher Immunreaktionen unter besonderer Berücksichtigung der Infektion mit Mycobacterium tuberculosis.
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2007.
Hessmann, Manuela
Wissenschaftlicher Jahresbericht 2007-2008
Division of Biophysics
Research Reports
Die Bedeutung des Rezeptors NKG2D und seiner
Adaptermoleküle DAP10 und DAP12 für die Zell-vermittelte Immunantwort nach der Infektion mit Mycobacterium tuberculosis.
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2008.
Wissenschaftlicher Jahresbericht 2007-2008
73
Research Group of Cellular Pneumology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Research Group of Cellular Pneumology
Head
PD Dr. Cordula Stamme
Postdoctoral Fellow
Dr. Christina Moulakakis
Scientific Staff
Alexandra Lange
Vicky Sender
Technician
Linda Lang
Research Reports
Surfactant protein A activation of atypical protein
kinase C ζ in IκB-α-dependent anti-inflammatory
immune regulation
Moulakakis C, Stamme C.
Fig. 25. SP-A fails to inhibit LPS-induced p65 nuclear translocation
in AKBI AM. AM from wild type (a-d) or AKBI (c-h) mice were left untreated or treated with 40 µg/ml SP-A (37°C, 1 h), exposed to media, or to 100 ng/ml LPS (1 h), and then analyzed by confocal microscopy. Color code: green: NF-κB p65; red: cell nuclei were
counterstained with propidium iodide. Overlays of single stainings
are shown. Images are representative of three independent experiments. Pixel densitometry of nuclear NFκ-B p65 was quantified
and statistically analyzed with one-way ANOVA and Newman-Keuls
post-hoc test. ** p < 0.001 (versus control), #p < 0.001 (versus LPS).
The pulmonary collectin surfactant protein (SP)-A
has a pivotal role in anti-inflammatory modulation
of lung immunity. The mechanisms underlying SP-Amediated inhibition of lipopolysaccharide (LPS)-induced NF-kB activation in vivo and in vitro are only
Fig. 26. PKCζ is vital for SP-A-mediated IB-κ stabilization and inhibition of LPS-induced NF-κB activity. (A-B) Pooled AM from either
four wild-type (A) or four PKCζ-deficient mice (B) were left untreated or treated with 40 µg/ml SP-A (1 h), and then exposed either
to media or to 100 ng/ml LPS (1 h). Cytosolic cell fractions were
immunoblotted for IκB-α (upper panel). Nuclear extracts of the
partially understood. We previously
demonstrated that SP-A stabilizes inhibitory κB-α (IκB-α, the primary regulator
of
NF-κB,
in
alveolar
macrophages (AM) both constitutively
and in the presence of LPS. Here we
show that in AM and PBMC from IκB-α
knockout/IκB-α knockin (AKBI) mice SPA fails to inhibit LPS-induced TNF-α production and p65 nuclear translocation,
confirming a critical role for IkB-a in SPA-mediated LPS inhibition. We identify
atypical (a) protein kinase C (PKC) ζ as
a pivotal upstream regulator of SP-Amediated IκB-α/NF-κB pathway modulation deduced from blocking experiments and confirmed by using AM from
74
Wissenschaftlicher Jahresbericht 2007-2008
Research Group of Cellular Pneumology
cells were analyzed by EMSA for NF-κB DNA binding activity (mid
panel). Data shown are representative of three independent experiments. Densitometric results are expressed as arbitrary units
(a.u.). Data were analyzed by one-way ANOVA with a post-hoc
Newman-Keuls test. * p < 0,01, ** p < 0,001 (versus control); #p <
0.01, ##p < 0.001 (versus LPS).
Research Reports
of SP-A by AMφ is a prerequisite for its modulation of
the IκB-α/NF-κB pathway. The inhibition of clathrincoated pit (CCP) formation and clathrin-coated vesicle (CCV) formation/budding abrogates SP-A-medi-
PKCζ-/- mice. SP-A transiently triggers aPKCThr410/403 phosphorylation, aPKC kinase activity,
ated IκB-α stabilization and SP-A-mediated inhibition
and translocation in primary rat AM. Co-immuno-
AMφ, as determined by Western analysis, fluores-
precipitation experiments reveal that SP-A induces
cence-activated cell sorting, confocal microscopy,
aPKC/p65 binding under constitutive conditions. To-
and EMSA. Actin depolymerization and inhibition of
gether the data indicate that anti-inflammatory
CCP formation further abolished SP-A-mediated inhi-
macrophage activation via IκB-α by SP-A critically
bition of LPS-induced TNF-α release, as determined
depends on PKCζ activity and thus attribute a nov-
by ELISA. In addition, SP-A-induced atypical PKCζ ac-
el, stimulus-specific signaling function to PKCζ in SP-
tivation was abolished by pretreatment of AMφ with
A-modulated pulmonary immune response.
CCV inhibitors as determined by in vitro immuno-
of LPS-induced NF-κB activation in freshly isolated rat
complex kinase assay. While CME is classically conCollaboration: Engelhardt J, Univ of Iowa, USA; Leit-
sidered as a means to terminate signaling, our results
ges M, Max-Planck-Institute of Experimental En-
demonstrate that SP-A uptake via CME by AMφ has
docrinology, Hannover; Schromm AB, Emmy-
to precede the initiation of SP-A signaling.
Noether-Group of Immunophysics, Seitzer U,
Division of Veterinary Infection Biology and Immunology, Research Center Borstel.
Supported in part by: Deutsche Forschungsgemeinschaft, grant STA 609/1-3.
Role of clathrin-mediated endocytosis of surfactant protein A by alveolar macrophages in intracellular signaling
Moulakakis C, Stamme C.
We recently provided evidence that anti-inflammatory macrophage activation, i.e. the inhibition of constitutive and signal-induced NF-κB activity by the pulmonary collectin surfactant protein (SP)-A, critically
involves a promoted stabilization of inhibitory κB
(IκB)-α the predominant inhibitor of NF-κB, via posttranscriptional mechanisms comprising the activation
of atypical (a) PKCζ. SP-A uptake and degradation by
alveolar macrophages (AMφ) occurs in a receptormediated, clathrin-dependent manner. However, a
mutual link between endocytosis of and signaling by
SP-A remains elusive. In the present study we investigated whether clathrin-mediated endocytosis (CME)
Fig. 27. CCV formation and budding are required for cell-association of SP-A with AMφ. Freshly isolated primary rat AMφ in suspension were incubated 2.5 mM amantadine (30 min), 2 µM
phenylarsine oxide (PAO) (30 min), 50 µM chlorpromazine (CPZ)
(1 h), or 10 µM CytoD (30 min) followed by the addition of 40 µg/ml
FITC-SP-A (1 h). Cells were washed and cell-association of FITC-SPA was measured by flow cytometry. Data are expressed as percent of total cell-associated SP-A ± SEM. Overlay of FACS histogram
is representative of three independent experiments. Data were analyzed by one-way ANOVA with a post-hoc Newman-Keuls test. *
p < 0.05, ** p < 0.01 (vs. cell-association of FITC-SP-A).
Wissenschaftlicher Jahresbericht 2007-2008
75
Research Group of Cellular Pneumology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
lavage, nuclear factor kappaB, (NF-kappaB)-activation in alveolar macrophages and circulating systemic mediators were monitored. Control patients
underwent bronchoalveolar lavage after intubation.
RESULTS: In the IPL, mediator concentrations increased with increasing end-expiratory pressure at
an EIP of 10 cm H2O, but decreased at 25 cm H2O
EIP. In patients, bronchoalveolar IL-6, monocyte
chemoattractant protein-1, and granulocyte monocyte-colony stimulating factor were increased by
ventilation regardless of the PEEP level. IL-6 and IL8 levels were moderately increased by PEEP but not
Fig. 28. Uptake of FITC-labeled SP-A by alveolar macrophages
(AMφ). Confocal images of (AMφ) without treatment (a), after treatment with (b) FITC-labeled SP-A (40 µg/ml), (c) amantadine (2.5
mM) prior to SP-A, and (d) CytoD (10 µM) prior to SP-A. Color code:
green: FITC-SP-A; red: actin; blue: DAPI.
zero end-expiratory pressure. Nuclear factor kappaB DNA binding activity in alveolar macrophages
and systemic mediator levels did not change. CONCLUSIONS: On the basis of the premise that cytokine levels may indicate mechanical stress, our
Supported in part by: Deutsche Forschungsgemein-
findings indicate that even low tidal volume venti-
schaft, grant STA 609/1-3, 1-4.
lation causes some stress. PEEP is beneficial at high
inspiratory pressure, but imposes moderate stress
at low inspiratory pressure.
Pulmonary cytokine responses during mechanical
ventilation of noninjured lungs with and without
Collaboration: Uhlig U, Uhlig S, Institute of Pharma-
end-expiratory pressure.
cology and Toxicology, University Hospital Aachen;
Stamme C.
Meier T, Lange A, Papenberg H, Ziemann M, Fentrop C, Schmucker P, Department of Anesthesiology,
BACKGROUND: Positive end-expiratory pressure
University Medical Center Schleswig-Holstein, Cam-
(PEEP) during mechanical ventilation may impose
pus Lübeck.
different degrees of stress on healthy lungs. On the
assumption that stress is reflected by cytokine production, we performed a translational study investi-
Theses
gating the effect of PEEP on bronchoalveolar and
systemic mediator levels in isolated perfused
Dissertation
mouse lungs (IPL) and in patients with healthy lungs.
Moulakakis, Christina
METHODS: (Part I) IPL were ventilated with end-ex-
Pulmonary Surfactant Protein A’s anti-inflammatory
piratory pressures of 0, 3, 6, or 10 cm H2O and end-
modulation of the IkB-a/NF-kB signal transduction
inspiratory pressure (EIP) levels of 10 or 25 cm H2O.
pathway
Interleukin (IL)-6 and macrophage inflammatory
Technisch-Naturwissenschaftliche Fakultät
protein-2 concentrations in the venous effluate were
Universität zu Lübeck, 2008.
monitored. (Part II) Patients (nonsmokers) scheduled for elective otorhinolaryngology surgery (duration>90 min) were randomized to receive either
ventilation with zero end-expiratory pressure or
PEEP (10 cm H2O). Mediators in bronchoalveolar
76
Wissenschaftlicher Jahresbericht 2007-2008
Abteilung Immunologie und Zellbiologie
Department of Immunology and Cell Biology
Fo r s c h u n g s z e n t r u m
Bo r s t e l
78
Wissenschaftlicher Jahresbericht 2007-2008
Abteilung Immunologie und Zellbiologie
Department of Immunology and Cell Biology
Abteilung Immunologie und Zellbiologie
Department of Immunology and Cell Biology
Wissenschaftlicher Jahresbericht 2007-2008
79
Zusammenfassung
Summary
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Abteilung
Immunologie und
Zellbiologie
Department of
Immunolgy and
Cell Biology
Head
Head
Prof. Dr. Dr. Silvia Bulfone-Paus
Prof. Dr. Dr. Silvia Bulfone-Paus
Secretary
Secretary
Annette Wallisch
Annette Wallisch
Die Abteilung Immunologie und Zellbiologie um-
The Department of Immunology and Cell Biology
fasst ein multi-disziplinäres Wissenschaftlerteam,
comprises a multi-disciplinary team of scientists do-
das die zellulären und molekularen Grundlagen
ing research on the cellular and molecular mecha-
der Infektion, der Allergie und des Tumorwachs-
nisms of infection, allergy and tumor growth. The
tums erforscht. Das breite Spektrum der wis-
broad scale of scientific interests and methodical
senschaftlichen Interessen und methodischen An-
approaches constitutes the precondition for the dy-
sätze bildet die Voraussetzung für die Dynamik,
namics, productivity and creativity of the depart-
Produktivität und Kreativität der Abteilung, die
ment which has committed itself not only to a deep-
sich letztendlich nicht nur dem tieferen Verständ-
er understanding of pathology, but also to the de-
nis der Pathologie, sondern auch der Entwicklung
velopment of new forms of therapy.
neuer Formen der Therapie verschrieben hat. Die
Abteilung hat einen übergreifenden und synergistischen Ansatz gewählt, in dem sowohl die
The department has chosen a comprehensive and
angeborene als auch die erworbene Immunität
synergetic approach in which both innate and ac-
berücksichtigt und soweit möglich, das murine mit
quired immunity are taken into account and, as far
dem humanen System verknüpft wird. Beide Sys-
as possible, the murine system is linked to the hu-
teme bestehen aus löslichen und zellulären Kom-
man one. Both systems consist of soluble and cel-
ponenten, die durch Rezeptor-vermittelte Mecha-
lular components which are regulated via receptor-
nismen reguliert werden und verschiedene Ebe-
mediated mechanisms and show different levels of
nen des ‚cross-talks’ und der ‚cross-regulation’
cross-talk and cross-regulation.
zeigen.
The division of Innate Immunity is investigating the
Die Laborgruppe Angeborene Immunität (H. Hei-
molecular mechanisms by which cow shed bacte-
ne) erforscht die molekularen Mechanismen, die
ria may decrease allergic symptoms in farmer’s
dazu führen, dass Kuhstall-Bakterien bei Kindern
children. It could be demonstrated that these bac-
in bäuerlichen Betrieben die Symptome der Aller-
teria strongly induce an allergy-protective TH1 im-
gie vermindern können. Es konnte gezeigt werden,
mune response via the activation of dendritic cells
dass durch diese Bakterien eine ausgeprägte Al-
to produce cytokines such as IL-12 and to modulate
lergie-Schutz TH1 Immunantwort induziert wird.
Notch ligand expression (JACI) (supported by the
Dies geschieht durch die Aktivierung von dendriti-
SFB/TR22). Jennifer Debarry, who worked on this
schen Zellen, welche Zytokine wie IL-12 produzie-
project for her doctoral thesis, was honored with
80
Wissenschaftlicher Jahresbericht 2005-2006
Zusammenfassung
Summary
ren und die die Notch-Liganden-Expression (JACI)
the Graduation prize in 2007, sponsored by the lo-
modulieren (gefördert durch den SFB/TR22). Jen-
cal government of the “Kreis Segeberg”. Further-
nifer Debarry, deren Promotionsarbeit sich mit die-
more, in a project in collaboration with Christoph
sem Thema beschäftigte, wurde hierfür in 2007 mit
Lange’s group, the expression and regulation of
dem Promotionspreis des Kreises Segeberg aus-
antimicrobial peptides in patients with COPD has
gezeichnet.
been investigated. A number of antimicrobial peptides in the lung have been detected and a correlation between the expression of human beta-de-
Außerdem wurde in einem weiteren Projekt, in Zu-
fensin1 (hBD-1) with the severity of the disease has
sammenarbeit mit der Gruppe von Christoph Lan-
been found. Currently, the epigenetic mechanisms
ge, die Expression und Regulation von antimikro-
regulating the expression of hBD-1 are under in-
biellen Peptiden bei Patienten mit COPD unter-
vestigation.
sucht. Einige antimikrobielle Peptide in der Lunge
konnten identifiziert und eine Korrelation zwischen
der Expression von humanem Beta-Defensin 1
The division of Cellular Immunology (A.J. Ulmer) is
(hBD-1) und der Schwere der Erkrankung festge-
engaged in elucidating the properties of bacterial
stellt werden. Zurzeit wird der Frage nachgegan-
cell wall components expressing pathogen associ-
gen, durch welche epigenetischen Mechanismen
ated
die Expression von hBD-1 moduliert wird.
lipopolysaccharide, lipopeptides, which are found
molecular
patterns
(PAMP).
Beside
in all bacteria species including Gram-negative and
Gram-positive bacteria, mycobacteria and myDie Laborgruppe Zelluläre Immunologie (A.J. Ul-
coplasma are focus of signal transduction and struc-
mer) erforscht die Mechanismen der immunologi-
ture/activity relationship studies. It has been
schen Aktivität von bakteriellen Zellwand Be-
demonstrated that lipopeptides, such as Pam2C-
standteilen, die Pathogen-assoziierte Strukturen
SK4 and also Pam3-MALP2, which signals through
(PAMP) exprimieren. Neben den Lipopolysaccha-
both, TLR2/1 and TLR2/6 heteromers activate a sim-
riden der Zellwand von Gram-positiven Bakterien
ilar downstream signaling pathway. Furthermore,
bilden die Lipopeptide den Schwerpunkt der For-
the Regulator of G-Protein Signaling 2 (RGS2) and,
schung, welche in allen Bakterienspezies wie
to a lesser degree the RGS1 were found to be also
Gram-negative Bakterien, Gram-positive Bakteri-
modulated by different TLR ligands (lipopetides and
en, Mykobakterien und Mykoplasmen gefunden
LPS) suggesting their important role in immunity.
werden. Es konnte gezeigt werden, dass Lipopeptide, wie Pam2C-SK4 und auch Pam3-MALP2,
welche sowohl durch TLR2/1 als auch durch TLR2/6
The adjuvant role of lipopeptides during develop-
Heterodimere, signalisieren, einen ähnlichen „Do-
ment of an immune response against an air borne
wnstream-Signalweg“ aktivieren. Außerdem konn-
allergen was investigated in a mouse model using
te gezeigt werden, dass der Regulator des G-Pro-
the Timothy grass pollen major allergen Phl p 1.
tein Signaling 2 (RGS2), und in einem geringeren
The mycoplasma lipopeptide FSL-1 was shown to
Ausmaß des RGS1, durch verschiedene TLR-Li-
boost the IgG2a Phl p 1-specific immunoglobulin in-
ganden (Lippeptide und LPS) moduliert werden.
dicating a Th1-type shift in presence of this adju-
Dies ist offenbar bei der Entwicklung der Immu-
vant. Furthermore, in cooperation with Arnd Pe-
nität von großer Bedeutung. Die adjuvante Rolle
tersen (Div of Clinical und Molecular Allergology),
der Lipopeptide bei der Entwicklung einer Immu-
the activation of lung epithelial cells to produce cy-
nantwort gegen „Air-borne“ Allergene wurde, un-
tokines and chemokines by Phl p 1 could be
Wissenschaftlicher Jahresbericht 2005-2006
81
Zusammenfassung
Summary
ter zu Hilfenahme des Timothy Grass Pollen Major
demonstrated, indicating that the bronchial epithel
Allergen Phl p 1, in einem Mausmodell untersucht.
is not only a physical barrier but is actively involved
Es konnte gezeigt werden, dass die Mykoplasma
in initiating a protective immune response against
Lipopeptide FSL-1, die Phl p 1-spezifische Immun-
pathogens.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
globulin Produktion von IgG2, deutlicher boosterte als die Produktion von IgG1. Dieses deutet auf
einen Shift in Richtung einer Th1-Immunantwort in
The division of Immunobiology (S. Bulfone-Paus)
Anwesenheit dieses Adjuvanz. In Kooperation mit
could demonstrate, employing the best-established
A. Petersen (Laborgruppe Klinische und Moleku-
murine model of sepsis (CLP model), an unexpect-
lare Allergologie) konnte die Aktivierung der Pro-
ed breach in mast cell -dependent, innate immune
duktion von Zytokinen und Chemokinen in Lunge-
defenses against gram-negative bacteria (Nature
nepithelzellen duch Phl p 1 nachgewiesen wer-
Medicine). Surprisingly, survival after septic peri-
den, was darauf hinweist, dass das Bronchu-
tonitis was significantly enhanced in IL-15 deficient
sepithel nicht nur eine physikalische Barriere,
mice. We could show that this survival advantage
sondern aktiv am Aufbau einer Immunantwort ge-
is mast cell-driven and IL-15-dependent. Murine
gen Invasoren beteiligt ist.
mast cells express constitutive and LPS-inducible IL15, and store IL-15 intracellularly, where it colocalizes with TNFα and proteases in mast cell granula.
Die Laborgruppe Immunbiologie (S. Bulfone-
IL-15-deficient mast cells exhibit markedly elevated
Paus) konnte mittels des erprobten murinen Mo-
chymase activity, leading to increased bactericidal
dells der Sepsis (CLP Model) eine unerwartete
activity and processing of neutrophil-recruiting
Bresche in der Mastzellen-abhängigen, angebo-
chemokines. By showing that intracellular IL-15 op-
renen Abwehr gegen Gram-negative Bakterien
erates as a specific negative transcriptional regu-
nachweisen (Nature Medicine). In IL-15 defizien-
lator of murine mast cell chymases, we provided
ten Mäusen war die Überlebensrate nach einer
the first evidence that defined mast cell protease
septischen Peritonitis deutlich gesteigert. Die
activities are transcriptionally regulated by an in-
Gruppe konnte nachweisen, dass dieser Vorteil
tracellularly retained cytokine. This newly de-
von Mastzellen gesteuert wird und IL-15 abhän-
scribed mechanism greatly affects an individual’s
gig ist. Murine Mastzellen exprimieren konstituti-
innate immune responses to septic infection and
ves und LPS-induzierbares IL-15, und speichern es
determines the chances of survival.
intrazellulär. Hier findet es sich zusammen mit TNFalpha und Proteasen in den Zell-Granula. IL-15
defiziente Mastzellen weisen eine deutlich ge-
While it is well-appreciated that the receptors for
steigerte Chymase Aktivität auf, welche zu einer
secreted cytokines transmit ligand-induced signals,
vermehrten bakteriziden Aktivität und Prozessie-
little is known about additional roles for cytokine re-
rung von Chemokinen führt, die Neutrophile re-
ceptor components in the control of ligand-trans-
krutieren. Durch den Nachweis, dass intrazellulä-
port and secretion. We could demonstrate that the
res IL-15 als ein spezifischer negativer transkrip-
IL-15 receptor alpha chain determines the mobi-
tionaler Regulator der murinen Mastzell-Chymase
lization and secretion of IL-15 therefore providing a
agiert, konnte erstmals der Beweis erbracht wer-
first evidence for cytokine chaperoning in the se-
den, dass bestimmte Aktivitäten der Mastzell-Pro-
cretory pathway by cytokine complexing with its re-
teasen transkriptionell durch ein intrazellulär ge-
ceptor (Mol Cell Biol). Erwin Duitman who worked
speichertes Zytokin reguliert werden. Dieser erst-
on the characterization of the IL-15/IL-15R trafficking
mals beschriebene Mechanismus hat großen Ein-
pathway was honored with the German Society for
82
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
fluss auf die individuelle angeborene Immunant-
Summary
Immunology poster prize in September 2008.
wort auf septische Infektionen und bestimmt somit die Überlebenschancen. Es ist bekannt, dass
die Rezeptoren für sezernierte Zytokine, Ligan-
The newly established division of Molecular Im-
den-induzierte Signale übermitteln. Die zusätzli-
munology (K. Lee) investigates molecular and cel-
chen Funktionen der Komponenten der Zytokin-
lular aspects of membrane trafficking in immune
Rezeptoren sind allerdings bislang recht uner-
function. The research focus is to understand the
forscht. Die Gruppe konnte zeigen, dass die IL-
roles of receptor endocytosis and the traffick-
15alpha Kette die Mobilisierung und Sekretion
ing/sorting machinery in apoptotic vs. survival sig-
von IL-15 verursacht. Hierdurch konnte erstmalig,
naling and its functional relevance for the immune
durch Bildung eines Komplexes aus dem Zytokin
system employing the death receptor Fas (CD95)
mit seinem Rezeptor, ein Zytokin-Chaperoning in
as a model system. Furthermore, the group is in-
den sekretorischen Signalwegen nachgewiesen
terested in the role of membrane trafficking in the
werden (MoL Cell Biol). Erwin Duitman, der die
context of an allergic reaction utilizing mast cells as
Charakterisierung des IL-15/IL-15R Trafficking Sig-
experimental model. The aim of the study is (1) to
nalwegs bearbeitete, wurde von der Deutschen
elucidate the molecular mechanisms responsible
Gesellschaft für Immunologie im September 2008
for the transport and fusion of mast cell granules to
mit dem Posterpreis ausgezeichnet.
the plasma membrane and (2) to investigate the
potential role of membrane trafficking in the development of mast cell mediated immune disor-
Die neu gegründete Laborgruppe Molekulare Im-
ders, with specific focus on allergy/asthma models.
munologie (K. Lee) untersucht die molekularen
und zellulären Aspekte der transmembranösen
Signaltransduktion bei immunologischen Funktio-
The division of Biochemical Immunology (F. Pe-
nen. Diese Gruppe geht der Frage nach, welche
tersen) places the main emphasis of its research on
Rolle die Rezeptor-Endozytose und die „Traf-
different aspects of the regulation, signal trans-
ficking/Sorting-Maschinerie“ in der Apoptose bzw.
duction and physiologic function of the CXC-
beim „Survival-Signaling“ spielt. Hierfür und zur
chemokine platelet factor 4 (PF-4, CXCL4), which is
Aufklärung der funktionellen Relevanz für das Im-
released by thrombocytes. The elucidation of the
munsystem wird der „Death Receptor“ FAS (CD95)
different signal pathways which participate in PF4-
eingesetzt. Außerdem wird anhand von Mastzel-
mediated neutrophil and monocyte activation, was
len die Rolle des Membran-Trafficking im Zusam-
greatly advanced during the last two years. The di-
menhang mit der allergischen Reaktion unter-
vision was able to identify sphingosine kinase 1
sucht. Ziel der Forschung ist (1): Darstellung der
(SphK1) as a central regulator of acute and de-
molekularen Mechanismen, die für den Transport
layed monocyte functions. SphK1, which becomes
und die Fusion von Mastzell-Granulomen zur Plas-
upregulated and activated upon CXCL4 stimula-
mamembran verantwortlich sind, sowie (2): Un-
tion, does not only regulate monocyte survival and
tersuchung der potentiellen Rolle des Membran-
cytokine release by controlling the activity of Erk,
Trafficking bei der Entwicklung von Mastzell-ver-
but, furthermore, also the generation of oxygen
mittelten Immunerkrankungen, insbesondere bei
radicals (ROS) (submitted). Interestingly, long-last-
Allergie/Asthma Modellen.
ing release of ROS by CXCL4-activated monocytes
does not only reflect a mechanism of acute defense
against microbial invaders but mediated also
In der Laborgruppe Biochemische Immunologie
apoptosis in endothelial cells (J Leukocyte Biol).
Wissenschaftlicher Jahresbericht 2007-2008
83
Zusammenfassung
Summary
(F. Petersen) werden schwerpunktmäßig verschie-
Recent progress has been made by the division in
dene Aspekte der Regulation, Signaltransduktion
the analysis of interactions between mast cells and
und physiologischen Funktion des von Thrombo-
neutrophils in allergic asthma (supported by the
zyten freigesetzten CXC Chemokins Plättchenfak-
SFB/TR22) which may reflect a novel axis in the
tor 4 (PF-4, CXCL4) untersucht. In den letzten zwei
pathology of the disease. Activated mast cells re-
Jahren hat die Aufklärung der verschiedenen Sig-
lease mediators leading to the attraction of neu-
nalwege, die an der von PF4-vermittelten neutro-
trophils while neutrophils secrete antimicrobial
philen und monozytären Aktivierung beteiligt sind,
peptides which are capable to activate mast cells.
große Fortschritte gemacht.
Beside allergic processes, this axis may play also
Fo r s c h u n g s z e n t r u m
Bo r s t e l
a role in the induction and maintenance of autoimmune diseases of the skin. This item is in the foDie Laborgruppe konnte Sphingosin-Kinase 1
cus of a recently launched project within the clus-
(SphK1) als einen zentralen Regulator der akuten
ter of excellence “Inflammation at Interfaces”.
und verzögerten Monozyten Reaktion identifizieren. SphK1 wird durch CXCL4 -Stimulation hoch-reguliert und beeinflusst nicht nur das Überleben
As a central aspect of research the division of Bio-
der Monozyten und die Zytokin-Freigabe durch
logical Chemistry (E. Brandt) is interested in the in-
Kontrolle der ERK-Aktivität, sondern auch die Ge-
teraction between mast cells, platelets and neu-
nerierung von Sauerstoff-Radikalen (ROS) (sub-
trophils in allergic and non-allergic inflammation.
mitted). Interessanterweise stellt eine lang anhal-
More specifically, communication of these cell pop-
tende Freisetzung von ROS durch CXCL4-aktivier-
ulations mediated by chemokines, proteases and
te Monozyten nicht nur einen Mechanismus der
antimicrobial peptides are in the focus of investi-
akuten Abwehr gegen mikrobiologische Invaso-
gation. Supported by the Transregio 22 and in co-
ren dar, sondern sie vermittelt auch die Apoptose
operation with the division of Biochemical Im-
in endothelialen Zellen (J Leukocyte Biol). Einen
munology it was discovered that the platelet-de-
weiteren Fortschritt konnte die Gruppe bei der
rived chemokine CXCL4 appears to have a gener-
Analyse der Interaktionen zwischen Mastzellen
al role as a regulator of human mast cell-secreted
und Neutrophilen beim allergischen Asthma ver-
proteases, in that it does not only inhibit process-
zeichnen (gefördert durch den Transregio 22). Hier
ing of chemokines by chymase but also the degra-
könnte sich eine neue Achse in der Pathologie die-
dation of several substrates (e.g. LL37) by tryptase.
ser Erkrankung abzeichnen. Aktivierte Mastzellen
Notably, cleavage by tryptase of the cathelicidin
setzen Mediatoren frei, die Neutrophile anlocken.
LL37 (a major neutrophil product) was found to ab-
Neutrophile ihrerseits sezernieren antimikrobielle
rogate its ability to induce mast cell degranulation
Peptide, welche in der Lage sind, Mastzellen zu
as well as its antimicrobial activity (J Immunol, un-
aktivieren. Außer bei den allergischen Prozessen,
der revision). In a project being aimed at elucidat-
ist diese Achse möglicherweise auch bei der In-
ing the impact of proteases on mast cell functions
duktion und Aufrechterhaltung von Autoimmuner-
the division could demonstrate the surface expres-
krankungen der Haut von Bedeutung. Dieses The-
sion of proteinase-activated receptor (PAR) –1 and
ma ist der Mittelpunkt eines kürzlich innerhalb des
intracellular expression of PAR-2 in human lung
Exzellenz Clusters „Entzündung an Grenzflächen“
mast cells as well as the induction of calcium sig-
gestarteten Projekts.
nals by synthetic and natural PAR-1 but not PAR-2
ligands.
Im Mittelpunkt der Forschung der Laborgruppe
84
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
Biologische Chemie (E. Brandt) steht die Untersu-
The division of Immune Cell-Analytics (M. Ernst)
chung der Interaktionen zwischen Mastzellen,
studies the contribution of central memory T-lym-
Plättchen und Neutrophilen bei der allergischen
phocytes in antigen-specific activation in vitro. Dif-
und nicht-allergischen Entzündung. Insbesondere
ferentiation, proliferation and secondarily release
interessiert sich diese Gruppe für die von Chemo-
of interferon gamma are the read-outs used. In par-
kinen, Proteasen und antimikrobiotischen Pepti-
ticular, the hypothesis that sleep and especially
den vermittelte Kommunikation dieser Zellpopu-
sleep-associated hormones secreted within the first
lationen. Gefördert durch den Transregio 22 und
half of the night (such as growth hormone and pro-
in Kooperation mit der Laborgruppe Biologische
lactin) may reinforce the function of memory T-lym-
Chemie wurde entdeckt, dass das thrombozytäre
phocytes is currently under investigation (support-
Chemokin CXCL4 anscheinend eine wichtige Funk-
ed by the SFB 654) utilizing cells of healthy donors
tion als Regulator bei von humanen Mastzellen se-
and of patients suffering from obstructive sleep ap-
zernierten Proteasen hat. Hierbei inhibiert es nicht
nea syndrome (OSAS) before and after therapy.
nur die Prozessierung von Chemokinen durch Kina-
Furthermore, in cooperation with the Division of
sen, sondern auch den Abbau von verschiedenen
Clinical Diseases, a new assay which allows a sen-
Substraten (z.B. LL37) durch Trypase. Bemerkens-
sitive/specific diagnosis of pulmonary tuberculosis
werterweise zeigte sich, dass dem Kathelicidin
in patients with repeated negative Ziehl-Neelsen
LL37 (ein wichtiges Produkt der Neutrophilen) die
stain in sputum samples (Am J Respir Crit Care
Fähigkeit eine Mastzellen-Gegranulation zu indu-
Med) was established by employing cell cultures
zieren, und auch die antimikrobielle Aktivität,
from the bronchoalveolar lavage stimulated with
nach Spaltung durch Trypase, verloren ging. In ei-
ESAT-6 and CFP-10 (M. tuberculosis-specific anti-
nem weiteren Projekt der Laborgruppe, das den
gens) and subjected to the interferon-gamma-spe-
Einfluss von Proteasen auf die Funktionen der
cific Elispot procedure.
Mastzellen untersucht, konnte die Oberflächen Expression des Proteinase-aktivierten Rezeptors
(PAR)-1 und die intrazelluläre Expression von PAR-
During the transition of the division of Tumor biolo-
2 in humanen Lungen-Mastzellen, so wie die In-
gy (J. Gerdes) to Immunoepigenetics (P. Singh) the
duktion von Calcium Signalen durch synthetischen
long-standing project to elucidate the function of
und natürlichen PAR-1 (jedoch nicht durch PAR-2 Li-
the proliferation-associated Ki-67 protein was con-
ganden), nachgewiesen werden.
cluded. It was found that pKi-67 not only physically
interacts with the chromatin of the rRNA genes, but
is also associated with the rRNA transcription fac-
Die Laborgruppe Immunzell-Analytik (M. Ernst) un-
tor UBF, indicating that the pKi-67’s likely role in cel-
tersucht die Funktion von Gedächtnis-T-Lympho-
lular proliferation processes may be founded upon
zyten bei der Antigen-spezifischen Aktivierung in
a role in ribosome biosynthesis. As part of a DFG-
vitro. Differenzierung, Proliferation und sekundäre
funded project it was confirmed that the loss of het-
Interferon-gamma-Freisetzung sind die von der
erochromatin protein 1 (HP1) results in a dramatic
Gruppe genutzten „Ablesesysteme“. Der Hypo-
genomic instability. Specifically, it could be shown
these, dass Schlaf und vor allem Schlaf-assoziier-
that the HP1
te Hormone (wie z.B. Growth Hormone und Pro-
ated with aberrant cerebral corticogenesis associ-
laktin), die Funktion von Gedächtnis-T-Lympho-
ated with genomic instability and that the proxi-
zyten verstärken können, wird zurzeit nachgegan-
mate cause of death is an acute respiratory failure
gen (gefördert durch den SFB 654). Hierzu werden
in newborn mice (JCB). Finally, the division has in-
Zellen von gesunden Probanden und von Patien-
vestigated the changing epigenotype of two
Wissenschaftlicher Jahresbericht 2007-2008
null mutation is lethal and is associ-
85
Zusammenfassung
Summary
Fo r s c h u n g s z e n t r u m
Bo r s t e l
ten
mit
obstruktivem
Schlafapnoe-Syndrom
(OSAS) vor und nach Therapie untersucht.
genes, CD14 and CD209, which are differentially
regulated during differentiation of monocytes into
dendritic cells. Monocyte differentiation leads to
transcription and hyperacetylation of the CD209
In Zusammenarbeit mit der Laborgruppe Klinische
promoter in dendritic cells with little affect on DNA
Infektiologie ist es gelungen, die ESAT-6 und
methylation; the CD14 epigenotype shows, to a
CFP-10 (M. tuberculosis-spezifische Antigene) sti-
lesser degree, the reverse where the gene is ex-
mulierten Zellkulturen aus der bronchoalveolären
pressed and its chromatin acetylated in monocytes
Lavage im Interferon-gamma-spezifischen Elispot-
and deacetylated in dendritic cells.
Verfahren einzusetzen und damit eine sensitiv/spezifische Diagnose der Lungentuberkulose
bei Patienten mit wiederholt negativer Ziehl-Neel-
The division of Veterinary Infection Biology and Im-
sen-Färbung in Sputum-Proben (Am J Respir Crit
munology (J. Ahmed) places the main emphasis of
Care Med) zu ermöglichen.
its investigations on the genetics as well as on cellular and molecular biological aspects of an infection with the intracellular protozoan Theileria. The
Während der Umgestaltung der Laborgruppe Tu-
goal of these internationally recognized studies is
morbiologie in die Laborgruppe Immunoepige-
the analysis and characterization of the pathogen-
netik (P. Singh) wurde das lang anstehende Pro-
host interaction, the development of effective vac-
jekt, die Funktion des Proliferations-assozierten Ki-
cines as well as diagnostic procedures. The divi-
67-Protein zu klären, verwirklicht. Es konnte ge-
sion achieved the successful application of two
zeigt werden, dass Ki-67 nicht nur physikalisch mit
projects in 2007 supported within the EU FP6 re-
dem Chromation der rRNA-Gene interagiert, son-
search program (ASEM-Dialog and ConFluTech),
dern auch mit dem rRNA Transkriptionsfaktor UBF
and in 2008 the funding of two further projects with-
assoziiert ist. Dies weist darauf hin, dass die Funk-
in the EU FP7 (POSTICK and Arbo-Zoonet). Further-
tion des pKi-67 im zellulären Proliferationsprozess
more, the division organized a total of 16 (seven in
wahrscheinlich auf einer Beteiligung in der ribo-
2007; nine in 2008) international workshops and
somalen Biosynthese basiert. Als Teil eines von
technical workshops and published the proceed-
der DFG geförderten Projekts konnte bestätigt
ings of three international meetings in Parasitology
werden, dass der Verlust des Heterochromatin
Research, Transboundary and Emerging Diseases
Protein 1 (HP1) in einer dramatischen genomi-
and Vaccine.
schen Instabilität resultiert. Insbesondere wurde
demonstriert, dass die HP 1
null Mutation letal
ist und mit einer aberranten zerebralen Cortico-
Networking: The Department of Immunology and
genese mit einer genomischen Instabilität assozi-
Cell Biology participates in the following coopera-
iert ist. Der Grund für den letztendlichen Tod ist, in
tive projects: DFG-Sonderforschungsbereiche (col-
neugeborenen Mäusen, ein akutes respiratori-
laborative research centers) 415, 617, 654, and Tran-
sches Versagen.
sregio 22 and Excellence Cluster “Inflammation at
Interfaces”; EU FP6 framework program (ConFluTech): Capacity building for the control of avian
Außerdem hat die Laborgruppe die wechselnden
influenza through technology transfer and training;
Epigenotypen von zwei Genen (CD14 und CD209)
(ASEM-Dialog): EU, China, and South-East Asia: Di-
untersucht. Während der Differenzierung von Mo-
alogue for the Development of Research Areas in
nozyten zu dendritischen Zellen werden diese auf
Animal Health of Mutual Interest. Marie Curie rein-
86
Wissenschaftlicher Jahresbericht 2005-2006
Division of Immunbiology
Research Reports
unterschiedliche Weise reguliert. Die Monozyten-
tegration grants. EU FP7: CURE, INFLA-CARE, PO-
Differenzierung führt zur Transkription und Hyper-
STICK and Arbo-Zoonet.
acetylierung des CD209-Promotors in dendritischen Zellen, mit geringem Effekt auf die DNA-Methylierung. Der CD14 Epigenotyp zeigt, in einem
weniger ausgeprägten Ausmaß, das Gegenteil.
Hier ist das Gen exprimiert und das Chromatin in
Monozyten acethyliert und in dendritischen Zellen
deacetyliert.
Im Focus der Laborgruppe Veterinärmedizinische
Infektiologie und Immunologie (J. Ahmed) stehen
Untersuchungen zur Genetik, sowie die zellulären
und molekularbiologischen Aspekte einer Infektion mit dem intrazellulären Protozoa Theileria. Das
Ziel dieser international anerkannten Studien ist
die Analyse und Charakterisierung der PathogenWirt- Interaktion, die Entwicklung wirkungsvoller
Impfstoffe, sowie diagnostischer Verfahren. Der
Laborgruppe ist es gelungen, zwei Projekte im FP6
des EU-geförderten Forschungsprogramms (ASEMDialog und ConFluTech) und in 2008 zwei weitere
Projekte im FP7 (POSTICK und Arbo-Zoonet), erfolgreich zu beantragen. Darüber hinaus organisierte die Laborgruppe insgesamt 16 (sieben in
2007; neun in 2008) internationale Workshops und
technische Workshops und publizierte die Sitzungsberichte von drei internationalen Meetings
in den Journals: Parasitology Research, Transboundary und Emerging Diseases und Vaccines.
Netzwerkbildung: Die Abteilung Immunologie und
Zellbiologie ist Partner folgender Verbundprojekte: DFG-Sonderforschungsbereiche 415, 617, 654
und Transregio 22; EU-6 Rahmenprogramm: (ConFluTech), Capacity building for the control of Avian influenza through technology transfer and training; (ASEM-Dialog), EU, China und Süd-Ost-Asien:
Dialog for the Development of Research Areas in
Animal Health of Mutual Interest. Marie Curie
Reintegrationsförderung. EU FP7: CURE, INFLA-CARE, POSTICK und Arbo-Zoonet.
Wissenschaftlicher Jahresbericht 2007-2008
87
Division of Immunbiology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Immunbiology
Head
show that IL-15 forms functional heterocomplexes
Prof. Dr. Dr. Silvia Bulfone-Paus
with soluble high affinity IL-15 receptor alpha (IL15Ralpha) chain in mouse serum and cell-condi-
Postdoctoral fellows
tioned medium, which prevents IL-15 detection by
Dr. Rajia Bahri
ELISA. We also demonstrate that two soluble IL-
Dr. Annalena Bollinger
15Ralpha (sIL-15Ralpha) sushi domain isoforms are
Dr. Elena Bulanova
generated through a novel alternative splicing
Dr. Erwin Duitman
mechanism within the IL-15Ralpha gene. These iso-
Dr. Orietta D’Orlando
forms potentiate IL-15 action by promoting the IL-15-
Dr. Niko Föger
mediated proliferation of the CTLL cell line and in-
Dr. Farhad Mirghomizadeh
terferon gamma production by murine NK cells,
Dr. Zane Orinska
which suggests a role in IL-15 transpresentation.
Graduate and Diploma/Master students
Diego Goyeneche-Patiño
Andre Jenckel
Rabea Langenhaun
Katharina Scholz
Technicians
Manuel Hein
Martina Hein
Marie-Luise Helms
Gesine Rode
Katrin Streek
Katrin Westphal
Kathleen Wilke
Guests and Trainees
Marvin Bloch
Romina Pritzkow
Research Reports
Soluble Interleukin IL-15Ralpha is generated by
alternative splicing or proteolytic cleavage and
forms functional complexes with IL-15
Bulanova E, Budagian V, Duitman E, Orinska Z,
Rückert R, Bulfone-Paus S.
Fig. 29. Alternatively spliced or shed sIL-15Ralpha forms heterocomplexes with IL-15. Different IL-15Ralpha isoforms may associate with
IL-15 intracellularly. IL-15·IL-15Ralpha sushi domain heterocomplexes
presumably follow a secretion route. Conversely, IL-15 associated with
IL-15Ralpha may be elaborated to the cell membrane, where IL-15 is
presented in trans by IL-15Ralpha and stimulates neighboring cells
expressing intermediate affinity IL-15Rbeta/gamma. Similarly, soluble
IL-15·IL-15Ralpha sushi domain complexes stimulate such cells, inducing proliferation of T cells and IFNgamma production by NK cells.
Transmembrane IL-15Ralpha molecules alone or together with IL-15
are subsequently shed by TACE, releasing sIL-15Ralpha alone or IL15·sIL-15Ralpha heterocomplexes, in which IL-15 is inactive. Diverse
extracellular stimuli, such as bacteria and viruses, might shift the fine
tuned balance between sushi domain and full-length IL-15Ralpha
molecules. Cells may favour production of distinct IL-15Ralpha isoforms via switching between them according to particular environmental changes and demands.
Interleukin 15 (IL-15) is a pleiotropic cytokine that is
Conversely, a full-length sIL-15Ralpha ectodomain
hardly detectable in biological fluids. Here, we
released by tumor necrosis factor-alpha-converting
88
Wissenschaftlicher Jahresbericht 2007-2008
Division of Immunbiology
Research Reports
enzyme (TACE)-dependent proteolysis inhibits IL-15
against Gram-negative bacteria by demonstrating
activity. Thus, a dual mechanism of sIL-15Ralpha
that MC protease activity is regulated by interleukin-
generation exists in mice, giving rise to polypep-
15 (IL-15). Mouse MCs express both constitutive and
tides with distinct properties, which regulate IL-15
lipopolysaccharide-inducible IL-15 and store it in-
function.
tracellularly. Deletion of IL-15 in mice markedly increases chymase activities, leading to greater MC
Collaboration: Krause H, Department of Urology,
bactericidal responses, increased processing and
Charité University Medicine, Berlin; Reiling N, Divi-
activation of neutrophil-recruiting chemokines, and
sion of Molecular Infection Biology, Research Cen-
significantly higher survival rates of mice after sep-
ter Borstel, Borstel.
tic peritonitis. By showing that intracellular IL-15 acts
as a specific negative transcriptional regulator of a
Supported in part by: Deutsche Forschungsgemein-
mouse MC chymase (mast cell protease-2), we pro-
schaft (SFB415).
vide evidence that defined MC protease activity is
transcriptionally regulated by an intracellularly retained cytokine. Our results identify an unexpected
IL-15 constrains mast cell-dependent antibacteri-
breach in MC-dependent innate immune defenses
al defenses by suppressing chymase activities.
against sepsis and suggest that inhibiting intracel-
Orinska Z, Mirghomizadeh F, Bulanova E, Buda-
lular IL-15 in MCs may improve survival from sepsis.
gian V, Bulfone-Paus S.
Collaboration: Brandt E, Nashkevich N, Schiemann
F, Schulmistrat J, Department of Immunology and
Cell Biology, Research Center Borstel, Borstel; GironMichel J, U542 Institut National de la Santé et de la
Recherche Médical (INSERM), Hôpital Paul Brousse,
Villejuif, France; Maurer M, Metz M, Department of
Dermatology and Allergy, Allergie-Centrum-Charité,
Charité–Universitätsmedizin Berlin, Berlin; Paus R,
Department of Dermatology, University Hospital
Schleswig-Holstein, University of Lübeck.
Supported in part by: grants from the Deutsche
Forschungsgemeinschaft
(SFB/TR22
and
MA
Fig. 30. Role of IL-15 in the regulation of chymase-mediated antimicrobial activity. IL-15, which shows increased expression on LPS
stimulation, inhibits transcription of Mcpt2. In the absence of IL-15,
transcription, intracellular storage and release of chymase on
stimulation are increased. The increase in intracellular chymase
activity leads to enhanced microbicidal activity, for example, by
the generation of larger amounts of antimicrobial peptides (AMPs)
from inactive precursors (pre-AMPs). Secreted chymases may exert their bactericidal activity directly or may function indirectly by
cleaving chemokine precursors and generating activated
chemokines to recruit the most potent neutrophil army.
1909/4-1/2).
Sepsis remains a global clinical problem. By using
Interleukin (IL)-21 is a T cell-derived cytokine which
the mouse cecal ligation and puncture model of
uses a heterodimeric receptor, composed of the
sepsis, here we identify an important aspect of mast
common gamma-chain (CD132) and an IL-21Ral-
cell (MC)-dependent, innate immune defenses
pha-chain. IL-21 activates lymphoid T and B cells,
Interleukin-21 stimulates antigen uptake, protease activity, survival and induction of CD4+ T
cell proliferation by murine macrophages.
Rückert R, Bulfone-Paus S, Brandt K.
Wissenschaftlicher Jahresbericht 2007-2008
89
Division of Immunbiology
Research Reports
modulates antibody production but also suppresses
components in the control of ligand-transport and se-
maturation of myeloid dendritic cells; however, its
cretion. Here, we show that IL-15 translocation into
role in the differentiation and function of other
the endoplasmatic reticulum occurs independently of
myeloid cells remains less clear. In this study we
the presence of IL-15Ralpha. Subsequently, however,
analysed IL-21/IL-21Ralpha effects on macrophage
IL-15 is transported through the Golgi apparatus on-
(MPhi) differentiation and function. MPhi could be
ly in association with IL-15Rα, and is then secreted.
generated readily from bone marrow with MPhi-
This intracellular IL-15/IL-15Ralpha complex presum-
colony-stimulating factor in the presence of IL-21 (des-
ably already is formed in the endoplasmatic reticu-
ignated IL-21MPhi) or from IL-21Ralpha-/- mice. IL-
lum and thus enables further trafficking of complexed
21Ralpha-/- mice had normal MPhi numbers, sug-
IL-15 through the secretory pathway. Just transfecting
gesting a non-essential role of both IL-21 and the IL-
IL-15Ralpha in cells, which transcribe, but normally do
21Ralpha
could
not secrete IL-15 suffice to induce IL-15 secretion. Thus,
demonstrate that mature MPhi express the IL-21Ral-
we here provide the first evidence of how a cytokine
pha and the common gamma-chain. However, short-
is chaperoned through the secretory pathway by
term IL-21 stimulation did not enhance MPhi prolifer-
complexing with its own high affinity receptor, and
ation but induced anti-apoptotic cell-cycle regulators
show that IL-15/IL-15Ralpha offers an excellent mod-
p21(waf1)/p27(Kip1) and expression of suppressors
el system for further exploration of this novel mecha-
of cytokine signalling (SOCS)2/SOCS3. Moreover, IL-
nism for the control of cytokine secretion.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
for
MPhi
generation.
We
21 enhanced phagocytosis by MPhi via IL-21Ralpha
signalling and supports protease activity and matrix metalloproteinase
12 expression. Stimulating MPhi with
IL-21 enhanced their capacity to induce antigen-specific CD4+ T cell
proliferation in dependence from the
IL-21Ralpha, which was not the case
for CD8+ T cells. Taken together, IL-21
plays a previously unrecognized role
in modulating innate and acquired
effector mechanisms of murine MPhi
by linking these different functions to
support CD4+ T cell-mediated immune responses.
How a cytokine is chaperoned through the secretory pathway by complexing with its own recep-
Fig. 31. Receptor chaperoning model of cytokine. Proposed model for the intracellular trafficking and IL-15Rα- dependent secretion
of IL-15 in the investigated cell lines. The abbreviations C, ER, GN
and E represent cytoplasm (C), endoplasmatic reticulum (ER), Golgi apparatus (GA) and environment (E).
tor: lessons from interleukin-15 (IL-15)/IL-15 receptor alpha.
Collaboration: Paus R, Department of Dermatology,
Duitman EH, Orinska Z, Bulanova E, Bulfone-Paus S.
University Hospital Schleswig-Holstein, University of
Lübeck.
While it is well-appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is
Supported in part by: Deutsche Forschungsgemein-
known about additional roles for cytokine receptor
schaft (SFB 415).
90
Wissenschaftlicher Jahresbericht 2007-2008
Division of Immunbiology
Research Reports
Theses
Diploma
Scholz, Katharina
Characterization of BTNL2, a Butyrophilin/B7-like
molecule: expression and regulation.
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2008.
Dissertation
Stelekati, Erietta
The role of mast cells in CD8+ T cell-mediated immune responses.
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2008.
Wissenschaftlicher Jahresbericht 2007-2008
91
Division of Cellular Immunology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Cellular Immunology
Head
RGS2 in a similar manner. Interestingly, the TLR3 lig-
Prof. Dr. Artur J. Ulmer
and poly(I:C) permanently up regulated RGS1 and
RGS2 expression indicating a different modulation by
Staff Scientists and Postdoctoral Fellows
Dr. Sabine Riekenberg
the MyD88- and TRIF-signalling pathway. This suggestion was confirmed by the use of MyD88-/- and
TRIF-/- BMDM. Inhibition of phosphorylation of p38 by
Graduate and Diploma Students
SB203580 and ERK by PD98059 as well as inhibition
Katja Farhat
of G -protein signalling by PTX indicates that these
Kristina Röschmann
signalling pathways were not involved in RGS1 and
Christine Warmbold
RGS2 modulation by TLR ligands. Our data lead to
the conclusion that modulation of RGS1 and RGS2 by
Technicians
different TLR ligands plays an important role in G-pro-
Carola Schneider
tein signaling during inflammatory and immunologi-
Suhad Al-Badri
cal reactions after bacterial and viral infections.
Research Reports
Activation of the MyD88-signal pathway and the
TRIF-signal pathway through Toll-like receptors
differently modulate Regulators of G-protein Signalling (RGS)
Riekenberg S, Farhat K, Ulmer AJ.
Regulators of G-protein signalling (RGS) accelerate
the GTPase activity of Gα subunits, thus driving G-proteins in their inactive GDP-bound form. This property
defines them as GTPase activating proteins (GAPs).
In our study we analyzed the effect of different Tolllike receptor (TLR) agonists on RGS1 and RGS2 expression
in
mouse
bone
marrow-derived
macrophages (BMDM) and J774 cells. After stimulation with TLR2/1 or TLR2/6 specific lipopeptide lig-
Fig. 32. Modulation of RGS1 and RGS2 mRNA by TLR2 and TLR4
ligands. Expression was measured after stimulation of BMDM with
100 ng/ml LPS, 100 nM FSL-1 and 100 nM Pam3C-SK4 for 0-24 h.
Specific RGS1 and RGS2 mRNA expression was determined by real-time PCR. The results show representative data from three independent experiments.
ands and the TLR4/MD2 ligand LPS, microarray
analyses showed most strong reduction of RGS2 mR-
Collaboration: Debarry J, Heine H, Div of Innate Im-
NA expression and strong modulation of RGS1. Real-
munity, Research Center Borstel; Jung G, Institute of
time PCR confirmed these modulations. In contrast to
Organic Chemistry, University of Tübingen, Tübin-
RGS2, which was always down regulated, RGS1 mR-
gen; Wiesmüller K-H, EMC microcollections GmbH,
NA was up regulated during the first 30 min after stim-
Tübingen.
ulation followed by down regulation (Fig. 32). Similar
results were also found in the mouse macrophages
Supported in part by: Deutsche Forschungsgemein-
cell line J774 and mouse dendritic cells. ODN1826, a
schaft (UL68/3-2).
ligand for intracellular TLR9, modulated RGS1 and
92
Wissenschaftlicher Jahresbericht 2007-2008
Division of Cellular Immunology
Research Reports
Ligand binding and signal transduction induced-
This indicates that Pam2C-SK4 is recognized by TLR2/1
by lipopeptides
as well as by TLR2/6 rather than by TLR2-homomers
Farhat K, Riekenberg S, Ulmer AJ.
(Fig. 33).
Toll-like receptors (TLRs) are primary triggers of the in-
In a further study we have compared the signal trans-
nate immune system by recognizing various microor-
duction pathways activated by the different TLR2
ganisms through conserved pathogen-associated mo-
dimers
lecular patterns (PAMPs). Among all TLRs, TLR2 is the
receptor for a functional recognition of bacterial
macrophages, using the synthetic LPs PamOct2C(VPGVG)4VPGKG, FSL-1 and Pam2C-SK4 to specifi-
lipopeptides (LPs) and is upregulated during various
cally target TLR2/TLR1 and/or TLR2/TLR6, respec-
disorders like chronic obstructive pulmonary disease
tively. Immunoblotting of MAP-kinases, usage of dom-
(COPD) and sepsis. This receptor is unique in its abili-
inant negative forms of adaptor molecules as well as
ty to form heteromers with either TLR1 or TLR6 to me-
microarray analyzes indicate that all dimers use the
diate intracellular signaling. According to the fatty
same signaling cascade to activate transcription fac-
acid pattern as well as the assembling of the polypep-
tors leading to an identical pattern of gene activa-
tide tail LPs can signal through TLR2 in a TLR1- or TLR6-
tion. In conclusion, LPs from mycoplasma, Gram-pos-
dependent manner. As shown recently there are also
itive and Gram-negative bacteria, although they may
di- and triacylated LPs, like Pam2C-SK4 and Pam3MALP2, which stimulate cells of TLR1-/- and of TLR6-/-
use different TLR2 dimers, induce the same signal
in
murine
bone
marrow
derived
transduction pathway during infection.
mice. Inhibition of TLR1-expression with TLR1-siRNA in
TLR6-/- cells and inhibition of TLR6-expression with
Collaboration: Debarry J, Heine H, Div of Innate Im-
TLR6-siRNA in TLR1-/- cells resulted in an marked re-
munity, Research Center Borstel; Lang R, Mages J, In-
duction of the response to Pam2C-SK4.
stitute of Medical Microbiology, Immunology and Hygiene,Technical University Munich; Jung G, Institute of
Fig. 33. Inhibition of lipopeptide-induced TNF release in mouse TLR1/- or TLR6-/- macrophages by TLR6- or TLR1-siRNA, respectively.
BMDM of (A) TLR1- or (B) TLR6-deficient mice were transfected with
25 nM of non target siRNA, (A) TLR6 siRNA, or (B) TLR1 siRNA. After
48 hours these cells were stimulated with 100 nM of FSL-1, Pam2CSK4, PamOct2C-(VPGVG)4VPGKG, or 100 ng/ml LPS for additional
24 hours. The TNF-α release into the culture supernatant was determined by ELISA. The results are expressed as % of TNF-α release obtained from stimulated non target siRNA-transfected cells.
Organic Chemistry, University of Tübingen; Wiesmüller K-H, EMC microcollections GmbH, Tübingen.
Supported in part by: Deutsche Forschungsgemeinschaft (UL68/3-2).
Wissenschaftlicher Jahresbericht 2007-2008
93
Division of Cellular Immunology
Research Reports
The adjuvant activity of FSL-1 during immunization
We found that FSL-1 exhibits mitogenic activity on
of mice with the Timothy grass polen major aller-
mouse spleen B-lymphocytes and induces TNF-alpha
gen Phl p 1 is TLR6-dependent
release in bone-marrow derived macrophages.
Farhat K, Röschmann K, Riekenberg S, Buwitt-
Whereas the activity of FSL-1 is absent in TLR6-defi-
Beckmann U, Ulmer AJ.
cient cells, the tri-acylated derivative Pam3-FSL-1
(Pam3C-GDPKHPKSPF) is TLR6-independent but
Bacterial lipopeptides (LP) are known to be strong
TLR1-dependent. Furthermore, TLR2, CD14 and
stimulators of the innate immune system and potent
MyD88 are involved in FSL-1 induced signal trans-
adjuvants of the adaptive immune system. The re-
duction. Additionally, we have investigated the ad-
ceptor for the recognition of LP is TLR2. TLR2 is
juvant activity of FSL-1 during immunization of mice
unique in its ability to form heteromers with either
with the Timothy grass pollen major allergen Phl p
TLR1 or TLR6 to mediate intracellular signalling. FSL-
1. Our results show that FSL-1 boosts the Phl p 1-spe-
1 (Pam2C-GDPKHPKSPF) represents a di-acylated
LP of Mycoplasma salvarium, which signals through
cific immunoglobulin production of both IgG1 and
TLR2 in a TLR6-dependent manner as determined in
Th1-immune responses (Fig. 34). Results with TLR6-
transfected human HEK293 cells. However, its re-
deficient mice indicate a central role of TLR6 in this
ceptor requirement during immune responses of
system. As a TLR6 polymorphism seems to play a
mice in vitro and in vivo has not been defined so far.
role in the pathogenesis of childhood asthma, this
Fo r s c h u n g s z e n t r u m
Bo r s t e l
IgG2a indicating an enhancement of both Th2- and
study might give insights into the importance of TLR
concerning the development of atopic diseases.
Collaboration: Seitzer U, Div. of Vet.-Infektiology u.
–Immunology, Research Center Borstel; Petersen A,
Div. of Clin. and Mol. Allergology, Research Center
Borstel; Jung G, Institute of Organic Chemistry, University of Tübingen; Wiesmüller K-H, EMC microcollections GmbH, Tübingen.
Supported in part by: Deutsche Forschungsgemeinschaft (Ul 68/3-1 and SFB/TR22, A3).
Activation respiratory epithelial cells by the Timothy grass pollen major allergen Phl p 1
Röschmann K, Farhat K, Ulmer AJ.
Fig. 34. Adjuvant effect of FSL-1 on the immunisation of mice with
Phl p1, the major allergen of Timothy grass Phleum pratense.
Groups of 5 mice were immunized intra peritonial with 10µg FSL-1
or 1µM Phl p 1 alone or in combination. Serum samples were collected and analyzed for Phl p 1 specific IgG1 and IgG2a on various days of the immunisation protocol. FSL-1 did not induce the production of Ig in any of the animals, while immunization with the allergen Phl p 1 resulted in an increase of IgG1. In contrast, in the
presence of FSL-1, administration of Phl p 1 induced much higher
amounts of the Th2-polarizing IgG1, indicating the adjuvant capacity of FSL-1. This effect is even more prominent concerning the
IgG2a titer. Phl p 1 alone did not induce any of the Th1-related antibody, whereas co-immunisation with FSL-1 resulted in a strong
IgG2a production.
94
Group 1 allergens from grass pollen (e.g. Phl p 1, the
major allergen of Timothy grass Phleum pratense)
cause IgE-reactivity in about 95 % of allergic subjects
and exist in all grass species. The respiratory epithelium represents a first line of contact of the immune system with airborne allergens. It functions as
physical barrier and is an important immunological
regulation system, which is also involved in allergic
Wissenschaftlicher Jahresbericht 2007-2008
Division of Cellular Immunology
Research Reports
reactions like asthma. The aim of this study was to
an indirect mechanism through which the allergen
investigate the interaction of Phl p 1 with human res-
may cross the epithelial barrier and attracts im-
piratory epithelium to elucidate the contribution of
munocompetent cells, thereby contributing to aller-
epithelial cells to the development of allergic reac-
gic responses in asthmatic airways.
tions.
Fig. 35. Effect of Phl p 1 and Der p 1 on cilia-driven particle transport in mouse tracheae ex vivo. 15-30 min after addition of the
beads to the isolated trachea a baseline of the velocity of particle transport has been come up and the allergens or control buffer
was added (first arrow) [(A) ctrl: 200 µl Hepes-Ringer solution, (B)
5 µM Phl p 1, (C) 1 µM Der p 1]. At the end of each experiment
100 µM ATP was added (second arrow) to test the vitality of the
ciliated epithelial cells. Representative results from at least 3 independent experiments are shown.
Collaboration: Petersen A, Div. of Clin. and Mol. Allergology, Research Center Borstel; König P, Institute
for Anatomy, University of Lübeck; Suck R, Allergopharma Joachim Ganzer KG, Reinbek.
Supported in part by: Deutsche Forschungsgemeinschaft (SFB/TR22, A3).
We found that Phl p 1 activates respiratory epithelial cells as measured by release of IL-8 and IL-6 and
induction of TGF-β mRNA. Phl p 1, in contrast to Der
Effects of microbial and allergic components on
p 1, an allergen from the house dust mite, does not
the immune system of the fruitfly Drosophila
exert proteolytic activity, as investigated by micro-
melanogaster
scopical observation and zymograms. In an ex vivo
Warmbold C, Röschmann K, Ulmer AJ.
model with mouse tracheae we were able to show
that Der p 1 but not Phl p 1 causes injure of ciliated
The fruitfly Drosophila melanogaster has been used
tracheal epithelium. In addition, Der p 1, in contrast
as a powerful model to study basic aspects of the
to Phl p 1, enhances the transportation velocity of
innate immune system. The two independent signal
particles by the tracheae, presumably by ATP re-
transduction pathways Toll and IMD are con-
leased from the injured epithelium (Fig. 35). We con-
stituents of the fruitfly`s immune system. The Toll-
clude that Phl p 1 affects tracheal epithelial cells
pathway is involved in signal transduction in re-
through a non-proteolytic activity. Enhancement of
sponse to infection with Gram-positive bacteria,
TGF-β expression induced by Phl p 1 together with
whereas the IMD-pathway is stimulated during in-
the increased release of IL-6 and IL-8 might provide
fection with Gram-negative bacteria. We have now
Wissenschaftlicher Jahresbericht 2007-2008
95
Division of Cellular Immunology
Research Reports
investigated whether isolated cowshed dusts as
we furthermore discovered that allergic proteins of
Fo r s c h u n g s z e n t r u m
Bo r s t e l
well as isolated bacteria and lipopeptides, which
Timothy grass pollen (Phleum pratense), namely Phl
have never been tested before in this system, are
p 1 and of housedust mite (Der p 1, Der-
able to activate the innate immune system. For our
matophagoides pteronyssinus) are also able to ac-
analysis we use the macrophage-like cell line SL2
tivate the Toll- and IMD-pathway in Drosophila (Fig.
isolated from embryos of the D.m. strain Oregon R.
36). By using RNAi-experiments and microarray-
The results of our studies are based on real-time
analyses we will confirm our current data.
PCR and on a luciferase reporter assays. In this assay a Firefly-luciferase is under the control of the
Collaboration: A. Petersen, Petersen A, Div. of Clin.
drosomycin-promotor as a read-out-system for the
and Mol. Allergology, Research Center Borstel;
Toll-pathway and the diptericin-promotor for the
Roeder T, Div. of Zoophysiology, Christian-Albrechts
IMD-pathway. The data are normalized by the con-
University Kiel.
stitutive Renilla-luciferase.
Supported in part by: Deutsche Forschungsgemeinschaft (SFB/TR22, A3 and A7).
Fig. 36. Effects of allergens on the immune system of Drosophila
melanogaster. The S2 cell line of the fruitfly is responsive to the major allergen of Timothy grass pollen (Phl p1). In this experiment we
were able to show that the signalling occurs via the Toll pathway
and this signalling is dose-dependent. In contrast, the IMD pathway is not involved.
In our experiments we found that isolated cowshed
dusts and bacteria excluding L.lactis G121 can activate the fruitfly`s immune system. In the human system L.lactis G121 leads to a delayed immune response. In addition to these bacterial components
96
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Immunology
Research Reports
Division of Molecular Immunology
Head
of Fas signaling can activate divergent signaling
Dr. Kyeong-Hee Lee
pathways to direct either cellular survival or apoptotic signals. These data suggest a different para-
Postdoctoral fellow
digm for transmembrane signaling and emphasizes
Dr. Gulnar Fattakhova
the role of the endocytic pathway for both receptor
degradation as well as signal propagation. Little in-
Graduate students
formation exists regarding the molecular elements
Xuan-Hung Nguyen
regulating the spatial and temporal compartmen-
Beate Schurek
talization in the endocytic pathway that contribute
to biologic responses. We have also identified im-
Technicians
mune specific regulators which are involved in re-
Patricia Prilla
ceptor endocytosis and membrane dynamics. By
studying the mechanisms by which our target molecules are regulated and how they control immune
Research Reports
cell signaling and function, we hope to better un-
Integrating molecular and cellular aspects of
derstand signaling pathways during normal im-
membrane trafficking and immune function
mune responses and how perturbations in these
Nguyen XH, Fattakhova G, Lee KH.
pathways can lead to immune deficiency or autoimmunity.
We are interested in the roles of receptor
endocytosis
and
the
traffick-
ing/sorting machinery in apoptotic vs.
survival signaling and its functional relevance for immune function. Receptor
endocytosis, in addition to serving as a
degradation pathway, can also target
activated receptors to the endocytic
compartment to facilitate signaling. This
mode of regulation can control the assembly of activated receptors with
downstream signaling molecules as
well as titrate signal intensity and thereby serves as
Fig. 37. Model for compartmentalization of Fas signaling.
a cellular mechanism to direct specific biologic responses. We are studying the function of the traf-
The internalization of Fas receptor is required for
ficking/sorting machinery in signal transduction and
FasL-mediated death inducing signaling complex
the molecular mechanisms that regulate endocytic
(DISC) assembly, caspase activation and apoptosis.
pathways employing the death receptor Fas (CD95)
However, in cells in which Fas internalization is dis-
as our model system. Our studies have demon-
abled, FasL stimulation activates both Erk and NFκB
strated that endosomal compartments are major
signaling pathways. Hence, the subcellular local-
sites for the initiation of Fas mediated death sig-
ization and internalization pathways of Fas play im-
naling complex formation and apoptosis. Moreover,
portant roles that control activation of distinct sig-
we have demonstrated that compartmentalization
naling cascades to determine divergent cellular
Wissenschaftlicher Jahresbericht 2007-2008
97
Division of Molecular Immunology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
fates, such as apoptosis versus cellular survival.
Furthermore, we are currently generating a mouse
deficient for this Rab5 regulator to elucidate the role
Collaboration: Marcus E. Peter, the Ben May Insti-
of this Rab5 regulator in membrane trafficking and
tute for Cancer research, University of Chicago,
mast cell function in an in vivo setting.
USA; Stefan Schütze, Institute of Immunology, UniCollaboration: Silvia Bulfone-Paus, Research Center
versity of Kiel, Germany.
Borstel, Germany.
Supported in part by: Fritz Thyssen Research Foundation and the German Research Foundation
Supported in part by: Marie Curie International
(DFG).
Reintegration Grant (FP7).
The role of membrane trafficking in mast cell function
Schurek B, Prilla P, Lee KH.
We are also interested in the role of membrane trafficking in the context of an allergic reaction utilizing
mast cells as experimental objects. Mast cells provide an excellent model system to study the physiological role of membrane trafficking. Mast cells
play a pivotal role in human allergic and inflammatory disease and intensively require specifically
regulated vesicle transport and membrane fusion
processes. Activated mast cells in response to triggering of the high affinity receptor for IgE, rapidly
reorganize lysosomal vesicles and secrete inflammatory mediators by a process called degranulation. Our research focus is to elucidate the molecular mechanisms responsible for the transport and fusion of mast cell granules to the plasma membrane.
We have established a lenti-viral based miRNA vector system which allows for an efficient transfection
of miRNA into primary mast cells and to generate
stable cell lines expressing gene specific miRNA.
Employing these genetic tools, our studies have indentified a regulator for the small GTPase Rab5.
The knock-down of the novel regulator in mast cells
results in an increased and sustained mast cell degranulation, indicating a novel function of Rab5 for
mast cell degranulation. Our on-going studies are
investigating the underlying mechanism by which
this molecule affects the trafficking and membrane
fusion of mast cell granules utilizing various mutants.
98
Wissenschaftlicher Jahresbericht 2007-2008
Division of Immune Cell-Analytics
Research Reports
Division of Immune Cell-Analytics
Head
Activated human leukocyte antigen-DR-positive cells,
Dr. Martin Ernst
as well as memory CD4CD45R0-positive T-cells, were expanded among cells of the BALF. Compared
Staff Scientists and Postdoctoral Fellows
with a group of control patients with alternative pul-
Dr. Franziska Schwartzkopff
monary pathologies, there was no significant difference in lymphocyte subpopulations. However,
Technicians
ESAT-6- and CFP-10-specific lymphocytes were more
Renate Bergmann (until December 2007)
concentrated, with a median BALF:peripheral blood
Franziska Daduna (since March 2008)
ratio of 9.9 and 8.9, respectively, in patients with PTB.
Erika Kaltenhäuser
Mycobacterium tuberculosis-specific T-cells are highly selectively compartmentalised at the site of infec-
Guests and Trainees
tion in active pulmonary tuberculosis.
Dr. José Fernando García, Barcelona, Spain (June
2007)
Collaboration: Jafari C*, Strassburg A, Greinert U*,
Kalsdorf B*, Lange C* (Division of Clinical Infectious
Diseases and Medical Clinic Borstel), Kirsten D#
Research Reports
(Krankenhaus Großhansdorf).
Local immunodiagnosis of pulmonary tuberculosis by enzyme-linked immunospot
Ernst M , Jafari C*, Strassburg A*, Greinert U*,
Rapid diagnosis of CNS tuberculosis by a T-cell in-
Kalsdorf B*, Kirsten D#, Lange C*.
terferon-γγ release assay on cerebrospinal fluid
Lymphocytes are crucial in the immune defence
mononuclear cells
Ernst M, Kösters K1, Nau R2, Bossink A3, Greiffen-
against Mycobacterium tuberculosis (MTB) infection.
The aim of the present study was to ascertain
dorf I1, Jentsch M1, Thijsen S3, Hinks T4, Lalvani
A4, Lange C5.
whether or not MTB-specific lymphocytes are selectively compartmentalised in the lungs of patients with
Central nervous system tuberculosis remains a clini-
minimal active pulmonary tuberculosis (PTB). Patients
cal diagnostic challenge. The ex vivo Mycobacteri-
with smear-negative MTB-culture-confirmed PTB were
um tuberculosis-specific enzyme-linked immunospot
prospectively recruited. Differential cell counts, im-
assay (ELISPOT) is a novel assay for the rapid de-
munophenotyping with monoclonal antibodies di-
tection of M. tuberculosis-specific T-lymphocytes in
rected against the cell surface markers CD4, CD8,
the peripheral blood. However, when performed on
CD4CD45RA, CD4CD45R0, CD38, human leukocyte
peripheral blood, this assay cannot distinguish bet-
antigen DR, CD19, CD3, CD57 and CD16 and MTB-
ween active tuberculosis or latent tuberculosis in-
specific enzyme-linked immunospot assays of peri-
fection. On the assumption that M. tuberculosis-spe-
pheral blood mononuclear cells and bronchoalveo-
cific T-lymphocytes migrate to sites of infection, we
lar lavage (BAL) mononuclear cells with 6-kDa early
were able to demonstrate high levels of M. tubercu-
secretory antigenic target (ESAT-6) and culture filtra-
losis-specific cells by ELISPOT in the cerebrospinal
te protein 10 (CFP-10) were performed. Among 12 pa-
fluid of a patient with tuberculous meningitis and in-
tients with culture-confirmed smear-negative PTB, no
tracerebral tuberculoma (Fig.38) four weeks before
differences were found in the distribution of total CD4
cerebrospinal fluid culture became positive for M. tu-
or CD8 T-cells in peripheral blood or BAL fluid (BALF).
berculosis by culture.
Wissenschaftlicher Jahresbericht 2007-2008
99
Division of Immune Cell-Analytics
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
assay (ELISPOT)) are more sensitive for the diagnosis of Mycobacterium tuberculosis (MTB) infection
than the tuberculin skin test (TST), but cannot distinguish active from latent MTB infection. The present
authors report a 38-yr-old female presenting with a
3-week history of malaise, dyspnoea, fevers and
coughing, who had received immunosuppressive
therapies over 8 months for mixed connective tissue
disease. Chest radiograph and thoracic computed
tomography showed ground glass opacities in both
Fig. 38. MTB specific ELISPOT with ESAT-6 and CFP-10 antigen on
mononuclear cells from the cerebrospinal fluid. For the MTB-specific
ELISPOT (T-SPOT.TB, Oxford Immunotec, Abingdon, UK), 2.5 x 105
mononuclear cells isolated from CSF are put in microtiter plate wells
in the presence of ESAT-6 and CFP-10 or PHA (positive control) or
in the absence of antigen (negative control). Antigen-reactive T-cells
in the wells release interferon-γ upon antigen contact. Subsequently interferon-γ is bound to an anti-interferon-γ-specific antibody at
the bottom of the wells. After counterstaining with a second soluble enzyme-conjugated anti-interferon-γ-specific monoclonal antibody and a color reaction, spots are visible on the bottom of the
plates at locations where single cells have produced interferon-γ
following antigen contact. Test results are available within 24 h. On
PBMCs the recommended cutoff for a positive response is 5 spot
forming cells (sfc)/250,000 mononuclear cells. There is no accepted cutoff value for extrasanguineous fluids currently.
lower lobes. The TST-induration was 0 mm and AFBs
or MTB nucleic acid was not detected on sputum
and bronchial secretions. However, TIGRAs performed on peripheral blood cells were reactive. A
high frequency of MTB-specific T-cells compatible
with the immunodiagnosis of active pTB was detected among bronchoalveolar lavage cells using ELISPOT. Antituberculous therapy was initiated 18 days
before MTB was discovered on sputum cultures. Detection of Mycobacterium tuberculosis-specific Tcells in the bronchoalveolar lavage using enzymelinked immunospot assay is a promising tool for the
Collaboration: 1Kösters K, Greiffendorf I, Jentsch M
(Klinikum Krefeld, Krefeld, Germany), 2Nau R (Uni-
diagnosis of active pulmonary tuberculosis in im-
versity Hospital of Göttingen, Göttingen, Germany),
3Bossink A, Thijsen S (Diakonessenhuis Utrecht,
bacilli smears.
Utrecht, The Netherlands), 4Hinks T, Lalvani A (Imperial College, London, UK), 5Lange C (Division of Clin-
Collaboration: Strassburg A*, Jafari C, Lotz W*,
ical Infectious Diseases and Medical Clinic Borstel).
and Medical Clinic Borstel).
Rapid diagnosis of pulmonary TB by BAL enzyme-
Effects of sleep on in vitro cytokine production af-
linked immunospot assay in an immunocompro-
ter polyclonal and antigen-specific stimulation
mised host - a case study
Schwartzkopff F, Marienfeld K, Ernst M.
munocompromised patients with negative acid-fast
Lange C* (Division of Clinical Infectious Diseases
Ernst M, Strassburg A*, Jafari C*, Lotz W* , Lange
C*.
Results of recent in vitro experiments concurred with
the notion that sleep supports the immunological
ABSTRACT: Immunocompromised patients with
synapse between APC and T-cell. Blood was taken
acid-fast bacilli (AFB) smear-negative active pul-
at 08:00 h after two nights of regular sleep (baseli-
monary tuberculosis (pTB) often present with non-
ne) and after one night of continuous wakefulness
specific clinical symptoms and findings. T-cell inter-
in 14 healthy subjects. PBMC were differentiated by
feron-c release assays (TIGRA) performed on who-
flow cytometry and stimulated with three antigen-
le blood (using ELISA) or peripheral blood mo-
specific stimuli (purified protein derivate/tuberculin
nonuclear cells (using enzyme-linked immunospot
(PPD), influenza A antigen, toxoplasma gondii anti-
100
Wissenschaftlicher Jahresbericht 2007-2008
Division of Immune Cell-Analytics
Research Reports
gen) and two polyclonal stimuli (phytohaemagglu-
39). Since antigen-specific IFN-γ release from T-cells
tinin (PHA) and anti-CD3) to assess IFN-γ production
is strictly dependent on appropriately functioning
in supernatants 96 hours later. While sleep compa-
APC, we assume that sleep deprivation hampers
red to continuous wakefulness did not influence PB-
the immunological synapse between these cells
MC subsets at this time point, polyclonal stimulati-
while T-cell function per se is rather enhanced.
on with PHA and anti-CD3 was significantly weaker
after regular sleep than after the night of continuous
wakefulness. This effect, however, was not evident
Fig. 39. IFN-γ production in supernatants 96 hours after polyclonal
and antigen-specific stimulation of PBMC at 08:00 h after two
nights of regular sleep or one night of continuous wakefulness.
p-values are indicated for Wilcoxon matched pairs signed-ranks
test of logarithmized data.
in PPD and influenza A antigen stimulated cultures.
Supported in part by: Deutsche Forschungsge-
In contrast, IFN-γ production in toxoplasma gondii
meinschaft (DFG), SFB 654 “Plasticity and Sleep”,
positive subjects was significantly higher after reg-
project C2.
ular sleep than after continuous wakefulness (Fig.
Wissenschaftlicher Jahresbericht 2007-2008
101
Division of Biological Chemistry
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Biological Chemistry
Head
granulation. Moreover, it became evident that pro-
Prof. Dr. Ernst Brandt
cessing dramatically affected the interaction of the
cathelicidin with bacteria and their products.
Staff Scientists and Postdoctoral Fellows
Dr. Florian Schiemann
Graduate and Diploma Students
Oranos Ghulam (since 08/2006)
Jan Schulmistrat (since 06/2006)
Technicians
Christine Engellenner
Gabriele Huß
Research Reports
Human mast cells degrade LL37 by tryptase
Schiemann F, Schulmistrat J, Brandt E.
The cathelicidin LL37 represents a potent antimicrobial and a cell-stimulating agent, most abundantly
Fig. 40. LL37 interacts with human lung mast cells.
Mast cells were incubated alone or in the presence
of increasing concentrations of LL37. After 30 min,
cell-free supernatants were harvested and incubated with a chromogenic substrate for hexosaminidase. Thereafter the reaction was stopped
and liberated free p-nitrophenol measured. The
percentage of hexosaminidase enzymatic activity
released was calculated relative to lysed cells run
in parallel. Shown is the mean ±SD of three independent experiments.
expressed in peripheral organs such as lung and skin
during inflammation. Since mast cells (MC) overtake
In contrast to the intact protein, LL37 fragements did
prominent immunmodulatory roles in these organs,
neither act microbicidal on gram-positive and –neg-
we wondered whether there exists interaction be-
ative bacteria nor did they neutralize the cell-stimu-
tween MC and LL37. Here we show for the first time
latory effect of LPS. Taken together our results sug-
that physiological concentrations of LL37 induce de-
gest that the intruiging two-step process initiated by
granulation in purified human lung MC (Fig. 40). As
LL37, consisting in mast cell acvtivation followed by
an intriguing consequence, LL37 rapidly undergoes
rapid mast cell-mediated degradation of the stimu-
limited cleavage by a released protease. Indeed,
lus in return, can be assigned a dead-end route,
blocking of degranulation by specific signal trans-
where the activating agent is finally removed and
duction inhibitors impaired cleavage of LL-37, demon-
no longer available for autocrine and paracrine ac-
strating that the cathelicidin induces its own pro-
tivation of cells.
cessing. The protease responsible for processing
was identified as tryptase by inhibitor studies and by
Collaboration: Petersen F, Mittelstädt J, Division of
comparison of the LL-37 fragments formed to those
Biochemical Immunology, Lindner B, Division of Im-
generated following incubation with the recombinant
munochemistry, Research Center Borstel.
enzyme. To get an impression of the functional consequences, we examined the resulting fragments for
Supported in part by: Deutsche Forschungsgemein-
typical functions exhibited by the fullsize LL37. In a
schaft, SFB/TR22 „Allergische Immunantworten der
first set of experiments we could clearly show that
Lunge“, Projekt A11.
these fragments fail to activate mast cells for de-
102
Wissenschaftlicher Jahresbericht 2007-2008
Division of Biological Chemistry
Research Reports
The platelet chemokine CXCL4 is an inhibitor of
Collaboration: Petersen F, Division of Biochemical
mast cell tryptase activity
Immunology, Research Center Borstel; Sommerhoff
Schiemann F, Brandt E.
CP, Department of Clinical Chemistry and Clinical
Biochemistry, Ludwig-Maximilians University, Mu-
In a previous study we showed that the platelet-de-
nich, Germany.
rived chemokine CXCL4 represents a strong regulator of mast cell chymase-mediated proteolysis,
Supported in part by: Deutsche Forschungsgemein-
by acting as a direct inhibitor of the enzyme. In or-
schaft, SFB/TR22 „Allergische Immunantworten der
der to elucidate whether CXCL4 would play a more
Lunge“, Projekt A11.
general role in the functional regulation of mast
cell specific enzymes, we investigated its impact
on the second major mast cell protease, i.e. on
Biology of proteinase-activated receptors on hu-
tryptase-mediated proteolysis. In initial experi-
man mast cells
ments we observed that CXCL4, within physiologi-
Ghulam O, Brandt E.
cally relevant concentrations, efficiently protected
the cathelicidin LL37 from degradation by tryptase
Proteinase-activated receptors (PARs) are widely ex-
as well as by degranulating mast cells (refer to an-
pressed in many immune and tissue cells, e.g. in
tecedent report above). This effect was based on
platelets, leukocytes, endothelial and vascular wall
the direct inhibition of tryptase catalytic activity
cells and also in the cells of the gastrointestinal tract.
rather than on protective protein-protein interac-
PARs are seven transmenbran proteins belonging to
tion of CXCL4 with the substrate LL37. This was
the G protein-coupled receptor family. They comprise
shown by corresponding experiments where CX-
four members (PAR-1 to 4) and have various physio-
CL4 likewise inhibited the cleavage of a chro-
logical roles, such as in hemostasis where they me-
mogenic peptide-substrate by tryptase. Raising
diate platelet activation, in tissue repair by promoting
questions as to the underlying mechanism(s), we
angiogenesis, as well as in inflammation by enhanc-
found that the efficiency of CXCL4 in counteracting
ing leukocyte adherence and proinflammatory cy-
the conversion of the chromogenic substrate was
tokine production by cells from a variety of tissues.
highly dependent on the presence of heparin as a
PARs are characterized by an unique mechanism of
co-factor, the inhibitory capacity of CXCL4 being
activation. Proteases, especially serine proteases are
negatively related to the concentration of heparin.
able to cleave the receptors at a specific site, there-
Against the background, that tryptase evolves its
by exposing a tethered ligand domain that binds to
catalytic activity only as a heparin-stabilized
and activates the receptors. Alternatively, some pro-
tetrameric molecular complex, these results strong-
teases may disarm the receptors by cleavage at a dif-
ly suggest that the inhibitory capacity of the cation-
ferent site. Recent research in mice suggests that ac-
ic CXCL4 is based on its high affinity to anionic gly-
tivation of proteinase-activated receptors at the sur-
cosaminogylcans. According to this model, com-
face of mast cells (MC) may play a role during in-
petition of CXCL4 for tryptase-associated heparin
flammation and the immune response. There are
results in the disintegration of the tetrameric mo-
initial hints that also in human MC PARs could be in-
lecular complex and in its concomitant inactiva-
volved in cell activation. We therefore set out to in-
tion. In general, our findings that CXCL4 counter-
vestigate whether human MC responses can be acti-
regulates two major proinflammatory mast cell
vated or modulated via ligands of PARs. In first ex-
specific proteases may indicate a special regula-
periments we observed that PAR-activating peptides
tory role for this chemokine during inflammatory
(PAR-APs: short synthetic peptides, representing the
processes.
proteolytically released tethered ligand sequences)
Wissenschaftlicher Jahresbericht 2007-2008
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Division of Biological Chemistry
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
for PAR-1 and PAR-4 are able to induce degranulation
Bacterial modification of CXCL7
of human primary skin and lung mast cells whereas
Schulmistrat J, Brandt E.
PAR-2-AP does not (Fig. 41). Interestingly, proteases
known to activate these receptors did not induce MC
The platelet-derived chemokine CXCL7 occurs in a
degranulation but nevertheless elicited intracelluar
Ca2+-signals, indicating the presence of these re-
variety of molecular forms that are generated
ceptors on human MC. Concerning human lung MC
crete the longest molecules termed platelet basic
we found expression of mRNA as well as protein for
protein (PBP, 94 aa) and its N-terminally truncated
PAR-1 and PAR-2, the former being exposed at the
derivative connective tissue-activating peptide III
cell membrane, the latter occuring exclusively intra-
(CTAP-III, 85 aa). N-terminal truncation of these pre-
cellularly (Fig. 41). At present we are investigating
cursors by leukocyte and/or mast cell proteases
what MC functions other than degranulation, e.g. cy-
leads to the formation of the active chemokine neu-
tokine and leukotriene production, might be regulat-
trophil-activating peptide 2 (NAP-2, 70aa), which
ed by PARs. We expect that investigating the modu-
causes neutrophil recruitment and activation by in-
lation of mast cell activity via PARs will contribute to
teraction with receptors CXCR-1 and CXCR-2. In ad-
the development of therapies for mast cell-depend-
dition to their function as chemokines, certain CXCL-
ent inflammatory or immune pathologies.
7-variants are also able to act as direct microbici-
through limited proteolysis. Platelets contain and se-
dal agents on bacteria and fungi. Due to these combined characteristics CXCL-7 plays an important
role as a first-line mediator of innate immunity in tissue injury and microbial invasion. As bacteria exhibit a broad spectrum of proteases, and moreover,
as recent studies have shown that these proteases
principally have the capability to cleave CXC
chemokines (e.g. CXCL-8), we were interested
whether bacteria may also proteolytically modify
CXCL7 and thereby generate cleavage products
with altered functional characteristics. In our present study we therefore investigated different strains
of gram-positive and gram-negative bacteria with
respect to their ability to modify CXCL7. For this purpose bacteria and PBP were coincubated for increasing periods of time and cell-free supernatants
were analysed by electrophoresis and Western blotting for the formation of cleavage products. As it
turned out several of these bacterial strains had a
high capacity to generate immuno-reactive fragments of the chemokine while others were comFig. 41. Surface expression and funcionality of PARs on human lung
mast cells. Either intact or permeabilized human lung mast cells
were stained with antibodies specific for PAR-1 or PAR-2 and recepor expression was evaluated by FACS analysis (A). Human lung
mast cells were incubated alone or in the presence of increasing
dosages of receptor-activating peptides for PAR-1 and PAR-2, and
degranulation of cells was assessed by measurement of hexosaminidase release (B).
104
pletely inactive. Purification of these cleavage products by immunoaffinity-chromatography and further
analysis by mass spectrometry revealed the formation of several molecular variants smaller in size
than CTAP-III. Some of these exhibited drastically altered bactericidal activity. Altogether our results
Wissenschaftlicher Jahresbericht 2007-2008
Division of Biological Chemistry
Research Reports
show that there exists a bidirectional relationship
between bacteria and antimicrobial agents in that
bactericidal molecules may not only impact bacteria, but the latter may also modify functions of antimicrobials by proteolytic processing.
Collaboration: Lindner B, Division of Immunochemistry, Research Center Borstel.
Wissenschaftlicher Jahresbericht 2007-2008
105
Division of Tumor Biology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Tumor Biology
Head
physiological function remains unknown. In a first at-
Prof. Dr. Johannes Gerdes (until April 2007)
tempt to elucidate the function of these proteins in
after 20,5 years at the FZB, I took advantage of ear-
vivo, we disrupted the murine Cbx1 gene, which en-
ly retirement regulations on 01. 05. 2008. Thus, for
codes the HP1β isotype, and show that the Cbx1 -
the very last time, my legendary farewell address:
null mutation leads to perinatal lethality. The new-
Vielen Dank!
born mice succumbed to acute respiratory failure,
und ... — Tschüss!
whose likely cause is the defective development of
–/–
neuromuscular junctions within the endplate of the
Dr. Prim B. Singh (since May 2008)
diaphragm. We also observe aberrant cerebral cortex development in Cbx1
–/–
mutant brains, which
Principle Investigators
have reduced proliferation of neuronal precursors,
Dr. Thomas Scholzen (until April 2008)
widespread cell death, and edema. In vitro cultures
Dr. Prim B. Singh (until April 2008)
of neurospheres from Cbx1
–/–
mutant brains reveal
a dramatic genomic instability. Our results demonStaff scientists and Postdoctoral Fellows
strate that HP1 proteins are not functionally redun-
Dr. Jeremy P. Brown (since September 2008)
dant and that they are likely to regulate lineage-
Dr. Jörn Bullwinkel
specific changes in heterochromatin organization.
Dr. Ramtin Rahmanzadeh (until February 2007)
Graduate and diploma students
Mustafa Billur
Hélène D. Kamdem (until March 2008)
Renja Romey (until June 2007)
Technicians
Bettina Baron-Lühr
Anja Lüdemann
Guest scientists and Trainees
Dr. Christian Bader (until May 2007)
David Gräser (until September 2007)
Kalicharan Patra (September 2008)
Vladimir Shteyn (June 2007 to January 2008)
Fig.42. A Giemsa-stained, mitotic chromosome spread from a
HP1β -/- animal possessing diplochromosomes (eg. at 2 o’clock).
Red: Telomeric probe.
Research Reports
β is required for development of the cerebral
HP1β
neocortex and neuromuscular junctions
Collaboration: Aucott R (Div. of Genetics and Ge-
Bullwinkel J, Billur M, Brown JP, Singh PB.
nomics, Roslin Institute, University of Edinburgh,
Scotland), Yu Y, Shi W, Fundele R (Dep. of Devel-
HP1 proteins are thought to be modulators of chro-
opment and Genetics, Uppsala University, Swe-
matin organization in all mammals, yet their exact
den), Menzel U, Kioussis D (Div. of Molecular Im-
106
Wissenschaftlicher Jahresbericht 2007-2008
Division of Tumor Biology
Research Reports
munology, National Institute of Medical Research,
London, England) , Wang G (School of Biological
Sciences, University of Liverpool, England), Reisert
I (Dep. of Anatomy and Cell Biology, University of
Ulm), Weimer J (Clinic of Obstetrics and Gynecology, Lab. of Oncology, UKSH, Kiel) Pandita RK,
Sharma GG, Pandita TK (Dep. of Radiation Oncology, Washington University School of Medicine, St.
Louis, USA).
The epigenotype of CD14 and CD209 during differentiation of monocytes to dendritic cells
Bullwinkel J, Gerdes J, Singh PB.
Fig. 43. Metaphase chromosomes of mouse embryonic fibroblasts
deficient for HP1α. DNA (blue), kinetochore protein cohesin (red)
and centromere protein CENP (green).
In the course of differentiation processes cells
change their gene expression profiles drastically.
Collaboration: Brümmendorf T, Braig M (Div. of
Using a model system, where monocytes are treat-
Telomer-/ Stem Cell-Biology, University Hospital Ep-
ed with GM-CSF and IL-4 to induce differentiation to
pendorf, Hamburg).
dendritic cells, we have studied changes in the
epigenotype of CD14. CD14 undergoes repression
Supported in part by: DFG grant SI 1209/2-1.
when monocytes differentiate into dendritic cells.
We found that active chromatin marks (histone
acetylation) are dramatically reduced upon differ-
Functional analysis of the Ki-67 protein
entiation whilst other epigenetic modifications, such
Romey R, Kamdem H, Gerdes J, Scholzen T.
as DNA methylation and repressive chromatin
marks, remain unchanged mainly unchanged.
Antibodies against the nuclear Ki-67 protein (pKi-67)
are widely used as a prognostic tool to determine
the growth fraction of tumor cells in cancer diag-
β
Regulation of genomic instability by HP1β
nostics. Previous studies performed in our labora-
Billur M, Singh PB.
tory suggested a role of pKi-67 in ribosomal RNA
(rRNA) synthesis. In this context we could show that
We are investigating the genomic instability ob-
the upstream binding factor (UBF), which is a key
served in the HP1β mutant mice using a variety of
regulator of rRNA synthesis, colocalized with a frac-
approaches including a study of the telomeres
tion of the nuclear pKi-67. To further analyze this as-
(measurement of telomere length and determina-
sociation we performed immunoprecipitations,
tion of protein constituents), the role of cohesins
clearly demonstrating an interaction of pKi-67 with
(SMC proteins, Sgo1 and Scc1) and the effect of the
both isoforms of UBF. Pulldown experiments with
mutations on senescence-associated heterochro-
bacterially expressed UBF and pKi-67 fragments fur-
matin foci (SAHF). To date, we have shown that
ther revealed that this interaction is direct and does
HP1β is not required to maintain telomere length
not depend on additional adapter proteins. With re-
and constitution. Cells that are null for HP1β escape
spect to pKi-67 the binding is mediated by the pro-
cellular senescence and our current work focusses
tein region encoded by the exons 8-10. To gain fur-
on this aspect of HP1β function.
ther insight into the physiological role of pKi-67, we
Wissenschaftlicher Jahresbericht 2007-2008
107
Division of Tumor Biology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
utilized RNA interference to knock down protein ex-
Kamdem, Hélène D.
pression. Small interfering RNAs (siRNAs) directed
Beitrag zum Verständnis der zellulären Funktion des
against the pKi-67 mRNA led to a profound reduc-
Ki-67-Proteins.
tion in cellular pKi-67 content within 48 hours while
Hochschule für Angewandte Wissenschaften
control RNAs had no effect. Interestingly, a small
Hamburg, 2008
fraction of pKi-67 appeared to be quite stable and
was present even after extended times of siRNA application. This fraction was located at the fibrillar
centers of the nucleoli during interphase and at the
corresponding nucleolar organizing regions during
mitosis. Although cells treated with siRNAs tended
to accumulate in the G0 phase, a substantial fraction was nevertheless able to enter S-phase, as was
evident by the incorporation of labeled deoxynucleotides. Moreover, we frequently observed cells in
different mitotic phases showing a strong reduction
in pKi-67 expression. These results argue against an
essential role of the protein for the passage through
S-phase or mitosis. Analysis of the rRNA synthesis
did not show an obvious difference in transcription
or processing of these ribosomal components. However, it has to be considered that the stable pKi-67
fraction resides at regions associated with rRNA
synthesis and may be sufficient to sustain pKi-67
function at these sites.
Theses
Dissertations
Rahmanzadeh, Ramtin
Untersuchungen zur Inaktivierung von Zellen und
Proteinen mit Hilfe optischer Adsorber.
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2007
Diploma
Romey, Renja
Untersuchungen zur Inhibition der Ki-67 Proteinexpression mittels RNA-Interferenz.
Mathematisch-Naturwissenschaftliche Fakultät
Christian-Albrechts-Universität zu Kiel, 2007
108
Wissenschaftlicher Jahresbericht 2007-2008
Division of Veterinary Infectiology and Immunology
Research Reports
Division of Veterinary Infectiology
and Immunology
Head
Research Reports
Prof. Dr. Jabbar Ahmed
Identification, molecular characterization and
subcellular localization of a Theileria annulata
Principle Investigators
parasite protein secreted into the host cell
Prof. Dr. Ulrike Seitzer
Schneider I, Haller D, Kullmann B, Beyer D, Ahmed JS, Seitzer U.
Graduate and Diploma Students
Jassim Abdo (since 27.07.08)
Awadia Ali
Silke Gerber
Diaeldin Hassan
Zhijie Liu
Heike Müller
Stefanie Renneker
Technicians
Doreen Beyer
Jessica Dobschanski
Birgit Kullmann
Guests and Trainees
Mohammed Abdigoudarzi, Razi Vaccine and Serum
Research Institute, Karaj, Tehran, Iran, 25.10.25.11.2007
Nuran Aysul, Veterinary Faculty Adnan Menderes
University, Aydin, Turkey, 04.05.-31.5.2008
Mohammed A. Bakheit, National Research Center
for Protozoan Diseases, Obihiro University for Agriculture and Veterinary Medicine, Japan 31.05.30.06.2008
Mehmet-Ali Türkyilmaz, Pendik Veterinary Research
and Control Institute, Istanbul, Turkey (16.12.31.12.2007, 1.01.-20.06.2008)
Tian Zhancheng, Lanzhou Veterinary Research Institute, Lanzhou, Gansu, China 5.3.–25.4.2008
Fig. 44. Analysis of subcellular localization of the Theileria annulata TaSE protein in infected bovine cells.
a) The signal for TaSE detection (red) shows a parasite associated staining (green: parasite membrane) but also away from the
parasite in small globular domains. TaSE is localized within the
parasite, in the parasite membrane (yellow signals) and outside
the parasite membrane in the host cell cytoplasm.
b) Co-localization of TaSE with the host cell tubulin network: Overlay image of tubulin (green) and TaSE (red) detection demonstrating a close association of TaSE with the host tubulin network
as well as points of co-localization (yellow signals).
c) Close up and overlay image of the tubulin (green) and TaSE-specific signals (red) from a parasite-free area of a cell. A very close
association TaSE to the tubulin network can be seen.
Size bars: a) 10 µm b) 10 µm c) 1 µm
Wissenschaftlicher Jahresbericht 2007-2008
109
Division of Veterinary Infectiology and Immunology
Research Reports
Intracellular leucoproliferative Theileria are unique
cations, management systems and age could be
as eukaryotic organisms that transform the immune
held as risk factors for T. annulata infection in North-
cells of their ruminant host. Theileria utilize the un-
ern Sudan, while for T. parva locations and seasons
controlled proliferation for rapid multiplication and
could be held as risk factors in Southern Sudan. The
distribution into host daughter cells. The equal dis-
results of this study will assist in the development of
tribution of the schizont into the daughter cells is
more effective control strategies for smallholder
thought to be accomplished by a tight association
dairy farms in the country.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
with the host cell mitotic apparatus. In this study, we
describe a highly conserved novel 37 kD Theileria
Collaboration: El Hussein AM, Central Veterinary Re-
annulata protein (TaSE). TaSE was found to be lo-
search Laboratories, Alamarat, Khartoum, Sudan;
calized inside the parasite, the parasite membrane
Kyule MN, Zessin KH, Faculty of Veterinary Medi-
and within the host cell cytoplasm. Moreover, it co-
cine, Free University of Berlin, Königsweg 67, D-
localized at distinct points with host cell micro-
14163 Berlin, Germany.
tubules, which was especially apparent during mitosis, where co-localization was found with the cen-
Supported in part by: International Foundation for
tromere, the mitotic spindle and the midbody. As-
Science, Stockholm, Sweden; Organisation of Is-
sociation of TaSE with the host cell tubulin network
lamic Conference Standing Committee on Scientific
was corroborated by coimmunoprecipitation and
and Technological Cooperation (COMSTECH), Is-
transient transfection experiments. This is the first
lamabad, Pakistan; Coordinated Action “Integrated
description of a theilerial protein co-localizing and
Consortium on Ticks and Tick-borne Diseases”
potentially interacting with a host cell protein. The
(ICTTD-3) Asian Component, Project number 510561.
distribution of TaSE during mitosis makes it a protein
to consider as playing a potential role for parasite
At least two genetically distinct large Babesia
distribution into daughter host cells.
species infective to sheep and goats in China
Schnittger L, Ahmed JS.
Supported in part by: ICTTD Concerted Action Project on Integrated Control of Ticks and Tick-borne
A fatal disease of sheep and goats in the northern
Diseases, contract no. IC18-CT95-0009).
part of China has been reported to be due to
Babesia ovis. However, some characteristics of the
causative agent in recent reports are not in accorDetermination of potential risk factors associated
dance with the original attributes ascribed to this
with Theileria annulata and Theileria parva in-
parasite. Therefore, the 18S small subunit ribosomal
fections of cattle in the Sudan
RNA (18S rRNA) genes of a number of Babesia iso-
Salih DA, Ahmed JS, Seitzer U.
lates in China were sequenced and compared with
that of other Babesia and Theileria species in an at-
A multi-variate logistic regression analysis was per-
tempt to clarify their taxonomic position. In the pres-
formed on two sets of data on the prevalence of
ent study, seven Babesia isolates were collected
Theileria annulata in Northern Sudan and Theileria
from distinct areas of northern China, and the 18S
parva in Southern Sudan, to determine the potential
rRNA genes were amplified and sequenced. The
risk factors that might affect the distribution of the
phylogenetic trees were inferred based on 18S
infections in those regions. The logistic regression
rRNA gene sequences of the Chinese ovine Babesia
model was fit with the tested risk factors for each
isolates and some of ovine Babesia and Theileria
disease, separately. The results indicated that lo-
species available in GenBank. In the phylogenetic
110
Wissenschaftlicher Jahresbericht 2007-2008
Division of Veterinary Infectiology and Immunology
Research Reports
tree, Babesia sp. isolates from Madang, Tianzhu,
Theileria buffeli (0.5 %), Theileria annulata (0.2 %),
Lintan, Ningxian, Hebei and Liaoning all grouped
Babesia bovis (1.7 %), and Babesia bigemina (0.3 %).
with B. motasi with 88.2-99.9 % identity, while
Mixed infections were detected in 406 samples
Babesia sp. Xinjiang grouped in a separate clade
(67.7 %) accounting for 17 different combinations.
between B. ovis and B. crassa with 79.7-81.2 % iden-
High infection of Theileria parva was reported
tity. The results indicated that there are at least two
among young calves compared to older cattle. The
distinct Babesia species groups-B. motasi and
highest prevalence of Theileria parva was reported
Babesia sp. Xinjiang, the latter was distinctly differ-
in the rainy season (October). The implications of
ent from other ovine Babesia isolates from China
these results on the epidemiology of tick-borne dis-
with less than 86.6% identity.
eases are discussed with emphasis on East Coast
fever.
Collaboration: Liu AH, Yin H, Guan GQ, Liu ZJ, Ma
ML, Dang ZS, Liu JL, Ren QY, Bai Q, Luo JX, Lanzhou
Collaboration: El Hussein AM, Central Veterinary Re-
Veterinary Research Institute, Lanzhou, Gansu, China.
search Laboratories, Alamarat, Khartoum, Sudan.
Supported in part by: National Natural Sciences
Supported in part by: International Foundation for
Foundation of China (No. 30571397) and the Natu-
Science, Stockholm, Sweden; Organisation of Is-
ral resource platform project (No. 2005DKA21104).
lamic Conference Standing Committee on Scientific
European Commission’s Inco-Dev Coordinated Ac-
and Technological Cooperation (COMSTECH), Is-
tion “Integrated Consortium on Ticks and Tick-borne
lamabad, Pakistan; Coordinated Action “Integrated
Diseases” (ICTTD-3) Asian Component, Project num-
Consortium on Ticks and Tick-borne Diseases”
ber 510561.
(ICTTD-3) Asian Component, Project number 510561.
Epidemiological studies on tick-borne diseases of
Status of tick distribution in Bangladesh, India
cattle in Central Equatoria State, Southern Sudan
and Pakistan
Salih DA, Seitzer U, Ahmed JS.
Seitzer U, Ahmed JS.
A herd-based study was carried out in Central Equa-
On a global basis, ticks transmit a greater variety
toria State, Southern Sudan, to study epidemiologi-
of pathogenic microorganisms, protozoa, rickettsi-
cal aspects of tick-borne diseases. Six herds of cat-
ae, spirochaets, and viruses than any other arthro-
tle situated in three different locations were select-
pods and are among the most important vectors of
ed and investigated every 3 months during the year
diseases affecting livestock, humans, and compan-
2005. Blood smears for Giemsa staining and blood
ion animals. Ticks and tick-borne diseases (TTBDs)
spots on filter paper for deoxyribonucleic acid ex-
affect 80% of the world cattle population and are
traction were collected from 600 apparently healthy
widely distributed throughout the world, particular-
indigenous cattle. A total of 69 (11.5 %) samples
ly in tropical and subtropical countries including In-
showed the presence of piroplasms in Giemsa-
dia, Pakistan, and Bangladesh. Ticks and tick-trans-
stained blood smears, and polymerase chain re-
mitted infections have coevolved with various wild
action increased the detection limit to 297 (49.5 %).
animal hosts, which constitute the reservoir hosts for
Using reverse line blot, it was possible to detect and
ticks and tick-borne pathogens of livestock, pets,
differentiate eight different piroplasms namely,
and humans. In this region, the livestock sector is
Theileria parva (71.2 %), Theileria mutans (73 %),
suffering from a number of disease problems
Theileria velifera (45.3 %), Theileria taurotragi (2.7 %),
caused by bacteria, viruses, fungi, and parasites.
Wissenschaftlicher Jahresbericht 2007-2008
111
Division of Veterinary Infectiology and Immunology
Research Reports
Among the parasitological problems, the damage
ue of 68.2% in May, declining in October and reach-
caused by TTBDs is considered very high, and the
ing the lowest value of 36.4% in January. The data
control of TTBDs has been given priority.
indicate that seroprevalence of anti-Theileria anti-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
bodies correlate to the known activities of its transCollaboration: Ghosh S, Bansal GC, Gupta SC, Ray
mitting vector and also imply that chemical therapy
D, Division of Parasitology, Indian Veterinary Re-
of animals should be performed during May to June
search Institute, Izatnagar 243122, India; Khan MQ,
in these Theileriosis-epidemic regions.
Irshad H, Animal Health Laboratories, Animal Sciences Institute, National Agricultural Research Cen-
Collaboration: Guo S, Mu YMa D, Yang S, Ge G,
tre, Islamabad, Pakistan; Shahiduzzaman M, De-
Fang B, Ga D, Animal Husbandry and Veterinary
partment of Parasitology, Faculty of Veterinary Sci-
Medicine Institute of Gannan Tibet Autonomous Re-
ence, Bangladesh Agricultural University, My-
gion, Hezuo, Gansu 747000, PR China; Liu Z,Ma M,
mensingh-2202, Bangladesh.
Luo J, Yin H, Lanzhou Veterinary Research Institute,
Lanzhou, Gansu, China.
Supported in part by: Coordinated Action “Integrated Consortium on Ticks and Tick-borne Dis-
Supported in part by: European Commission’s Inco-
eases” (ICTTD-3) Asian Component, Project number
Dev Research Program “Molecular and immuno-
510561.
logical characterization of merozoite antigens and
their encoding genes of a Theileria species highly
pathogenic for small ruminants in China: applica-
Serological investigation of ovine theileriosis by
tion for the development of diagnostics and vac-
ELISA in Gannan Tibet Region of Gansu Province
cine.”, Project number: ICA-1999-30151; Coordinated
in China
Action “Integrated Consortium on Ticks and Tick-
Seitzer U, Ahmed JS.
borne Diseases” (ICTTD-3) Asian Component, Project number 510561.
The Gannan Tibet Autonomous Region is the
biggest and most important stock-raising region of
Gansu Province, People’s Republic of China. Many
A survey of ticks (Acari:Ixodidae) on cattle, sheep
sheep and goats from this region suffer from Thei-
and goats in the Dohuk Governorate, Iraq
leria challenge each year, resulting in inestimable
Seitzer U, Ahmed JS.
economical losses. A first large-scale serological investigation by enzyme-linked immunosorbent assay
A survey on hard ticks affecting cattle, sheep, and
of ovine theileriosis in this region was performed by
goats was done in Dohuk Governorate from March
testing a total of 1,165 sera samples collected from
2005 until February 2006 in three areas (Dohuk sur-
seven districts of three counties. The results showed
rounding area, Barwary Balla near Turkish border,
that seroprevalence of reactive antibodies varied
and Eqre). The species collected from cattle were
from 27.8 to 83.3%, whereby the average was 70.1%
Hyalomma anatolicum anatolicum and Hyalomma
in the disease-endemic areas. Another 44 sera sam-
anatolicum marginatum, while the species collect-
ples collected every month from July 2004 to June
ed from sheep and goats were Rhipicephalus bur-
2005 from a stable flock of sheep in Biandu Town of
sa, Rhipicephalus turanicus, Haemaphysalis parva,
Lintan County were analysed to evaluate the sea-
and Hyalomma spp. The occurrences of the ticks in
sonal kinetics of anti-Theileria antibodies. The re-
the three areas were different; in Dohuk surround-
sults indicated that anti-Theileria antibody sero-
ing area they occured at the beginning of March
prevalence rose from March, reaching a peak val-
and disappeared in the middle of April, while in the
112
Wissenschaftlicher Jahresbericht 2007-2008
Division of Veterinary Infectiology and Immunology
Research Reports
Barwary area the ticks occured at the end of March
and distribution of tick differentiation protocols and
and disappeared at the end of May. In Eqre, the
diagnostic tools.
peaks of infestation occurred after harvesting of cereals in the middle of June until end of July.
Collaboration: Alp H, Aksin M, Pendik Veterinary
Control and Research Institute, Pendik, Istanbul,
Collaboration: Omer LT, Dohuk Research Center,
Turkey.
College of Veterinary Medicine, University of Dohuk,
Dohuk, Iraq; Kadir MA, College of Medicine, Uni-
Supported in part by: Coordinated Action “Inte-
versity of Kirkuk, Iraq.
grated Consortium on Ticks and Tick-borne Di
Supported in part by: Coordinated Action “Integrated Consortium on Ticks and Tick-borne Dis-
Development and evaluation of a loop-mediated
eases” (ICTTD-3) Asian Component, Project number
isothermal amplification method for diagnosis of
510561.
tropical theileriosis
Salih DA, Liu Z, Bakheit MA, Ali AM, Seitzer U, Ahmed JS.
Current status of ticks in Asia.
Ahmed JS, Seitzer U.
A loop-mediated isothermal amplification (LAMP)
assay was developed and evaluated for diagno-
Ticks and tick-borne diseases (TTBD) pose a major
sis of tropical theileriosis. A set of six primers was
constraint for the development and improvement of
designed based on the unique gene of Theileria
the livestock industry. They cause economical loss-
annulata (Theileria annulata strain Ankara hypo-
es by decreasing milk production, effecting weight
thetical protein (GeneDB TA04795). The protocol
loss, and increasing risk for bacterial, viral, and fun-
for the reaction was setup and the specificity and
gal infections. It has been reported that 80% of
sensitivity of the assay were established. The
1,200 million cattle are at risk for TTBDs causing a
specificity experiment showed that LAMP primers
global annual loss of US$7,000 million. Ticks are cur-
amplified T. annulata DNA successfully, while no
rently considered to be second only to mosquitoes
amplification was seen for Theileria parva, Theile-
as vectors of human infectious diseases in the
ria mutans, Theileria sergenti, Theileria sinensis,
world. There are more than 850 species recognized
Babesia bovis as well as bovine genomic DNA
with approximately 180 in the family Argasidae (soft
and water control. When the sensitivity of LAMP as-
ticks) and the others in the family Ixodidae (hard
say was compared with that of conventional PCR
ticks). In Asia, the economical losses due to TTBDs
a 10-fold higher sensitivity was found, with a de-
is great; however, the knowledge on Asian ticks is
tection limit of 10 pg/microl of genomic DNA iso-
scarce and needs intensive studies regarding their
lated from a T. annulata-infected cell line. The
geographical distribution, ecology, and diseases
LAMP product was confirmed by restriction diges-
transmission. To close this gap, the Asian compo-
tion and staining with SYBR Green I. Furthermore,
nent of the Integrated Consortium on Ticks and Tick-
the LAMP assay was applied for the diagnosis of
Borne Diseases (ICTTD-3) organized a meeting held
T. annulata in field samples and compared with re-
from 26th-28th April 2006 in Pendik, Istanbul, Turkey
verse line blot (RLB), demonstrating that results of
on the characterization of Asian ticks. Besides the
the LAMP assay corresponded to those of RLB.
knowledge dissemination, this meeting resulted in
These results indicate that the LAMP assay is rap-
a number of achievements such as the establish-
id and simple to run, cost-effective, sensitive and
ment of working groups for epidemiological studies
specific and has potential usefulness for applica-
Wissenschaftlicher Jahresbericht 2007-2008
113
Division of Veterinary Infectiology and Immunology
Research Reports
tion in epidemiological studies on T. annulata in-
with a calculated sensitivity and specificity of 77.4 %
fection of cattle.
and 100%, respectively. Thus the test proved its suit-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
ability for the diagnosis of tropical theileriosis and
Collaboration: El Hussein AM, Central Veterinary Re-
has application for use in serological surveys to
search Laboratories, Alamarat, Khartoum, Sudan;
monitor the prevalence of the disease or identify
Unger H, Viljoen G, Animal Production and Health Sec-
carrier animals with high specificity.
tion, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy
Agency, Vienna International Centre,
Wagramer Strasse 5, P.O. Box 100, A1400 Vienna, Austria
Supported in part by: International
Atomic
Energy
Agency
(IAEA)
through Technical Co-operation
project no. SUD/05/029 entitled
“Characterization and quality assured production of an attenuated Theileria annulata vaccine”. IAEA fellowship (SUD/07020).; European Commission’s Inco-Dev Specific Support Action “ Evaluation and improvement of integrated
Fig. 45. Specificity of cELISA established with rTaSP antigen and monoclonal antibody 1C7. Only the positive control and different sera
from T. annulata infected animals are positive, all other tested sera
from animals with different infections, including T. parva, were negative. Dotted line: established threshold value of the cELISA.
livestock disease control measures through distribution of molecular diagnostic tools, evaluation of
Collaboration: Bakheit MA, National Research Cen-
disease situation, training and capacity building in
ter for Protozoan Diseases (NRCPD), Obihiro Uni-
Asia” (INCOME), Project number PL 515915.
versity for Agriculture and Veterinary Medicine, Inada-cho, Obihiro, 080-8555, Hokkaido, Japan; Geysen
D, Department of Animal Health, Institute of Tropical
Development of a competitive ELISA for detection
Medicine, Nationalestraat 155, 2000 Antwerp, Bel-
of Theileria annulata infection
gium; Shiels B, Tait A, Parasitology Group, Division
Renneker S, Kullmann B, Gerber S, Dobschanski
of Infection and Immunity, Institute of Comparative
J, Ahmed JS, Seitzer U.
Medicine, Glasgow University Veterinary School,
Bearsden Road, Glasgow G61 1QH, UK.
In previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant
Supported in part by: European Commission’s Inco-
antigen and successfully used to develop and vali-
Dev Specific Support Action “Evaluation and im-
date a recombinant-protein-based ELISA for the de-
provement of integrated livestock disease control
tection of circulating antibodies in serum of T. an-
measures through distribution of molecular diag-
nulata-infected animals. In this study, the same anti-
nostic tools, evaluation of disease situation, training
gen was used to develop a competitive ELISA
and capacity building in Asia“ (INCOME), Project
(cELISA) using a monoclonal antibody that was
number PL 515915.
found to bind to TaSP. The cELISA accurately differentiated T. annulata-infected from uninfected animals and demonstrated a satisfactory performance
114
Wissenschaftlicher Jahresbericht 2007-2008
Division of Veterinary Infectiology and Immunology
Research Reports
Evaluation of Theileria annulata recombinant im-
nostic tools, evaluation of disease situation, training
munodominant proteins for the development of
and capacity building in Asia“ (INCOME), Project
ELISA
number PL 515915.
Seitzer U, Beyer D, Kullmann B, Ahmed JS.
A number of Theileria annulata genes have been
Improved diagnosis for nine viral diseases con-
cloned, sequenced and expressed, including TaSP,
sidered as notifiable by the world organization
TaD, TaSE and TamtHSP70. Several recent publica-
for animal health
tions document the suitability of the recombinant
Ahmed JS.
TaSP protein for use in the diagnosis of tropical theileriosis. To investigate whether TaD, TaSE or TamtH-
Nine viral diseases included in the World Organi-
SP70 elicit a humoural immune response in the T.
zation for Animal Health list of notifiable diseases
annulata-infected host and to assess the potential
(former list A) were chosen for their contagiousness
of these proteins for development of diagnostics, a
and high capacity of spreading to improve their di-
total of 156 field sera from Sudan and 49 negative
agnosis using new and emerging technologies. All
sera from Germany were investigated in ELISA for
the selected diseases—foot-and-mouth disease,
the presence of specific antibodies against these
swine vesicular disease, vesicular stomatitis, classi-
recombinant proteins in comparison to TaSP. Anti-
cal swine fever, African swine fever, bluetongue,
bodies against TaD and TaSE were found to be
African horse sickness, Newcastle disease and
present, whereas no antibody response could be
highly pathogenic avian influenza—are considered
detected against the recombinant TamtHSP70.
as transboundary diseases, which detection causes
Highest titres were found to be present against the
the prohibition of livestock exportation, and, thus, it
TaSP protein, with antibody titres against TaD and
leads to high economical losses. The applied di-
TaSE being in general somewhat lower. Correlation
agnostic techniques can fall into two categories: (i)
analysis showed a significant correlation of TaSP
nucleic-acid detection, including padlock probes,
and TaSE and of TaSE and TaD antibody titres, how-
real-time PCR with TaqMan, minor groove binding
ever not between TaSP and TaD. In conclusion, the
probes and fluorescence energy transfer reaction
infected bovine host was shown to produce anti-
probes,
bodies against three of the four recombinant T. an-
Cleavase/Invader assay or the loop-mediated am-
isothermal
amplification
like
the
nulata proteins tested, all three having been de-
plification technology and the development of rap-
scribed or predicted to be parasite membrane pro-
id kits for ‘mobile’ PCR and (ii) antigen-antibody de-
teins. The outstanding performance of the TaSP pro-
tection systems like simplified and more sensitive
tein for detection of T. annulata infection in indirect
ELISA tests. Besides, internal controls have been im-
ELISA was confirmed.
proved for nucleic acid-detecting methods by using
an RNA plant virus—Cowpea Mosaic Virus—to en-
Collaboration: Bakheit MA, National Research Cen-
sure the stability of the RNA used as a positive con-
ter for Protozoan Diseases (NRCPD), Obihiro Uni-
trol in diagnostic real-time RT-PCR assays. The de-
versity for Agriculture and Veterinary Medicine, In-
velopment of these diagnosis techniques has re-
ada-cho, Obihiro, 080-8555, Hokkaido, Japan.
quired the joint efforts of a European consortium in
which nine diagnostic laboratories and an SME who
Supported in part by: European Commission’s Inco-
have collaborated since 2004 within the European
Dev Specific Support Action “Evaluation and im-
Union-funded Lab-on-site project.
provement of integrated livestock disease control
measures through distribution of molecular diag-
Collaboration: Rodriguez-Sanchez B, Sanchez-Viz-
Wissenschaftlicher Jahresbericht 2007-2008
115
Division of Veterinary Infectiology and Immunology
Research Reports
caino JM, Animal Health Department, Veterinary Fac-
tion was confirmed through EcoRI restriction enzyme
ulty, Universidad Complutense de Madrid, Avda
digestion analysis and sequencing. The assay was
Puerta de Hierro s/n, 28040 Madrid, Spain; Uttenthal
proven sensitive since specific amplification was ob-
A, Rasmussen TB, Department of Virology, National
tained from 0.1 pg DNA of T. luwenshuni or T. uilen-
Veterinary Institute, Technical University of Denmark,
bergi. The LAMP assay was evaluated by testing 86
Lindholm,
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Denmark;
field samples in comparison to the reverse line blot
Hakhverdyan M, Belak S, Department of Virology,
method, showing a sensitivity and specificity of 66.0%
National Veterinary Institute, Ulls väg 2B, SE-751 89
and 97.4%, respectively. These results indicate that
Uppsala, Sweden; King DP, Ferris NP, Ebert K, Reid
the LAMP assay is rapid and simple to run, cost ef-
SM, Department of Exotic Disease Control, Ash Road,
fective, sensitive, and specific and has potential use-
Pirbright, Surrey, GU24 0NF, UK; Kiss I, Veterinary In-
fulness for application in diagnostics of and epi-
stitute of Debrecen, 2, Pf. 51, H-4002 Debrecen, Hun-
demiological studies on T. luwenshuni and T. uilen-
gary; Brocchi E, Cordioli P, Department of Research,
bergi infection of small ruminants.
DK-4771
Kalvehave,
Istituto Zooprofilattico Sperimentale della Lombardia
e dell’Emilia Romagna, Via Bianchi 7/9, 25124 Bres-
Collaboration: Bakheit MA, National Research Cen-
cia, Italy; Hjerner B, McMenamy M, Department of
ter for Protozoan Diseases (NRCPD), Obihiro Uni-
Veterinary Science, The Queen’s University of Belfast,
versity for Agriculture and Veterinary Medicine, In-
Stoney Road BT4 3SD Belfast, UKMcKillen J, Veteri-
adacho, Obihiro, 080-8555, Hokkaido, Japan; Luo J,
nary Sciences Division, Agri–food and Biosciences In-
Yin H, Hou J, Lanzhou Veterinary Research Institute,
stitute, Stormont, Belfast, BT4 3SD, UK.
Lanzhou, Gansu, China.
Supported in part by: Lab-on-site Project of the
Supported in part by: “863” Project (2006
European Commission, project number: SSPECT-
AA10A207), Specific Fund for Sino-Europe Coopera-
2004-513645.
tion, MOST, and EU-funded Coordinated Action “Integrated Consortium on Ticks and Tick-borne Diseases” (ICTTD-3), Project number 510561, National
Development of loop-mediated isothermal am-
Natural Sciences Foundation of China ( 30571397),
plification (LAMP) assay for rapid diagnosis of
International Cooperation Fund of NSFC, Beijing,
ovine theileriosis in China
China. ZL is the recipient of a DAAD scholarship.
Liu Z, Salih DA, Ahmed JS, Seitzer U.
Loop-mediated isothermal amplification (LAMP) is a
Existence of splicing products in homologues of
novel nucleic acid detection method in which the tar-
Theileria lestoquardi clone-5 gene in T. annulata
get deoxyribonucleic acid (DNA) can be efficiently
and T. parva
amplified with high specificity and sensitivity under
Kullmann B, Ahmed JS, Seitzer U.
isothermal conditions using a set of either four or six
specific primers. In this study, we have identified a
Clone 5 has been described as an immunogenic pro-
conserved sequence for Theileria luwenshuni (UTR-
tein and was used to establish an ELISA for malignant
lu8) and for T. uilenbergi (UTRu6) suitable for de-
theileriosis. Molecular characterization of the gene
signing a set of six primers for the simultaneous de-
product revealed alternative splicing at the single in-
tection by LAMP of these pathogens causing theile-
tron resulting in two mRNA transcripts, translating in-
riosis in sheep and goats in China. LAMP was per-
to a long and a short protein form.2 Homologues of
formed at 63 degrees C, and the amplified DNA was
clone 5 exist in Theileria annulata and T. parva ac-
detectable within 15 min. The specificity of the reac-
cording to the available annotated GenBank se-
116
Wissenschaftlicher Jahresbericht 2007-2008
Division of Veterinary Infectiology and Immunology
Research Reports
quences, showing however only the long protein
“863” Project (2006AA10A207), Specific Fund for
forms in these parasites (GenBank accession num-
Sino–Europe Cooperation, MOST, and European
bers CAI73679, EAN33624).The present study aimed
Union-funded Coordinated Action “Integrated Con-
to determinewhether two splice variants of homo-
sortium on Ticks and Tickborne Diseases” (ICTTD-3),
logues of clone 5 occur in T. annulata and T.parva.
Project number 510561. ZL is the recipient of a DAAD
scholarship.
Collaboration: Bakheit MA, National Research Center for Protozoan Diseases (NRCPD), Obihiro University for Agriculture and Veterinary Medicine, In-
Comparison of the molecular structure of the TaSP
ada-cho, Obihiro, 080-8555.
gene of Theileria annulata from Sudanese isolates
Supported in part by: Coordinated Action “Inte-
Ali AM, Ahmed JS, Seitzer U.
grated Consortium on Ticks and Tick-borne Diseases” (ICTTD-3) Asian Component, Project number
The polymorphic region of the Theileria annulata sur-
510561.
face protein TaSP was cloned and sequenced from
different isolates of cattle and cell lines from different areas of Sudan. Amino acid sequence alignment
Small-scale expressed sequence tag analysis of
revealed a high diversity showing amino acid and
Theileria sp. (China)
length polymorphism, both within and between par-
Liu Z, Ahmed JS, Seitzer U.
asite isolates. The generation of TaSP diversity may
allow the evasion of host immunity by the parasite
Recently, Theileria sp. (China) has been designat-
since TaSP is a highly antigenic parasite protein.
ed as T. luwenshuni [formerly Theileria sp. (China 1)]
and T. uilenbergi [formerly Theileria sp. (China 2)].
Collaboration: D Hassan, EL Hussein ARM, Central
A cDNA library of T. uilenbergi merozoites was con-
Veterinary Research Laboratories, Al Amarat, Khar-
structed and subjected to random sequencing.
toum, Sudan; SM Hassan, Faculty of Veterinary Med-
Among the obtained sequences were three highly
icine, University of Khartoum, Khartoum North, Sudan;
identical cDNA clones, indicating a gene family.
MM Mukhtar, Institute of Endemic Diseases, Univer-
Bioinformatic analyses indicated these genes con-
sity of Khartoum, Khartoum, Sudan; M Bakheit, Na-
tain signal peptides and encode potential im-
tional Research Center for Protozoan Diseases (NR-
munogenic proteins. The presence of tandemly
CPD), Obihiro University for Agriculture and Veteri-
arranged and additional variants of these genes
nary Medicine, Inada-cho, Obihiro, 080-8555.
was shown. Analysis of one recombinantly expressed clone revealed immunoreactivity for serum
Supported in part by: DAAD, European Union (ICA4-
from Theileria-infected animals. No cross-reaction
1999-30151), Animal Resources Bank (Sudan), IAEA
with serum of T. lestoquardi-, Babesia motasi-, or
NO.SUD04/027 entitled “Control of ticks and tick-
Anaplasma ovis-infected animals was observed, in-
borne diseases in the Sudan”.
dicating a potential antigen for development of
serological diagnostic tools.
Immune response of Theileria sp. (China) infectCollaboration: Z Dang, J Hou, J Luo, H Yin, Lanzhou
ed sheep to recombinant Theileria proteins
Veterinary Research Institute, Lanzhou, Gansu, China.
Seitzer U, Beyer D, Kullmann B, Miranda J, Ahmed JS.
Supported in part by: Supported in part by the
Sheep and goats in northwest China suffer from
Wissenschaftlicher Jahresbericht 2007-2008
117
Division of Veterinary Infectiology and Immunology
Research Reports
theileriosis from infection with Theileria sp. (China),
their encoding genes of a Theileria species highly
resulting in large economic losses. To investigate
pathogenic for small ruminants in China: applica-
the immune response to infection with Theileria sp.
tion for the development of diagnostics and vac-
(China), parameters of cellular and humoral immu-
cine.”, (ADDAV), Project number: ICA-1999-30151.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
nity of experimentally infected sheep against two recombinantly expressed Theileria proteins were investigated. The in vitro proliferative response of
Influence of subculturing on gene expression in a
blood mononuclear cells to a recombinant T. annu-
Theileria lestoquardi infected cell line
lata membrane protein and a recombinant Theile-
Ali AM, Beyer D, Bakheit MA, Kullmann B, Salih
ria sp. (China) homologue to T. annulata surface
DA, Ahmed JS, Seitzer U.
protein, both putative membrane proteins, was significantly elevated and significant amounts of spe-
In this study potential molecularmarkers for identifi-
cific immunoglobulinswere produced against both.
cation of attenuation in a Theileria lestoquardi-infected cell line to be used in vaccination trials were
identified. Two markers associated with attenuation
in Theileria annulata vaccine strainswere analyzed
(metalloproteinase activity and TNFα mRNA expression). The result showed a decreased activity
ofMMP9 and decreased mRNA expression of TNFα
with increasing passage number. Suppression subtractive hybridization was used to identify potential
new markers of attenuation. Random screening revealed nine differentially expressed genes, one
from the parasite and eight from the host. Quantitative real time-PCR confirmed mRNA expression of
the parasite vacuolar H+ATPase to be downregulated at higher passages.
Fig. 46. Proliferative response of experimentally infected animals
23 days p.i. to different antigens. a) Average values of four animals each per group. b) Data for four individual sheep each of tick
or blood infected animals are shown. Averages are depicted with
standard deviations. *p<0.05 compared to respective uninfected
control animal (→). rTaD: recombinant TaD protein; rTcSP: recombinant TcSP protein; merozoite: Theileria sp. (China) merozoite
crude antigen. Tick infected: animals infected by attraction of 200
Haemaphysalis qinghaiensis ticks. Blood infected: animals infected with T. sp. (China) infected blood.
Collaboration: M Bakheit, National Research Center for Protozoan Diseases (NRCPD), Obihiro University for Agriculture and Veterinary Medicine, Inada-cho, Obihiro, 080-8555.
Supported in part by: EU-funded AsianComponent
of the Inco-Dev ICTTD-3 Coordination Action Project
(Project No. 510561). AA is the recipient of a schol-
Collaboration: Liu Z, Yin H, Lanzhou Veterinary Re-
arship from the Sudanese Government; DAS is the
search Institute, Lanzhou, Gansu, China; Miranda J,
recipient of an IAEA fellowship (SUD/07020).
cInstituto de Biologia Experimental e Tecnologica/
Instituto de Tecnologia Quimica e Biologica / Universidade Nova de Lisboa, Oeiras, Portugal.
Supported in part by: European Commission’s IncoDev Research Program “Molecular and immunological characterization of merozoite antigens and
118
Wissenschaftlicher Jahresbericht 2007-2008
Division of Biochemical Immunology
Research Reports
Division of Biochemical Immunology
Head
However, the intracellular signaling pathways acti-
PD Dr. Frank Petersen
vated by CXCL4 are not well understood. Over the
past decade, it has become abundantly clear that
Staff Scientists and Postdoctoral Fellows
sphingolipids and their metabolites are key signal-
Dr. Brigitte Kasper
ing molecules. Sphingolipids are ubiquitous com-
Dr. Jessica Mittelstädt
ponents of cell membranes and their metabolites
Dr. Xinhua Yu
ceramide, sphingosine, and sphingosine-1-phosphate (S1P) have important physiological functions,
Graduate and Diploma Students
including regulation of cell growth and survival. In
Roland Gross (until 10/07)
the present study we investigated the role of sphin-
Lisette Leonhardt
gosine kinase 1 in CXCL4-induced cytokine release
Janne Wessel (until 04/08)
and monocyte survival. We could show that CXCL4-
Geske Woller (until 10/07)
induced mRNA expression of IL-6, TNF, and MCP-1
as well as the release of the corresponding proteins
Technicians
is strongly reduced in the presence of pharmaco-
Cindy Hass
logical SphK inhibitors (SKI and DMS). Furthermore,
Diana Heinrich
pretreatment of the cells with SKI reverts CXCL4-mediated rescue from apoptosis. CXCL4-induced
monocyte survival is accompanied by inhibition of
Research Reports
caspases which is controlled by SphK1 and its
The role of sphingosine kinase 1 in CXCL4-in-
down-stream element Erk. Taken together, these da-
duced cytokine release and monocyte survival
ta demonstrate the critical role of SphK1/S1P in CX-
Kasper B, Petersen F.
CL4 signaling in monocytes and suggest that SphK1
is a potential therapeutic target to suppress proin-
CXCL4 (Platelet factor 4; PF4) belongs to the family
flammatory responses induced by CXCL4.
of CXC chemokines, and shares 30 % to 60 % sequence identity and typical structural properties
Collaboration: Brandt E, Division of Biological
with other CXC chemokines, including the neu-
Chemistry, Research Center Borstel; Schütze S, De-
trophil-activating peptide-2 (NAP-2; CXCL7), inter-
partment of Immunology, UKSH, Campus Kiel.
leukin-8 (IL-8; CXCL8), or interferon (IFN)-inducible
protein 10 (IP-10; CXCL10). However, with regard to
Supported in part by: DFG SFB 415 “Spezifität und
its receptors, signal transduction, and biological
Pathophysiologie von Signaltransduktionswegen”,
functions, CXCL4’s role within the family of
Teilprojekt B6.
chemokines appears to be rather exceptional. Unlike other chemokines, which affect only a limited
set of target cells, CXCL4 was reported to be active
CXCL4-induced oxygen radical formation in hu-
on a variety of different cell types including ba-
man monocytes is regulated by multiple sig-
sophils, T cells, NK cells, neutrophils, and mono-
nalling pathways
cytes. On the latter cells, CXCL4 induces a complex
Kasper B, Petersen F.
spectrum of activities including the expression and
release of proinflammatory cytokines/chemokines
Monocytes and macrophages can respond to
and the protection from spontaneous apoptosis.
proinflammatory stimuli by a variety of acute and
Wissenschaftlicher Jahresbericht 2007-2008
119
Division of Biochemical Immunology
Research Reports
delayed defense mechanism such as oxygen radi-
treated monocytes SphK1 mRNA becomes signifi-
cal (ROS) formation, phagocytosis and killing of mi-
cantly up-regulated and that CXCL4 induces SphK1
croorganisms, or the presentation of antigens.
enzyme activity as well as its translocation to the
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Fig. 47. Effect of SphK inhibitor (SKI) or SphK1-specific siRNA on CXCL4-induced ROS formation. A: Freshly isolated monocytes were preincubated for 20 min at 37°C in the presence or absence of increasing concentrations SKI, or DMSO as solvent control. Subsequently, cells
were stimulated with 4 µM CXCL4 and ROS formation was quantified by chemiluminescence in the presence of luminol and recorded for
60 min. Individual assay backgrounds were determined in samples of unstimulated cells in the presence or absence of inhibitors run in
parallel and were subtracted. Either data (relative light units; RLU) from one representative experiment are given (inset), or data were
integrated over 60 min and shown as mean ± SD of four independent experiments. Asterisk (*) indicates statistically significant differences (P < .04) between inhibitor-treated samples and untreated controls. B-D: GM-CSF generated macrophages were transfected with
either SphK1-specific siRNA or non-silencing control-siRNA. (B) Cell viability (annexin V/PI staining), and transfection efficiency was tested 24 h after transfection using Cy5-labeled non-silencing control siRNA. (C) After 24 h, total RNA was isolated, transcribed into cDNA
and SPHK1 expression was tested by RQ-PCR. Relative quantification was performed using RelQuant software and data were presented as normalized ratio of SPHK1 versus HPRT. D: Alternatively, 24 h after transfection ROS formation was determined in these cells following stimulation with 4 µM CXCL4 as described above. Data are shown as mean ± SD of eight independent experiments. Asterisk (*)
indicates statistically significant differences (* P < .005, ** P < .0003) between SphK1-siRNA and control-siRNA treated samples.
However, only limited information exists on the spe-
cell membrane. Furthermore, using specific SphK in-
cific signaling pathways which are involved in the
hibitors (SKI) we could demonstrate that preincu-
control of these biological responses. Recently, we
bation of the cells with SKI resulted in a significant
could show that CXCL4-induced formation of ROS is
and dose-dependent reduction of CXCL4-mediated
controlled by a rapid activation of PI3K and p38
respiratory burst by 47% at 0.33 µM SKI to 98 % at
MAPK. Here, we report for the first time that sphin-
27 µM SKI (Fig. 47A). These data provided first evi-
gosine kinase 1 (SphK1) plays a key role in CXCL4-
dence that activation of SphK is involved in the gen-
triggered signaling. We demonstrate that in CXCL4-
eration of ROS in monocytes. To strengthen our re-
120
Wissenschaftlicher Jahresbericht 2007-2008
Division of Biochemical Immunology
Research Reports
sults with SphK inhibitors we next used siRNA knockdown strategy to verify these data. ROS production
induced by CXCL4 has been shown in monocytes as
well as in macrophages. Since for technical reasons
monocytes could not be used for knockdown experiments, GM-CSF-generated macrophages were
used instead. In a first approch we tested cell viability (annexin V/PI staining), and transfection efficiency 24 h after transfection using Cy5-labeled nonsilencing control siRNA (Fig. 47B). After 24 h 77 ±
14 % are viable and 57 ± 12% of the cells were positive for Cy5-labeled siRNA. More importantly, treatment of macrophages with SphK1-specific siRNA resulted in 37 % decreased SphK1 mRNA expression
(Fig. 47C), and 45% reduction in CXCL4-mediated
ROS production after 24 h (Fig. 47D). Finally, although monocyte responses induced by CXCL4
could be partially mimicked by the addition of high
dosages of exogenous sphingosine-1-phosphate
(S1P), CXCL4-signals were transduced independently from S1P receptors. Taken together, our data
indicate that SphK1 is a central signalling element
in CXCL4-induced monocyte activation which is involved in the regulation of short term (ROS production) as well as long term functions (cytokine production and cell survival).
Collaboration: Brandt E, Division of Biological
Chemistry, Research Center Borstel; Schütze S, Department of Immunology, UKSH, Campus Kiel.
Supported in part by: DFG SFB 415 “Spezifität und
Pathophysiologie von Signaltransduktionswegen”,
Teilprojekt B6.
Regulation of T cell chemotaxis by CXCL4
Woller G, Leonhardt L, Petersen F.
Directed migration of cells along a chemotactic
gradient is a fundamental cellular process involved e.g. in host defense, tissue development,
and wound repair. Surprisingly, although a broad
spectrum of different mediators is able to induce
Fig. 48. Effect of CXCL4 on T cell chemotaxis. A: CXCL4 lacks chemotactic activity on T cells. PHA-generated T cell blasts (105/well) were
placed in the upper compartment of a Boyden chamber system and
migration was determined towards increasing concentrations of native CXCL4, recombinant CXCL4, or recombinant CXCL11. B./C: CXCL4
inhibits CXCL11 and CXCL9-induced chemotaxis. Increasing concentration of CXCL11(I-TAC; B), CXCL9 (MIG; C) or medium alone (dashed
line) were placed in the lower compartments of a 48-well microchamber and the migration of T cells (105/well) was determined.
In a parallel set 4 µM CXCL4 was added to the cells in the upper wells.
D: CXCL4 does not modulate CXCL10-induced chemotaxis. In a parallel set of experiments migration of T cells towards CXCL10 (IP10) in
the presence or absence of CXCL4 was determined. Data are given
as mean + SD obtained from 4 or 5 independent experiments. Statistically significant differences between samples receiving CXCL11
alone or in combination with CXCL4 are indicated (*, P < 0.05).
Wissenschaftlicher Jahresbericht 2007-2008
121
Division of Biochemical Immunology
Research Reports
a chemotactic response, only very few regulators
allelic gene variant of CXCL4 was discovered and
or inhibitors of this function are known. CXCL4
the corresponding protein, termed CXCL4L1, has
(platelet factor 4; PF4), a platelet-derived CXC-
been detected recently in supernatants of activat-
chemokine, modulates long-term immunregulatory
ed platelets. While the N-termini of CXCL4 and CX-
functions in T cells but lacks the capacity to induce
CL4L1 are identical, both variants differ in three
chemotaxis in these cells (Fig. 48A). However, in
amino acid residues within the C-terminal a-helix.
the current study we are able to show for the first
Since biological functions and biochemical prop-
time, that CXCL4 acts as a potent inhibitor of T cell
erties of CXCL4L1 are largely unknown, we ana-
chemotaxis induced by CXCR3 ligands CXCL11
lyzed the capacity of the C-terminal peptides 47-70
and CXCL9, but not CXCL10 (Fig. 48B-D). CXCL4 did
of CXCL4 and CXCL4L1 to regulate cellular func-
neither interfere with ligand binding to CXCR3 nor
tions of human monocytes, neutrophils, and T cells.
with ligand-induced internalization or calcium sig-
Comparable to full-size CXCL4, both partial struc-
naling of CXCR3. By several lines of evidence we
tures were able to promote exocytosis in neu-
could rule out the participation of known CXCL4-re-
trophils, to generate ROS production in monocytes
ceptors, like proteoglycans or CXCR3-B, in CXCL4-
as well as to inhibit chemotaxis in T cells. Interest-
mediated inhibition of chemotaxis. Interestingly, CX-
ingly, CXCL4L1(47-70) displayed a significant high-
CL4, but not CXCL11, activates the beta2-integrin
er potency in the induction of monocyte and neu-
LFA-1, suggesting that reduced chemotaxis involves
trophils functions as compared to CXCL4 or its cor-
an enhanced adhesion of the cells. This more gen-
responding C-terminal peptide. These differences
eral mechanism would explain our further finding
suggest that not only the partial structures but al-
that CXCL4 also inhibits the chemotaxis of T cells
so the native molecules differ in their capacity to
and neutrophils induced by the CXCR3-independent
induce cellular effects. Thus, despite to their prin-
ligand CXCL12 (SDF-1a). Taken together, our results
cipal similar biological functions the individual ex-
identify CXCL4 as the first chemokine which acts as
pression of both variants could play a role in the
an inhibitor rather than an inducer of chemotaxis on
fine-tuning of an inflammatory response.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
T cells and neutrophils in vitro.
Supported in part by: DFG SFB 415 “Spezifität und
Supported in part by: DFG SFB 415 “Spezifität und
Pathophysiologie von Signaltransduktionswegen”,
Pathophysiologie von Signaltransduktionswegen”,
Teilprojekt B6.
Teilprojekt B6.
Crossregulation of immune responses by mast
Activation of human phagocytes and T cells by
cells and dendritic cells
variants of platelet factor 4 (CXCL4)
Mittelstädt J, Schiemann F, Petersen F.
Wessel J, Petersen F.
Mast Cells are well known for their pivotal role in
The platelet-derived chemokine CXCL4 is known as
allergic as well as autoimmune diseases. Howev-
a potent activator of human phagocytes inducing
er, today it is clear that mast cells are key players
adhesion and exocytosis in neutrophils as well as
in the innate immune response and participate in
phagocytosis and the generation of reactive oxy-
a wide range of chronic and acute inflammatory
gen species (ROS) in monocytes. Furthermore, CX-
diseases. It is commonly accepted that investiga-
CL4 has the capacity to prevent monocyte from un-
tions limited on these cells alone are not sufficient
dergoing apoptosis and to promote their differen-
to understand their impact and physiological func-
tiation into macrophages. A few years ago, a non-
tion. Within this view, we focused our investigations
122
Wissenschaftlicher Jahresbericht 2007-2008
Division of Biochemical Immunology
Research Reports
on the cellular interaction of mast cells and den-
Cellular interactions of human mast cells and neu-
dritic cells. Beside enzymes, mast cells are capa-
trophil granulocytes in allergic asthma
ble to release a variety of cytokines and
Leonhardt L, Petersen F.
chemokines, which may affect DC functions and
their differentiation. Dendritic cell activation and
Asthma is characterized as a chronic inflammation
maturation can be mediated by LPS or cytokines
of the airways which involves various cell types in-
like TNF-alpha and is accompanied by expression
cluding eosinophils, neutrophils and mast cells.
of costimulatory molecules as well as cytokines.
This inflammatory process is seen as directly
Since mast cells as well as DC are in the first line
causative for the asthma pathology in terms of air-
of defence of the host against microbial invaders,
way hyperreactivity and airway remodelling. Al-
we asked whether both cell type could interact
though eosinophilia of the lung is a hallmark of the
which each other resulting in a cross-regulatory
allergic inflammation, disappointing results from
process. In a first approach, where we analysed
anti-IL-5 as wells as IL-12 studies revealed that the
consequences during coculture of immature DC
accumulation of eosinophils alone cannot explain
(iDC) with human primary lung mast cells, we nei-
the pathology of asthma. Today it is clear, that oth-
ther observe relevant changes in DC phenotype
er cell types including neutrophils and mast cells
nor the induction of cytokine release or modulation
also contribute to the disease. However, the un-
of T cell responses by DC. In a more indirect ap-
derlying pathomechanisms as well as pathophys-
proach, we investigated the effect of mast cell pro-
iological role of these cells in asthma remain to be
teases on the maturation of DC in presence of LPS
elucidated. Following our hypothesis, activated
and the cathelicidin LL37, an antimicrobial peptide
mast cells are essential for neutrophil accumula-
abundantly present in microbial infections. Inter-
tion and activation in the allergic lung. Further-
estingly, LL37 does not directly modulate DC func-
more, the interaction between both cell types
tions, but has the capacity to prevent LPS-induced
could play a key role in the structural and func-
maturation of iDC. By contrast, we could show that
tional alteration of the airways. In first experiments
LL37 acts as a potent activator of MC inducing the
we could show that Fc-eRI-activated human lung
release of mast cell proteases such as tryptase.
mast cells release immediately (30 min) and de-
Most intriguing, blocking of LPS-induced matura-
layed (4-24h) mediators which are capable to in-
tion by LL37 was completely abrogated in the pres-
duce a chemotactic response in neutrophils. Cur-
ence of mast cells or mast cell tryptase. We could
rently, we are working on the identification of these
show that tryptase released by LL37-activated
mediators by HPLC-chromatography in combina-
mast cells is responsible for a rapid degradation
tion with nano-ESI-FT mass spectrometry. These tri-
of the AMP into biological inactive fragments
als will be accompanied by transcriptome analy-
which leads to the reconstitution of DC activation
sis of activated mast cells. In the long run we will
by LPS. Our findings suggest an indirect interplay
evaluate relevant mediators in sputum and lavage
between mast cells and dendritic cells in a com-
fluids of patients suffering from allergic and non-
plex inflammatory situation.
allergic asthma. We hope that our approaches do
not only provide a better understanding of the
Collaboration: Brandt E, Division of Biological
pathological processes in asthma but will help to
Chemistry, Research Center Borstel.
improve diagnosis and development of new strategies for the treatment of the disease.
Supported by in part by: DFG PE 967/1-1.
Collaboration: Brandt E, Division of Biological
Chemistry, Research Center Borstel; Lindner B, Divi-
Wissenschaftlicher Jahresbericht 2007-2008
123
Division of Biochemical Immunology
Research Reports
sion of Immunochemistry, Research Center Borstel.
mast cells are involved in the pathogenesis of ex-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
perimental EBA and if MCs-targeted treatment may
Supported in part by: DFG SFB/TR 22 “Allergische
serve as a potential therapeutic strategy of EBA. Fi-
Immunantworten der Lunge“, Teilprojekt A11.
nally, by using corresponding knock-out animals, we
aim to identify cytokines playing an essential role
in EBA. From our approaches we expect the develInvestigating the molecular mechanism of tissue
opment of novel strategies in the diagnosis and
damage in autoimmunity to type VII collagen us-
treatment of autoimmune dermatoses.
ing experimental models
Collaboration: Zillikens D, Ludwig R, Department of
Yu X, Petersen F.
Dermatology, UKSH, Campus Lübeck; Bulfone-Paus S,
Epidermolysis bullosa acquisita (EBA) is an autoim-
Division of Immunobiology, Research Center Borstel.
mune bullous disease caused by autoantibodies to
type VII collagen, the main constituent of anchoring
Supported in part by: Cluster of Excellence „Inflam-
fibrils in dermal-epidermal junction. Recently, in vi-
mation at Interfaces”; Research Area H: IRN Au-
vo mouse models of EBA have been established, by
toimmunity to Type VII Collagen.
active immunization of type VII antigen or passive
transfer of autoantibody to type VII collagen. As an
autoantibody-mediated autoimmune disease with
Theses
well characterized autoantigen and ingenious disease modelling, EBA emerges as a unique model
Diploma
for investigating the pathogenesis of autoantibody-
Wessel, Janne
mediated autoimmune diseases. With the recently
Aktivierung humaner Leukozyten durch Varianten
established mouse models, investigators demon-
des CXC-Chemokins Plättchenfaktor 4
strated that some biological possesses are essen-
Technisch-Naturwissenschaftliche Fakultät
tial in the development of the disease, including
Universität zu Lübeck, 2008.
generation of tissue-bound autoantibodies, activation of complement system, generation of reactive
Dissertation
oxygen species (ROS) by neutrophils and neutrophil
Gross, Roland
degranulation. Despite of those significant findings,
Direkte und indirekte Effekte thrombozytärer Che-
the pathogenesis of the diseases is still largely un-
mokine auf humane Mastzellen.
clear. Therefore, identifying biological events in-
Technisch-Naturwissenschaftliche Fakultät
volved in the development of EBA and further char-
Universität zu Lübeck, 2007.
acterizing their molecular mechanisms will help to
explore the pathogenesis of the disease as well as
Woller, Geske
to search for novel therapeutic targets. In addition,
Regulation der Chemotaxis humaner T-Zellen durch
clarity of the pathogenesis of EBA will also help to
das thrombozytäre Chemokin Plättchenfaktor 4.
elucidate the pathogenesis of other anti-body me-
Technisch-Naturwissenschaftliche Fakultät
diate inflammatory diseases in general. To under-
Universität zu Lübeck, 2007.
stand the molecular mechanism responsible for
neutrophil-mediated tissue damage we will investigate mediators, signalling pathways and receptors
involved in the control of neutrophil activation. Furthermore we will address the question whether
124
Wissenschaftlicher Jahresbericht 2007-2008
Division of Innate Immunity
Research Reports
Division of Innate Immunity
Head
nificant increase only in Defensin Beta-1 (DEFB1)
PD Dr. Holger Heine
mRNA expression in bronchial biopsy samples of
COPD patients that weakly but significantly corre-
Staff Scientists and Postdoctoral Fellows
lates with disease severity (Fig. 49A, B).
Dr. Thomas Scholzen (since 04/2008)
Dr. Jennifer Debarry (07/2007 - 02/2008)
Graduate and Diploma Students
Jennifer Debarry (until 06/2007)
Ellen Andresen
Karina Stein
Technicians
Ina Goroncy
Katrin Sprenger
Research Reports
Molecular basis of increased DEFB1 gene expression in the lung of COPD patients
Andresen E, Heine H.
Ongoing inflammatory immune responses in the
lung are characteristic for the pathophysiology of
10 healthy volunteer controls and 35 individuals
Fig. 49. Study of DEFB1 gene expression in biopsy samples. Here,
we tested the hypothesis that the DEFB1 mRNA is differently regulated and expressed in the peripheral lung during pathogenesis
of COPD. (A) In biopsies obtained from 10 healthy controls and 32
COPD patients, the DEFB1 mRNA expression was analyzed by
quantitative real-time PCR and normalized to 2-microglobulin expression. The one-tailed hypothesis was tested using unpaired ttest from log transformed data; all data is shown as mean ± SEM.
DEFB1 mRNA expression is significantly increased in biopsies of
COPD patients. (B) Spearman correlation between the DEFB1 mRNA expression and the percent of predicted FEV1/VC (N = 41). The
DEFB1 mRNA showed a weak but significant correlation with the
disease severity.
with stage I-IV COPD (defined by an FEV1/VC < 70
% and FEV1 < 80 % of predicted) were recruited at
The first discovered human DEFB1 has been large-
the Medical Clinic of the Research Center Borstel.
ly considered as a constitutively expressed de-
The study was approved by the Ethical Committee
fensin, but recent observations suggest some sort of
of the Medical faculty of the University of Lübeck.
DEFB1 gene regulation in a variety of cell types.
Following written informed consent bronchoscopy
However, the molecular mechanisms responsible
was performed according to national guidelines
for the upregulation of DEFB1 gene expression in
with epithelial cell biopsies and bronchoalveolar
COPD are still unknown. Alterations in gene ex-
lavage (BAL) with 300 ml of normal saline. Of all de-
pression can be caused by epigenetic program-
fensins investigated, our data demonstrated a sig-
ming leading to changes in the chromatin state (hi-
Chronic Obstructive Pulmonary Disease (COPD). Defensins are components of the innate host response
against bacterial infections and regulators of inflammation and immunity. In the present study, we
tested the hypothesis that defensins are differently
regulated and expressed in the peripheral lung tissue during pathogenesis of COPD. For this purpose,
Wissenschaftlicher Jahresbericht 2007-2008
125
Division of Innate Immunity
Research Reports
stone modifications and DNA methylation). We
(Fig. 50B). Altogether, our results support the hy-
demonstrated modulation of DEFB1 gene expres-
pothesis that the observed alterations in DEFB1
sion by using inhibitors of human histone deacety-
gene expression in COPD are a consequence of
lases (HDACi) or DNA methylation suggests an im-
epigenetic changes, although definitive proof will
portant role for epigenetic mechanisms in the
require additional studies. The understanding of
DEFB1 gene regulation (data not shown). Further-
the biological function and interplay of epigenetic
more, bisulfite sequencing analysis of DNA methy-
mechanisms in DEFB1 gene expression might be an
lation revealed that the increased DEFB1 mRNA lev-
important contribution to the understanding of the
el found in biopsies of COPD patients correlates
pathogenesis of COPD.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
with the decreased methylation of distinct CpG sites
at the DEFB1 promoter (Fig. 50A).
Collaboration: Lange C, Div. Clinical Infectious Diseases, Research Center Borstel.
Supported in part by: DFG, SFB617, project A23.
Investigation of allergy-protective mechanisms by
microarray analysis of human dendritic cells stimulated with four different bacterial strains
Stein K, Debarry J, Heine H.
The incidence of allergic diseases is worldwide increasing, especially in industrialized regions. A
growing number of publications indicate that farming environment in early childhood reduces the occurrence of allergic reactions later in life. Recently,
we showed that the cowshed isolates Lactococcus
lactis G121 and Acinetobacter lwoffii F78 prevent allergic immune responses in a mouse asthma model. However, the molecular mechanisms modulating
Fig. 50. Epigenetic studies in DEFB1 gene regulation. (A) Bisulfite
sequencing analysis of specific promoter CpG sites among biopsies from 4 healthy controls and 4 COPD patients. Positions of individual CpG sites relative to the transcriptional start are indicated. Increased DEFB1 mRNA level found in biopsies of COPD patients correlates with the decreased methylation of distinct CpG
sites at the DEFB1 promoter. (B) ChIP assays were performed with
chromatin from BAL fluid cells of 11 study subjects. Spearman correlation between the DEFB1 mRNA expression and H3K4me3
showed a strong positive correlation with the establishment of the
active histone modification in the vicinity of the transcriptional start
site of the DEFB1.
allergic reactions in humans are only poorly understood. Thus, we stimulated human dendritic cells
(DC) with these bacteria and two other reference
strains, Escherichia coli F1111 9-41 and Bacillus sub-
tilis DSM618, and prepared microarray analysis after 3, 6 and 12 hrs of stimulation. The results showed
that a total number of 3623 probe sets in L. lactis-,
4254 sets in A. lwoffii- and 4625 sets in E. coli-treated DCs were regulated at least three-fold but only
1939 probe sets in B. subtilis-treated DCs. Scatter-
Moreover, histone H3K4 trimethylation (H3K4me3)
was profoundly increased in the vicinity of the tran-
plot analysis revealed that the induced expression
scriptional start site of DEFB1 and showed a strong
comparable, with already a high number of regu-
positive correlation with DEFB1 gene expression
lated genes after 3 hrs. In contrast, nearly no genes
126
pattern of E. coli and A. lwoffii stimulated DCs is
Wissenschaftlicher Jahresbericht 2007-2008
Division of Innate Immunity
Research Reports
were regulated in L. lactis-treated DCs after 3 or 6
ther characterized and identified using blocking
hrs and only a moderate number after stimulation
experiments.
with B. subtilis. However, after 12 hrs of stimulation,
L. lactis-treated DCs showed an expression pattern
similar to that induced by A. lwoffii and E. coli
whereas DCs treated with B. subtilis differed clearly. Furthermore, cluster analysis confirmed a close
relation between A. lwoffii-, E. coli- and L. lactis-treated DCs after 12 hrs whereas B. subtilis-treated DCs
formed a separate cluster. Overall, the analysis of
gene expression induced by cowshed bacteria patterns leads to a better understanding of the molecular mechanism preventing allergic immune responses.
Collaboration: Hanuszkiewicz A, Holst O, Dept. of
Immunochemistry and Biochemical Microbiology,
Research Center Borstel.
Supported in part by: DFG, SFB/Transregio 22, project A2.
The allergy-protective properties of Acinetobac-
ter lwoffii F78 are imparted by its lipopolysaccharide
Debarry J, Stein K, Heine H.
An increasing number of epidemiological studies
Fig. 51. TLR4/lipid A antagonist compound 406 blocks the TH1polarizing effects of A. lwoffii F78. Human moDCs were stimulated with indicated stimuli in presence or absence of C406. (A) After 16 hours of stimulation the concentration of IL-12 was measured in the supernatant. (B) After 16 hours of stimulation these
DCs were co-cultured with autologous naïve T cells and were restimulated after 5 days. The induced IFN-γ release was measured
in the supernatant after additional 20 hours. Presented data
(mean values ± SD) are representative for at least 3 independent experiments with moDCs from different healthy donors.
show that exposure to farming environment during
ment of allergic reactions later in life (“hygiene hy-
We could show that A. lwoffii F78 induced a TH1polarizing program in human dendritic cells which
pothesis”). Also, it had been shown that certain
led to TH1-differentiation. In addition, a positive in-
bacteria from this environment may have allergy-
fluence on the TBet/GATA3 level could be detect-
protective properties. In the present study we fur-
ed. Preincubation with the TLR4 antagonist com-
ther characterized one of these bacteria, namely
pound 406 inhibited activation by A. lwoffii F78
Acinetobacter lwoffii F78, with regard to the bac-
(Fig. 51) and the isolated LPS of A. lwoffii F78 pro-
early childhood strongly influences the develop-
teria-induced signaling and possible mechanisms
moted induction of IL-12 and IFN-γ as well as the
of allergy protection. The impact of A. lwoffii F78
whole bacteria (Fig. 52). In summary, we found ev-
on human monocyte-derived dendritic cells espe-
idence that the allergy-protecting effects of
cially with respect to their THelper cell polarization
A. lwoffii F78 are due to the activation of a TH1-po-
capacity was investigated by ELISA and Real-time
larizing program in human dendritic cells and that
PCR experiments as well as confocal microscopy.
the LPS of A. lwoffii F78 is responsible for these
The molecule responsible for these effects was fur-
beneficial effects.
Wissenschaftlicher Jahresbericht 2007-2008
127
Division of Innate Immunity
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
ing environment during early childhood concomitant with increased exposure to microbial substances can lead to a decreased risc of developing
allergies in later life. Recently, we showed that the
Gram+ cowshed isolate Lactococcus lactis G121
prevents allergic immune responses in a mouse allergy model. However, receptors and signalling
pathways involved in immune cell activation by this
bacterium are mainly unknown. L. lactis-stimulated
human monocyte-derived dendritic cells (DCs)
showed an induction of IL-12p70, the main TH1-polarizing cytokine, which is known to inhibit the allergy-associated TH2-driven immune response. Stimulation of mouse bone marrow-derived Toll-like receptor (TLR) 2 deficient cells revealed no major role
for this receptor in L. lactis-mediated activation.
However, when we preincubated human DCs with
inhibitors of phagocytosis (Cytochalasin D) or endosomal acidification (Bafilomycin A1, Fig. 53), L.
lactis–induced secretion of IL-12 was almost completely blocked.
Fig. 52. A. lwoffii F78-LPS is responsible for the TH1 polarization.
Human moDCs were stimulated with indicated stimuli. (A) After 16
hours of stimulation the concentration of IL-12 was measured in the
supernatant. (B) After 16 hours of stimulation these DCs were co
cultured with autologous naïve T cells in a ratio of 1:10 (DC:T cell)
and were restimulated with PMA and Ionomycin after 5 days. The
IFN-γ release was measured in the supernatant after additional 20
hours. Data are representative for at least 3 independent experiments with moDCs from different healthy donors.
Collaboration: Hanuszkiewicz A, Holst O, Dept. of
Immunochemistry and Biochemical Microbiology,
Research Center Borstel.
Supported in part by: DFG, SFB/Transregio 22, project A2.
Fig. 53. Influence of the inhibitor for phagosomal maturation
Bafilomycin A1 on IL-12 cytokine secretion after stimulation with L.
lactis. Human monocyte-derived dendritic cells were preincubated with Bafilomycin A1 for 30 min followed by treatment with the
indicated stimuli. After 20 hrs of stimulation IL-12p70 release in the
supernatant was measured by ELISA. Lipopolysaccharide (LPS)
treatment and stimulation with the acidification-dependent TLR7
ligand CL097 were used as controls.
This strongly indicates the importance of uptake
and endosomal maturation for activation by L. lacIntracellular recognition of the cowshed bacteria
tis, and, thus, the involvement of intracellular re-
L. lactis G121 is required to induce an allergy pre-
ceptors. However, stimulation of mouse bone mar-
ventive immune response
row-derived dendritic cells from TLR-KO animals
with L. lactis showed no implication of the intracel-
Stein K, Debarry J, Heine H.
lullar TLRs 3, 7 or 9. Despite the fact that MyD88-KO,
An increasing number of studies indicate that farm-
128
in contrast to Trif-KO, macrophages could not be ac-
Wissenschaftlicher Jahresbericht 2007-2008
Division of Innate Immunity
Research Reports
tivated after stimulation with L.lactis G121. Interest-
Diploma
ingly, immortalized mouse bone marrow-derived
Stein, Karina
macrophages deficient for the intracellular receptor
Einfluss von Mustererkennungsrezeptoren auf die
NOD2 showed a strong decrease of cytokine in-
Regulation der Immunantwort dendritischer Zellen
duction upon L. lactis-treatment (Fig. 54), despite the
nach Stimulation mit Kuhstallbakterien.
fact that MDP, the minimal ligand for NOD2, does
Mathematisch-Naturwissenschaftliche Fakultät
not activate the cells by itself. Overall, identifying
Christian-Albrechts-Universität zu Kiel, 2008.
the molecular mechanism of the activation of immune cells by L. lactis G121 will lead to a better understanding of the molecular mechanism preventing allergic immune responses.
Fig. 54. Cytokine release of Wildtype and NOD2 deficient cells after L. lactis treatment. Wildtype (WT) immortalized mouse bone
marrow-derived macrophages or cells deficient for NOD2 were
stimulated with L. lactis and Lipopolysaccharide (LPS) as control.
After 20 hrs TNF-α release in the supernatant was measured by
ELISA.
Collaboration: Hanuszkiewicz A, Holst O, Dept. of
Immunochemistry and Biochemical Microbiology,
Research Center Borstel.
Supported in part by: DFG, SFB/Transregio 22, project A2.
Theses
Dissertation
Debarry, Jennifer
Mögliche Mechanismen der Allergieprävention:
dendritische Zellen als Regulatoren der durch Stallbakterien ausgelösten allergieprotektiven Immunantwort.
Mathematisch-Naturwissenschaftliche Fakultät
Christian-Albrechts-Universität zu Kiel, 2007.
Wissenschaftlicher Jahresbericht 2007-2008
129
Abteilung Klinische Medizin
Department of Clinical Medicine
Fo r s c h u n g s z e n t r u m
Bo r s t e l
130
Wissenschaftlicher Jahresbericht 2007-2008
Abteilung Klinische Medizin
Department of Clinical Medicine
Abteilung Klinische Medizin
Department of Clinical Medicine
Wissenschaftlicher Jahresbericht 2007-2008
131
Zusammenfassung
Summary
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Abteilung
Klinische Medizin
Department of
Clinical Medicine
Leiter/Head
Leiter/Head
Prof. Dr. Peter Zabel
Prof. Dr. Peter Zabel
Sekretariat/Secretary
Sekretariat/Secretary
Sylvia Kempf
Sylvia Kempf
Insgesamt ist es der Abteilung Klinische Medizin
During the last two years, the Department of Clini-
auch in den vergangenen 2 Jahren gelungen,
cal Medicine succeeded in further reinforcing the
durch Optimierung der Patientenversorgung, Im-
clinical and translational pulmonary research at the
plementierung des neuen Bereiches „Experimen-
Research Center Borstel through the optimisation of
tellen Pneumologie“, Einwerbung zusätzlicher
the patient care, the implementation of the new sec-
Drittmittel, Entwicklung innovativer Methoden für
tion “Experimental Pneumology”, the development
Forschung und Diagnostik sowie Generierung neu-
of innovative methods for research and diagnostics
er Kooperationen und Verbundprojekte die Klini-
as well as the creation of new cooperations and col-
sche und Translationale Lungenforschung am FZB
laborative projects. Most of the research projects
weiter zu stärken. Dabei sind die meisten For-
dedicated to the Borstel mission are correspond-
schungsprojekte unserer Borsteler Mission ent-
ingly structured interdisciplinarily and comprehen-
sprechend interdisziplinär und abteilungsüber-
sively with respect to the different departments and
greifend angelegt und haben so eine einzigarti-
thus resulted in a unique connection between basic
ge Verbindung von Grundlagenwissenschaft und
science and the clinic which is further to be culti-
Klinik erreicht, die es weiter zu pflegen und aus-
vated and extended.
zubauen gilt.
This Borstel expertise became especially apparent
49th Annual Congress
for Pneumology and
Ventilation Medicine
2. Deutscher
Allergie-Kongress
In zwei von der Abteilung organisierten großen
through two big congresses organised by the de-
Kongressen konnte diese Borsteler Expertise be-
partment: Wolf-Meinhard Becker was the president
sonders sichtbar gemacht werden: Wolf-Meinhard
Becker war Kongresspräsident des „2. Gemeinsa-
of the “Second Joint German Allergy Congress” in
Lübeck from 26th to 29th September 2007. This suc-
men Deutschen Allergie-Kongress“ vom 26. - 29.
cessful congress met with an extremely positive re-
September 2007 in Lübeck. Bei den über 1000 Kongressteilnehmern stieß dieser erfolgreiche Kon-
sponse of the more than 1000 participants. For the
first time Lübeck then hosted the 49th Annual Con-
gress auf eine äußerst positive Resonanz. Vom 9.
gress of the “German Society for Pneumology and
bis 12. April 2008 war dann der 49. Jahreskongress
der „Deutschen Gesellschaft für Pneumologie und
Ventilation Medicine” presided by Peter Zabel from
9th to 12th April 2008. With nearly 3000 participants
Beatmungsmedizin“ mit Peter Zabel als Kongres-
the congress was not only the biggest one this city
spräsident zum ersten Mal in Lübeck zu Gast. Mit
had ever experienced but also one of the most vis-
nahezu 3000 Teilnehmern war der Kongress in Lü-
ited of the Society. It reflected the whole range of
beck nicht nur der größte, den diese Stadt je er-
pneumology with 13 plenary sessions, 56 symposia,
lebt hat, sondern auch einer der meistbesuchten
3 hot topic sessions, 8 workshops, 28 industrial sym-
unserer Gesellschaft. Mit 13 Plenarsitzungen, 56
posia, 19 post-graduate courses and more than 400
132
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Kapitel
Symposien, 3 Hot Topic Sitzungen, 8 Workshops,
scientific abstracts. The leader of the Federal state
28 Industriesymposien, 19 PG-Kursen und über 400
of Schleswig-Holstein, Peter Harry Carstensen,
wissenschaftlichen Abstracts spiegelte der Kon-
opened the congress by expressing his respect for
gress die ganze Breite der Pneumologie wider.
the outstanding performances of the Research Cen-
Der Ministerpräsident von Schleswig-Holstein, Pe-
ter Borstel in the field of pulmonary research. At the
ter Harry Carstensen, eröffnete den Kongress und
beginning of the congress, a top-class international
bekundete dabei seinen Respekt für die hervor-
ERS (European Respiratory Society) symposium with
ragenden Leistungen des Forschungszentrums
the subject “State of the Art in Tuberculosis” carried
Borstel auf dem Gebiet der Lungenforschung. Ein
out in our manor house directly integrated Borstel
hochkarätiges internationales ERS-Symposium
into the congress events and was one of its high-
zum Thema „State of the Art in Tuberculosis“ am
lights.
Beginn des Kongresses im unserem Herrenhaus
bezog auch Borstel direkt in das Kongressgeschehen ein und war einer der Höhepunkte des
Over the last two years, the Medical Clinic of Bors-
Kongresses.
tel has been able to further consolidate its position
as a highly competent hospital specialized in pulmonary, infectious and allergic diseases and in ad-
Die Medizinische Klinik Borstel hat auch in den ver-
dition to optimise the patient care. This develop-
gangenen zwei Jahren ihre Position als hochkom-
ment is especially due to the cooperation practised
petente pneumologische, infektiologische und al-
on all levels with the Medical Clinic III of the Uni-
lergologische Fachklinik ausbauen können und
versity Clinic of Schleswig-Holstein (UK S-H), Campus
die Patientenversorgung weiter optimiert. Dazu
Lübeck, which is also directed by Peter Zabel and
trägt besonders auch die in allen Ebenen geleb-
has resulted in a complementary university con-
te Kooperation mit der Medizinischen Klinik III des
nection of the patient care in Borstel.
UK S-H, Campus Lübeck bei, die ebenfalls von Pe-
Besides the permanent exchange of assistants con-
ter Zabel in Personalunion geleitet wird, und zu ei-
tinuing their education between these two clinics,
ner komplementären universitären Anbindung der
this cooperation is also used for common scientific
Krankenversorgung in Borstel geführt hat.
projects, clinical studies and joint projects with oth-
Neben dem ständigen
Ärzte-Team
Austausch von Assistenten in Weiterbildung
Medical Team
zwischen diesen beiden
Kliniken wird diese Kooperation auch für gemeinsame
wissen-
schaftliche Projekte, klinische Studien und Verbundprojekte
mit
anderen Kliniken und
Instituten der Universität Lübeck genutzt.
Die gemeinsame Zertifizierung
als
einziges
„Zentrum für Klinische
Wissenschaftlicher Jahresbericht 2007-2008
133
Zusammenfassung
Summary
Infektiologie“ in Schleswig-Holstein von der Deut-
er clinics and institutes of the University of Lübeck.
schen Gesellschaft für Infektiolologie und die ge-
The common certification as the only “Center for
meinsame ambulante Versorgung allergologi-
Clinical Infectiology” in Schleswig-Holstein by the
scher Patienten durch den Ausbau der interdiszi-
German Society of Infectiology and the joint ambu-
plinären Allergiesprechstunde in Kooperation mit
lant treatment of allergy patients by the extension
der Hautklinik, der HNO-Klinik, der Pädiatrie und
of the interdisciplinary allergy consulting hour in co-
der Med. Klinik III am Campus Lübeck belegen die-
operation with the Clinics of Dermatology, ENT, Pae-
se Synergien eindrucksvoll. Im Jahr 2007 konnte an
diatrics, and the Medical Clinic III at the Campus
der Medizinischen Klinik Borstel nach langen Ver-
Lübeck give impressive evidence of these synergies.
handlungen endlich ein Medizinisches Versor-
In the year 2007, a Medical Treatment Center (MVZ)
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Gründung des MVZ
gungszentrum (MVZ) gegründet werden, das die
could finally be founded at Borstel after long ne-
Praxen für Pathologie (E. Vollmer) und Lungen- und
gotiations, which organisationally unites the Prac-
Bronchialheilkunde (U. Greinert) organisatorisch
tices for Pathology (E. Vollmer) and Bronchopul-
zusammenführt und eine Sicherung der ambulan-
monary Medicine (U. Greinert) and serves as a
ten Krankenversorgung in Borstel bedeutet. Diese
guarantee for the ambulant patient treatment in
neue Versorgungsstruktur kann zusammen mit dem
Borstel. This new structure for patient care is able to
seit 2005 bestehenden Klinischen Studienzentrum
ensure the execution of the multiple clinical studies
(Leiter: C. Lange) an der Medizinischen Klinik Bor-
at the Medical Clinic in Borstel together with the
stel die Durchführung der vielfältigen klinischen
Clinical Study Center (head: C. Lange) existing since
Studien gewährleisten. In den vergangenen zwei
2005. Within the past two years, the economic situ-
Jahren ist die ökonomische Situation der Klinik
ation of the clinic remained pleasantly stable de-
trotz abschmelzender Budgets und weiterer großer
spite further decreasing budgets and other severe
Belastungen durch die Wandlungen im Gesund-
financial burdens due to the modifications in the
heitssystem bei gleichzeitig notwendiger Qua-
health system besides mandatory quality improve-
litätssteigerung und fortschreitender Leistungsver-
ment and a further rise in efficiency. This is only pos-
dichtung erfreulich stabil. Dies gelingt nur dank
sible owing to the particular commitment and the
des besonderen Einsatzes und der hohen Motiva-
high motivation of the members of the clinical staff
tion der Mitarbeiter der Klinik, denen an dieser
who have especially to be thanked here.
Stelle ganz besonders gedankt werden muss.
The restructuring of the Department of Clinical Med-
Appointment: Heinz Fehrenbach, Experimental
Pneumology (W3),
University of Lübeck
Die Neustrukturierung der Abteilung Klinische Me-
icine with the further reinforcement of translational
dizin mit der weiteren Stärkung der translationa-
pulmonary research was decisively pushed by the
len Lungenforschung wurde im Jahre 2008 durch
appointment of Heinz Fehrenbach for the W3 pro-
die Berufung von Heinz Fehrenbach auf die im
fessorship for Experimental Pneumology within the
Rahmen des Exzellenz-Clusters „Inflammation at
framework of the Excellence Cluster “Inflammation
Interfaces“ neu geschaffene W3-Professur für Ex-
at Interfaces” in the year 2008. Heinz Fehrenbach
perimentelle Pneumologie entscheidend voran-
had up to then headed a group of researchers in
getrieben. Heinz Fehrenbach war bis dahin Leiter
Marburg supported by the BMBF and brings a num-
einer BMBF-geförderten Forschergruppe in Mar-
ber of complex animal models for the research of
burg und bringt eine Reihe komplexer Tiermodel-
inflammation and regeneration processes in the
le zur Erforschung der Entzündungs- und Regene-
lung to Borstel. Since August 2008, the former labo-
rationsvorgänge in der Lunge mit nach Borstel. Zu
ratory group of “Clinical Immunopharmacology”,
seinem Bereich gehört ab August 2008 auch die
which was headed by P. Zabel, also belongs to his
134
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
ehemalige von P. Zabel geleitete Laborgruppe
section and is now dedicating itself to “Functional
„Klinische Immunpharmakologie“, die sich jetzt
Inflammation Research”, headed by Hans-Peter
unter der Leitung von Hans-Peter Hauber der
Hauber. Above that, the field of “Experimental Pneu-
„Funktionellen Entzündungsforschung“ widmet.
mology” recently comprises the research groups of
Der Bereich „Experimentelle Pneumologie“ um-
“Cellular Pneumology” (head: C. Stamme) and
fasst darüber hinaus neu die Forschergruppen
“Barrier Integrity” (head: I. Lautenschläger) so that
„Zelluläre Pneumologie“ (Leiterin: C. Stamme)
the tandem formation striven for of experimental
und „Barriereintegrität“ (Leiter: I. Lautenschläger),
and clinical pneumology has now been translated
so dass jetzt die angestrebte Tandembildung aus
into action.
Experimenteller und Klinischer Pneumologie umgesetzt ist.
During the years 2007/08, a multitude of national
and international studies/cooperations were exIm NRZ (Leiterin: S. Rüsch-Gerdes) wurden in den
tended in the National Reference Center (NRZ;
Jahren 2007/08 eine Vielzahl nationaler und inter-
head: S. Rüsch-Gerdes) in order to meet the dra-
nationaler Studien/Kooperationen erweitert, um
matically changed world-wide resistance develop-
der dramatisch veränderten weltweiten Resisten-
ment of M. tuberculosis (cooperation or study part-
zentwicklung von M. tuberculosis Rechnung zu tra-
ners: Robert Koch Institute (RKI), health authorities,
gen (Kooperations- bzw. Studienpartner: Robert
WHO, Médecins Sans Frontières (MSF), Internation-
Koch Institut (RKI), Gesundheitsämter, WHO, Mé-
al Committee of the Red Cross (ICRC), and The
decins Sans Frontières (MSF), International Com-
Foundation for Innovative New Diagnostics (FIND)).
mittee of Red Cross (ICRC) und The Foundation for
In addition, the cooperation with the “Global Fund”
Innovative New Diagnostics (FIND)). Darüber hin-
to build up laboratories and to establish networks
aus wurde die Zusammenarbeit mit „Global
in selected countries with high resistance rates has
Fund“ zum Aufbau von Laboratorien und Etablie-
been intensified. The presence and the spreading
rung von Netzwerken in ausgewählten Ländern
of extremely resistant tuberculosis strains (XDR-TB)
mit hohen Resistenzraten intensiviert. Das Vor-
put the research of the resistance origin of reserve
kommen und die Verbreitung von extrem resisten-
antibiotics such as Linezolid into the focus. The char-
ten Tuberkulosestämmen (XDR-TB) hat die Erfor-
acterization of the first Linezolid-resistant strains oc-
schung der Resistenzentstehung von Reserveanti-
curred phenotypically and molecular-biologically at
biotika wie Linezolid in den Fokus gerückt. Die
the NRZ. In order to reveal possible resistance
Charakterisierung der ersten Linezolid-resistenten
mechanisms, Linezolid-resistant strains were gener-
Stämme erfolgte phänotypisch und molekularbio-
ated and typed in vitro for the first time. Evaluations
logisch im NRZ. Um mögliche Resistenzmechanis-
of new diagnostic procedures also represent an im-
Linezolid-resistant
strains were generated
and typed in vitro for the
first time
men aufzudecken, wurden erstmals in vitro Line-
portant principal topic at the NRZ. On the one hand,
XDR-TB Stämme
zolid-resistente Stämme erzeugt und typisiert. Eva-
a new hybridisation strip test, the MTBDRplus, for
luierungen neuer diagnostischer Verfahren stellen
the molecular biological determination of resist-
ebenfalls einen wichtigen Schwerpunkt im NRZ
ance against Rifampicin and Isoniazid of the M. tu-
dar. Zum einen wurde ein neuer Hybridisierungs-
berculosis complex was evaluated. On the other
streifentest, der MTBDRplus, zur molekularbiolo-
hand, a new procedure for the direct determination
gischen Resistenzbestimmung gegenüber Rifam-
of M. tuberculosis in the sputum and the simultane-
picin und Isoniazid von M. tuberculosis-Komplex
ous testing for Rifampicin resistance was tested in
evaluiert. Zum anderen wurde in einer internatio-
an international multi-center study (coordinated by
nalen Multi-Center-Studie (koordiniert durch FIND)
FIND) for the first time. In this course the new real-
Wissenschaftlicher Jahresbericht 2007-2008
135
Zusammenfassung
Summary
ein neues Verfahren zum direkten Nachweis von
time PCR-based procedure is tested in comparison
M. tuberculosis im Sputum und der simultanen Te-
with conventional and alternative molecular bio-
stung auf Rifampicin-Resistenz erstmalig erprobt.
logical techniques.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Dabei wird das neue, auf real time-PCR basierende Verfahren gegen konventionelle und alternative molekularbiologische Techniken getestet.
During the last two years, the division of “Molecular
Mycobacteriology” (head: S. Niemann) was able to
successfully implement its research concept with
TB-Genomforschung mit
modernen
ultraschnellen Hochdurchsatz Systemen
world-wide first online
database for the
automated identification
of clinical isolates
Die Molekulare Mykobakteriologie (Leiter: S. Nie-
the focus on “Tuberculosis Epidemiology”, “Spread-
mann) konnte in den letzten zwei Jahren ihr For-
ing and Origin of Resistances”, and “Population
schungskonzept mit den Schwerpunkten „Tuberku-
Structure and Virulence of Clinical M. tuberculosis
lose Epidemiologie“, „Verbreitung und Entstehung
Complex Isolates“. Three new extramurally funded
von Resistenzen“, und „Populationsstruktur und Vi-
projects were granted (BMBF and EU FP7) in this ex-
rulenz von klinischen M. tuberculosis Komplex Iso-
tremely competitive field which allow international-
laten“ erfolgreich umsetzen. So wurden in diesem
ly embedded long-term research. A BMBF grant
äußerst kompetitiven Feld drei neue Drittmittel-
with a main emphasis on genome research with
projekte eingeworben (BMBF und EU FP7), die
modern ultra-fast high-throughput systems is espe-
langfristige Forschungsarbeiten mit internationaler
cially worth mentioning. Within the framework of this
Einbettung ermöglichen. Besonders erwähnens-
project the genomes of more than 10 clinical iso-
wert ist hierbei eine Schwerpunktförderung des
lates will be decoded in order to analyse the high-
BMBF für die Genomforschung mit modernen Ul-
ly interesting question of micro-evolution of clinical
traschnellen Hochdurchsatz Systemen. Im Rahmen
isolates for example in infection chains or in the
dieses Projekts werden die Genome von mehr als
case of a resistance development. The basic results
10 klinischen Isolaten entschlüsselt, um die hoch-
concerning the global population structure of tu-
interessante Fragestellung der Mikroevolution von
berculosis pathogens were used for the establish-
klinischen Isolaten z.B. in Infektketten oder bei Re-
ment of the world-wide first online database for the
sistenzentwicklung zu analysieren. Die grundle-
automated
genden Erkenntnisse zur globalen Populati-
(www.MIRU-VNTRplus.org). Pioneering papers in
onstruktur von Tuberkulose Erregern wurden darü-
high-ranking journals on the origin of extremely re-
ber hinaus zur Etablierung der weltweit ersten On-
sistant tuberculoses and the therapy of MDR pa-
line-Datenbank zur automatisierten Identifizierung
tients represented scientific highlights.
klinischer
Isolate
genutzt
identification
of
clinical
isolates
(www.MIRU-VNTR-
plus.org). Wissenschaftliche Höhepunkte waren
wegweisende Arbeiten in hochrangigen Journalen
The main topics of research in the division of “Clin-
zur Entstehung „Extrem Resistenter Tuberkulosen“
ical Infectiology” (head: C. Lange) were the devel-
unter Therapie von MDR-Patienten
opment and validation of immunological methods
for the quick differentiation of active and latent infections with M. tuberculosis (together with the divi-
TBNET
Forschungsschwerpunkte der „Klinischen Infektio-
sion of “Immunocellular Analytics” at the RCB) and
logie“ (Leiter: C. Lange) waren die Entwicklung
the evaluation of clinical cohorts of patients with
und Validierung immunologischer Methoden zur
multiresistant and extensively resistant tuberculosis
raschen Differenzierung aktiver und latenter In-
(XDR-Tb) in Germany and Europe. The division of
fektionen mit M. tuberculosis (gemeinsam mit der
Clinical Infectiology is scientifically closely connect-
Immunzell-Analytik am FZB) und die Evaluation
ed with other European teams via the TBNET (Tu-
136
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
stenter und extensiv-resistenter Tuberkulose (XDR-
berculosis Network European Trialsgroup), an as-
Tb) in Deutschland und Europa. Über das TBNET
sociation with more than 160 members in 28 coun-
(Tuberculosis Network European Trialsgroup), ei-
tries founded in 2006. Having the overall charge in
nem 2006 gegründeten Studienverbund mit mehr
the TBNET and funded by the European Commis-
als 160 Mitgliedern in 28 Ländern, ist die Klinische
sion, the Clinical Infectiology will build up a data-
Infektiologie mit anderen europäischen Arbeits-
base on multiresistant tuberculosis during the next
gruppen wissenschaftlich eng verbunden. Feder-
years. A German-wide project on host and
führend im TBNET wird die Klinische Infektiologie
pathogen determinants for resistance and disease
in den kommenden Jahren mit Mitteln der Eu-
progression in tuberculosis, supported by the BMBF,
ropäischen Kommission eine klinische Datenbank
represents the division of Clinical Infectiology in
zur multiresistenten Tuberkulose aufbauen. In ei-
clinical and basic research. Projects on the co-in-
nem Deutschland-weiten Projekt zu Wirts- und Pa-
fection of M. tuberculosis and HIV, supported by the
thogen-Determinanten zur Resistenz und Krank-
Hector foundation in Weinheim and the DFG, and
heitsprogression bei der Tuberkulose, welches
on the role of antimicrobial peptides in COPD, sup-
vom BMBF gefördert wird, ist die Klinische Infek-
ported by the DFG, have successfully been able to
tiologie in der Klinischen- und Grundlagenfor-
be carried on in the current year.
schung vertreten. Projekte zur Koinfektion von M.
tuberculosis und HIV, die von der Hector-Stiftung
in Weinheim und der DFG gefördert werden, und
In the Division of “Clinical and Experimental Pathol-
zur Rolle antimikrobieller Peptide bei der COPD,
ogy” (head: E. Vollmer) the examinations of the ex
welches von der DFG gefördert wird, konnten im
vivo tissue culture model were further extended;
laufenden Jahr erfolgreich fortgeführt werden.
they allow functional studies in the human system.
This model is unique and the result of a very fruitful
continuous interdisciplinary cooperation. Mean-
In der „Klinischen und Experimentellen Patholo-
while procedures have been established to carry
gie“ (Leiter: E. Vollmer) wurden die Untersuchun-
out transcriptome analyses of tissue materials from
gen am ex-vivo Gewebekulturmodell weiter aus-
this model which already resulted and will further
gebaut; sie erlauben funktionellen Studien im hu-
result in the discovery of new molecular relation-
manen System. Dieses Modell ist einzigartig und
ships in the course of pulmonary diseases. The ex-
Ergebnis einer sehr fruchtbaren kontinuierlichen in-
pression and the regulation of the chemokine re-
terdisziplinären Kooperation. Zwischenzeitlich
ceptor CXCR7 in non-small-cell lung carcinomas for
wurden Verfahren etabliert, um Transkriptomana-
example have thus been described. In addition, the
lysen an Gewebematerialien aus diesem Modell
identification of a so far unknown pulmonary TGF-β
durchzuführen, welche bereits zur Entdeckung neu-
receptor was possible, which is up-regulated in the
er molekularer Zusammenhänge im Verlauf von
case of an infection and obviously connected with
Lungenerkrankungen geführt haben und auch wei-
pulmonary fibrosis or emphysema formation. Apart
ter führen werden. Beispielsweise wurde so die Ex-
from this, the establishment of proteome-wide
pression und Regulation des Chemokinrezeptors
analysis procedure took place which contributes to
CXCR7 in nichtkleinzelligen Lungenkarzinomen be-
the elucidation of disease-relevant pathomech-
schrieben. Es gelang weiter die Identifizierung ei-
anisms in human lung tissues as additional complex
nes bislang in der Lunge unbekannten TGF-ß-Re-
read-out possibilities.
zeptors, welcher bei Infektion herauf reguliert wird
und augenscheinlich in Zusammenhang mit pulmonaler Fibrosierung bzw. Emphysembildung
In cooperation with groups from the Netherlands,
Wissenschaftlicher Jahresbericht 2007-2008
137
ex-vivo Gewebekulturmodell: Transkriptomanalysen
Zusammenfassung
Summary
steht. Darüber hinaus ist die Etablierung proteom-
the USA, and England, the division of “Cellular Al-
weiter Analyseverfahren erfolgt, welche als zu-
lergology” (head: H. Haas) succeeded for the first
sätzliche komplexe „Read Out“-Möglichkeit zur
time in demonstrating that the glycoprotein Omega-
Aufklärung krankheitsrelevanter Pathomechanis-
1, which is secreted by Schistosoma eggs, triggers
men in humanen Lungengeweben beitragen.
a T-helper type 2 response in vitro and in an animal
Fo r s c h u n g s z e n t r u m
Bo r s t e l
model. This effect, which may be achieved both with
natural and recombinant Omega-1, could also be
The 3-D structure of
IPSE/alpha-1 was
elucidated by means of
NMR and X-ray crystal
structural analysis.
Die Gruppe „Zelluläre Allergologie“ (Leiter: H.
detected in IL-4 receptor knock-out animals and thus
Haas) konnte in Zusammenarbeit mit Gruppen
is not linked to the function of the IL-4 receptor. The
aus den Niederlanden, USA und England erstmals
ability for the induction of the Th2-response makes
zeigen, dass das von Schistosomen-Eiern sezer-
Omega-1 an interesting candidate molecule for
nierte Glykoprotein Omega-1 in vitro und im Tier-
therapeutic application in diseases accompanying
modell eine T-Helfer Typ 2-Antwort auslöst. Dieser
a pathological Th1-phenotype. This is why a patent
Effekt, der sowohl mit natürlichem als auch re-
has been applied for for Omega-1. The European
kombinantem Omega-1 zu erzielen ist, war auch
patent was granted for IPSE/alpha-1, an im-
in IL-4-Rezeptor-knockout-Tieren nachweisbar und
munomodulatory molecule, which is also secreted
ist somit nicht an die Funktion des IL-4-Rezeptors
by Schistosoma eggs. The granting in Japan and
gekoppelt. Die Fähigkeit zur Induktion der Th2-Ant-
the USA is to come shortly. The 3-D structure of
wort macht Omega-1 zu einem interessanten Kan-
IPSE/alpha-1 was elucidated by means of NMR and
didatenmolekül für die therapeutische Anwen-
X-ray crystal structural analysis.
dung bei Erkrankungen, die mit einem pathologischen Th1-Phänotyp einhergehen. Für Omega-1
wurde daher ein Patentantrag eingereicht. Für IP-
The application for the project “In vitro culture of
SE/alpha-1, ein ebenfalls aus Schistosomen-Eiern
Schistosoma mansoni – culture dish instead of mam-
sezerniertes immunmodulatorisches Molekül, wur-
mal final host“ was positively approved by the
de das Europäische Patent erteilt, die Zuteilung in
BMBF. It is the aim of the project to optimise the in
Japan und den USA steht kurz bevor. Die 3-D-Struk-
vitro culture of Schistosoma in such a way that the
tur von IPSE/alpha-1 wurde mittels NMR und Rönt-
larvae of the parasite completely develop in vitro
genkristallstruktur-Analyse aufgeklärt.
until the adult stage inclusive of the laying of ripe
infectious eggs. The in vitro culture is a highly effective instrument for parasitological and immuno-
In vitro-Kultur von
Schistosoma mansoni
Vom BMBF wurde der Antrag „In vitro-Kultur von
logical questions allowing to test the effect of po-
Schistosoma mansoni – Kulturschale statt Säuge-
tential new pharmaceuticals against Schistosoma
tierendwirt“ positiv begutachtet. Ziel des Vorha-
using a high-throughput method and thus to note
bens ist es, die in vitro-Kultur von Schistosomen so
morphological modifications of the parasite after
zu optimieren, dass sich die Larven des Parasiten
drug addition in real-time. This project will lastingly
in vitro komplett bis zum Erwachsenenstadium in-
reduce the number of animal experiments for the re-
klusive Ablage reifer, infektiöser Eier entwickeln.
search of Schistosoma.
Die in vitro-Kultur ist ein hoch-effektives Instrument
für parasitologische und immunologische Fragestellungen, das es u.a. ermöglicht, den Effekt
The award of the internationally announced Kanert
potentieller neuer Pharmaka gegen Schistosomen
prize to the research project “Detection of lipid
im high-throughput-Verfahren zu testen, und dabei
adducts at the panallergens of the peanut (Arachis
morphologische Veränderungen des Parasiten
hypogaea) Ara h 8, Ltp, and Oleosin and investi-
138
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
nach Wirkstoffzugabe in Echtzeit zu registrieren.
gation of their contribution to allergenicity” was an
Dieses Vorhaben wird die Anzahl der Tierversuche
important milestone for the laboratory group of
zur Erforschung von Schistosomen nachhaltig re-
“Molecular and Clinical Allergology” (head: W.-M.
duzieren.
Becker). The prize was handed over to W.-M. Becker and A. Petersen in Davos in September 2008. The
topic of the prize marked the beginning of the new
Ein wichtiger Meilenstein für die Laborgruppe
research field of comparative examinations on the
„Molekulare und Klinische Allergologie“ (Leiter:
interaction and processing of allergens (aeroaller-
W.-M. Becker) war die Zuerkennung des interna-
gens and nutrient allergens) in the respiratory and
tional ausgeschriebenen Karnert-Förderpreises
gastro-intestinal tract (lung and colon) (cooperation
mit dem Forschungsprojekt: Nachweis von Lipi-
with I. Lautenschläger and S. Bade). The common
daddukten an die Panallergene der Erdnuss (Ara-
participation of the teams of “Molecular and Clini-
chis hypogaea) Ara h 8, LTP und Oleosin und Untersuchung ihres Beitrags zur Allergenität. Der
cal Allergology” and “Mucosa Immunology” on the
3rd Joint German Allergy Congress 2008 in Erfurt, at
Preis wurde im September 2008 W.-M. Becker und
which three of nine poster/seminar prizes were
A. Petersen) in Davos überreicht. Die Thematik
awarded to the colleagues of the Research Center
des Preises markierte den Beginn des neuen For-
Borstel, represented another highlight of the year
schungsfeldes der vergleichenden Untersuchun-
2008. In cooperation with T. Goldmann (Clinical and
gen zur Interaktion und Prozessierung von Aller-
Experimental Pathology) the uptake of aeroaller-
genen (Aeroallergenen und Nahrungsallergenen)
gens (grass allergens) could also be detected on
im Respirations- und Gastrointestinaltrakt (Lunge
pulmonary biopsy material for the first time in 2008.
und Darm, Kooperation mit I. Lautenschläger und
By means of microarray analysis, a gene expression
S. Bade). Ein weiterer Höhepunkt des Jahres 2008
cluster could subsequently be determined through
war die gemeinsame Teilnahme der Arbeitsgrup-
activation with the grass pollen major allergen Phl
pen „Molekulare und Klinische Allergologie“ und
p 1. The characterization of the relevant signal trans-
„Mukosaimmunologie“ am 3. Gemeinsamen Deut-
duction is the subject of current investigations.
Kanert Forschungspreis
schen Allergie-Kongress 2008 in Erfurt, auf dem
den Mitarbeitern des Forschungszentrums Borstel
drei von neun Poster-/ Vortragspreisen zuerkannt
Within the period 2007/2008 covered by this report,
wurden. In Zusammenarbeit mit T. Goldmann (Kli-
a number of extramural grant applications were ap-
nische und Experimentelle Pathologie) konnte in
proved for the laboratory group of “Mucosa Im-
2008 die Aufnahme von Aeroallergenen (Grä-
munology” (head: A. Frey): (i) a DFG application be-
serallergenen) erstmals auch an Lungenbiopsie-
longing to the program 1313 with the main empha-
material nachgewiesen werden. Mittels Mikroar-
sis on “Biological Responses to Nanoscale Parti-
ray konnte anschließend das Genexpressionsmu-
cles“ within the cooperation “PARENTRY – How
ster durch die Aktivierung mit dem Gräserpollen-
particles enter the body: investigating particle-bar-
Majorallergen Phl p 1 bestimmt werden. Die
rier interactions at the intestinal mucosa” with the
Charakterisierung der relevanten Signaltransduk-
University of Lübeck and the University of Hamburg,
tion ist Gegenstand laufender Untersuchungen.
(ii) the application made for the Division of Clinical
Medicine within the framework of the pact for research and innovation “Recognition and classifica-
Im Berichtszeitraum 2007/2008 wurden für die La-
tion of antigens at mucosal border areas”, and (iii)
borgruppe „Mukosaimmunologie“ (Leiter A. Frey)
an application within the program “Imaging in dis-
eine Reihe von Drittmittelanträgen bewilligt: (i)
ease processes” being part of the research focus
Wissenschaftlicher Jahresbericht 2007-2008
139
Patent: Division of
Mucosa Immunology
Zusammenfassung
Summary
ein DFG-Antrag im Schwerpunktprogramm 1313
“Biomedical techniques” at the University of Lübeck.
„Biological Responses to Nanoscale Particles“ im
The first patent applied for by the laboratory group
Verbund „PARENTRY - How particles enter the bo-
of “Mucosa Immunology” in 2005 was granted (“Kit
dy: investigating particle-barrier interactions at
for highly sensitive detection assays“ (DE 10 2005
the intestinal mucosa“ mit der Universität zu Lü-
051976)). The patent relates to the system based on
beck und der Universität Hamburg, (ii) für die Ab-
2,4-dichlorophenoxy acetic acid which may be used
teilung Klinische Medizin im Rahmen des Paktes
for example for the labelling and highly sensitive
für Forschung und Innovation gestellte Antrag „Er-
detection of proteins, peptides or nucleic acids. An
kennung und Klassifizierung von Antigenen an mu-
efficient national protection for the technology de-
kosalen Grenzflächen“ und (iii) ein Antrag im
veloped at the Research Center could thus be
Schwerpunktprogramm „Bildgebung bei Krank-
achieved until 2025. The laboratory group of “Mu-
heitsprozessen“ im Forschungsschwerpunkt „Bio-
cosa Immunology” was able to establish and to
medizintechnik“ der Universität zu Lübeck. Das er-
strengthen numerous industrial cooperations (for
ste, von der Laborgruppe Mukosaimmunologie in
example with Boehringer Ingelheim Pharma Ltd. &
2005 angemeldete Patent („Kit for highly sensitive
Co. KG, Karl Storz Ltd. & Co. KG, Atto-Tec Ltd., R-Bio-
detection assays“ (DE 10 2005 051976)) wurde er-
Pharm AG, the Society for Silica Microsystems Ltd.,
teilt. Das Patent betrifft das auf 2,4-Dichlorphe-
Laser and Medicine Technology Ltd. Berlin). Fur-
noxyessigsäure basierende System, welches bei-
thermore, the preparations for first translational
spielsweise zur Markierung und zum hochsensiti-
projects on the subjects of “allergy origin” and
ven Nachweis von Proteinen, Peptiden oder Nu-
“chronic inflammatory respiratory diseases” could
kleinsäuren eingesetzt werden kann. Damit
be concluded.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
konnte wirksamer nationaler Schutz für die am Forschungszentrum entwickelte Technologie bis 2025
Rat model: isolated
perfused small intestine
erlangt werden. Die Laborgruppe Mukosaimmun-
The research group of “Barrier Integrity” (head: I.
ologie konnte zahlreiche Industriekooperationen
Lautenschläger) succeeded in completely establish-
etablieren und festigen (z.B. mit Boehringer In-
ing the isolated perfused small intestine of the rat, a
gelheim Pharma GmbH & Co. KG, Karl Storz
new method for the examination of barrier functions
GmbH & Co. KG, Atto-Tec GmbH, R-BioPharm AG,
of this important surface organ. The intestine model
Gesellschaft für Silizium-Mikrosysteme mbH, Laser-
allows to analyse the barrier, transport, and immuno-
und Medizin-Technologie GmbH Berlin). Des Wei-
functions in the isolated organ in detail via the on-line
teren wurden die Vorbereitungen für erste trans-
access on the decisive compartments (vascular bed,
lationale Projekte zu den Themen „Allergieent-
lumen, lymph, and intestinal tissue). Most recent se-
stehung“ und „chronisch-entzündliche Atemwegs-
ries of experiments show that the intestine in this
erkrankungen“ abgeschlossen.
model now remains stable for at least 4 hours. The
previously available period of time for the observation of different physiological and pathophysiologi-
In der Forschergruppe „Barriereintigrität“ (Leiter:
cal mechanisms thus nearly doubles and may be ex-
I. Lautenschläger) gelang die vollständige Eta-
tended onto the level of gene regulation and gene
blierung des isoliert perfundierten Dünndarms der
products (protein level). By means of this model, the
Ratte, einer neuen Methode zur Untersuchung von
project “Recognition and classification of antigens at
Barrierefunktionen dieses wichtigen Oberflächen-
mucosal border areas” (see above), which is sup-
organs. Das Darmmodell ermöglicht über den On-
ported by the pact for research and innovation in co-
line-Zugriff auf die entscheidenden Kompartimen-
operation with the Clinic of Anaesthesiology of the
te (Gefäßbett, Lumen, Lymphe und Darmgewebe)
UK S-H, Campus Kiel, will decisively be promoted.
140
Wissenschaftlicher Jahresbericht 2007-2008
Zusammenfassung
Summary
detailliert die Barriere-, Transport- und Immun-
The research group of “Cellular Pneumology“
funktionen im isolierten Organ zu analysieren.
(head: C. Stamme) was especially successful in ex-
Neueste Versuchsreihen zeigen, dass der Darm in
tramural fund-raising during the reporting period.
diesem Modell nun über mindestens 4 Stunden
Two DFG applications (1. “Pulmonary C-type lectin
stabil bleibt. Dadurch verdoppelt sich annähernd
regulation of the immune balance to bacterial
das bislang realisierte Zeitfenster zur Beobach-
lipopolysaccharide“, 2. “Endosomal signalling in in-
tung unterschiedlicher physiologischer und pa-
nate immunity“) with a total volume of 600,000 Euro
thophysiologischer Mechanismen, die nun auch
were approved. In addition, further support was
auf die Ebene der Genregulation und der Gen-
granted by the University of Lübeck within the main
produkte (Proteinebene) erweitert werden kann.
research focus of Biomedical Techniques, “Respira-
Mit Hilfe dieses Modells wird das durch den Pakt
tion and Ventilation”. Also the members of the
für Forschung und Innovation geförderte Projekt
group received special personal awards: C.
„Erkennung und Klassifizierung von Antigenen an
Moulakakis concluded her doctoral thesis on the
mukosalen Grenzflächen“ (s.o.) in Kooperation mit
subject of “Pulmonary Surfactant Protein A’s anti-in-
der Klinik für Anästhesiologie des UK S-H, Campus
flammatory modulation of the IkB-a/NF-kB signal
Kiel entscheidend befördert.
transduction pathway” with the overall mark 1.2
and for this was awarded the PhD prize of the district of Bad Segeberg and C. Stamme was “Invited
Die Forschergruppe „Zelluläre Pneumologie“ (Lei-
Chair” at the American Thoracic Society (ATS) Con-
terin: C. Stamme) war im Berichtszeitraum im Be-
gress in Toronto, Canada, in May 2008.
reich der Drittmitteleinwerbung besonders erfolgreich. So wurden 2 DFG-Anträge (1. “Pulmonary
C-type lectin regulation of immune balance to
The laboratory group of “Functional Inflammation
bacterial lipopolysaccharide”, 2. “Endosomal sig-
Research” (head: H.-P. Hauber) puts its main em-
nalling in innate immunity”) mit einem Gesamtvo-
phasis on the pathomechanisms of mucus hyper-
lumen von 600 TEuro bewilligt. Zusätzlich erfolgte
secretion in chronic inflammatory respiratory dis-
eine Weiterförderung durch die Universität zu Lü-
eases (COPD, cystic fibrosis, bronchial asthma) and
beck im Rahmen des Schwerpunktes Biomedizin-
the possibility of therapeutic influences on them as
technik ‚Atmung und Beatmung’. Auch die Mit-
well as on the pathomechanisms of ventilation-in-
glieder der Gruppe erhielten ganz persönliche
duced lung damage. These questions are of great
Anerkennungen: C. Moulakakis schloss ihre Pro-
clinical relevance and reflect important problems of
motion zum Thema „Pulmonary Surfactant Protein
the patients treated in the Medical Clinic (Weaning
κB-α
α/NFA’s anti-inflammatory modulation of the Iκ
Center) so that the findings revealed though inno-
κB signal transduction pathway“ mit der Gesamt-
vative models may be translationally verified and
note 1,2 ab und erhielt dafür 2008 den Promo-
further developed in the clinical situation. The mod-
tionspreis des Kreises Segeberg zuerkannt und C.
el of the explanted human mucosa (explant model)
Stamme war „Invited Chair“ beim Amercian Thor-
from the upper respiratory tract could further be de-
acic Society (ATS) Congress in Toronto, Canada,
veloped. After the mucosa had been stimulated
im Mai 2008.
with bacterial components such as LPS or PAM3 as
well as cytokines (interleukin-4 and interleukin-13
among others) during the past years, it is now pos-
Die Laborgruppe „Funktionelle Entzündungsfor-
sible for the first time to carry out a stimulation with
schung“ (Leiter: H.-P. Hauber) befasst sich schwer-
living bacteria (Chlamydiae and Pseudomonas
punktmäßig mit den Pathomechanismen der Mu-
aeruginosa). By means of this stimulation the ex-
Wissenschaftlicher Jahresbericht 2007-2008
141
Promotionspreis
des Kreises Segeberg:
Christina Moulakakis
Zusammenfassung
Summary
kushypersekretion bei chronisch entzündlichen
plant model may now be used for examinations of
Atemwegserkrankungen (COPD, Cystische Fibro-
the early interaction between pathogen and host in
se, Asthma bronchiale) und ihrer therapeutischen
the upper respiratory tract and complements the
Beeinflussbarkeit sowie den Pathomechanismen
model of infection of the lower respiratory tract in
des beatmungsinduzierten Lungenschadens. Die-
human lungs (D. Drömann, J. Rupp). The ventilation
se Fragestellungen sind von großer klinischer Re-
model of the mouse was also further developed so
levanz und reflektieren wichtige Probleme der in
that intratracheal provocation with bacterial com-
der Medizinischen Klinik behandelten Patienten
ponents became possible. In this way, the early
(Weaning Zentrum), so dass die in innovativen
phase of ventilation-associated pneumonia could
Modellen gewonnen Erkenntnisse translational in
be simulated in an animal model for the first time.
der klinischen Situation überprüft und weiterent-
The expansion of this ventilation mouse model on-
wickelt werden können.
to pre-damaged lungs (by infection, emphysema or
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Model of infection of the
lower respiratory tract in
human lungs
fibrosis) will convey important insights in the understanding of the clinically relevant ventilation situaDas Modell der explantierten humanen Mukosa
tions.
(Explant model) aus den oberen Atemwegen
konnte weiterentwickelt werden: Nachdem in den
vergangenen Jahren die Mukosa mit bakteriellen
Bestandteilen wie LPS oder PAM3 sowie Zytokinen
(u. a. Interleukin-4 und Interleukin-13) stimuliert
wurde, es ist jetzt erstmals möglich eine Stimulation mit lebenden Bakterien (Chlamydien und Pseu-
domonas aeruginosa) durchzuführen. Durch die
Stimulation mit lebenden Bakterienstämmen kann
das „Explant model“ nun zu Untersuchungen der
frühen Interaktionen zwischen Erreger und Wirt im
oberen Respirationstrakt verwendet werden und
ergänzt das Modell der Infektion des unteren Respirationstraktes an humanen Lungen (D. Drömann, J. Rupp). Das Beatmungsmodell der Maus
wurde ebenfalls weiter entwickelt, so dass die intratracheale Provokation mit bakteriellen Bestandteilen möglich wurde. Auf diese Weise konnte erstmals im Tiermodell die Frühphase einer beatmungs-assoziierten Pneumonie simuliert werden. Die Ausweitung dieses Beatmungsmodells in
der Maus auf vorgeschädigte Lungen (durch Infektion, Emphysem oder Fibrose) wird wichtige Erkenntnisse zum Verständnis der klinisch relevanten
Beatmungssituationen vermitteln.
142
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical Immunopharmacology
Research Reports
Division of Clinical Immunopharmacology
Head
model of explanted human mucosal tissue from the
Prof. Dr. Peter Zabel (until 08/08)
upper airways. Calu-3 cells and explanted mucosa
PD Dr. Hans-Peter Hauber (since 08/08)
were stimulated with different concentrations of CP.
Mucus protein expression and mucin gene tran-
Principle Investigator
scription (MUC5AC) were quantified using PAS stain-
PD Dr. Karoline I. Gaede
ing and real time PCR. CP significantly increased
mucus protein and MUC5AC mRNA expression in
Staff scientists and postdoctoral fellows
Calu-3 cells and in the explant model (P < .05). In
Dr. Daniel Droemann
further experiments dexamethasone (DEX) was
Dr. Markus Blaukovitsch
added at different concentrations to CP-stimulated
Dr. Stefanie Heinemann
Calu-3 cells and explanted tissue. DEX significantly
reduced CP-induced mucus protein and MUC5AC
Technicians
mRNA expression in a dose dependent manner
Jessica Hofmeister
(P < .05). DEX-mediated suppression of CP-induced
Simone Ross
mucus expression could be partly antagonized by
Carmen Schöne
addition of the glucocorticoid receptor antagonist
RU 486. Our data suggest that CP can directly in-
Trainees
duce mucus expression in epithelial cells. This effect
Franziska Ganzert
can at least in part attenuated by glucocorticos-
Özke Köz
teroids. Further studies will have to define the exact
Melanie Röpcke
intracellular pathway (eg. MAP kinase).
Romina Pritzkow
Frederike Sührk
Svenja Schulz
Meriam Winterhoff
Research Reports
Chlamydia pneumoniae induces mucus expression that can be attenuated by glucocorticos-
Fig. 55. Epithelial mucus protein expression (red cells) in explanted mucosal tissue from upper airways after 24 h without (A) and
in the presence of Chlamydia pneumoniae (15 cfu/well). PAS staining. Original magnification x 200.
teroids
Collaboration: Goldmann T, Vollmer E, Division of
Hauber HP, Zabel P.
Clinical and Experimental Pathology, Research Center Borstel; Wollenberg B, ENT-Department, Univer-
Chlamydia pneumoniae (CP) can lead to acute and
sity Hospital Schleswig-Holstein, Campus Lübeck;
chronic infection of the lung. Mucus hypersecretion
Rupp J, Department of Microbiology, University Hos-
can often be found in chronic inflammatory lung dis-
pital Schleswig-Holstein, Campus Lübeck.
ease. Although CP induces inflammatory changes in
the airway mucosa it is unclear whether CP can directly stimulate mucus expression. To address this
question the effect of CP on mucus expression was
investigated using in vitro cell culture techniques
(Calu-3, a mucoepidemoid cell line) and an ex vivo
Wissenschaftlicher Jahresbericht 2007-2008
143
Division of Clinical Immunopharmacology
Research Reports
Inhibition of cyclooxygenase-2 reduces bacterial
cause lung injury (ventilator-induced lung injury,
and allergic induced mucus expression in human
VILI). Moroever, ventilated patients are at high risk
airway mucosa
of developing infections of the lower airways (ven-
Heinemann S, Zabel P, Hauber HP.
tilator-associated pneumonia, VAP). We used a
Fo r s c h u n g s z e n t r u m
Bo r s t e l
model of mechanically ventilated mice (C57/Bl6) to
Mucus hypersecretion is frequently observed in
investigate the effect of inhaled lipooplysaccharide
chronic inflammatory lung disease. Excessive mucus
(LPS) and lipopetide (PAM3) on ventilated lungs.
production leads to airway obstruction and supports
We hypothesized to simulate an early phase of VAP.
bacterial infection and colonization of the lung. At
In addition we wanted to compare the effects of LPS
present no specific mucus regulating drug is avail-
and PAM3 since PAM3 has not been investigated
able. Cyclooxygenase(COX)-2 is an important for
very well until yet. Mice were mechanically ventilat-
the generation of prostaglandins (PGs). PGs play an
ed for 120 min. After 30 min intratracheal challenge
important role as mediators in bacterial and aller-
was carried out with sterile saline solution (sham),
gic inflammation. Explanted mucosal tissue from up-
LPS or PAM3. Lung function parameters as well as
per airway mucosa was stimulated with Th2 type cy-
neutropil influx and proinflammatory cytokine ex-
tokines interleukin(IL)-4, IL-9, and IL-13 as well as with
pression (TNFα and MIP-2) were measured. Me-
lipopolysaccharide (LPS) and lipopetide (PAM3). Ep-
chanical ventilation alone significantly increased
ithelial mucus expression was using PAS staining.
resistance and significantly decreased compliance
Th2 type cytokines, LPS and PAM3 significantly in-
over time (P < .05). This effect was augmented by
duced mucus expression (P < .05). By adding the
sham challenge. PAM3 but not LPS significantly in-
COX-2 specific inhibitor rofecoxib bacterial and al-
creased resistance compared to sham (P < .05). Me-
lergic induced mucus expression could be signifi-
chanical ventilation alone significantly increased
cantly decreased (P < .05). Our data suggest that in-
TNFα and MIP-2 mRNA expression compared to
hibition of COX-2 may offer a therapeutic option to
spontaneously breathing animals (P < .05). Both LPS
reduce mucus hypersecretion. Ongoing studies will
and PAM3 significantly increased the numbers of
have to define which PGs are most important for and
neutrophils as well as TNFα and MIP-2 mRNA ex-
which PG receptors are mainly involved in inducing
pression in the lungs compared to sham (P < .05). LPS
mucus expression. Moreover, the effects of blocking
had a stronger effect compared to PAM3 (P < .05).
COX-1 and COX-2 will be compared.
Our data show that LPS as well as lipopetide
(PAM3) augment inflammation in ventilated lungs.
Collaboration: Goldmann T, Vollmer E, Division of
PAM3 but not LPS increases resistance. The under-
Clinical and Experimental Pathology, Research Cen-
lying mechanisms are under current investigation.
ter Borstel; Wollenberg B, ENT-Department, University Hospital Schleswig-Holstein, Campus Lübeck;
Rupp J, Department of Microbiology, University Hospital Schleswig-Holstein, Campus Lübeck
Comparison of the effect of LPS and PAM3 on ventilated lungs
Hauber HP, Zabel P.
Invasive mechanical ventilation is a life saving technique. However, mechanical ventilation itself can
144
Fig. 56. Histology sections of ventilated mouse lungs without (A)
and with intratracheal application of sterile saline (B), LPS (C) and
PAM3(D). Note the increase in neutrophil numbers after challenge
with either LPS or PAM3. HE staining. Original magnification x200.
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical Immunopharmacology
Research Reports
the cytospins. Neutrophils dominated the inflammatory cells in
both OSA patients and healthy
control subjects. The numbers of
lymphocytes were significantly
increased in OSA patients compared to controls (P < .05). In
addition the numbers of lymphocytes increased with severity of OSA. Our data show that
PHAL can be used to investigate local inflammatory cell
profiles in upper airways. Further studies are warranted to
compare the inflammatory cell
counts in the PHAL of OSA patients before and after therapy
with continous positive airway
Fig. 57. MIP-2 and TNFα mRNA expression in spontaneously breathing animals (w/o) and in ventilated mice without (control) and after challenge with sterile saline (sham), LPS or PAM3. Bars indicate
mean values+SEM. *: P < .05 vs w/o. **: P < .05 vs control. +: P <
.05 vs sham. §: P < .05 vs +PAM3.
pressure (CPAP). Moreover, differential cell counts
in the PHAL of OSA patients will have to be compared to heavy snorers without OSA.
Collaboration: Karp D, Division of Inflammation and
Collaboration: Rüller S, Müller E, Sleep Disorder
Regeneration, Research Center Borstel.
Unit, Medical Clinic, Research Center Borstel.
Pharnygeal lavage as a new tool to evaluate lo-
New aspects in obstructive sleep apnea: Coher-
cal inflammation in patients with obstructive
ences with iron metabolism
sleep apnea
Eberhardt F, Zabel P.
Hauber HP, Zabel P.
Study Objectives: Hepcidin and ferritin are both
Obstructive sleep apnea (OSA) is characterized by
known to be influenced by hypoxia, a key feature of
recurrent episodes of apnea and snoring. Previous
the obstructive sleep apnea syndrome (OSAS). Nev-
studies have demonstrated that apnea and hypox-
ertheless, potential effects of therapeutic improve-
ic stress are associated with systemic inflammation.
ments in oxygenation levels on iron metabolism have
Limited data from the literature also indicate that lo-
not been examined in OSAS so far. We assumed the
cal inflammation is present in the upper airways of
main parameters of iron regulation to be sensitive to
patients with OSA. However, sampling of biopsies
improvements in oxygenation levels by continuous
from the upper airways is uncomfortable for pa-
positive airway pressure (CPAP) therapy. Design: Fol-
tients. Therefore we evaluated a new technique, the
low-up study over three months of CPAP therapy.
pharyngeal lavage (PHAL). Patients gargled with
Setting: The sleep laboratory of the Pulmonary Re-
sterile saline solution, cytospins were made and dif-
search Center Borstel, Germany
ferential cell counts were prepared. Squamous ep-
Patients: A group of 56 patients with newly diag-
ithelial cells were the most prominent cell group in
nosed OSAS.
Wissenschaftlicher Jahresbericht 2007-2008
145
Division of Clinical Immunopharmacology
Research Reports
Interventions: Indices of cardiorespiratory polygra-
come a valuable technique to assess airway in-
phy and plasma concentrations of prohepcidin, fer-
flammation and severity of disease in asthmatic
ritin, IL-8 as inflammation marker, and further pa-
subjects and to adjust anti-inflammatory treatment.
rameters of iron regulation were assessed before
However, other detection of exhaled in NO in other
and after three months of scheduled CPAP-therapy.
inflammatory airway disease (eg. pneumonia) has
Results: Cardiorespiratory parameters improved in-
not been studied very well yet.
dicating a successful therapy (p<0.001 for all). Pro-
Patients with community acquired pneumonia (CAP)
hepcidin concentrations increased during CPAP-ther-
were recruited and fractionated exhaled NO
apy (p<0.001) and were correlated with improve-
(FENO) of the alveolar compartment was detected
ments in mean oxygen saturation (MOS, R=0.349,
using the chemiluminescence Analysator CLD 88 sp
p=0.01) and decreases in IL-8 concentrations (R=-
(Eco medics). Detection of neutrophil activation
0.503, p<0.001). Ferritin concentrations decreased
markers CD11b and CD66b was done by FACS
during therapy (p<0.001) with this drop being asso-
analysis in induced sputum. FENO-levels in hospi-
ciated with rises in MOS (R=-0.315, p=0.023) and
talized patients with CAP on admission day com-
decrements in IL-8 (R=0.507, p<0.001). Moreover,
pared to healthy individuals were significantly high-
CPAP-therapy decreased concentrations of iron,
er (P < 05) as well as on admission day compared
transferrin receptor, and lactoferrin (p<0.02 for all)
to a follow up examination five days later (P < .05).
and increased concentrations of UIBC (p=0.01).
In addition we found a significant correlation be-
Conclusions: Three months of successful CPAP-thera-
tween FENO and CD11b and CD66b expression on
py affected most parameters of iron regulation with
neutrophils in induced sputum (P < .05). Further-
prohepcidin and ferritin being correlated to im-
more lower FENO-levels were demonstrated in pa-
provements in mean oxygen saturation and inflam-
tients who received an additional glucocorticoide
mation. Our data indicate a so far neglected clini-
therapy. We conclude that FENO is an adequate
cally relevant relationship between iron metabolism
technique to assess the severity of airway inflam-
and oxygenation levels.
mation and to monitor the response to antibiotic
Fo r s c h u n g s z e n t r u m
Bo r s t e l
treatment in non-severe CAP.
Collaboration: J. Klement, A. Peters (Departments of
Internal Medicine I), M. Voss (Psychiatry and Psy-
Collaboration: Jungnitz C, Dalhoff K, Schaaf B, Med-
chotherapy) and C. Benedict, J. Born, K.M. Oltmanns
ical Clinic III, University Hospital Schleswig-Holstein
(Neuroendocrinology) University of Luebeck; A.J. Ghio
Campus Lübeck
(United States Environmental Protection Agency,
North Carolina, USA); B. Schultes (Interdisciplinary
Obesity Center, Kantonsspital St. Gallen, Switzerland)
Nontypeable Haemophilus influenzae infection
of human lung tissue induces proinflammatory cy-
Supported in part by: Deutsche Forschungsgemein-
tokine response via Toll-like receptor 2 and EGF-
schaft (SFB 654, C2)
receptor
Drömann D, Zabel P.
Exhaled nitric oxide detection for monitoring of
Pulmonary presence of the facultative intracellular
airway inflammation in community acquired
pathogen nontypeable Haemophilus influenzae
pneumonia
(NTHI) is associated with acute infection and has
Drömann D, Zabel P.
been implicated as an infectious trigger in chronic
obstructive pulmonary disease. In addition NTHI
Measurement of exhaled nitric oxide (NO) has be-
146
represents a predominant cause of bacterial pneu-
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical Immunopharmacology
Research Reports
monia and is able to activate EGFR signaling by
fect phenocopy of sarcoidosis, it might be misdiag-
NTHI-derived EGF-like factor. Vital lung specimens
were infected with a suspension of 106 colony form-
nosed as sarcoidosis. In the current it was hypothe-
ing units (cfu) x ml-1 over a periode of 24h. Presence
tients. In a prospective case study, sarcoidosis pa-
of NTHI DNA was detected using in situ hybridiza-
tients were evaluated for potential beryllium expo-
tion (ISH) in in vitro infected lung tissue. CXCL-8 and
sure. In those patients in whom beryllium exposure
TNF
expression was determined by ELISA. Toll-like
was confirmed and beryllium hypersensitivity
receptor- (TLR) 2 mRNA was analyzed by realtime-
demonstrated, the diagnosis of sarcoidosis was re-
PCR. Expression of the MAP-kinases p38 and
jected and corrected to CBD. In 84 patients seen for
p42/44 was determined by western blot.
re-evaluation or making a diagnosis of sarcoidosis,
NTHI DNA was found to be located in 60-75% of
beryllium exposure was recognised and a diagno-
alveolar macrophages (AM) and 15-25 % of epithe-
sis of CBD was made in 34 out of 84 patients. The
lial cells (AEC) in in vitro infected lung tissues. NTHI
time lag between clinical diagnosis of sarcoidosis
infection increased TLR2 mRNA expression and in-
and the final diagnosis of CBD ranged 0-18 yrs (me-
duced a strong release of CXCL-8 (NTHI 325±170 vs
dian 3 yrs) and the mean (range) age at time of di-
control 55±43 µg/ml) and TNF
(NTHI 304±190 vs
agnosis of CBD was 43.9(25-80) yrs. Beryllium-con-
control 39±28 pg/ml) in in vitro infected lung speci-
taminated workplaces causing disease encom-
mens (P < .01). In addition increased expression of
passed a wide spectrum of industries and technical
the MAP-kinases p38 and p42/44 was observed af-
trades in which beryllium-exposure is generally not
ter infection. Blockade of TLR2 and EGFR by mono-
perceived as a health hazard. In conclusion, chron-
clonal antibody as well as specific inhibition of the
ic beryllium disease still belongs to the spectrum of
MAP-kinases by SB203580 and UO126 significantly
differential diagnoses of granulomatous disorders.
sised that CBD exists in cohorts of sarcoidosis pa-
decreased the proinflammatory cytokine response.
In conclusion AM and AEC serve as host cells for pri-
Collaboration: Müller-Quernheim J., Prasse A. and
mary NTHI infection. The strong proinflammatory cy-
Zissel G. Dept. of Pneumology, Medical University
tokine expression in human lung tissue is by medi-
Hospital Freiburg, Freiburg, Germany; Fireman E.
ated by TLR2 and EGFR via the p38 and p42/44
Dept. of Pulmonary and Allergic Diseases, Tel Aviv
MAP-kinase pathway.
Sourasky Medical Center, Tel Aviv, Israel.
Collaboration: Rupp J, Department of Microbiology,
Supported in part by: Deutsche Forschungsgemein-
University Hospital Schleswig-Holstein Campus
schaft, grant MU 692/7-1.
Lübeck; Goldmann T, Division of Clinical and Experimental Pathology, Research Center Borstel; Xu
F, Limmer S, Dalhoff K, Medical Clinic III, University
CC chemokine ligand-2 (CCL2) gene polymor-
Hospital Schleswig-Holstein Campus Lübeck.
phism and protein expression in sarcoidosis
Gaede KI.
Diagnoses of chronic beryllium disease within co-
CC chemokine ligand-2 (CCL2), also known as
horts of sarcoidosis patients
monocyte chemoattractant protein (MCP)-1 is ex-
Gaede KI.
pressed by monocytes and human alveolar epithelial cells type-2 and many others. CCL2 exhibits
An increase in chronic beryllium disease (CBD) has
chemotactic activity towards macrophages/ mono-
been suggested due to higher industrial use of
cytes as well as lymphocytes and induces the pro-
beryllium alloys. Since occupational CBD is a per-
duction of inflammatory cytokines affecting granu-
Wissenschaftlicher Jahresbericht 2007-2008
147
Division of Clinical Immunopharmacology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
loma formation. Its role in the pathogenesis of sar-
Theses
coidosis is under discussion. A biallelic A/ G polymorphism in the distal regulatory region of the CCL2
Habilitation
gene at position –2518 is known to affect the level
Dr. med. Hauber, Hans-Peter
of CCL2 expression in response to inflammatory
Für das Fach ‚Innere Medizin’
stimuli. This study investigated the influence of the
Medizinische Fakultät
CCL2 (-2518) gene polymorphism on alveolar and
Universität Lübeck, 2007.
systemic protein expression, susceptibility to sarcoidosis and its clinical phenotypes. The study population consisted of 123 patients with sarcoidosis
(radiograph type I: n = 20; II: n = 60; III: n = 35;
IV: n = 8) and 119 age matched healthy controls. The
CCL2 polymorphism was genotyped by a PCR-restriction fragment length polymorphism method.
CCL2 protein expression in serum, bronchoalveolar
lavage fluid and supernatants of LPS stimulated
and unstimulated cells of the bronchoalveolar
lavage and the blood was determined by ELISA.
The genotype distribution and the allele frequency
of patients with sarcoidosis showed no significant
differences as compared with controls. Basically,
sarcoidosis patients produced higher levels of CCL2
than controls. The LPS induced CCL2 production by
blood cells correlated significantly with the number
of G alleles in patients with active sarcoidosis
(p < 0.001) and in patients with type II sarcoidosis
(p < 0.05) Interestingly, disease activity evaluated by
increased serum levels of angiotensin converting
enzyme (sACE) correlated significantly with the number of G alleles patients with type I sarcoidosis
(p<0.05). Increased CCL2 production by blood cells
observed in G positive patients with active sarcoidosis is in line with G allele associated sACE concentrations. In summary, our study gives good evidence for a role of the CCL2 G allele as a disease
modifier influencing inflammatory responses and
radiographic type.
Collaboration: Müller-Quernheim J. and Zissel G.
Dept. of Pneumology, Medical University Hospital
Freiburg, Freiburg, Germany.
Supported in part by: Deutsche Forschungsgemeinschaft, grant MU 692/7.
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Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical and Experimental Pathology
Research Reports
Division of Clinical and Experimental
Pathology
Head
Research Reports
Prof. Dr. Dr. Ekkehard Vollmer
What’s going on in human lung tissues?
A human ex vivo model system for pulmonary re-
Principle Investigator
search
PD Dr. Torsten Goldmann
Goldmann T, Schultz H, Lang DS, Kähler D, Stellmacher F, Vollmer E.
Staff Scientists
Dr. Holger Schultz
On the path to enlighten at least some of the com-
Dr. Florian Stellmacher
plex events taking place within pathologic changes
Dr. Jürgen Galle
during pulmonary disorders, we have developed a
Dr. Dagmar S. Lang
novel model system which facilitates to gain insights
into the underlying molecular background. Here we
Graduate and Diploma Students
want to present some current results employing this
Andreas Zyzik
model, which is the result of a continuous fruitful co-
Dipl. Ing. Daniel Kähler
operation of the disciplines biology, pathology, pul-
Mahdi Abdullah
monary medicine, microbiology, allergology, and
Uwe Mertens
surgery.
Sebastian Marwitz
Janine Radtke
Technicians
Angela Kelp
Heike Kühl
Maria Lammers
Waltraud Martens
Heidi Scheibel
Jasmin Tiebach
Rolf Warneke
Tanja Zietz
Secretary
Fig. 58. Schematic overview.
Gabriele Cornehls
Iris Jonas
Methodologically based on the HOPE-technique
Monika Wolgast
which has been brought to applicability in the Clin.
& Exp. Pathology in Borstel, this model uses fresh human lung tissues which are subjected to a short term
tissue culture. During cultivation, these tissues undergo a whole variety of different stimulations before being fixed by the HOPE-technique. After paraffin-embedding these paraffin embedded speci-
Wissenschaftlicher Jahresbericht 2007-2008
149
Division of Clinical and Experimental Pathology
Research Reports
mens are ready to be analyzed by a complete pan-
Small Cell Lung Cancer (NSCLC), with the astonish-
el of molecular and histologic techniques. We have
ing finding of TLR9 to be functionally active ex-
shown that the tissues respond to the stimulations
pressed here, we faced our interest in equal meas-
with remarkable changes in the expression of RNA
ure towards pulmonary infections. An elementary
and proteins as well.
series of experiments dated back to 2003 showed
Fo r s c h u n g s z e n t r u m
Bo r s t e l
that mycobacteria (BCG) are capable of infecting
human lung tissues within the model, that they infect
only certain alveolar macrophages (Fig. 59A, B),
and that this infection seems to have an influence
onto the expression of TLR2 by human alveolar
macrophages (Fig. 59C, D).
This prompted us for further research using different
pathologically relevant infectious agents as, for exFig. 59. Ziehl-Neelsen staining of ex vivo infected human alveolar
macrophages using the STST model and BCG.
B: PCR control of the ex vivo infected lung tissues (1, 4: non infected lungs; 2, 3: infected lungs; Ex and -: controls).
C, D: Detection of TLR2 in ex vivo infected or non infected human
lung tissues (C: non infected, D: infected with BCG).
ample Chlamydia pneumoniae in the course of
COPD (Fig. 60). As a relatively new field we have
now entered allergy, where the effects of different
allergens are being studied in human lung tissues.
As an example we show the uptake of Phlp1 in the
human alveolar compartment (Fig. 61).
Fig. 60. Immunohistochemical staining of Toll-like receptor (TLR)-2
(A) and TLR4 (B) in chronic obstructive pulmonary disease (COPD)
lung tissue (original magnification, x400). (C) TLR2 mRNA expression in controls, Chlamydia pneumoniae (Cpn)+ and Cpn– COPD
(Cpn+, n = 4; Cpn–, n = 4), and in vitro–infected COPD lung tissue
(n = 4). *p < 0.05 compared with controls, Cpn+, and Cpn– COPD
lung tissue. (D) IL-8 secretion in lung tissue after Cpn infection over
24 hours ± blocking of TLR2 and TLR4 by monoclonal antibody. *p
< 0.05 compared with Cpn infection, Cpn infection + isotype antibody and Cpn + anti–TLR4 antibody.
Fig. 61. Immunohistochemical detection of the ex vivo uptake of
Phlp1 in the alveolar compartment of human lung tissues using a
monoclonal antibody and polymer detection with visualization by
HRP and AEC (400x).
By successful application of transcriptome and proteome analyses using archived tissues from this
Currently, this unique system is broadly applied,
model, we are now capable to complexly describe
and demanded to be used, to address problems of
the underlying molecular events during stimulation,
different scientific sectors such as infection, allergy
which also delivers information about novel relevant
and tumor-immunology. After successful initiation of
target-genes in human pulmonary disorders (for ex-
the model, which was designated STST (Short Term
ample the discovery of CXCR7 in NSCLC).
Stimulation of Tissues), mainly for studying immuno-
Taken together, we are assured that this unique in-
logical events during ex vivo stimulation of Non
terdisciplinary model system will continue to take
150
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical and Experimental Pathology
Research Reports
substantial part within the illumination of molecular
key regulator of apoptosis, cleaved caspase-3 was
events in the course of lung diseases by providing
evaluated immunohistologically and by reverse tran-
both functional insights and diagnostic information
scriptase – polymerase chain reaction (RT-PCR); the
as well.
TUNEL labeling assay was additionally performed.In
Collaboration: Drömann D, Dalhoff K, Zabel P, Med.
chemo-sensible CPC-N cells (SCLC) Ki-67 expression
Clinic Borstel, Research Center Borstel and Med. Clin-
was consistently reduced by more than 50 % follow-
ic III, UK-SH Campus Lübeck; Rupp J, Med. Microbiol-
ing chemotherapy, whereas caspase-3 expression
ogy, UK-SH Campus Lübeck; Petersen A, Molecular &
was consistently induced up to 30 % in response to
Clin. Allergology, Research Center Borstel; Branscheid
each anticancer agent. In contrast, in the chemo-re-
D, Hospital Großhansdorf, Center for Lung Diseases
sistant A549 cell line (AC) the Ki-67-index of 90 % re-
and Thoracic Surgery; Ullmer A, Röschmann K, Cellu-
mained nearly unaffected and both caspase-3 pro-
lar Immunology, Research Center Borstel.
tein levels and gene expression were unaltered by the
different chemotherapeutic agents. In addition, one
vital specimen of a patient with chemo-sensitive SCLC
STST (Short term stimulation of tissues): analysis of
and four different samples of human breast cancer tis-
molecular events during lung cancer chemotherapy
sues consistently revealed reduced proliferation and
Lang DS, Schultz H, Vollmer E, Goldmann T.
clearly increased protein levels as well as enhanced
gene expression (in SCLC) of cleaved caspase-3 in re-
To date, no effective chemotherapeutic treatment for
sponse to the individual cytotoxic drugs. Under the
non-small cell lung cancer (NSCLC) exists. Therefore,
chosen culture conditions characteristic differences re-
this type of tumor is characterized by a poor progno-
lated to the histological type of tumor became evident
sis. With regard to the high cellular heterogeneity of
in NSCLC specimens without considerable effects on
NSCLC, experimental data are necessary to elucidate
viability or functionality of the tumor cells: untreated
the molecular mechanisms underlying tumor behav-
adenocarcinomas (AC) exhibited lower proliferation
ior in detail, thus providing the base for development
rates than squamous cell carcinomas (SCC) and af-
of individual and more efficient anticancer treatment
ter cultivation, only SCC showed a statistically signifi-
regimens. Therefore, we used an ex vivo tissue culture
cant enhancement of expression of caspase-3.
model (STST: Short-Term Stimulation of Tissues) in combination with the novel HOPEtechnique (Hepes - Glutamic acid buffer mediated Organic solvent Protection Effect). To
analyze
the
effects
of
conventional
chemotherapy in STST we applied carboplatin, vinorelbine and gemcitabine. A series
of cell culture experiments using A549
(NSCLC), CPC-N (SCLC, small-cell lung cancer), HeLa, and HEK cell lines was performed for means of comparison. Two different chemo-susceptible types of cancer
(breast cancer and SCLC) were also included. We studied possible chemotherapy-induced alterations of multiple known relevant biomarkers in 41
samples of NSCLC. Expression of Ki-67 and BrdU-uptake were assessed by immunohistochemistry. As a
Fig. 62. Individual distribution patterns of Ki 67 protein in human
NSCLC specimens of both adenocarcinoma type (upper panel AC) and of squamous cell carcinoma type (lower panel D-F) in the
absence (RPMI) or presence of 3 different cytotoxic drugs following 16h culture period.
Wissenschaftlicher Jahresbericht 2007-2008
151
Division of Clinical and Experimental Pathology
Research Reports
In response to the individual anticancer drugs, prolif-
ed by this investigation. In NSCLC, the observed
erative activity was always effectively abrogated in all
drug-induced effects upon a number of multiple bio-
tumor samples. Despite a high variability in the Ki-67
markers are in good agreement with existing data
response patterns among the individual NSCLC tissue
from both experimental and clinical studies. How-
specimens, differences in Ki-67 labeling index, which
ever, the consistent differences in the responsive-
were characteristic for the histological type of NSCLC
ness of the human NSCLC samples in STST that
remained evident under chemotherapy with SCC ex-
were closely related to the histological type of tis-
hibiting much less responsiveness to the same drugs
sue have not yet been reported in detail. The
(Fig. 62).
chemo-sensitivity of SCLC and breast cancer that
Fo r s c h u n g s z e n t r u m
Bo r s t e l
was consistently evident following short-term cultiIn addition, AC exhibited higher expression of
vation was in marked contrast to the effects seen in
cleaved caspase-3 levels when compared to SCC in
the more chemo-resistant NSCLC specimens, thus re-
response to chemotherapeutical treatment condi-
vealing the close correlation of our experimental
tions that were not statistically significant (Fig. 63).
data to the situation in vivo. This experimental ap-
Likewise, transcription of caspase-3 in four different
proach should also be applicable for other types of
NSCLC samples (SCC n = 3, AC n = 1) did not exhibit
cancer and it should also be possible to elucidate
a significant upregulation in response to any of the
molecular mechanisms within tumor-free tissues,
cytotoxic drugs, which is in marked contrast to the
e.g. the events taking place upon corticosteroid
highly responsive SCLC.
treatment within the human lung.
Collaboration: Zabel P, Drömann D, Med. Clinic Borstel, Research Center Borstel and Med.
Clinic
III,
UK-SH
Campus
Lübeck; Branscheid D, Hospital
Großhansdorf, Center for Lung
Diseases and Thoracic Surgery;
Ressmeyer A, Martin C, Pulmonary
Pharmacology,
Re-
search Center Borstel; Zeiser T,
Dept. of Gynecology, Asklepios
Hospital, Bad Oldesloe.
Fig. 63. Individual distribution patterns of activated caspase-3 protein in human NSCLC specimens of both adenocarcinoma type (upper panel A-C) and of squamous cell carcinoma type (lower panel D-F) in the absence (RPMI) or presence of 3 different cytotoxic
drugs following 16h culture period.
The chemokine receptor CXCR7 is consistently expressed in human Non Small Cell Lung Cancer
The major focus of this study was to examine the re-
and tumor-free lung tissues with possible regula-
liability of a novel ex vivo tissue model (STST) and
tion upon chemotherapy
to evaluate multiple aspects of human lung tumor
Goldmann T, Radtke J, Marwitz S, Lang DS,
behavior in response to conventional chemothera-
Schultz H, Vollmer E.
py. With such an experimental approach, not only
the complexity within the tissue is maintained but al-
Various chemokine receptors are expressed by ma-
so the heterogeneity among individual patients can
lignant tumors and have been associated with tu-
be studied directly, which was clearly demonstrat-
mor progression and metastasis. A recent study
152
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical and Experimental Pathology
Research Reports
demonstrated the promotion of tumor growth in
Lower panel: RT-PCR targeting CXCR7 and GAPDH mRNA in human NSCLC after STST:
a: medium controls; b: stimulation with carboplat and gemcitabine.
M=pBr322 Msp1; upper signals: A 257 bp fragment of GAPDH; lower signals: CXCR7.
B, C, D) In situ hybridization targeting mRNA of CXCR7 in human
lung tissues using a digoxigenin-labeled DNA-probe and new-fuchsine as a chromogen:
B) Adenocarcinoma of the lung (400x)
C) Alveolar macrophages (400x)
D) Alveolar epithelial cell type II, see arrow (1000x).
breast and lung cancer by CXCR7, formerly known
as orphan receptor CMKOR1/RDC1 utilizing cell
lines and animal models. CXCR7-expression in human breast and lung cancer specimens, as well as
in the tumor vasculature was shown on the proteinlevel by immunohistochemistry. Here we employed
human lung tissues treated with the novel HOPEtechnique to analyze the overall expression of CR-
Stimulation of tumor tissues with physiological con-
CX7 on the mRNA-level in Non Small Cell Lung Can-
centrations of carboplat and gemcitabine (N=30)
cer (NSCLC) and tumor-free lung tissues. Addition-
was performed within the human model system des-
ally, the effect of chemotherapy upon the transcrip-
ignated STST (Short Term Stimulation of Tissues) in
tion of CRXR7 was addressed, employing a
comparison to corresponding tissues, which re-
currently established human model system. Apply-
ceived cultivation without stimulation. Here we
ing RT-PCR with a specific primer set spanning an
found an up-regulation of CXCR7-transcription upon
amplicon of 156bp (5’–TGCTGGACATCTTCTCCATC-
chemotherapy in 13.4% of the cases, down-regula-
3’ forward, 5’-CTTCATCAGCTCCTACCT-3’ reverse),
tion in 33.3%, and no regulation in 53.3%, as deter-
we found transcription of CXCR7 in all of the 30
mined by semiquantitative RT-PCR in relation to
NSCLC samples analyzed. A set of 10 examples is
GAPDH (exemplified in the lower panel of figure 1a;
shown in Fig. 64A. Specificity of the RT-PCR was ver-
note up-regulation of CXCR7 in sample 4 upon
ified by sequencing of the PCR-products. The results
chemotherapy).Taken together, our study verifies
of RT-PCR were then validated by in situ hybridiza-
the recently reported results on the mRNA-level in a
tion (N=21) using a double stranded DNA-probe la-
comparably large amount of specimens. However,
beled with digoxigenin, which revealed transcripts
although some signals were occasionally observ-
of CXCR7 within the tumor cells of 17 samples (Fig.
able, we did not detect strong expression within the
64B) with only some occasional signals in the tumor-
tumor-associated vasculature on the RNA-level. The
associated vasculature. Moreover, 20 specimens of
effects of chemotherapy onto CXCR7 are heteroge-
tumor–free human lungs were also subjected to in
neous. Nevertheless, the same holds true for the
situ hybridization and all revealed transcripts local-
overall characteristics of the tumors themselves and
ized mainly in alveolar macrophages (Fig. 64C),
their behavior upon chemotherapeutic interventions
and in cells, which reflect the morphology of alveo-
within the patients. Therefore, the effects of
lar epithelial cells type II (Fig. 64D).
chemotherapy onto CXCR7 might be valuable hints
towards an understanding of the broad chemotherapy-resistance found in NSCLC. We have additionally shown a consistent transcription of CXCR7 within tumor free lung tissues suggesting a possible new
function of this receptor in non-malignant processes within the human lung.
Collaboration: Drömann D, Med. Clinic III, UK-SH
Campus Lübeck; Branscheid D, Hospital GroßhansFig. 64. A) Upper panel: RT-PCR targeting CXCR7 and GAPDH mRNA in human NSCLC:
M=pBr322 Msp1; upper signals: A 257 bp fragment of GAPDH; lower signals: CXCR7.
dorf, Center for Lung Diseases and Thoracic Surgery.
Wissenschaftlicher Jahresbericht 2007-2008
153
Division of Clinical and Experimental Pathology
Research Reports
On the role of Transketolase-like enzyme 1 (TKTL1)
cubated for 30 min. in a 1:100 dilution. We used an
human lung tissues and Non Small Cell Lung Cancer
enzyme-polymersystem (ZytochemPlus HRP Polymer
Schultz H, Kähler D, Vollmer E, Goldmann T.
Kit, Zytomed Systems) for sensitive detection with
Fo r s c h u n g s z e n t r u m
Bo r s t e l
permanent AEC (Zytomed Systems) as chromogen.
It has recently been shown that TKTL1 particularly
Tissue arrays were counterstained by incubation in
influences total transketolase activity and cell pro-
Mayer’s haemalum for 5 minutes. Negative-controls
liferation by providing the ability of tumors to de-
were included in each staining series under omis-
grade glucose through the anaerobic transketolase-
sion of primary antibody. The immunohistochemical
dependant pentose phosphate pathway. TKTL1 mR-
stainings showed elevated expression of TKTL1 in
NA silencing (via small interfering anti-TKTL1-mRNA
human lung cancer: 40.9% expressed TKTL1 weakly
constructs) leads to inhibition of cell proliferation in
(score 1), 38.6% moderately (score 2), 17.1% strong-
colorectal cancer; protein overexpression and a sig-
ly (score 3), and 3.4% of the tumors were TKTL1-neg-
nificant correlation to Her2 overexpression was
ative (score 0). Breast cancer specimen stainings
found in breast cancer cells where 89 % expressed
were scored 0: none; scored 1: 32%; scored 2: 36%;
TKTL1 and 45% showed strong expression. Gastric
scored 3: 32%. In general, adenocarcinomas were
tumors and granulosa cell tumors of the ovary were
more often strongly positive (score 3) than the squa-
also found to express high amounts of TKTL1 (36.9 %;
mous cell carcinomas (22.1 % for adenocarcinomas
81 %). Further, with renal cancer another carcinoma
and 12.2% for squamous cell carcinomas). Staining
displayed intensively elevated transketolase activi-
of the HOPE-fixed specimens showed comparable
ty due to TKTL1-upregulation. Proliferation-influenc-
results; these results verify findings in the five cases
ing activities of TKTL1 might play a role in a variety
of adenocarcinomas of the lung previously de-
of cancers. Hence we investigated a capacious col-
scribed. Signals were recognized in the cytoplasm
lective of 88 formalin-fixed non-small cell lung can-
and occasionally in nuclei of tumor cells.
cer (NSCLC)-tissues (39 adenocarcinomas; 49 squamous cell carcinomas) by immunohistochemistry to
describe TKTL1 protein expression in human lung
carcinomas. A collective of 24 breast cancer specimens was also included in the study (21 invasiveductal; 1 tubular; 1 lobular; 1 mucinous carcinoma).
Further, we challenged a potential correlation of TKTL1 to age, sex, TNM-classification and grading as
well as TTF1 and SPA expression. Tissues were fixed
with formalin and paraffin embedded. Additionally
we investigated 40 HOPE (Hepes Glutamic Acid
Buffer Mediated Organic Solvent Protection Effect)fixed NSCLC-tissue probes. For increased inter specimen comparability and even staining quality we utilized Tissue Microarrays (TMAs) using an MTA1
Fig. 65. Examples of TKTL1-stained A) adenocarcinomas; B) squamous cell carcinomas; C) breast cancer specimens; D) TKTL1-positive macrophages in the neighbourhood of an adenocarcinoma
and E) TKTL1-positive AEC II cells in the neighbourhood of an adenocarcinoma (arrows).
(Manual Tissue Arrayer 1) device (Alphametrix). For
There was a conspicuous amount of signal-contain-
homogeneous high-throughput staining conditions
ing alveolar macrophages and alveolar epithelial
the tissues were stained automatically (Autostainer
cells type II (AEC II cells), observed in the close
480, Medac, Germany). TMAs were deparaffinized
neighborhood of the NSCLC (Fig. 65 D, E) as well as
and rehydrated; primary antibody (anti-TKTL1;
in completely healthy parts of lung tissue (not
clone: JFC12T10; Zytomed Systems, Berlin) was in-
shown). Examples of stained tumor types, alveolar
154
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical and Experimental Pathology
Research Reports
macrophages and AEC II cells are shown in figure
sic sciences continuously contributes to direct scien-
8 A-E. No staining was found in negative controls.
tific output. This requires intensive collaborations with
Comparison of TKTL1-expression to several clinical
other hospitals and provides indispensable sources
parameters revealed that there is no significant cor-
of material for various research activities.
relation between TKTL1-appearance and age, sex,
We thank these colleagues of the different hospitals
TNM-classification parameters or tumor grading.
we cooperate with in routine diagnostic services for
Further, no correlation could be defined concerning
their confidence in our work. In cooperation with the
SPA and TTF1 expression.
National Reference Center for Mycobacteria we
have analyzed different tissues infected with NTB My-
A high variability of TKTL1-expression has been de-
cobacteria and found curious strains by sequencing,
scribed in several tumor types. There is proven evi-
like M. tilburgii, M. neoaurum or other acid fast germs
dence that TKTL1-associated or -inducing, abnormal
like Rhodococcus equi. As a molecular technique cru-
glucose degradation is increased in tumors. But TK-
cial for the decision regarding targeted therapy of
TL1 is also a key enzyme in the healthy organism
colorectal carcinomas with an Anti-EGF-R regimen,
whose activation-status influences the balance of
we have established the analysis of K-ras mutations
anaerobic glucose- or oxygen-focussed bioenergy-
out of formalin-fixed, paraffin-embedded tissues
obtainment. Natural appearance is proven through
again in close cooperation with the National Refer-
positive detected macrophages and AEC II cells (Fig.
ence Center for Mycobacteria Molecular techniques
65 D, E) as well as signals in healthy lung tissues.
are increasingly becoming important in routine di-
Therefore tests on cancer cells or cancer tissues
agnostics, the application of which - due to the use
alone are not capable to gain insights into the com-
of formalin as a fixative - is restricted to a large de-
plex situation in patients subjected to diets or other
gree. In cooperation with our clinical partners we
treatments. We have shown that a large portion of
have now continued to implement the HOPE-tech-
NSCLC overexpresses TKTL1; moreover there was a
nique into routine pathology as a base for improved
significant expression in non malignant cells of the
diagnostics and therapeutics, especially within the di-
lungs. Although it is tempting to speculate of a po-
agnostics necessary to apply targeted therapies, to
tential therapeutic benefit by modulation of TKTL1-ac-
the welfare of patients.Also embodied in clinico-
tivity in the future, further studies are necessary to in-
pathological interactions where the diagnostic ex-
vestigate the real amount of effects during TKTL1-tar-
pertise of tissue analyzes is indispensable, we are
geted diets or therapies within the human organism.
engaged in several scientific multicenter studies concerning NSCLC and are an active member of the Eu-
Collaboration: Zabel P, Med. Clinic Borstel, re-
ropean Study Group on Rare Lung Diseases. We
search Center Borstel and Med. Clinic III, UK-SH
have continued the work as a reference center for the
Campus Lübeck; Branscheid D, Hospital Großhans-
quality control in molecular pathology detecting my-
dorf, Center for Lung Diseases and Thoracic Sur-
cobacteria by the Professional as well as the Scien-
gery; Zeiser T, Dept. of Gynecology, Asklepios Hos-
tific Society of German Pathologists.
pital, Bad Oldesloe.
Theses
Scientific observations in diagnostic pathology
Vollmer E, Goldmann T, Schultz H, Galle J, Stell-
Diploma
macher F.
Kähler, Daniel
Hochdurchsatzanalysen molekularer Biomarker an
Routine pathology as a link between clinical and ba-
humanen Lungenkarzinomgeweben.
Wissenschaftlicher Jahresbericht 2007-2008
155
Division of Clinical and Experimental Pathology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Technisch-Naturwissenschaftliche Fakultät
Universität Lübeck, 2008.
Bachelor of Science
Marwitz, Sebastian
Molekularbiologische Analyse von Entzündungsmediatoren in humaner Lunge.
Studiengang Molecular Life Sciences
Universität zu Lübeck, 2008.
Radke, Janine
Molekularbiologische Gewebeuntersuchungen von
Genregulationen an humanen Nichtkleinzelligen
Lungenkarzinomen.
Studiengang Molecular Life Sciences
Universität zu Lübeck, 2008.
156
Wissenschaftlicher Jahresbericht 2007-2008
Division of Mycobacteriology and
National Reference Center for Mycobacteria
Research Reports
Division of Mycobacteriology and
National Reference Center for Mycobacteria
Head
Dr. Sabine Rüsch-Gerdes
Research Reports
Worldwide emergence of extensively drug-resist-
Deputy
ant tuberculosis
PD Dr. Elvira Richter
Rüsch-Gerdes S.
Principle Investigator
Multidrug-resistant tuberculosis (MDR TB) has been
Dr. Doris Hillemann
documented in nearly 90 countries and regions
worldwide. Treatment for MDR TB patients requires
Secretary
use of second-line drugs for >24 months. To facilitate
Birgit Voß
treatment of MDR TB in resource-limited countries,
where most TB cases occur, the World Health Or-
Graduate and Diploma Students
ganization (WHO) and its partners developed the
Janina Kolb
Green Light Committee, which helps ensure proper
use of second-line drugs, to prevent further drug re-
Technicians
sistance. Nonetheless, the Green Light Committee
Kristine Beuck
encountered numerous anecdotal reports of MDR
Manuela Dorn
TB cases with resistance to most second-line drugs.
Gudrun Heinonen
Once a strain has developed resistance to second-
Silvia Höllger
line drugs, these new TB strains are even more dif-
Margrit Kernbach
ficult to treat with existing drugs. Mycobacterium tu-
Anne-Kathrin Landgraf
berculosis strains that are resistant to an increasing
Kirsten Ott
number of second-line drugs used to treat MDR TB
Ilse Radzio
are becoming a threat to public health worldwide
Frauke Schaefer
(Fig. 66).
Birte Schlüter
Daniela Stephan
Tanja Struwe-Sonnenschein
Ann-Kathrin Witt
Research Reports
Guests and Trainees
approx. 110 national and international guests attended the NRC for
advanced training in 2007 and
2008
Fig. 66. XDR-TB cases world-wide, www./WHO/HTM/TB/2008.394,
Anti-tuberculosis drug resistance in the world, Fourth global report.
In this study the Network of Supranational Reference
Wissenschaftlicher Jahresbericht 2007-2008
157
Division of Mycobacteriology and
National Reference Center for Mycobacteria
Research Reports
Laboratories was surveyed for M. tuberculosis iso-
multidrug-resistant and extensively drug-resistant tu-
lates that were resistant to second-line anti-TB drugs
berculosis, resistance to the injectable drug capre-
during 2000–2004. Extensively drug-resistant TB (XDR
omycin was an independent predictor for therapy
TB) was defined as MDR TB with further resistance to
failure in this cohort. As Mycobacterium tuberculo-
>3 of the 6 classes of second-line drugs. Of 23 eligi-
sis drug resistance is increasing worldwide, there is
ble laboratories, 14 (61 %) contributed data on 17,690
an urgent need for novel interventions in the fight
isolates, which reflected drug susceptibility results
against tuberculosis.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
from 48 countries. Of 3,520 (19.9 %) MDR TB isolates,
347 (9.9 %) met criteria for XDR TB. Further investiga-
Collaboration: WHO Collaborating Centre for TB
tion of population-based trends and expanded ef-
and Lung Diseases, Fondazione S. Maugeri, Care
forts to prevent drug resistance and effectively treat
and Research Institute, via Roncaccio 16, 21049,
patients with MDR TB are crucial for protection of
Tradate, Italy. [email protected]
public health and control of TB.
Collaboration: various members of the Suprana-
Multidrug-resistant tuberculosis treatment out-
tional Reference Laboratories Network.
comes in Karakalpakstan, Uzbekistan: treatment
complexity and XDR-TB among treatment failures
Rüsch-Gerdes S.
Resistance to second-line injectables and treatment outcomes in multidrug-resistant and exten-
A pilot programme to treat multidrug-resistant TB
sively drug-resistant tuberculosis cases
(MDR-TB) was implemented in Karakalpakstan,
Rüsch-Gerdes S.
Uzbekistan in 2003. This region has particularly high
levels of MDR-TB, with 13% and 40% among new and
No information is currently available on the influ-
previously treated cases, respectively. The study de-
ence of injectable second-line drugs on treatment
scribes the treatment process and outcomes for the
outcomes of multidrug-resistant (MDR) and exten-
first cohort of patients enrolled in the programme,
sively drug-resistant (XDR) tuberculosis (TB) patients.
between October 2003 and January 2005. Con-
To investigate this issue, a large series of MDR- and
firmed MDR-TB cases were treated with an individ-
XDR-TB cases diagnosed in Estonia, Germany, Italy
ualised, second-line drug regimen based on drug
and the Russian Federation (Archangels Oblast) be-
susceptibility test results, while suspected MDR-TB
tween 1999 and 2006 were analysed. All study sites
cases were treated with a standardised regimen
performed drug susceptibility testing for first- and
pending susceptibility results. Of 108 MDR-TB pa-
second-line anti-TB drugs, laboratory quality assur-
tients, 87 were started on treatment during the study
ance and treatment delivery according to World
period. Of these, 33 (38%) were infected with strains
Health Organization recommendations. Out of
resistant to at least one second-line drug at base-
4,583 culture-confirmed cases, 240 MDR- and 48
line, but none had initial ofloxacin resistance. Treat-
XDR-TB cases had a definitive outcome recorded
ment was successful for 54 (62%) patients, with 13
(treatment success, death, failure). Among MDR-
(15%) dying during treatment, 12 (14%) defaulting
and XDR-TB cases, capreomycin resistance yielded
and 8 (8%) failing treatment. Poor clinical condition
a higher proportion of failure and death than capre-
and baseline second-line resistance contributed to
omycin-susceptible cases. Resistance to capre-
treatment failure or death. Treatment regimens
omycin was independently associated with un-
were changed in 71 (82%) patients due to severe ad-
favourable outcome (logistic regression analysis:
verse events or drug resistance. Adverse events
odds ratio 3.51). In the treatment of patients with
were most commonly attributed to cycloserine,
158
Wissenschaftlicher Jahresbericht 2007-2008
Division of Mycobacteriology and
National Reference Center for Mycobacteria
Research Reports
ethionamide and p-aminosalicylic acid. Extensively
to generate and subsequently characterize linezol-
drug resistant TB (XDR-TB) was found among 4 of
id resistant M. tuberculosis strains by determination
the 6 patients who failed treatment and were still
of MIC-values, analysis of growth characteristics,
alive in November 2006. While acceptable treat-
and mutations in the putative target gene. Ten in vit-
ment success was achieved, the complexity of treat-
ro-selected linezolid-resistant Mycobacterium tu-
ment and the development of XDR-TB among treat-
berculosis mutants were isolated and character-
ment failures are important issues to be addressed
ized. Strains with mutations in the 23S rDNA gene
when considering scaling up MDR-TB treatment.
(G2576T and G2061T) showed decelerated growth
and higher levels of MIC (minimal inhibitory con-
Collaboration: Cox H, Macfarlane Burnet Institute
centration)-values (16 and 32µg/ml) compared to
for Medical Research and Public Health, Mel-
those without mutations in the 23S rDNA (MIC-values
bourne, Australia; Kalon S, Allamuratova S,
4 and 8µg/ml).
Médecins Sans Frontières, Tashkent, Uzbekistan;
Sizaire V, Médecins Sans Frontières, London, United
Kingdom; Tigay ZN,4 Karimovich HA, Ministry of
Evaluation of the GenoType® MTBDRplus Assay
Health, Karakalpakstan, Uzbekistan; Kebede Y,
for rifampicin and isoniazid susceptibility testing
Mills C, Médecins Sans Frontières, Amsterdam, The
of Mycobacterium tuberculosis strains and in clin-
Netherlands.
ical specimens
Hillemann D, Richter E, Rüsch-Gerdes S.
In vitro selected linezolid-resistant Mycobacteri-
The worldwide increase of multidrug-resistant tu-
um tuberculosis mutants
berculosis points out how the importance of timely
Richter E, Hillemann D, Rüsch-Gerdes S.
identification of resistant Mycobacterium tuberculo-
sis (MTBC) strains to achieve effective disease manLinezolid has been a valid alternative drug in the
agement and prevention of its spreading. Recently,
management of multidrug resistant tuberculosis. It
nonradiometric fully automated systems for resist-
belongs to an entirely new class of antibiotics, the
ance screening were introduced with technical and
oxazolidinones, and has a spectrum of activity
safety advantages. However, the time for resistance
against virtually all important gram-positive
testing still is around 7 to 10 days beginning from
pathogens. Linezolid interacts with the domain V of
the positive culture. The most rapid results could be
the 23S rRNA, which is part of the peptidyltrans-
achieved by direct testing of patient specimens with
ferase center of the ribosome although not inhibiting the peptidyltransferase activity. So far, the first
fast molecular methods. DNA strip assays targeting
rpoB plus katG (GenoType® MTBDR; Hain Life-
in vitro generated linezolid-resistant mycobacteria
science, Nehren, Germany) were developed and
were M. smegmatis clones. Within these, two class-
evaluated for MTB cultures and spear-positive spec-
es of mutants were found. Mutants of the first class
imens. DNA strip assays are based on a multiplex
had alterations in the domain V of 23S rRNA
PCR in combination with a reverse hybridization. In
(G2447T, according to Escherichia coli numbering
order to enlarge the detection capacity of drug re-
system), high MIC-values of ≥64 µg/ml, and a de-
sistance, a new GenoType® MTBDRplus assay was
creased growth rate in culture. The other mutants
developed including a broader variety of rpoB
showed wild-type growth characteristics in cultures,
gene mutations and inhA gene mutations. Covering
lower MIC-values of 4-8 µg/ml, and no mutation in
mutations in the regulatory region of inhA, it can be
the 23S rRNA, pointing to a non-ribosomal mecha-
expected that additional INH-resistant strains can
nism of resistance. In the present study, we aimed
be detected. The aim of the present study was to
Wissenschaftlicher Jahresbericht 2007-2008
159
Division of Mycobacteriology and
National Reference Center for Mycobacteria
Research Reports
determine the sensitivity and accuracy of the new
The MDR isolate 2 showed RMP resistance in both
MTBDRplus assay in comparison to the MTBDRas-
assays (MTBDR: omission of rpoB WT5 and positive
say for the detection of the presence of INH and
for rpoB MUT3; MTBDRplus: omission of rpoB WT8
RMP resistance-associated mutations in rpoB, katG,
and positive for rpoB MUT3). Concerning INH re-
and inhA from culture specimens and directly from
sistance isolate 2 had to be interpreted as INH sus-
smear-positive clinical specimens. Compared to the
old MTBDR assay the new GenoType® MTBDRplus
whereas the MTBDRplus indicated INH resistance
assay enhanced the detection of isoniazid resist-
(omission of the inhA WT1 probe, positive for the in-
ance from 102 (87.8%) to 106 (90.2%) of 116 INH resistant strains. Thus, the new GenoType® MTBDR-
hA MUT1 probe).
plus assay represents a reliable and upgraded tool
Collaboration: Allerheiligen V, Hain Lifescience
for the detection of INH and RMP resistance in
GmbH, Germany.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
ceptible (katG WT probe) by the MTBDR assay,
strains or directly from smear-positive specimens.
Mycobacterium bohemicum and cervical lymphadenitis in children
Richter E.
Members of the genus Mycobacterium are well-established causes of granulomatous lymphadenitis in
children. M. bohemicum was first described in 1998
in a patient with Down syndrome. The organism is
characterized by a unique 16S rRNA gene sequence
and has been isolated from humans, animals, and
the environment. We report on 4 cases of cervical
lymphadenitis caused by M. bohemicum that occurred in 4 children (2 boys, 2 girls) from Austria during 2002–2006. From all patients, parts of the exFig. 67. Representative patterns of a pansusceptible (lanes 1) and
an MDR strain (lanes 2), which had an inhA C15T mutation in the
regulatory region of inhA obtained with the new MTBDRplus assay
(B) but not the MTBDR (A). The positions of the oligonucleotides
and the marker lines are given. The targeted genes and specificity are shown from left to right as follows. The MTBDR assay (A):
conjugate control; amplification control; M. tuberculosis complexspecific control; rpoB amplification control; rpoB wild-type probes
located in the hotspot region WT1-5; rpoB mutant probes (Mut1,
2A, 2B, and 3) with mutations in codons rpoB D516V, H526Y, H526D,
S531L; katG amplification control; katG codon 315 wild-type probe;
katG codon 315 mutation probes (MUT1 and MUT2) with AGC-toACC (S315T1) and AGC-to-ACA (S315T2) exchanges. The MTBDR
plus assay (A) has additional three rpoB WT probes (resulting in
WT 1-8) and targets the regulatory region of the inhA gene with
the inhA amplification control; inhA gene wild-type probes WT1
spanning region -9 to -22 and WT from -1 to -12; inhA mutant probes
MUT1, 2, 3A and 3B with mutations -15C/T, -16A/G, -8T/C and -8T/A.
cised lymph nodes were directly used for culture
and M. bohemicum was identified by DNA
sequencing of part of the 16S rRNA gene. Nontuberculous mycobacterial cervical lymphadenitis is
most frequently caused by M. avium (80%), M. mal-
moense, M. kansasii, M. lentiflavum, M. haemophilum, and M. scrofulaceum. Because the phenotypic characteristics of M. bohemicum closely resemble those of M. scrofulaceum, these species can
easily be misidentified by analysis of biochemical
and cultural features only. M. bohemicum infection
has only been reported in 5 patients worldwide. The
high rate of 4 cases of M. bohemicum occurring in
The pansusceptible isolate 1 was positive with the
Austria as a relatively small country (8 million in-
wild-type rpoB and katG probes of the MTBDR, and
habitants) could be an indication, that infections
additionally inhA wild-type probes of MTBDRplus.
with M. bohemicum may be more common than
160
Wissenschaftlicher Jahresbericht 2007-2008
Division of Mycobacteriology and
National Reference Center for Mycobacteria
Research Reports
previously thought. More such cases may be dis-
the Escherichia coli numbering system) and pheno-
covered as a result of improved microbiologic di-
typic characteristics group the species among the
agnostic techniques. We believe that M. bo-
slowly growing mycobacteria.It was proposed to
hemicum should be listed among the species that
name the new species Mycobacterium alsiense,
induce nontuberculous mycobacterial infections.
pertaining to the Isle of Als (Denmark), the location
Collaboration: Huber J, Eberl, R, Zenz,W, Medical
of the hospital to which the first patient was admit-
University of Graz, Graz, Austria; Binder L Elisa-
ted.
bethinen Hospital, Linz, Austria, Maaß, M, UniversiCollaboration: Engel R, Research Center Borstel,
ty Hospital Salzburg, Salzburg, Austria.
Structural Biochemistry; Tortoli E, Regional Reference Center for Mycobacteria, Careggi Hospital,
Mycobacterium alsiense, a novel, slowly growing
50134 Florence, Italy; Kristiansen JE, Hendricks, O,
species isolated from two patients with pul-
Department of Research and Department of Clini-
monary disease
cal Microbiology, Sygehus Sønderjutland/ Sønder-
Richter E, Hillemann D.
borg, University of Southern Denmark, 6400 Sønderborg, Denmark; Fischer A, Schubert, S, Institute
A previously undescribed, slowly growing My-
for Medical Microbiology and Virology, University
cobacterium species was isolated from pulmonary
Hospital Schleswig-Holstein, 24105 Kiel.
specimens of two patients, one from Denmark and
one from Italy. The phenotypic characteristics of the
new strain were poorly distinctive. The strain grew
Occurrence and clinical relevance of Mycobac-
at 25°C to 37°C, but not at 45°C, with a weak yel-
terium chimaera sp. nov., Germany
low pigment under both light and dark conditions
Richter E.
on Löwenstein-Jensen and Stonebrink medium. It remained unpigmented on solid Middlebrook 7H10
Bacteria of the Mycobacterium avium complex
medium. Mycolic acid methyl esters were analyzed
(MAC) play an important role among infections
by thin-layer chromatography and revealed the
caused by nontuberculous mycobacteria. MAC con-
presence of - and keto-mycolic acids, with methoxy-
sists of the 2 well-established species, M. avium
mycolic acids being present in minor quantities, a
(which has 4 subspecies) and M. intracellulare, as
common pattern among mycobacterial species.
well as several other closely related mycobacteria.
Fatty acid methyl esters were identified by
Recently, a new species derived from the group of
gas/liquid
spectrome-
unnamed members of the MAC has been defined.
try.High-performance liquid chromatography re-
It combines features characteristic of different MAC
vealed an identical profile characterized by a sin-
members and has been named M. chimaera sp.
gle, late cluster of peaks grossly resembling My-
nov.. Molecular genetic standard tools in clinical mi-
cobacterium palustre and Mycobacterium lacus.
crobiologic laboratories do not differentiate MAC
However, phenotypic characteristics alone do not
members. These tools merely provide a rough clas-
chromatography-mass
allow a clear identification of a new strain. The iso-
sification in M. intracellulare and M. avium and/or
lates showed unique 16S rRNA internal transcribed
the MAC group as a whole. Currently, a detailed
spacers and hsp65 sequences: the 16S rRNA was
genotyping of MAC is restricted to research labo-
most closely related to Mycobacterium szulgai and
ratories. Nevertheless, several studies have shown
Mycobacterium malmoense. Both genotypic char-
that certain serotypes or genotypes were associat-
acteristics including the presence of a long helix 18
ed with different clinical manifestations of MAC in-
at positions 451 to 482 of 16S rRNA (according to
fection concerning the patient groups affected, the
Wissenschaftlicher Jahresbericht 2007-2008
161
Division of Mycobacteriology and
National Reference Center for Mycobacteria
Research Reports
localization and course of disease, and the antimi-
Krautwald-Junghanns ME, Clinic for Birds and Rep-
crobial drug resistance patterns. Retrospective mo-
tiles, Faculty of Veterinary Medicine, University of
lecular genetic analysis of 166 Mycobacterium in-
Leipzig, Leipzig, Germany
Fo r s c h u n g s z e n t r u m
Bo r s t e l
tracellulare isolates showed that 143 (86%) strains
could be assigned to Mycobacterium chimaera sp.
nov. Of 97 patients from whom M. chimaera sp. nov.
NRL activities
was isolated, only 3.3% exhibited mycobacterial
Rüsch-Gerdes S, Richter, E, Hillemann D, Niemann S.
lung disease, whereas all M. intracellulare isolates
caused severe pulmonary infections.
In recent years the global situation concerning drug
resistance has changed dramatically. In order to
Collaboration: Schweickert B, Petrich A, Göbel UB,
combat this, the NRL has begun as cooperation
Buchholz P, Moter A, Charité-Universitätsmedizin,
partner/study site to enlarge the activities for the en-
Berlin, Germany; Goldenberg O, Transgenomic Ltd.,
hancement of better diagnostic tools for the rapid
Glasgow, Scotland, UK.
detection of drug-resistant TB. Especially in collaboration with FIND (The Foundation for Innovative
New Diagnostics, Geneva) the improvement, evalu-
Transmission of tuberculosis between men and
ation and implementation of new diagnostic meth-
pet birds: a case report
ods was forced in the last years. The NRL is one of
Richter E.
the international multi-center-study sites (coordinated by FIND) to evaluate new assays for the detec-
This case report describes an infection of My-
tion and identification of M. tuberculosis as well as
cobacterium tuberculosis in an African Grey parrot
for resistance testing. Concerning the improvement
(Psittacus erithacus erithacus) kept as a pet bird. Di-
of TB-Surveillance in Germany the NRL focussed
agnosis was confirmed by microbiologic and patho-
mainly on the investigation of the epidemiology of
logic results, and indicated a human-avian trans-
TB. By combining classical and modern molecular
mission. Clinical signs included sublingual nodules
epidemiological tools the longitudinal studies in
resulting in anorexia and signs of osteolysis in the
Hamburg, Schleswig-Holstein, and most recently in
long bones. Proliferation consisted of several nod-
Baden-Württemberg were continued. These studies
ules with small greenish-caseous foci in cross-sec-
were performed in close cooperation with the pub-
tion and revealed a severe granulomatous inflam-
lic health offices in the respective regions. Several
mation without intralesional acid-fast rods. M. tu-
aspects of TB epidemiology were addressed, e.g.
berculosis was cultured from a pooled sample of
trends in resistance rates, outbreaks, and emer-
sublingual and liver nodules, and was confirmed via
gence of particular M. tuberculosis strains. Further-
polymerase chain reaction. Transmission between
more, the molecular strain typing results will be con-
men and parrot was proved by spoligotyping pat-
nected with the epidemiological data of the RKI.
tern analysis. The absence of facial skin lesions and
Quality control gets more and more importance in
acid-fast rods within the tubercles is contrary to pre-
laboratory diagnostic. Therfore, the NRL was asked
vious publications of tuberculosis in birds. This dis-
to take the lead in the so called “Ringversuch” from
ease in a common pet bird species is of zoonotic
INSTAND e.V. in Düsseldorf. Since beginning of 2008
importance, and those parrots with close contact to
the NRL is responsible for the preparation of spec-
owners suffering from tuberculosis may serve as a
imens and samples for microscopy, primary detec-
potential reservoir for human infection.
tion, identification of mycobacteria, and susceptibility testing. After successful establishment of the
Collaboration: Schmidt V, Schneider S, Schlomer J,
162
first series now twice a year all samples for app.
Wissenschaftlicher Jahresbericht 2007-2008
Division of Mycobacteriology and
National Reference Center for Mycobacteria
Research Reports
250 national and international laboratories were
produced, the results assessed, laboratories certificated and consulted concerning quality improvement. Furthermore, in its function as a Supranational Reference Laboratory for DST, the NRL has broadened its activities concerning inter-laboratory proficiency testing. Meanwhile the NRL performs quality
assurance for more than 10 National Reference
Laboratories of other countries (e.g. Austria, Kazakhstan, Serbia, and Uganda). One further focus is
the support of laboratories implementing new methods by training and attending. In this connection the
availability of the proficiency testing samples provided by the NRL aided laboratories in new method
performance and implementation.
Theses
Habilitation
Dr. rer. nat. Richter, Elvira
für das Fach ‚Mikrobiologie’
Medizinische Fakultät
Universität zu Lübeck, 2008.
Wissenschaftlicher Jahresbericht 2007-2008
163
Division of Molecular Mycobacteriology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Molecular Mycobacteriology
Head
Main Results: For the TST, 40.4% (243/601) of contacts
PD Dr. Stefan Niemann
were positive at a 5-mm cutoff, whereas only 66 (11%)
were QFT positive. QFT positivity, but not TST, was as-
Staff Scientists and Postdoctoral Fellows
sociated with exposure time (P < 0.0001). Six contacts
Dr. Silke Feuerriegel
progressed to TB disease within the 2-year follow-up.
All were QFT positive and had declined preventive
Graduate and Diploma Students
treatment, equating to a progression rate of 14.6%
Claudia Plinke
(6/41) among those who were QFT positive. The pro-
Susanne Homolka
gression rate for untreated TST-positive subjects was
Caterina Sibilia
significantly lower (P < 0.003), at 2.3% (5 of 219), and
Stefanie Schweinhuber
one subject who progressed was TST negative. Conclusions: Results suggest that QFT is a more accurate
Technicians
indicator of the presence of LTBI than the TST and pro-
Lisa Dost
vides at least the same sensitivity for detecting those
Tanja Ubben
who will progress to active TB. The high rate of pro-
Petra Vock
gression to active TB of those who are QFT positive
(14.6%), which is far greater than the 2.3% found for
Guests and Trainees
those who are TST positive, has health and econom-
Claudio Köser, University of Cambridge
ic implications for enhanced TB control, particularly if
this higher progression rate is seen in studies of other at-risk populations.
Research Reports
Predictive value of a whole blood IFN-gamma
Collaboration: Roland Diel, School of Public Health,
assay for the development of active tuberculosis
University of Düsseldorf, Düsseldorf, Germany; Robert
disease after recent infection with Mycobacteri-
Loddenkemper, German Central Committee against
um tuberculosis
Tuberculosis, Lungenklinik Heckeshorn, HELIOS,
Niemann S.
Klinikum Emil von Behring, Berlin, Germany; Karen
Meywald-Walter, Public Health Department Hamburg-
Rationale: Numerous studies have been published on
Mitte, Hamburg, Germany; Albert Nienhaus, Institution
the new Mycobacterium tuberculosis (MTB)–specific
for Statutory Accident Insurance and Prevention in the
IFN-gamma release assays. However, their prognos-
Health and Welfare Services, Hamburg, Germany.
tic value for progression from latent tuberculosis infection (LTBI) to active TB has yet to be established.
Objectives: To compare the QuantiFERON-TB Gold In-
Molecular evidence of a microepidemy among
Tube assay (QFT) with the tuberculin skin test (TST) in
Hungarian homeless patients with tuberculosis in
recently exposed close contacts of active TB cases
Budapest due to a newly identified local My-
with respect to their development of TB disease with-
cobacterium tuberculosis lineage
in 2 years. Methods: Close contacts (n = 601) of MTB-
Niemann S.
positive source cases underwent both TST and QFT
testing and were subsequently observed for 103
In a retrospective study that included 66 homeless
(±13.5) weeks. Risk factors for MTB infection were eval-
tuberculosis patients representing 47.1% of all home-
uated by multivariate analysis. Measurements and
less cases notified in Budapest in 2002 in a subset
164
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Mycobacteriology
Research Reports
of 12 (18.1 %) patients a locally emerged lineage
demiological link, were perfectly matched by MIRU-
which led to a microepidemy was identified and de-
VNTR typing with the 24 loci. Two IS6110 clusters were
fined by IS6110 fingerprinting, spoligotyping and
split by differences into 6 to 12 MIRU-VNTR loci, clear-
mycobacterial interspred repetitive unit typing.
ly supporting the absence of a link, as judged by con-
These data may serve as a reference to better mon-
tact tracing data. In contrast, only one MIRU-VNTR clus-
itor and understand the patterns and transmission
ter, grouping what were probably epidemiologically
dynamics of tuberculosis in this at risk population in
unlinked isolates, was split by IS6110 RFLP. However,
Hungary. The findings also indicate that tuberculo-
these isolates were also distinguished by spoligotyp-
sis control and prevention steps among the home-
ing. Both the optimized 24-locus and 15-locus sets thus
less need to be strengthened.
showed a comparable to slightly better predictive value, especially when combined with spoligotyping,
Collaboration: Csaba Ködmön, National Public Heal-
than the current gold standard IS6110 RFLP for the
th and Medical Officers Service, Miskolc, Hungary;
study of tuberculosis transmission in Hamburg. Be-
Cristina M. Gutierrez, Centre National de Reference
cause the epidemiological characteristics of this set-
des Mycobacteries, Institute Pasteur, Paris, France;
ting are similar to those of many developed countries,
Cristophe Sola, Unité de la Tuberculose et des Myco-
these results support the wide applicability of this re-
bactéries, Institut Pasteur de Guadeloupe, Pointe-á-
al-time genotyping approach for population-based
Pitre, Guadeloupe; Judit Lukács, Ákos Somoskövi; De-
studies of M. tuberculosis transmission
partment of Respiratory Medicine, School of Medicine, Semmelweis University, Budapest, Hungary.
Collaboration: Sabine Rüsch-Gerdes, National Reference Center for Mycobacteria, Research Center
Borstel, Borstel, Germany; Mara Cardoso Oele-
Assessment of an optimized mycobacterial inter-
mann, Philip Supply, Vincent Vatin, Camille Locht, In-
spersed repetitive-unit-variable-number tandem-
stitut Pasteur de Lille, Lille, France; Roland Diel,
repeat typing system combined with spoligoty-
School of Public Health, University of Düsseldorf,
ping for population-based molecular epidemio-
Düsseldorf, Germany; Walter Haas, Department for
logy studies of tuberculosis
Infectious Disease Epidemiology, Robert Koch-Insti-
Niemann S.
tut, Berlin, Germany.
An optimized set of 24 mycobacterial interspersed
Supported in part by: Germany Ministry of Health,
repetitive-unit-variable-number tandem-repeat (MIRU-
Berlin, Germany; the Robert Koch Institute, Berlin,
VNTR) loci, including a discriminatory subset of 15 loci,
Germany; INSERM and Institut Pasteur de Lille,
has recently been defined for the typing of Mycobac-
France; and the European Community (grant QLK2-
terium tuberculosis. Here, we evaluated the perform-
CT-2000-00630).
ances of this MIRU-VNTR typing system in combination
with spoligotyping for the detection of transmission
chains in a population-based study comprising 91% of
Evaluation and strategy for use of MIRU-VNTRplus,
culture-confirmed tuberculosis patients reported in
a multifunctional database for online analysis of ge-
2003 in Hamburg, Germany. Of the 154 isolates in-
notyping data and phylogenetic identification of
vestigated, more than 90% had high IS6110 copy num-
Mycobacterium tuberculosis complex isolates
bers (6). IS6110 restriction fragment length polymor-
Niemann S.
phism (RFLP) typing resulted in 13 clusters, 5 of which
had a confirmed epidemiological link. All five, as well
Because of its portable data, discriminatory power,
as six of the eight IS6110 clusters with no identified epi-
and recently proposed standardization, mycobac-
Wissenschaftlicher Jahresbericht 2007-2008
165
Division of Molecular Mycobacteriology
Research Reports
terial interspersed repetitive-unit-variable-number
The results can easily be exported. In the present
tandem-repeat (MIRU-VNTR) typing has become a
study, we evaluated the database consistency and
major method for the epidemiological tracking of My-
various analysis parameters both by testing the ref-
cobacterium tuberculosis complex (MTBC) clones.
erence collection against itself and by using an ex-
However, no public MIRU-VNTR database based on
ternal population-based data set comprising 629 dif-
well-characterized reference strains has been avail-
ferent strains. Under the optimal conditions found, lin-
able hitherto for easy strain identification. Therefore,
eage predictions based on typing by 24-locus MIRU-
a collection of 186 reference strains representing the
VNTR
primary MTBC lineages was used to build a data-
spoligotyping were verified in >99% of the cases. On
base, which is freely accessible at http://www.MIRU-
the basis of this evaluation, a user strategy was de-
VNTRplus.org. The geographical origin and the drug
fined, which consisted of best-match analysis fol-
susceptibility profile of each strain were stored to-
lowed, if necessary, by tree-based analysis. The
gether with comprehensive genetic lineage informa-
MIRU-VNTRplus database is a powerful tool for high-
tion, including the 24-locus MIRU-VNTR profile, the
resolution clonal identification and has little equiva-
spoligotyping pattern, the single-nucleotide- and
lent in terms of functionalities among the bacterial
large-sequence-polymorphism profiles, and the
genotyping databases available so far.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
analysis
optionally
combined
with
IS6110 restriction fragment length polymorphism fingerprint. Thanks to flexible import functions, a single
Collaboration: Caroline Allix-Béguec, Genoscreen, 1,
or multiple user strains can be analyzed, e.g., for lin-
rue du Professeur Calmette, Lille 59019 Cedex,
eage identification with or without the use of refer-
France; Dag Harmsen, Thomas Weniger, Department
ence strains, by best-match or tree-based analyses
of Periodontology, University Hospital Münster,
with single or combined marker data sets.
Waldeyerstrasse 30, Münster, Germany; Philip Supply, Institut Pasteur de
Lille, 1, rue du Professeur Calmette, Lille
59019 Cedex, France.
Supported in part by:
INSERM, the Institut
Pasteur de Lille, Lille,
France, the Germany
Ministry of Health and
the German Federal
Ministry for Education
and Research (BMBF)
within
the
PathoGenomikPlus
Network.
Fig. 68. Phylogenetic composition of the Mycobacterium tuberculosis complex. A radial tree was constructed by using the 24-locus MIRUVNTR typing data for the reference strains in the MIRU-VNTRplus database and 187 genotypes with unassigned or T spoligotypes from
the Brussels population-based collection by using the neighbour-joining algorithm and categorical distance coefficient. The positions of
Brussels genotypes with unassigned or T spoligotypes are indicated by yellow circles. Lineages containing exclusively T-spoligotype strains
or lineages corresponding to well-characterized, classical clades of the Euro-American superlineage (such as the Haarlem lineage) comprising T-spoligotype strains are denoted T-specific and T-aspecific, respectively. EAI, East African-Indian; West Afri 2, West African 2 (M.
africanum 2); West Afri 1, West African 1 (M. africanum 1); Tur, Turkish; BJ, Beijing; CAS, Central Asian; LAM, Latin American-Mediterranean.
The data were analyzed by using the MIRU-VNTRPlus Webseite (www.miru-vntrplus.org). Originally published in JCM 2008 46:2692-2699.
166
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Mycobacteriology
Research Reports
Emergence and transmission of second line drug
Risk of acquired drug resistance during short-
resistant Mycobacterium tuberculosis complex
course directly observed treatment of tuberculo-
strains in Germany
sis in an area with high levels of drug resistance
Niemann S, Rüsch-Gerdes S.
Niemann S, Rüsch-Gerdes S.
Background. Drug resistant tuberculosis (TB) has be-
Background. Data on the performance of stan-
come a serious challenge for global TB control.
dardized short-course directly observed treatment
High rates of resistant and multidrug resistant (MDR,
(DOTS) of tuberculosis (TB) in areas with high levels
resistance to at least isoniazid and rifampin) M. tu-
of drug resistance and on the potential impact of
berculosis complex (MTBC) strains have been doc-
DOTS on amplification of resistance are limited.
umented in several “hot spot” areas around the
Therefore, we analyzed treatment results from a
world. Additionally, increasing rates of second line
cross-sectional sample of patients with TB enrolled
drug resistance have been reported and an out-
in a DOTS program in an area with high levels of
break of an extensively resistant (XDR, defined as
drug resistance in Uzbekistan and Turkmenistan in
MDR plus resistance to any fluoroquinolone and in-
Central Asia.
jectable drugs) strain in South Africa has attracted
Methods. Sputum samples for testing for suscepti-
world wide attention. Therefore, representative da-
bility to 5 first-line drugs and for molecular typing
ta on the prevalence and spread of second line
were obtained from patients starting treatment in 8
drug resistant TB are urgently needed to develop
districts. Patients with sputum smear results positive
more effective TB control strategies.
for TB at the end of the intensive phase of treatment
Methods. Second line drug resistance testing and
and/or at 2 months into the continuation phase
molecular typing (IS6110 DNA fingerprinting and
were tested again.
spoligotyping) will be performed for MDR strains.
Results. Among 382 patients with diagnoses of TB,
Results. In the study period, more than 700 MDR
62 did not respond well to treatment and were
strains were notified at the NRC. So far, a significant
found to be infected with an identical Mycobacteri-
number of second line drug resistant strains could
um tuberculosis strain when tested again; 19 of
be determined. In year 2005, 40% of the MDR strains
these patients had strains that developed new or
showed additional resistance to prothionamide,
additional drug resistance. Amplification occurred
10% were additionally resistant to amikacin, and
in only 1.2% of patients with initially susceptible or
12% showed additional resistance to capreomycin.
monoresistant TB strains, but it occurred in 17% of
DNA fingerprinting revealed that an increasing
those with polyresistant strains (but not multidrug-re-
number of strains belonged to the Beijing genotype.
sistant strains, defined as strains with resistance to
Transmission of second line resistant strains could
at least isoniazid and rifampicin) and in 7% of those
be documented. Further analyses are in progress.
with multidrug-resistant strains at diagnosis. Overall,
Conclusions. Our preliminary data already docu-
3.5% of the patients not initially infected with mul-
mented a significant number of second line drug re-
tidrug-resistant TB strains developed such strains
sistance among MDR strains investigated. The Bei-
during treatment. Amplification of resistance, how-
jing genotype, which has been shown to be a ma-
ever, was found only in polyresistant Beijing geno-
jor cause of resistant TB in several high incidence
type strains.
settings, is also frequent among MDR strains from
Conclusions. High levels of amplification of drug re-
Germany.
sistance demonstrated under well-established
DOTS program conditions reinforce the need for implementation of DOTS-Plus for multidrug-resistant TB
in areas with high levels of drug resistance. The
Wissenschaftlicher Jahresbericht 2007-2008
167
Division of Molecular Mycobacteriology
Research Reports
strong association of Beijing genotype and amplifi-
ofloxacin resistance amplification, while strains
cation in situations of preexisting resistance is strik-
from 4 patients displayed different DNA fingerprints
ing and may underlie the strong association be-
during treatment, 3 of which matched those of co-
tween this genotype and drug resistance.
hospitalised patients. A final patient with ofloxacin
Fo r s c h u n g s z e n t r u m
Bo r s t e l
resistance was found to have a mixed infection durCollaboration: Helen S. Cox, Australian Internatio-
ing treatment, also potentially indicating reinfection.
nal Health Institute, University of Melbourne, Aus-
Pre-existing second line resistance and severe clin-
tralia; Gabit Ismailov, Juan Daniel Orozco, Daribay
ical condition were associated with amplification of
Doshetov, Médecins Sans Frontières and Ministry of
ofloxacin resistance, while poor treatment adher-
Health, Nukus, Karakalpakstan; Lucie Blok, Méde-
ence was not found to be a significant contributor.
cins Sans Frontières, Amsterdam.
Conclusions: Ofloxacin resistance and XDR-TB
emerging during treatment for MDR-TB was caused
Supported in part by: Robert-Koch-Institute (Berlin,
by both amplification and exogenous nosocomial
Germany), the European Union Concerted Action
reinfection. The emergence of XDR-TB during sec-
project (QLK2-CT-2000-00630), and the World Health
ond line MDR-TB treatment is alarming and has im-
Organization.
plications for the scale up of such treatment.
Development of ofloxacin and XDR tuberculosis
during MDR-TB treatment is caused by resistance
amplification and exogenous reinfection
Sibilia K, Schmidt S, Rüsch-Gerdes S, Niemann S.
Background: While there is an urgent need to scale
up the provision of treatment for multidrug resistant
tuberculosis (MDR-TB) internationally, limited data
exists on the emergence of extensively drug resistant TB (XDR-TB) during currently recommended second line treatment.
Method: The development of ofloxacin resistance
and XDR-TB during MDR-TB treatment with a regimen containing at least 5 drugs to which the strain
was presumed to be susceptible was assessed in a
cohort of 87 patients treated in Karakalpakstan,
Uzbekistan. Isolates from patients at baseline and
Fig. 69. IS6110 DNA fingerprint patterns of the 4 isolates displaying ofloxacin resistance amplification during therapy (A, patient 9)
and those from patient 14 potentially reinfected with a second
strain from patient 10 during his stay in the hospital in Karakalpakstan (Uzbekistan) (B). Resistance is displayed by a black box. Abbreviations: H=Isoniazid, R=Rifampicin, E=Ethambutol, P=Pyrazinamide, S=Streptomycin, Ofx=Ofloxacin, Cm=Capreomycin,
Eto=Ethionamide, Cs=Cycloserine, PAS=p-aminosalicylic acid,
Am=Amikacin. Originally published in N Engl J Med. 2008
359(22):2398-400.
during treatment underwent drug susceptibility test-
Collaboration: Helen S. Cox, Burnet Institute for
ing and DNA fingerprinting.
Medical Research and Public Health, Melbourne,
Results: While none of the 87 patients displayed
Australia; Stobdan Kalon, Jonny Polonsky, Médecins
ofloxacin resistance at baseline, isolates from 18
Sans Frontières, Karakalpakstan, Nukus, Uzbekistan;
patients (21%) developed ofloxacin resistance dur-
Zinaida N. Tigay, Hamraev A. Karimovich, Ministry
ing treatment, with 10 (11%) defined as XDR-TB. On-
of Health, Nukus, Karakalpakstan, Uzbekistan; Clair
ly 5 (28%) of these patients were successfully treat-
Mills, Médecins Sans Frontières, Amsterdam, The
ed. Isolates from 13 patients had identical DNA fin-
Netherlands.
gerprints
168
throughout
treatment
representing
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Mycobacteriology
Research Reports
Supported in part by: German Ministry of Health.
Health and the Robert-Koch-Institute, Berlin, Germany.
Tuberculosis ethambutol resistance: Concor-
Analyses of mutations conferring resistance to
dance between phenotypic and genotypic test re-
second-line drugs in Mycobacterium tuberculosis
sults
strains from a high incidence region
Plinke C, Rüsch-Gerdes S, Niemann S.
Feuerriegel S., Rüsch-Gerdes S, Niemann S.
Mutations in the embB306 codon of Mycobacterium
Background. Resistance to second-line drugs (fluoro-
tuberculosis are potential markers for detecting re-
quinolones and injectable aminoglycosides) has be-
sistance to ethambutol. However, more recently, em-
come a serious problem for the treatment of My-
bB306 mutations have been found in ethambutol
cobacterium tuberculosis infections. Resulting XDR-tu-
susceptible isolates and an association with broad
berculosis implies an enormous threat for tuberculosis
drug resistance rather than ethambutol resistance
control worldwide. It is therefore of great importance
has been reported. To further investigate this ques-
to analyze the genetic basis of clinical resistance.
tion, we analyzed the association between em-
Methods. M. tuberculosis strains from a high-inci-
bB306 mutations and phenotypic ethambutol re-
dence region in Uzbekistan (Karakalpakstan) which
sistance among isolates from a cross-sectional drug
displayed resistance to ofloxacin (n = 56) and to
resistance survey carried out in Karakalpakstan,
capreomycin and/or amikacin (n = 111), respective-
Uzbekistan. Among 197 strains analyzed, 40 (20%)
ly, were sequenced concerning the resistance de-
had an embB306 mutation, out of which 32 were
termining regions (gyrA, gyrB, rrs, tlyA). 25 of the
EMB resistant also. Among the eight strains with dis-
strains showed resistance to both second-line drugs
crepant results, one was found to have an embB306
and were therefore XDR-TB.
wild type sequence and seven were tested EMB re-
Results. The most common mutation seen among
sistant after re-analysis. All of these had an in-
ofloxacin resistant strains is the substitution of as-
creased ethambutol MIC, however, for three strains
partic acid to glycine at codon 94 (32.1 %) followed
it was below the critical concentration of 2 µg/ml.
by the substitution of alanine to valine at codon 90
Furthermore, in four strains we confirmed the pres-
(30.4 %) in the gyrA region. 17.9 % of the ofloxacin re-
ence of heteroresistant mixed populations which
sistant strains display substitutions of aspartic acid
might also influence conventional ethambutol test-
other than glycine at codon 94 and changes of ser-
ing.Our results confirm that embB306 mutations are
ine for proline at codon 91 in gyrA, respectively. Two
useful markers for predicting ethambutol resistance.
ofloxacin resistant strains show SNPs in gyrB at
Discrepancies between molecular and phenotypic
codon
ethambutol resistance test results are most likely
543 (GCG ACG), respectively, that have not been re-
caused by problems with conventional susceptibili-
ported previously. Concerning resistance to amikacin
ty testing mainly due to the small increase of the
and capreomycin the most common mutation seen is
MIC observed in some ethambutol resistant strains.
the substitution of nucleotide adenine to guanine at
485
(CGT TGT)
and
at
codon
position 1401 (67.6 %) in rrs followed by a change of
Collaboration: Helen S. Cox, Macfarlane Burnet Insti-
cytosine to thymine at position 1402 (2.7 %).
tute for Medical Research and Public Health, Aus-
Conclusion. This is the first study analyzing
tralia; Stobdan Kalon, Médecins Sans Frontières,
M. tuberculosis strains from a high-incidence region
Uzbekistan; Daribay Doshetov, Ministry of Health,
for fluoroquinolone and aminoglycoside resistance
Karakalpakstan, Uzbekistan. Funding: Parts of this
determining mutations. In addition to the previous-
work were supported by the Germany Ministry of
ly described mutations, new SNPs in gyrB and tlyA
Wissenschaftlicher Jahresbericht 2007-2008
169
Division of Molecular Mycobacteriology
Research Reports
were detected, whose impact needs to be further
population structure of MTBC strains shows an in-
elucidated. Sequence analysis in general is of great
triguing diversity. M. africanum is still a significant
importance for the rapid detection of XDR strains.
cause of TB in the study region; however, the ma-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
jority of strains belong to the M. tuberculosis Euro
Collaboration: Helen Cox, Macfarlane Burnet Insti-
American lineage further divided in six sublineages.
tute for Medical Research and Public Health, Australia; Stobdan Kalon, 3Médecins Sans Frontières,
Karakalpakstan, Uzbekistan; Zinaida Tigay, Ministry
of Health, Tashkent, Uzbekistan; Clair Mills, Médecins
Sans Frontières, Amsterdam, The Netherlands.
High genetic diversity among Mycobacterium tuberculosis complex strains from Sierra Leone
Homolka S, Rüsch-Gerdes S, Niemann S.
Pathogens of the Mycobacterium tuberculosis complex (MTBC) remain one of the leading infectious
killers worldwide. Among tuberculosis (TB) high incidence regions, Sub Saharan Africa is particularly
affected with approx. 1.6 million new cases every
year. Besides this dramatic situation, data on the diversity of MTBC strains causing this epidemic in this
area are only sparsely available. Here we analyzed
Fig. 70. Population structure of clinical M. tuberculosis complex isolates from Sierra Leone. Minimum spanning tree based on the diversity of MIRU-VNTR data. Circles show the different clonal complexes identified (maximum neighbour distance: 4 changes; minimum size: 2 MIRU-VNTR types) by the set of 24 loci among the 97
MTBC strains analyzed. The size of each circle is proportional with
the number of MIRU-VNTR types belonging to a particular complex. Colours indicate strain previous classification (Table 2). EAI,
M. tuberculosis East African Indian; LAM, M. tuberculosis Latin
American Mediterranean. Originally published in BMC Microbiology 2008, 8:103.
the population structure of MTBC strains from Sierra Leone with a special focus on the prevalence of
Collaboration: Erik Post, Barbara Oberhauser, Ger-
M. africanum. A total of 97 strains isolated from
man Leprosy and TB Relief Association, Würzburg,
smear positive cases registered for re-treatment in
Germany; Abu Garawani George, Lars Westman,
the Western Area and Kenema districts in years
National Leprosy/TB Reference Laboratory, Free-
2003/2004 were investigated by susceptibility test-
town, Sierra Leone; Foday Dafae, National Program
ing (first line drugs) and molecular typing (IS6110
Manager for Tuberculosis and Leprosy, Freetown,
DNA fingerprinting, spoligotyping, and MIRU-VNTR
Sierra Leone.
typing). Among the strains analyzed, 32 were re-
Funding: Parts of this work were supported by the
sistant to isoniazid, and 11 were multidrug resistant
Germany Ministry of Health and the German Fed-
(at least resistant to isoniazid and rifampin). The
eral Ministry for Education and Research (BMBF)
most prominent genotype found, was M. africanum
within the PathoGenomikPlus Network.
West African-2 (n=17), followed by M. tuberculosis
Haarlem (n=14), LAM (n=11), M. africanum West
African-1 (n= 6), M. tuberculosis EAI (n=4), Beijing
Pulmonary tuberculosis: Virulence of Mycobacteri-
(n=4), S (n=4) and Cameroon genotype (n=4). Fur-
um africanum and relevance in HIV co-infection
thermore, two new genotypes Sierra Leone-1 and -
Kubica T, Rüsch-Gerdes S, Niemann S.
2 were found, that represent 7 % and 10 % of all isolates, respectively. In conclusion, resistance rates in
Although Mycobacterium africanum is being isola-
Sierra Leone have reached an alarming level. The
ted in a significant proportion of cases of pul-
170
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Mycobacteriology
Research Reports
monary tuberculosis in West Africa, its pathogenic
versity of Science and Technology, Kumasi, Ghana;
potential remains a matter of discussion. Recent re-
Margaret Amanua Chinbuah, John Gyapong, Ivy
ports leave the question of whether M. africanum
Osei, Health Research Unit, Ghana Health Service,
causes more severe pathology than M. tuberculosis
Accra, Ghana.
or resembles opportunistic pathogens and might
gain importance in the course of the HIV pandem-
Supported in part by: German Federal Ministry of
ic. Patients with pulmonary tuberculosis associated
Education
with M. africanum (n = 556) and M. tuberculosis
Genome Research Network (NGFN1; Project
(n = 1350) were studied in Ghana, West Africa, and
01GS0162).
and
Research,
German
National
compared regarding self-reported signs and symptoms, chest radiography, HIV status, mycobacterial
drug resistance and mycobacterial clustering as de-
Origin, spread and demography of the My-
termined by spoligotyping and IS6110 fingerprints.
cobacterium tuberculosis complex
The rate of M. africanum infections was similar in
Wölbeling F, Kubica T, Niemann S.
HIV-positive (27 %) and HIV-negative (30 %) patients.
M. africanum clustered less than M.
tuberculosis (21 % vs 79 %; OR, 0.38;
95% CI, 0.3–0.5; p < 0.001) corresponding to its lower prevalence
(29 % vs 70 %). Clinically and radiographically, no significant differences were found except that M.
africanum caused lower-lobe disease less frequently than M. tuber-
culosis (OR, 0.39; 95% CI, 0.2–0.7;
pc = 0.01), whereby this association
applied to HIV-negative patients
only. No difference in virulence, as
assessed by the severity of radiological presentation, was found
when the two M. africanum subtypes West African 1 and West African 2 were compared. In the population studied, M. africanum
closely resembled M. tuberculosis in pathology and
cannot be considered an opportunistic pathogen.
Collaboration: Christian G. Meyer, Thorsten Thye,
Rolf D. Horstmann, Department of Molecular Medicine, Bernhard Nocht Institute for Tropical Medicine,
Hamburg, Germany; Genevieve Scarisbrick, De-
Fig. 71. Mycobacterium tuberculosis complex (MTBC) evolutionary
scenario (out of Mesopotamia). The main migration events are
numbered and correspond to: 1, M. prototuberculosis, the ancestor of the MTBC, this bacterium reached the Fertile Crescent some
40,000 years ago by sea or land; 2 and 3, two distinct basal lineages arose, EAI and LAM and spread out of Mesopotamia some
10, 000 years ago; 4, 5 and 6, later on (8–5000 years ago) derived
populations from clade 1 followed main human migration patterns
to Africa, Asia and Europe, giving rise to locally adapted tubercle
strains and further diversifications. Note that the depicted borders
are “artificial” and are used for the demonstration. Global movements and intercontinental exchanges tend to blur this phylogenetic signal though strong enough to be detected nowadays. Originally published in Plos Pathog 2008 4(9): e1000160.
partment of Radiology, Komfo Anokye Teaching
Hospital, Kumasi, Ghana; Edmund N.L. Browne, El-
The evolutionary timing and spread of the My-
lis Owusu-Dabo, Department of Community Health,
cobacterium tuberculosis complex (MTBC), one of
School of Medical Sciences, Kwame Nkrumah Uni-
the most successful groups of bacterial pathogens,
Wissenschaftlicher Jahresbericht 2007-2008
171
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Research Reports
remains largely unknown. Here, using mycobacter-
High functional diversity in Mycobacterium tuber-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
ial tandem repeat sequences as genetic markers,
culosis driven by genetic drift and human de-
we show that the MTBC consists of two independ-
mography
ent clades, one composed exclusively of M. tuber-
Homolka S, Niemann S.
culosis lineages from humans and the other composed of both animal and human isolates. The lat-
Mycobacterium tuberculosis infects one third of
ter also likely derived from a human pathogenic lin-
the human world population and kills someone
eage, supporting the hypothesis of an original
every 15 seconds. For more than a century, scien-
human host. Using Bayesian statistics and experi-
tists and clinicians have been distinguishing be-
mental data on the variability of the mycobacterial
tween the human- and animal-adapted members
markers in infected patients, we estimated the age
of the M. tuberculosis complex (MTBC). However,
of the MTBC at 40,000 years, coinciding with the ex-
all human-adapted strains of MTBC have tradi-
pansion of “modern” human populations out of
tionally been considered to be essentially identi-
Africa. Furthermore, coalescence analysis revealed
cal. We surveyed sequence diversity within a glob-
a strong and recent demographic expansion in al-
al collection of strains belonging to MTBC using
most all M. tuberculosis lineages, which coincides
seven megabase pairs of DNA sequence data. We
with the human population explosion over the last
show that the members of MTBC affecting humans
two centuries. These findings thus unveil the dy-
are more genetically diverse than generally as-
namic dimension of the association between hu-
sumed, and that this diversity can be linked to hu-
man host and pathogen populations.
man demographic and migratory events. We further demonstrate that these organisms are under
Collaboration: Sabine Rüsch-Gerdes, National Ref-
extremely reduced purifying selection and that, as
erence Center for Mycobacteria, Research Center
a result of increased genetic drift, much of this ge-
Borstel, Borstel, Germany; Thierry Wirth, Falk Hilde-
netic diversity is likely to have functional conse-
brand, Ecole Pratique des Hautes Etudes, Muséum
quences. Our findings suggest that the current in-
National d’Histoire Naturelle, UMR-CNRS 5202, Dé-
creases in human population, urbanization, and
partement Systématique et Evolution, Paris, France ;
global travel, combined with the population ge-
Caroline Allix-Béguec, Institut Pasteur de Bruxelles,
netic characteristics of M. tuberculosis described
Laboratoire Tuberculose et Mycobactéries, Brussels,
here, could contribute to the emergence and
Belgium; Kristin Kremer, Dick van Soolingen, Na-
spread of drug-resistant tuberculosis.
tional Institut of Public Health and Environment,
Bilthoven, The Netherlands; , Camille Locht, Philip
Collaboration: Ruth Hershberg, Mikhail Lipatov,
Supply, Institut Pasteur de Lille, Lille, France; Sylvain
Hadar Sheffer, Dmitri A. Petrov, Marcus W. Feldman,
Brisse, Institut Pasteur, Genotyping of Pathogens
Department of Biology, Stanford University, Stan-
and Public Health, Paris, France; Axel Meyer,
ford, California, United States of America; Peter M.
Lehrstuhl für Zoologie und Evolutionsbiologie, De-
Small, Institute for Systems Biology, Seattle, Wash-
partment of Biology, University of Konstanz, Kon-
ington, United States of America; Jared C. Roach,
stanz, Germany.
Seattle Children’s Hospital Research Institute, Seattle, Washington, United States of America; Kristin
Supported in part by: Germany Ministry of Health and
Kremer, Mycobacteria Reference Unit (CIb-LIS), Na-
the German Federal Ministry for Education and Re-
tional Institute of Public Health and the Environment,
search (BMBF) within the PathoGenomikPlus Network.
Bilthoven, The Netherlands; Sebastien Gagneux,
MRC National Institute for Medical Research, London, United Kingdom.
172
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular Mycobacteriology
Research Reports
Supported in part by: Wellcome Trust and by Na-
will be further explored.
tional Institutes of Health (NIH grant AI34238), by
Conclusions. The genetic diversity among MTBC
NIH grant GM28016, by the German Ministry of
strains appears to be higher than previously antici-
Health and Ministry for Education and Research
pated and might have functional consequence as
(BMBF) within the PathoGenomikPlus Network,
already indicated by differences in virulence model
by NIH grant 5K08AI056092, by the Medical
systems.
Research Council, UK and by NIH grant HHSN266200700022C.
Collaboration: Sabine Rüsch-Gerdes, National Reference Center for Mycobacteria, Research Center
Borstel, Borstel, Germany; Norbert Reiling, Kerstin
Genetic and biological diversity of clinical
Walter, Stefan Ehlers, Molecular Infection Biology,
Mycobacterium tuberculosis complex strains
Research Center Borstel, Borstel, Germany; Corde-
Homolka S, Niemann S.
lia Arndt-Sullivan, Klaus-Peter Pleissner, Stefan Kaufmann, 3Max Planck Institute for Infection Biology,
Background. Pathogens of the Mycobacterium tu-
Berlin, Germany.
berculosis complex (MTBC; causative agents of tu-
Supported in part by: German Ministry of Health
berculosis [TB]) remain one of the leading infectious
and Ministry for Education and Research (BMBF)
killers worldwide. Only recently, fine molecular typ-
within the PathoGenomikPlus Network.
ing has indicated that the genetic heterogeneity
among MTBC isolates, albeit relatively low, influences the transmissibility and virulence of clinical
ALOX5 variants associated with susceptibility to
isolates as well as the induced immune response.
human pulmonary tuberculosis
However, only limited information about the under-
Niemann S.
lying pathogenomic mechanisms is available. In
this work we have used a combination of different
The 5-lipoxygenase (ALOX5)-derived lipid media-
experimental approaches to further investigate the
tors leukotrienes and lipoxins have regulatory func-
genetic and biological diversity of clinical MTBC iso-
tions in inflammation by modulating activities of im-
lates.
Results. Based on the analysis of a collection of 176
mune cells and cytokine production. Recently, it was
shown in ALOX5–/– mice that host control of My-
strains we were able to define main phylogenetic
cobacterium tuberculosis is regulated by 5-lipoxy-
lineages that show a geographic sub-structuring
genase (5-LO). ALOX5 polymorphisms were geno-
with distinct African, Asian and an African-European
typed in 1916 sputum-positive patients with pul-
branching. The genetic diversity was further ex-
monary tuberculosis (TB) from Ghana and in 2269
plored by multi locus sequence typing that revealed
exposed, apparently healthy controls. Polymor-
lineage specific SNPs with potential functional im-
phisms of a variable number of tandem repeats
plications. To investigate pathogenetic properties of
(VNTR) of the ALOX5 promoter and of the exonic
strains of different lineages, a standardized culti-
non-synonymous variant g.760G>A were analysed
vation system was established that allows a com-
by fragment length determination and fluorescence
parative transcriptome and proteome analysis as
resonance energy transfer, respectively, and DNA
well as investigations in virulence model systems.
sequencing. Mycobacterial lineages of >1400 iso-
First experiments e.g. with murine macrophages
lates were differentiated biochemically and genet-
and in a low dose aerosol infection mouse model
ically. Carriers of one variant (n repeats 5) and one
already revealed pathogenomic differences in in-
wild-type VNTR allele (n = 5) or of the exonic allele
dividual clinical strains of particular genotypes that
g.760A had a higher risk of TB [Pcorrected = 0.026,
Wissenschaftlicher Jahresbericht 2007-2008
173
Division of Molecular Mycobacteriology
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Fo r s c h u n g s z e n t r u m
Bo r s t e l
odds ratio (OR) 1.19 (95 % CI 1.04–1.37) and
Pcorrected = 0.026, OR 1.21 (95 % CI 1.04–1.41), respectively]. The association of the exonic variant
was stronger in infections caused by the mycobacterial lineage M. africanum West-African 2
[Pcorrected = 0.024, OR 1.70; (95 % CI 1.2–2.6)]. Determination of haplotypes revealed the strongest
associaton with TB for the ‘non-5/760A’ haplotype
compared
with
the
‘non-5/760G’
haplotype
(P = 0.003, OR 1.50). Our observation of an association of ALOX5 variants with susceptibility to TB contributes evidence of the importance of 5-LO products to the regulation of immune responses to M. tu-
berculosis.
Collaboration: Sabine Rüsch-Gerdes, National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany; Florian Herb, Thorsten Thye, Rolf
D. Horstmann, Christian G. Meyer, Department of Molecular Medicine, Bernhard Nocht Institute for Tropical
Medicine, Hamburg, Germany; Edmund N.L. Browne,
Ellis Owusu-Dabo, Margaret A. Chinbuah, Department
of Community Health, School of Medical Sciences,
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana; John Gyapong, Ivy Osei, Health
Research Unit, Ghana Health Service, Accra, Ghana;
Oliver Werz, Department of Pharmaceutical Analytics,
Institute of Pharmacy, Eberhard Karls University, Tuebingen, Germany.
Supported in part by: German Federal Ministry of
Education
and
Research,
German
National
Genome Research Network (NGFN1; Project
01GS0162; NGFN2, NIE-S17.
Theses
Bachelor of Science
Schweinhuber, Stefanie
Genomische Diversität klinischer Mycobacterium tuberculosis Komplex Isolate
Studiengang Molecular Life Science
Universität zu Lübeck, 2007.
174
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular and Clinical Allergology
Research Reports
Division of Molecular and Clinical
Allergology
Head
Research Reports
Dr. Wolf-Meinhard Becker
Degradation, processing and transmission of the
major grass pollen allergen Phl p 1 at the respi-
Associated Physician
ratory interphase
Dr. Gabriele Berger (Clinical and Paediatric Aller-
Petersen A, Becker W-M.
gology)
The respiratory epithelium forms the first line of conPrinciple Investigators
tact and interaction between airborne particles,
Prof. Dr. A. Petersen
e.g. pollen, their released proteins and the innate
Dr. F. Schocker (since 01.09.2008)
immune system. At this level the induction of immune responses is initiated. We focused on the fate
Graduate and Diploma Students
of timothy grass pollen allergen Phl p 1 and its mo-
Susanne Krause (née Riecken) (until 10/2008)
lecular modifications after contact with the respira-
Sandara Rennert
tory interface. To investigate the influence of mucosal secretion, immunoblotting and zymography
Technicians
was performed. The allergen uptake and transmis-
Marisa Böttcher
sion by epithelial cells were studied by FACS analy-
Stefanie Fox (until 05/2008)
sis, confocal microscopy and determinations of cy-
Maren Hohn (since 9/2008)
tokine release and immunohistology. Mucosal se-
Daniela Warneke
cretions, e.g. nasal secretions of patients suffering
from bronchial inflammations or allergy to aeroal-
Guest and Trainees
lergens, revealed proteolytic activity. Also super-
Florian Wölbeling, Master student, Life sciences at
natants from mast cells or neutrophils, a situation
the University of Lübeck, Master practical course
similar to acute inflammation, cause a partial cleav-
(6 weeks, 11/06 – 2/07): Untersuchungen zum Nach-
age of the Phl p 1. To analyze the allergen uptake
weis von Erdnussallergen-Fragmenten in der Mut-
by epithelial cells, two cell lines were used that rep-
termilch.
resent different regions of the lung: A549 (derived
Anika Gallinger, Master student, Life sciences at the
from alveolar pneumocytes) and Calu-3 (from the
University of Lübeck, Master practical course
upper respiratory epithelium). Both cell lines were
(6 weeks, 11/07 – 2/08): Strukturelle Untersuchung
activated by Phl p 1 as demonstrated by the release
proteolytischer Fragmente des Gräserpollenaller-
of IL-8 and IL-6. A549 cells take up the allergen into
gens Phl p 1.
vesicles, which are not associated to lysosomes
Jana Schlenk, Diploma student, Biology at the Uni-
(macropinocytosis). Afterwards the allergen is re-
versity of Kiel, Practical course (6 weeks, 8/07 –
leased into the culture medium in a time-dependent
10/07): Untersuchung der RNase-Aktivität des Erd-
manner. In contrast, Calu-3 cells revealed colocal-
nussallergens Ara h 8.
ization of Phl p 1 and lysosomes as demonstrated
Dr. Marija Gavrovic-Jankulovic, Faculty of Chemistry,
by using confocal microscopy (probably accessory
Dept. Biochemistry, University of Belgrade, Serbia
antigen presenting cells). Additionally, Calu-3 cells
(11/08)
express MHCII molecules on the cell surface, which
is a prerequisite for antigen processing and presentation.
Wissenschaftlicher Jahresbericht 2007-2008
175
Division of Molecular and Clinical Allergology
Research Reports
To examine the allergen uptake under more physio-
ized concerning their structure, immunogenicity and
logical conditions, biopsies were taken from different
function. For estimation of the molecular mass, ex-
regions of the human lung, incubated with Phl p 1
tract and Phl p 1 samples were analysed by gelfil-
and immunostained. Only low amounts of antigen
tration. We determined complexes of up to 500 kDa,
were taken up in the alveolar regions, while the al-
but about 50% of the proteins still revealed molecu-
lergen uptake of the epithelial cells in the bronchial
lar masses of 60 - 10 kDa. The immunogenicity was
sections was stronger. However, macrophages in the
determined by Western blotting using patients’ sera
alveolar space showed a considerable antigen up-
and monoclonal antibodies. The binding patterns of
take. The secretions of the respiratory tract cause an
the HCHO treated samples were quite similar to
incomplete fragmentation of allergens which may fa-
those of the untreated controls, but the intensity was
cilitate the allergen uptake by epithelial cells. Our re-
diminished. Performing the CD203c basophil acti-
sults reveal differences in the uptake and transmis-
vation test, again no significant differences were ob-
sion of the allergen in the two cell lines. While the ep-
served compared to the untreated samples. After
ithelial cells of the upper airways probably degrade
KCNO treatment the binding patterns were dra-
and process the allergens, cells of the lower airways
matically changed and showed a severe decrease
seem to transmit the unprocessed allergens. Addi-
in intensity. Reactivity was only detectable with mon-
tionally, we are in progress to perform comparative
oclonal antibodies harbouring linear epitopes. No
studies on primary alveolar epithelial cells (AEC-II)
binding was observed with patients’ sera. The
isolated from human lung.
CD203c assay resulted in a 100fold less activation,
Fo r s c h u n g s z e n t r u m
Bo r s t e l
when the allergoids were used instead of the unCollaboration: Röschmann K, Ulmer AJ, Division of
treated controls. After HCHO or KCNO treatment
Cellular Immunology, Goldmann T, Division of Clin-
about 50% of the protein content was polymerised
ical and Experimental Pathology, Research Center
to allergoid complexes of molecular masses up to
Borstel; Blume C, ZAUM-Center of Allergy and Envi-
500 kDa. By use of KCNO the allergens were much
ronment, Division of Environmental Dermatology
more modified than by use of HCHO and differed
and Allergy, Munich.
individually among the different allergens. Our
studies establish the molecular basis to investigate
Supported in part by: DFG-SFB TR 22, project A3.
the uptake of allergoids by antigen presenting cells.
Collaboration: Ernst M, Immune Cell Analytics, ReStructural, immunological and functional charac-
search Center Borstel, Borstel; Bellinghausen I,
terization of grass pollen allergoids
König B, Saloga J, Dermatology Department, Uni-
Petersen A, Becker, W-M.
versity of Mainz, Mainz.
Allergoids are often used for specific immunother-
Supported in part by: DFG (Pe 491/8).
apy instead of allergen extracts. They are structurally modified by chemical treatment to reduce allergenicity but retain immunogenicity. In order to de-
Recombinantly produced banana lectin isoform:
termine the influence of allergoids on the uptake by
a valuable tool for glycoproteomics and a potent
antigen presenting cells and the further processing,
modulator of the proliferation response in CD3+,
we have produced allergoids of timothy grass
CD4+ and CD8+ populations in human PBMCs
pollen and the major allergen Phl p 1 by treatment
Petersen A.
with fomaldehyde (HCHO) or potassium cyanate
(KCNO). Afterwards, the allergoids were character-
176
Lectins as carbohydrate binding proteins have
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular and Clinical Allergology
Research Reports
been employed in various biological assays for de-
Ara h 8 – analysis of natural and recombinant
tection and characterization of glycan structures on
preparations
glycoproteins, including clinical biomarkers in dis-
Riecken S, Petersen A, Becker W-M.
ease states. A mannose specific banana lectin
(BanLec) is unique in its specificity for internal
Peanuts belong to the classical or true food aller-
α1,3 linkages as well as β1,3 linkages at the re-
gens which can directly sensitize patients and elic-
ducing termini. The immunomodulatory potential of
it mild to severe allergic reactions. A subgroup of
natural BanLec was recognized by a strong im-
grass and birch pollen allergic patients reacts to
munoglobulin G4 antibody response and T cell mi-
peanuts with an OAS. This may be caused by a
togen activity in humans. To explore its applicabili-
cross-reactivity between the peanut profilin Ara h 5
ty in glycoproteomics and its modulatory potential,
and Ara h 8, which is a Bet v 1 homologous protein.
the gene of banana lectin was cloned, sequenced
Both allergens were found by DNA-technology. The
and a recombinant protein was produced in E. coli.
aim of our study is to identify natural Ara h 8 in
Protein sequencing as well as mass spectrometry of
peanut extract, to develop a strategy to purify it
the recombinant BanLec (rBan Lec) and its tryptic
from peanut extract and to characterize it by N-ter-
in-gel digest were performed. The specificity in gly-
minal sequencing. A three step purification protocol
can structure detection was examined with five pro-
was developed for the isolation of Ara h 8 from
tein extracts rich in glycoprotein content, as well as
roasted peanuts. In a first step, size exclusion chro-
horseradish peroxidase glycoprotein. The im-
matography from peanut extract was performed.
munomodulatory potential was assessed by an in-
The Ara h 8 containing fractions were analyzed by
hibitory assay and a human T cell proliferation as-
SDS-PAGE and immunoblotting. Afterwards, a re-
say. The obtained cDNA revealed a novel banana
duction and alkylation was carried out, followed by
lectin isoform with an open reading frame of 426 nu-
ion exchange chromatography. The amino terminus
cleotides, encoding a cytoplasmatic protein of 141
was determined by microsequencing. Besides,
amino acids. The molecular mass of rBanLec de-
Ara h 8 was analyzed on the level of gDNA and cDNA.
termined by EST FT-MS and N-terminal sequencing
The recombinant protein was expressed as an His-
confirmed the cDNA at the protein level. The novel
tagged fusion protein in E. coli and afterwards pu-
rBanLec isoform induced a strong proliferation re-
rified using metal chelating chromatography. The
sponse in CD3+, CD4+ and CD8+ populations in hu-
natural and recombinant proteins were compared
man PBMCs. The recombinant BanLec is a useful
by immunoblotting with patients’ sera. The combi-
reagent for glycoproteomics and lectin microarrays
nation of several purification methods led to ho-
with a potential for modulation of the immune re-
mogenous natural Ara h 8. Using size exclusion
sponse.
chromatography Ara h 8 was successfully isolated
from other peanut proteins. Only a cross contami-
Collaboration: Gavrovic-Jankulovic M, Bobic S, Fac-
nation with Ara h 6 was observed. Due to the fact
ulty of Chemistry, University of Belgrade, Belgrade,
that Ara h 6 contains several cysteine residues
Serbia; Paulsen K, Institute of Medical Microbiology
which form disulfide bonds, the subsequent reduc-
and Immunology, University of Aarhus, Aarhus, Den-
tion, alkylation and ion exchange chromatography
mark; Brckalo T, Molecular Immunopathology Unit,
led to pure Ara h 8. In comparison to the purified
Universitat Pompeu Fabra (DCEXS), Barcelona,
Ara h 8 the recombinant protein showed the same
Spain; Lindner B, Division of Immunochemistry, Re-
reactivity with patients’ sera in immunoblotting. We
search Center Borstel, Borstel, Germany.
developed a method for the purification of natural
Ara h 8 from peanut extract. The N-termini determined by protein sequencing and deduced from the
Wissenschaftlicher Jahresbericht 2007-2008
177
Division of Molecular and Clinical Allergology
Research Reports
cDNA sequence are absolutely identical. However,
but peanut LTP as sensitizing allergen cannot be ex-
the sequences differed by two amino acids from the
cluded. The clinical relevance of peanut LTP re-
published Ara h 8 isoform (ACC: AY328088). This in-
mains an open question until peanut-allergic pa-
dicates that we found a new isoform of Ara h 8. On
tients are challenged with the full panel of peanut
the molecular level, we confirmed the already pub-
allergens.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
lished isoform as well, but the new isoform seems
to be the most dominant isoform in peanut extracts.
Collaboration: Mari A, Quaratino D, Zennaro D,
Center for Clinical and Experimental Allergology,
Collaboration: Jappe U, Paul-Ehrlich-Institut, Division
IDI-IRCCS, Rome, Italy; Reese G, Vieths S, Division of
of Allergology.
Allergology, Paul-Ehrlich-Institut, Langen, Germany.
Supported in part by: Freundeskreis ForschungsIdentification of a Lipid Transfer Protein (LTP)
zentrum Borstel und Sparkassenstiftung Südholstein.
in peanut extract and cloning of two LTP isoallergens
Riecken S, Petersen A, Becker W-M.
Detection of peanut antigens/allergens in breast
milk
The presence of a LTP in peanut has been hypoth-
Schocker F, Petersen A, Becker W-M.
esized but has never been confirmed. The aim was
to identify LTP in peanut extract and characterize its
Peanut allergy is one of the most severe food aller-
physicochemical and immunological properties.
gies because the risk for life-threatening reactions
We tried to identify peanut LTP by use of cross-re-
and also because it is rarely outgrown. According
acting rabbit anti-Cor a 8 antiserum, sera from
to the literature, allergic reactions to peanut can oc-
peach and peanut-allergic patients and by N-ter-
cur during first known exposure which means that
minal sequencing Peanut LTP was identified in an
earlier peanut exposure must have taken place.
acidic extract of peanut meal at 8 kDa by im-
There is some evidence suggesting that peanut ex-
munoblotting and by N-terminal sequencing. The
posure during lactation can trigger sensitization as
IgE reactivity of two cloned and expressed isoaller-
peanut protein was found in breast milk following
gens was demonstrated with sera from peach and
maternal dietary ingestion (Vadas et al., JAMA
peanut-allergic patients by immunoblotting. The im-
2001; 285:1746-1748). Other research groups claim
munoblot analysis with patients’ sera showed IgE-
that there is no link beween maternal peanut avoid-
reactivity to natural LTP in 68% (n=25) of patients al-
ance and the development of peanut allergy in the
lergic to peanut and peach (Pru p 3 positive) and
breast fed baby, and that consuming peanuts
24% (n=17) of peach-allergic patients (Pru p 3 posi-
would increase the development of tolerance.
tive), which are asymtomatic to peanut. Only a weak
Since 1998, advice in the UK has been that pregnant
LTP reactivity was detectable in 29% (n=24) of pa-
and lactating women should avoid eating peanuts
tients with peanut allergy (Pru p 3 negative).For the
if the familiy is at risk of an atopic disease. How-
first time peanut LTP is described as a novel aller-
ever, just recently, the House of Lords has demand-
gen with immunological and molecular biological
ed to withdraw the existing UK advice because the
methods. The N-terminal sequence data allowed
prevalence of peanut allergy has continued to rise
cloning and production of recombinant LTP from
despite the avoidance recommendations. What-
peanut. Based on clinical data we suggest that the
ever the reason for these findings could be, it re-
main sensitization of peanut allergic patients to
mains clear that investigations are needed to elu-
peanut LTP is caused by cross reactivity to Pru p 3,
cidate whether or not avoidance of peanuts while
178
Wissenschaftlicher Jahresbericht 2007-2008
Division of Molecular and Clinical Allergology
Research Reports
breast feeding is necessary. In the light of these con-
Stipends
tradicting views our project is very challenging. We
Riecken, Susanne
aim to identify peanut antigens/allergens in breast
Freundeskreis Forschungszentrum Borstel und Spar-
milk after peanut consumption and hope to learn
kassenstiftung Südholstein
more about how peanut allergens are processed
during their passage through the gastrointestinal
tract. Likewise we intend to get more information
about possible protecting sIgA and IgG antibodies
in breast milk and serum samples of our recruited
lactating mothers. In the first step, we spiked milk
samples of mothers, who avoided peanuts during
lactation with extract of commercially available
peanuts and analysed the samples by using SDSPAGE and immunoblot techniques. By this means
we were able to detect up to 2,5 µg peanut protein
in 15 µg breast milk using a rabbit antibody directed against one of the major allergens, the Ara h 2.
Using the 2D electrophoresis techniques we could
even detect amounts of 200 ng peanut protein in
150 µg breast milk which offers further investigations of peanut allergens for subsequent MALDI
analysis. Thus, we established qualitative and quantitaive tools to detect peanut proteins in breast milk
to validate these results in samples after peanut
consumption.
Collaboration: Ahrens F, Childrens’ Hospital Altona,
Pediatric Pneumology and Allergology, Hamburg.
Supported in part by: DFG (SCHO-828/2-1).
Theses
Dissertation
Riecken, Susanne
Untersuchungen zum Allergenrepertoire der Erdnuss: Molekulare Charakterisierung von Ara h 7, Ara
h 8, Oleosin und LTP
Technisch-Naturwissenschaftliche Fakultät
Universität Lübeck, 2008.
Wissenschaftlicher Jahresbericht 2007-2008
179
Division of Cellular Allergology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Cellular Allergology
Head
co-evolution between worm and host characterized
PD Dr. Helmut Haas
by the endeavour of the worm to dampen the defence of the host without harming him, and the en-
Principal Investigators
deavour of the host, to get rid of the undesired guest
PD Dr. Jürgen van der Bosch (until 02/08)
without causing “collateral damage”. Due to their
Dr. Gabriele Schramm
immunomodulatory properties, parasitic worms are
attractive candidates for further elucidating the me-
Postdocs
chanism of Th2 induction and for identify-
Dr. Lina Meyer (since 09/08)
ing/characterizing anti-inflammatory effector molecules for designing novel strategies to fight allergy
Graduate Students
and autoimmune disorders. The Division of Cellular
Astrid Mewes (until 12/07)
Allergology focuses on Schistosoma mansoni, a parasitic fluke with high immunomodulatory capacity.
Diploma and Bachelor students
We have sequenced and/or further characterised
Rabea Langenhaun
three major immunogens from S. mansoni eggs:
Katrin Marszalek
omega-1, IPSE/alpha-1 and kappa-5. While omega1 and IPSE/alpha-1 are important immunomodula-
Technicians
tory molecules, the biological role of kappa-5 has
Daniela Barths
still to be elucidated. With the elaboration of an ef-
Achim Gronow
fective in vitro culture protocol, we got, moreover, an
Heike Rohweder (since 02/08)
important tool for schistosome research in our
Maria Pfohl (since 09/08)
hands.
Birte Hanisch (since 10/08)
Guests and Trainees
Omega-1, a glycoprotein secreted by Schistoso-
Anna Gilicze (Semmelweis University, Budapest)
ma mansoni eggs, drives Th2 responses
Dr. Franco Falcone (University of Nottingham)
Schramm G, van der Bosch J, Haas H.
Ahmed Fawzy Abou Karima (University of Southampton)
The outcome of immune responses (i.e. health or di-
Stefanie Neubert
sease) in the course of infectious or autoimmune disorders is critically dependent on the differentiation
of polarized T helper (Th) cell response. It is, there-
Research Reports
fore, of prime importance to understand the mecha-
In contrast to allergens, parasitic worms are obli-
nisms governing Th subset differentiation. While
gate and potent inducers of a T helper type 2 (Th2)
many microbial molecules that induce the differen-
and IgE response. On the other hand, namely in the
tiation of Th1 cells (e.g. LPS, CpG) are well-characte-
course of chronic worm infections, Th2 (and also
rized, very little is known about the molecules that re-
Th1) responses are down-regulated by worm-deri-
sult in the development of Th2 responses. Helminth
ved highly active anti-inflammatory molecules. Thus,
parasites belong to the most potent natural stimuli for
parasitic worms – despite being inducers of a Th2
the induction of Th2-polarized immune responses.
response – possess an anti-allergic/anti-inflamma-
Among those, Schistosoma mansoni eggs and so-
tory effect. This seeming paradox results from the
luble extracts derived thereof (SmEA) are well known
180
Wissenschaftlicher Jahresbericht 2007-2008
Division of Cellular Allergology
Research Reports
and widely studied stimuli for Th2 polarization in vitro
mansoni lead to the induction of a Th2 response
and in vivo. In this process dendritic cells (DCs) have
and later on during chronic infection to a general
been shown to play an important role. However, the
downregulation of inflammatory responses inclu-
specific Th2-inducing components have remained
ding Th2. We have previously isolated and recom-
elusive. In a collaborative approach, we could de-
binantly expressed a molecule from S. mansoni
monstrate that omega-1, a glycoprotein secreted
eggs that triggers the release of IL-4 and IL-13 from
from S. mansoni eggs and present in SmEA, modu-
human and murine basophils. This molecule was
lates human monocyte-derived DCs to drive Th2 po-
called IL-4-inducing principle from Schistosoma
larization with similar characteristics as whole SmEA.
mansoni eggs (IPSE/alpha-1). The fact that IL-4 as
Furthermore, using IL-4 dual reporter mice we could
well as IL-13 are key cytokines for the development
show that both natural and recombinant omega-1
of a Th2 response and are, moreover, involved in
alone are sufficient to generate Th2 responses in vi-
regulatory/anti-inflammatory mechanisms, e.g. via
vo even in the absence of IL-4R signaling. Thus ome-
alternative activation of macrophages, suggests
ga-1, a single component of SmEA, conditions DCs for
that IPSE/alpha-1 plays a role in this respect. To ob-
the efficient, IL-4R-independent priming of Th2 res-
tain additional information on the function and the
ponses. Having a single molecule with a strong Th2
mechanism of action of IPSE/alpha-1, the three-di-
inducing activity will help to unravel the specific pa-
mensional structure of this molecule was addres-
thways governing the instruction of Th2 responses.
sed. Since similarity searches revealed no sequen-
Considering the translational relevance, the identifi-
ces with significant homology, the three-dimensio-
cation of omega-1 as a potent Th2-inducing adjuvant
nal structure of IPSE/alpha-1 was analysed by NMR
provides novel opportunities for the development of
and X-ray crystallography in a collaborative ap-
therapeutic interventions of Th1-mediated diseases
proach. This analysis required IPSE/alpha-1 at high
(patent application has been filed).
amounts/concentrations which could be realised
neither with the natural molecule due to its low
Cooperation: Bart Everts, Hermelijn H. Smits, Cor-
availability nor with the recombinant E. coli-ex-
nelis H. Hokke, Alwin J. van der Ham, Maria Yaz-
pressed material because of its tendency to self-ag-
danbakhsh, Department of Parasitology, Leiden
gregate. However, upon deletion of the positively
University Medical Centre, 2333 ZA Leiden, The
charged C-terminus (containing a nuclear localisa-
Netherlands; Markus Mohrs, Georgia Perona-
tion sequence and a cysteine residue responsible
Wright, Katja Mohrs, Trudeau Institute, Saranac
for the dimerisation of this molecule), we were ab-
Lake, New York 12983, United States; Colin M.
le to express and refold a highly soluble and sta-
Fitzsimmons, Department of Pathology, University of
ble IPSE/alpha-1 monomer. Following C13-/N15-la-
Cambridge, Cambridge CB2 1QP, UK; Michael J.
beling, the 3D structure of this molecule could be
Doenhoff, School of Biology, University of Notting-
determined by solution NMR spectroscopy. Fur-
ham, Nottingham NG7 2RD, UK.
thermore it was crystallized and a native X-ray
data set was recorded to 1.66Å. Analysis revealed
Supported in part by: DFG SFB/Transregio 22 / A12.
that IPSE/alpha-1 has two Greek key motifs
(each containing four anti-parallel
-sheets) and
belongs to the family of the extremely staDetermination of the three-dimensional structure
ble / crystallins, present for example in high con-
of IPSE/alpha-1
centrations in the eye lens (Fig. 72). We expect from
Schramm G, Barths D, Haas H.
targeted mutations based on the structural informations valuable help for elucidating the mecha-
Infections with the parasitic helminth Schistosoma
nism of action of this molecule.
Wissenschaftlicher Jahresbericht 2007-2008
181
Division of Cellular Allergology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
ted. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa5 with IgE and IgG isotype antibodies, but against
IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one
of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.
Fig. 72. Structure of an IPSE/alpha-1-Monomer containing two
Greek key motifs (Ribbon Model).
Cooperation: J.V. Hamilton, Institute of Biological Environmental and Rural Sciences, Aberystwyth Uni-
Collaboration: Michael Sattler, Helge Meyer, Kostas
versity, Ceredigion, SY23 3DA, UK; C.I.A Balog, M.
Tripsianes Technical University Munich; Jochen
Wuhrer, A.M. Deelder, C.H. Hokke, Department of
Müller-Dieckmann, Hubert Mayerhofer, Georgios N.
Parasitology, Center of Infectious Diseases, Leiden
Hatzopoulos EMBL, Hamburg; Christoph Müller-
University Medical Center, 2300 RC Leiden, The
Dieckmann, ESRF, Grenoble, France.
Netherlands, V. Wippersteg, Institute for Genetics,
Heinrich-Heine-University, D-40225 Duesseldorf, Ger-
Supported in part by: DFG SFB/Transregio 22 / A12.
many, S. Beckmann, C.G. Grevelding, Institute of
Parasitology, University of Giessen, D-35392
Giessen, Germany, E. Weber, GALMED GmbH , D-
Molecular characterisation of kappa-5, a major
06097 Halle, Germany, N.W. Brattig, Bernhard
antigenic glycoprotein from Schistosoma manso-
Nocht Institute for Tropical Medicine, University of
ni eggs
Hamburg, D-20359 Hamburg, Germany, D.W.
Schramm G, Gronow A, Goldmann T, Haas H.
Dunne, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK, M.J. Doenhoff,
The major immunopathological consequences of
School of Biology, University of Nottingham, Not-
infection with Schistosoma mansoni, a T helper type
tingham NG7 2RD UK.
2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage
Supported in part by: DFG SFB/Transregio 22 / A12.
of the parasite. Three antigens of S. mansoni eggs,
termed IPSE/alpha-1, omega-1 and kappa-5, have
been found to be the primary targets of the egg-di-
In vitro culture of Schistosoma mansoni – culture
rected antibody response of the host. Here, we re-
dish instead of mammalian host
port on the isolation, cloning and characterisation
Herrmann K, Falcone FH, Barths D, Haas H.
of kappa-5. Apart from an uncharacterised mRNA
sequence in S. japonicum, no significant similarities
Approx. 200 million people worldwide are suffering
of kappa-5 to known sequences from other species
from Schistosomiasis partly with life-threatening con-
were found. In contrast to IPSE/alpha-1 and omega-
sequences and still far more are endangered from
1, which have been found only in eggs, kappa-5 was
it. The affected countries, among them potent new-
present in miracidia as well as in eggs at the mR-
ly industrialising economies such as China, South-Af-
NA and protein levels. In eggs, isoforms of kappa-
rica and Brazil, have a genuine interest in an effec-
5 were observed with both three and four fully oc-
tive treatment and prevention of this disease. The
cupied N-glycosylation sites, while in miracidia on-
development and optimisation of the in vitro cultu-
ly one isoform with four N-glycans could be detec-
re of schistosomes shall pro-actively and lastingly
182
Wissenschaftlicher Jahresbericht 2007-2008
Division of Cellular Allergology
Research Reports
reduce/prevent the foreseeable flood in animal ex-
With the in vitro culture a highly effective tool for pa-
periments for studying these parasites. We have es-
rasitological and immunological studies is availa-
tablished in our lab an in vitro method for maturing
ble which among others allows testing potential
larvae (schistosomula) of Schistosoma mansoni up
new drugs and vaccines against schistosomes by
to the adult stage including pairing and deposition
high-throughput procedures. In contrast to animal
of immature eggs (Fig. 73). Using our culture proto-
experiments, the in vitro culture allows continuous
col, we were furthermore able to keep adult schi-
monitoring of the parasite including access to each
stosome pairs in vitro for more than 18 months at
developmental stage as well as real time recording
unimpaired motility. Based on these results and sup-
of morphological changes following e.g. addition of
ported by a grant (BMBF 0315277; one post-doc,
drugs.
one technician), we aim now at further optimising
the culture conditions to obtain full egg maturation
Collaboration: Cornelis H. Hokke, Department of
in vitro and, thus, to completely replace experi-
Parasitology, Leiden University Medical Center,
mental mammalian final hosts (e.g. mice, golden
2300 RC Leiden, The Netherlands; Christoph G.
hamsters) by the culture dish. In parallel, the tech-
Grevelding, Institute for Parasitology, Justus Liebig
nique of cryoconservation (i.e. gentle freezing) of li-
University Giessen, D-35392 Giessen, Germany.
ve schistosomes will be optimised along with establishment of a schistosome bank. This will allow the
Supported in part by: BMBF 0315277.
need-based access to infectious stages of the parasite and makes the permanent maintenance of
the parasite life cycle in laboratory animals redun-
Theses
dant.
Diploma
Marszalek, Katrin
Partielle
Deglykosylierung
von
HEK-IPSE/alpha-1 für die Kristallisation
Julius-Maximilians-Universität,
Würzburg, 2007.
Langenhaun, Rabea
Etablierung einer cDNA-Expressionsbank aus basophilen Granulozyten
Freie Universität Berlin, 2007.
Dissertation
Mewes, Astrid
Untersuchung eines neuartigen Mechanismus der
Fig. 73. Schistosomula were cultured in vitro to adult parasites including pairing after 6 weeks and deposition of immature eggs (resembling a string of pearls) after 8 weeks. At the upper right mature eggs isolated from infected murine liver are shown. Our aim
is “closing the loop” (symbolized by the red arrow) by optimising
our culture protocol to obtain full egg maturation in vitro including
complete development of miracidia (life cycle stage that hatches
from eggs and infects water snails as intermediate hosts).
Aktivierung basophiler Granulozyten durch das sekretorische Schistosomen-Ei-Produkt, IPSE/alpha-1
Technisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, 2007.
Wissenschaftlicher Jahresbericht 2007-2008
183
Division of Mucosaimmunology
Research Reports
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Division of Mucosaimmunology
Head
The stabilities of three murine B cell epitopes from the
PD Dr. Andreas Frey
model antigen hen egg ovalbumin in murine intestinal fluid (IntFl) were determined. While all three oval-
Staff Scientists
bumin-epitopes were degraded with half-lifes in the
Dr. Steffen Bade
range of milliseconds when present as free peptides,
Dr. Barbara Frey
their conjugation to cholera toxin B subunit (CTB) as
Dr. Niels Röckendorf
carrier protein prolonged their half-lifes up to 12 minutes. The corresponding RI peptides were not deg-
Graduate Students
raded by murine IntFl. Balb/c-mice were immunized
Naho Fujimoto
intraperitoneally or intragastrically with either a na-
Fabian Reuter
tural or an RI peptide conjugated to CTB. While i.p.
Katrin Wehry
application of natural and RI peptide-conjugates resulted in similar IgG- and IgA-titers for each peptide,
Technicians
by i.g. immunization RI peptides induced higher an-
Alheidis Ehmke
ti-peptide IgG-titers or IgA-titers than their natural
Hanne Krüger
counterparts (Fig. 74). Only one natural peptide,
Imke Wysokinski
which also showed the highest in vitro-stability in IntFl, could induce a weak IgA-response at all. However, antibodies induced by RI peptides failed to rec-
Research Reports
ognize the native protein ovalbumin.
Influence of antigen stability on mucosal immune
responses
Reuter F, Bade S, Frey A.
The induction of a protective secretory IgA response
at the intestinal mucosa requires the delivery of an
antigen to this very site. For immunization purposes
this means that a vaccine should be administered
orally or intragastrically and that it must to reach the
inductive sites of the mucosal lymphoid tissue in the
gut in uncompromised form. Hence, the digestive stability of vaccines is of utmost importance for successful immunizations. Yet, especially vaccines based on
peptidic antigens may suffer considerable degradation in the proteolytic environment of the gut. It may
be possible to stabilize such peptidic antigens by
converting them into their retro-inverse (RI) configuration. Such RI peptides, which are composed solely
Fig. 74. Immune response at mucosal surfaces after intragastrical
immunization. Immunoglobulin A titers (mean ± SEM; N=6-7) in fecal extracts of mice immunized with either CTB-conjugated natural
(nat) Ova-peptides or CTB-conjugated retro-inverse (RI) Ova-peptides were determined against the peptides used for immunization.
Statistical analysis of the results (one-way ANOVA and Bonferroni
post hoc test) revealed significant differences between the groups
immunized with the respective conjugates of natural and RI peptides (***: p<0.001; **: p<0.01); n.b., not detectable.
of D-amino acids, are not prone to proteolytic degradation, but their capability to induce a protective
We conclude that stabilization of antigens against
immune response at the intestinal mucosa needs to
proteolytic degradation is a prerequisite for suc-
be investigated.
cessful oral vaccinations, yet maintaining the natu-
184
Wissenschaftlicher Jahresbericht 2007-2008
Division of Mucosaimmunology
Research Reports
ral antigen conformation is just as critical for indu-
and protein-enriched simIF over 3h but (iii) are par-
cing protective immunity by peptide-based vacci-
tially destroyed in simIF that lacks additional pro-
nes. While digestion-resistant RI peptides were ab-
tease substrate. We assume that the proteins of na-
le to induce high antibody-titers against themselves,
tIF are preferred substrates for the intestinal pro-
they did not sufficiently mimic the structure of the
teases and thus can protect vaccine-targeting li-
epitopes that is displayed in the context of the na-
gands from destruction.
tural protein.
Collaboration: Hirst TR, John Curtin School of MedCollaboration: Hirst TR, John Curtin School of Med-
ical Research, The Australian National University,
ical Research, The Australian National University,
Canberra, Australia.
Canberra, Australia.
Supported in part by: Deutsche ForschungsgemeinSupported in part by: Deutsche Forschungsgemein-
schaft (DFG), grant Fr 958/2-4 and Bundesministeri-
schaft (DFG), grant Fr 958/2-4, and Bundesministe-
um für Bildung und Forschung (BMBF), grant 01 KO
rium für Bildung und Forschung (BMBF), grant 01 KO
0113.
0113.
Degradation of food allergens by digestive proProtection of potential vaccine targeting ligands
teases
against intestinal proteolysis
Bade S, Reuter F, Frey A.
Reuter F, Bade S, Frey A.
Food allergies manifest as immunoglobulin E-mediEndowing mucosal vaccines with ligands that target
ated defense reactions against certain food pro-
antigen to mucosal lymphoid tissues may improve
teins and may be caused by a lack of mucosal im-
immunization efficacy provided that the ligands
mune tolerance against the antigen in question. A
withstand the proteolytic environment of the gas-
faulty tolerance induction could be due to prema-
trointestinal tract until they reach their destination.
ture destruction or incomplete formation of tolero-
We have investigated whether and how three re-
genic T cell epitopes in the gut lumen.
nowned ligands - Ulex europaeus agglutinin I and
To determine the luminal stability of potential aller-
the B-subunits of cholera toxin and E. coli heat-labi-
gen-derived T cell epitopes in the murine intestinal
le enterotoxin - master this challenge. We assessed
fluid we used our novel protease susceptibility as-
the digestive power of natural murine intestinal fluid
say for peptides and standardized murine intestinal
(natIF) using assays for trypsin, chymotrypsin and
fluid. Major allergens from food classified by the Eu-
pancreatic elastase along with a test for nonspeci-
ropean Union Allergen Hitlist (crustaceans, eggs,
fic proteolysis. The natIF was compared with simu-
fish, peanut, soybean, milk/-products, tree nuts, se-
lated murine intestinal fluid (simIF) that resembled
same, mustard, celery, wheat) were synthesized in
the trypsin, chymotrypsin and elastase activities of
form of nested 10mer and 16mer peptides covering
its natural counterpart but lacked or contained al-
the entire amino acid sequences of the respective
bumins as additional protease substrates. The li-
allergens. The mean peptide half-lifes determined
gands were exposed to the digestive fluids and de-
in this set-up ranged from 52 µs to about 10 min for
gradation was determined. The studies revealed
the 10mers and from 55 µs to 42 s for the 16mers.
that (i) the three pancreatic endoproteases consti-
Although most peptides were degraded within mil-
tute only one third of the total protease activity of
liseconds, several peptides withstood proteolysis
natIF and (ii) the ligands resist proteolysis in natIF
long enough to theoretically survive at least parti-
Wissenschaftlicher Jahresbericht 2007-2008
185
Division of Mucosaimmunology
Research Reports
ally the murine small intestinal passage. It remains
many experimental set-ups, and facilitates multiple-
to be elucidated if these comparably stable aller-
labeling of biomolecules.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
gen-derived fragments constitute potential tolerogenic murine T cell epitopes.
Collaboration: Franek M, Department of Biotechnology, Veterinary Research Institute, Brno, Czech Re-
Collaboration: Becker WM, Division of Molecular
public.
and Clinical Allergology, Research Center Borstel,
Germany; Franek M, Department of Biotechnology,
Supported in part by: Bundesministerium für Bildung
Veterinary Research Institute, Brno, Czech Republic.
und Forschung (BMBF), grants 01 KO 0113 and 13 N
8473.
Supported in part by: Bundesministerium für Bildung
und Forschung (BMBF), grant 01 KO 0113; Deutsche
Forschungsgemeinschaft (DFG), Schwerpunktpro-
Nanoparticles at mucosal interfaces
gramm 1089 „Novel vaccination strategies“, grant
Bade S, Röckendorf N, Frey A.
Fr 958/4-1; Deutsche Forschungsgemeinschaft (DFG)
grant Fr 958/2-4.
With the advent of nanotechnology, nanoscale particles (nanoparticles, NPs) have come into widespread use in basic research as well as in everyday
Biolabeling with 2,4-dichlorophenoxyacetic acid
applications, ranging from advanced functional ma-
(2,4-D)-derivatives
terials for high-tech applications in mechanical en-
Bade S, Röckendorf N, Frey A.
gineering, electronics and catalysis over functional
coatings to suntan lotions and new drugs. This om-
Current analytic and diagnostic methodologies re-
nipresence carries a high risk of unvoluntary expo-
quire highly-sensitive, versatile and robust tags for
sure to nanoparticulate materials and requires a
single- or multiple-labeling of biomolecules. We ha-
thorough investigation of the physiological events
ve developed a new labeling and detection system
and consequences connected with the potential
based on derivatives of 2,4-dichlorophenoxyacetic
uptake and incorporation of engineered nanopar-
acid (2,4-D) and monoclonal anti-2,4-D antibodies
ticles. As the digestive tract is one of the key sites
that meets these requirements.. It can be used for
at which the body comes into contact with foreign
a large variety of biomolecules such as (gly-
matter we investigate the interactions of NPs with
co)proteins, peptides and nucleic acids. When its
the different layers and cells of the small intestinal
sensitivity of detection is compared with that of the
mucosa, identify pathways along which NPs enter
established systems digoxigenin (DIG)/anti-DIG
the mucosa, and quantify the exit routes of NPs to
and biotin/streptavidin, the performance of 2,4-D is
distant tissues. To investigate the interaction of syn-
at least comparable to if not better than that of its
thetic NPs with digestive juice and the pre-epitheli-
competitors. Equipment of 2,4-D-derivatives with ali-
al mucus layer of the intestinal barrier, we employ
phatic spacers considerably increases their affinity
colloidal nanocrystals composed of fluorescent co-
towards the anti-2,4-D antibodies and raises the
res and stabilized by a layer of surfactants/ligands
sensitivity of detection up to 1,600fold compared
attached to their surface. Different synthetic, water-
with their respective non-spacered 2,4-D-derivati-
soluble core/shell (CdSe/CdS or CdSe/ZnS) and
ves. Hence, the detection of about 100 amoles spa-
core/shell-alloy/shell (CdSe/CdZnS/ZnS) nanopar-
cered 2,4-D-tag is possible. The 2,4-D-based labe-
ticles were incubated with murine intestinal fluid in
ling system augments the field of indirect labeling
order to check if interaction with intestinal constitu-
systems, outperforms commonly used systems in
ents from the digestive juice may change the
186
Wissenschaftlicher Jahresbericht 2007-2008
Division of Mucosaimmunology
Research Reports
nanoparticles’ optical characteristics. Indeed, fluo-
rescent dye at the sphingosine moiety of the mole-
rescence of most core/shell particles was consid-
cule was prepared by a convenient one pot syn-
erably impaired in the presence of murine intestin-
thesis. The labeled GM1 permitted the detection of
al fluid (Fig. 75). However, our investigations also re-
the natural ganglioside GM1 ligand Escherichia co-
vealed that by choosing appropriate particle-ligand
li heat-labile enterotoxin subunit B (EtxB) in pmole
combinations the fluorescent properties of the na-
quantities on a solid support. When an epitope-
noparticles could be retained regardless of their in-
mapping of several ganglioside binding proteins
cubation in mucosal secretions. Further studies now
and protein fragments was performed by screening
focus on the identification of optimal ligand-nano-
a cellulose membrane-bound synthetic library of 64
particle combinations that are useful for bio-ima-
16mer peptides with the new probe, several pepti-
ging applications at mucosal interfaces.
des displaying ganglioside GM1 affinity could be
identified. The labeled glycolipid represents a versatile tool for various biochemical investigations.
Collaboration: Hirst TR, John Curtin School of Medical Research, The Australian National University,
Canberra, Australia.
Supported in part by: Bundesministerium für Bildung
und Forschung (BMBF), grants 13 N 8473 and 01 KO
0113.
Fig. 75. Mean emission spectra of core-shell (CdS/ZnSe) nanoparticles after different treatments. Nanoparticles were incubated in
water (red), physiological buffer (green) or murine intestinal fluid
(mIntFl, blue) for 90 min at room temperature and fluorescence
spectra were recorded.
Peptide based optical contrast agents for targeting of intestinal malignancies
Collaboration: Weller H, Institute of Physical Chem-
Röckendorf N, Wehry K, Fujimoto N, Frey A.
istry, University Hamburg, Germany; Gebert A, Institute of Anatomy, University Lübeck, Germany;
Intestinal tumors exhibit cell surface properties that
Hüttmann G, Institute of Biomedical Optics, Univer-
differ from neighboring healthy epithelia and thus
sity Lübeck, Germany; Bings N, Institute of Anor-
allow tumor cell-specific molecular targeting. The
ganic and Analytical Chemistry, University Mainz,
accessibility of cell surface receptors such as gang-
Germany.
lioside GM1 is such a discriminatory characteristic.
Although ganglioside GM1 is expressed in the api-
Supported in part by: Deutsche Forschungsgemein-
cal membrane of all intestinal epithelial cells it can
schaft (DFG), grant Fr 958/5-1.
be reached by particle-conjugated ligands on tumor cells only. In order to exploit this phenomenon
we want to develop a nanoparticulate optical con-
Identification of GM1 binding sequence motifs by
trast agent equipped with a peptidic GM1 binding
screening a synthetic peptide library with a new-
ligand. For identification of ligand peptides a novel
ly developed fluorescent ganglioside GM1 deriv-
screening platform was devised where potential
ative
ganglioside GM1-binding peptides are generated
Röckendorf N, Bade S, Frey A.
on glass capillary plates using microfluidic noncontact arraying techniques and screened in situ for
A ganglioside GM1 probe bearing a dark-red fluo-
binding of fluorophore-labeled GM1. These three-di-
Wissenschaftlicher Jahresbericht 2007-2008
187
Division of Mucosaimmunology
Research Reports
mensional supports are easy to handle and show
“molecular evolution” of peptides with defined pro-
better sensitivity than either flat glass or membrane
perties. A population of peptides is prepared by
supports because of their large inner surface and
techniques which allow the automated synthesis of
low interference with readout systems. A custom flu-
up to 2,400 peptides simultaneously on cellulose
orescence reader was designed to comply with the
membranes or resin supports.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
specific optical behaviour of peptide arrays synthesized on microcapillary plates. This reader uses
a small numerical aperture for excitation and a large numerical aperture for detection in epifluorescence-mode. Background noise from fluorescence
and Raman scattering is reduced by time-gated
photon counting. Peptides showing affinity to ganglioside GM1 will be conjugated to a nanoparticulate carrier bearing a fluorescent dye. The resulting
optical contrast agent shall be used for fluorescence endoscopic intestinal tumor screening.
Collaboration: Bürger M, Gesellschaft für Silizium
Mikrosysteme mbh, Dresden, Germany; Helfmann J,
Laser- und Medizin-Technologie GmbH, Berlin, Germany.
Supported in part by: Bundesministerium für Bildung
und Forschung (BMBF), grant 13 N 8473.
Identification of peptide sequences with desired
properties by using genetic algorithms
Röckendorf N, Frey A.
The use of small synthetic peptides as specific ligands is a versatile approach in many areas of the
life sciences. Due to their small size and robust nature peptidic ligands offer several advantages over
proteinacious ligands such as monoclonal antibodies. They diffuse more rapidly, are less antigenic,
can be rendered protease resistant and experience less steric hindrance upon target binding. Yet,
Fig. 76. Affinity improvement of peptides optimized by a molecular evolution process. The 25 best sequences taken from generation 10 show an up to 150fold higher affinity towards a fluorescently
labelled ganglioside GM1 derivative than the 25 best lead peptides. Left: optimization of L-peptides (red), right: optimization of Dpeptides (green).
the identification of short peptide sequences with
high specific affinity to molecular targets is a chal-
The peptides are then screened for binding to the
lenging task since the tremendous diversity of pept-
molecular target of interest, ranked according to
ide sequences renders the usual high throughput
their performance and are crossed and mutated in
techniques quite inappropriate. We therefore devi-
silico using a specially designed genetic algorithm.
sed an approach for a directed, quality-driven
With this cooperative in silico / in vitro optimization
188
Wissenschaftlicher Jahresbericht 2007-2008
Division of Mucosaimmunology
Research Reports
methodology a stepwise refinement of the peptides
cer and ranks second in cancer induced deaths in
is attained so that in the end a ligand is obtained
Germany. However, colorectal cancer is curable in
that should perfectly meet the requirements for the
about 90 % of all cases if recognized early.
application of interest. Various peptides exhibiting
The basic screening tests for the diagnosis of colo-
different specific properties, for example a high bin-
rectal cancer are the fetal occult blood test (FOBT)
ding affinity towards ganglioside GM1 or specific
and colonoscopy. Whereas the FOBT is readily ac-
binding to a defined locus on the surface of the bac-
cepted, patients are often reluctant to undergo co-
terial lectin FimH, were designed this way. Lead
lonoscopy. Therefore it is desirable to develop al-
peptides for targeting of an intracellular tumormar-
ternative means for the reliable detection of early
ker were identified. We could show that an evoluti-
pathological changes of the intestinal wall such as
on of peptide sequences assembled from L- as well
the presence of small polyps. To visualize such ear-
as from D-amino acid building blocks is possible.
ly pathological changes a biological marker is ur-
The performance of the optimization process was
gently required. We observed in the course of a stu-
driven to an up to 1.8 fold increase in relative pep-
dy on intestinal antigen uptake that an intestinal tu-
tide suitability or “fitness” per generation, an expo-
mor cell line lacked a mature glycocalyx, a dense
nential growth in peptide fitness was observed (Fig.
network of highly glycosylated proteins and lipids
76). Starting from lead peptides with diverse se-
that are anchored in the epithelial cell membrane.
quences, a consensus sequence was approached
Hence, we set out to investigate whether the ab-
after several rounds of breeding, and the genetic
sence of a glycocalyx can serve as a general mar-
algorithm used converged after 10 generations to
ker for intestinal neoplasia.
local fitness optima.
Collaboration: Schubert P, R-Biopharm AG, Darmstadt, Germany; Lindhorst TK, Christiana Albertina
University of Kiel, Germany; Bürger M, Gesellschaft
für Silizium Mikrosysteme mbh, Dresden, Germany;
Helfmann J, Laser- und Medizin-Technologie GmbH,
Berlin, Germany.
Supported in part by: Bundesministerium für Bildung
und Forschung (BMBF), grant 13 N 8473.
The intestinal glycocalyx: a biological marker for
the early detection of cancer
Wehry K, Bade S, Röckendorf N, Frey A.
Fig. 77. Comparison of the glycocalyx of healthy and cancerous
epithelium from human colon. Apical cell surfaces were examined
by TEM after treatment of the tissue for glycocalyx visualisation. A:
healthy intestinal epithelium with regular, closely packed microvilli (MV) covered with a thick, continuous glycocalyx (G); B-D:
(pre)cancerous intestinal epithelium (B: adenocarcinoma, C: polyp,
D: carcinoma in situ) without a glycocalyx and few to no microvilli; bar: 1 µm.
Tissue samples from patients diagnosed with colorectal cancer and patients diagnosed with diverti-
According to the German cancer register (GEKID;
culitis were analysed for the presence and archi-
Gesellschaft der epidemiologischen Krebsregister
tecture of the glycocalyx using a special tissue stai-
in Deutschland e.V.) 73,250 people in Germany de-
ning procedure and transmission electron micros-
veloped colorectal cancer in 2004 (16.8 % of all can-
copy (TEM). In healthy tissue areas most epithelial
cer incidences) and 27,782 people died of this dis-
cells had microvilli, which were covered with a thick,
ease (13.3 % of all cancer induced deaths). Thus, co-
continuous glycocalyx (Fig. 77A). By contrast, all
lorectal cancer is the second most diagnosed can-
cancerous regions of the tissues (adenocarcinoma,
Wissenschaftlicher Jahresbericht 2007-2008
189
Division of Mucosaimmunology
Research Reports
polyps and carcinoma in situ) showed dedifferenti-
blocking reagent which is intended to prevent non-
ated cells without a glycocalyx and few to no mi-
specific binding of sample components to solid
crovilli (Fig. 77, B-D). Only in some cases thin glyco-
phase immunoassay supports.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
calyx fragments were visible, but never a thick, continuous surface coat. Statistical evaluation of the da-
Supported in part by: Bundesministerium für Bildung
ta revealed that the thickness of the glycocalyx can
und Forschung (BMBF), grant 13 N 8473.
be used to discriminate early pathological changes
(polyps), carcinoma in situ and adenocarcinoma
from healthy tissue with high reliability.
Theses
Collaboration: Vollmer E, Division of Clinical and Ex-
Dissertation
perimental Pathology, Research Center Borstel,
Reuter, Fabian
Germany; Fröschle G, Division of General, Visceral,
Verdauungsstabilität von Antigenen im Dünndarm -
Accident and Vascular Surgery, Asklepios Clinic Bad
Bedeutung für orale Immunisierungen
Oldesloe, Germany.
Mathematisch-Naturwissenschaftliche Fakultät
Christian-Albrechts-Universität zu Kiel, 2008.
Supported in part by: Bundesministerium für Bildung
und Forschung (BMBF), grant 13 N 8473.
Rasschaert, K.
Identification of a novel F4 receptor involved in endocytosis and transcytosis. Faculty of Verterinary
Novel tools for the analysis of mucus components
Medicine
and glycoproteins
Ghent University, Belgium, 2008.
Wehry K, Fujimoto, N, Frey A.
Albers, E.
Diagnostic as well as therapeutic approaches to-
Entwicklung verdauungsresistenter Peptidantigene
wards disorders of the gastrointestinal or respira-
für orale Impfstoffe. Medizinische Fakultät
tory tracts by necessity involve dealing with the mu-
Westfälische Wilhelms-Universität Münster, 2007.
cus layer and other carbohydrate-rich structures at
the respective surfaces. The analysis of those glycomolecules requires special assays and reagents
Stipends
which we are in the progress of establishing. Spe-
Fujimoto, Naho
cifically, monoclonal antibodies directed against
„NESACS-GDCh Exchange Program”, Boston, USA,
the peptide backbone of one main component of
2007.
intestinal and airway mucus, the highly glycosylated
mucin protein MUC5AC, have been generated. The
Patents
antibodies recognize human MUC5AC in a variety
Bade S, Röckendorf N, Franek M, Gorris H-H, Frey A
of solid phase assay set-ups and crossreact with the
(2008). Kit for highly sensitive detection assays.
respective mucins of other species, thus represen-
Granted German Patent; DE 10 2005 051946 C1.
ting valuable tools for the analysis of human mucous samples and for investigations in animal mo-
Frey A, Helfmann, J, Schmidt, MA, Müller, G (2008).
del systems. Since the usually employed blocking
Method and device for investigating substance li-
reagents on protein- or detergent-basis may be of
braries. Granted United States Patent 10/239,986.
little efficiency in the analytics of carbohydrate-rich
components, we have also developed a new
190
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
Division of Clinical Infectious Diseases
and Clinical Studies
Head
linked-immunospot in response to purified protein
PD Dr. Christoph Lange
derivate (PPD) and mycobacterial specific antigens
early-secretory-target (ESAT)-6 and culture-filtrate-
Staff scientists and postdoctoral fellows
protein (CFP)-10 by peripheral blood mononuclear
Barbara Eker
cells (PBMC) and bronchoalveolar-lavage mo-
Dr. Ulf Greinert
nonuclear cells (BALMC) of patients with pulmonary
Dr. Gunar Guenter
sarcoidosis, smear-negative tuberculosis and con-
Dr. Claudia Jafari
trols. Release of IFN-γ in response to ex vivo contact
Dr. Barbara Kalsdorf
with PPD, ESAT-6 or CFP-10 by BALMC and PBMC
were comparable among patients with sarcoidosis
Graduate and Diploma students
and controls (PBMC p=0.2326; BALMC p=0.1767)
Ellen Andresen
and were less frequently observed in both groups
Tim Grüber
compared to patients with tuberculosis (BALMC
Robert Hörster
p<0,05; PBMC p<0.0001). Within PBMC the immun-
Benjamin Krummel
ophenotype of sarcoidosis-patients differed from
Leonhard Leidl
patients with tuberculosis, as well as from controls,
Heike Sarrazin
while within BALMC it resembled the one of patients
Janine Wolske
with tuberculosis. In contrast to patients with tuberculosis, the frequency of mycobacteria-specific local
Technicians, Study nurses
and systemic immuneresponses is not elevated in
Francisca Danuda
patients with sarcoidosis when compared to con-
Andrea Glaewe
trols. The immunophenotype represents the local re-
Lenka Krabbe
semblance of the granulomatous reaction underly-
Waltraud Wappler
ing tuberculosis and sarcoidosis, while showing systemical difference. These observations do not support a role of an infection with Mycobacterium
Research Reports
tuberculosis in the pathogenesis of sarcoidosis.
Antimycobacterial immune responses in patients
with pulmonary sarcoidosis
Hoerster R, Jafari C, Strassburg A, Greinert U,
Kalsdorf B, Lange C.
Sarcoidosis is a multisystem granulomatous disease of unknown origin. Pathogenetic involvement of
Mycobacterium tuberculosis has frequently been discussed in the etiology of sarcoidosis, however, studies still remain contradictory. We addressed the
question of mycobacterial involvement in the pathogenesis of sarcoidosis by analysing cellular im-
Fig. 78. Tuberculosis and sarcoidosis can rapidly be distinguished
by assaying M. tuberculosis specific immune responses in bronchoalveolar-lavage mononuclear cells.
mune responses to mycobacterial antigens. We examined the interferon (IFN)-γ production by enzyme-
Collaboration: Gaede KI, Div. of Clin. Immunophar-
Wissenschaftlicher Jahresbericht 2007-2008
191
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
macology, Research Center Borstel; Ernst M, Div. of
counts, immunophenotyping with monoclonal anti-
Immune Cell-Analytics, Research Center Borstel;
bodies against cell surface markers CD4, CD8,
Kirsten D, Hospital Großhansdorf.
CD4CD45RA, CD4CD45R0, CD38, HLADR, CD19,
Fo r s c h u n g s z e n t r u m
Bo r s t e l
CD3, CD57, CD16 and MTB-specific enzyme linked
immunospot assays (ELISPOT) with early-secretoryLocal immunodiagnosis of pulmonary tuberculo-
antigenic-target-6 (ESAT-6) and culture-filtrate-pro-
sis by enzyme-linked immunospot
tein-10 (CFP-10) of peripheral blood mononuclear
Jafari C, Strassburg A, Greinert U, Kalsdorf B,
cells (PBMCs) and broncho-alveolar lavage mo-
Lange C.
nonuclear cells (BALMCs) were performed.
Lymphocytes are crucial in the immune defense
Among 12 patients with culture-confirmed smear-ne-
against Mycobacterium tuberculosis (MTB) infec-
gative pTB, no differences were found in the distri-
tion. We ascertained whether MTB-specific lym-
bution of total CD4 or CD8 T cells in peripheral
phocytes are selectively compartmentalised in the
blood or BAL. Activated HLA DR+ cells as well as
lungs of patients with minimal active pulmonary tu-
memory CD4CD45R0+ T cells were expanded
berculosis (pTB).
among cells of the BAL. Compared with a group of
control patients with alternative pulmonary
pathologies, there was no significant difference in lymphocyte subpopulations. However, ESAT-6 and CFP-10 specific lymphocytes were concentrated with a median of
9.9 and 8.9 times more in BAL compared to
peripheral blood in patients with pTB. MTBspecific T cells are highly selectively compartmentalised at the site of infection in active pTB.
Collaboration: Ernst M, Ernst M, Div. of Immune Cell-Analytics, Research Center Borstel; Kirsten D, Hospital Großhansdorf.
Rapid diagnosis of pulmonary tuberculosis by BAL-ELISPOT in an immunocompromised host
Strassburg A, Jafari C, Lange C.
Fig. 79. A novel test for the rapid diagnosis of acid-fast bacilli
smear negative pulmonary tuberculosis, M. tuberculosis specific Tlymphocytes which are recruited to the lungs in active pulmonary
tuberculosis can rapidly be detected by enzyme-linked immunospot on mononuclear cells from the bronchoalveolar-lavage.
a) ELISPOT on blod mononuclear cells; b) ELISPOT on BAL mononuclear cells.
Immunocompromised patients with acid-fast bacilli
(AFB) smear-negative active pulmonary tuberculosis
(pTB) often present with nonspecific clinical symptoms and findings. T-cell interferon-c release assays (TIGRA) performed on whole blood (using ELI-
We prospectively recruited patients with smear-ne-
SA) or peripheral blood mononuclear cells (using
gative, MTB culture-confirmed pTB. Differential cell
enzyme-linked immunospot assay (ELISPOT)) are
192
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
more sensitive for the diagnosis of Mycobacterium
Rapid diagnosis of tuberculous meningitis by enu-
tuberculosis (MTB) infection than the tuberculin skin
meration of cerebrospinal fluid antigen-specific
test (TST), but cannot distinguish active from latent
cells
MTB infection. The present authors report a 38-yr-old
Lange C.
female presenting with a 3-week history of malaise,
dyspnoea, fevers and coughing, who had received
Hospital in-patients with suspected tuberculous
immunosuppressive therapies over 8 months for
meningitis (TBM), predominantly in India.
mixed connective tissue disease. Chest radiograph
We determined whether interferon-gamma (IFN-γ)
and thoracic computed tomography showed gro-
secreting Mycobacterium tuberculosis antigenspe-
und glass opacities in both lower lobes. The TST-in-
cific T-cells are present in the cerebrospinal fluid
duration was 0 mm and AFBs or MTB nucleic acid
(CSF) of patients with TBM and to evaluate the fea-
was not detected on sputum and bronchial secreti-
sibility of CSF enzyme-linked immunospot (ELISpot)
ons. However, TIGRAs performed on peripheral
for the diagnosis of active TBM. This was a pros-
blood cells were reactive. A high frequency of MTB-
pective blinded hospital-based study. The overnight
specific T-cells compatible with the immunodiagno-
ELISpot assay detected M. tuberculosis antigen-spe-
sis of active pTB was detected among bronchoal-
cific IFN-gamma secreting T-cells in CSF from nine
veolar lavage cells using ELISPOT. Antituberculous
of 10 prospectively recruited patients with TBM, and
therapy was initiated 18 days before MTB was dis-
zero of seven control patients with meningitis of
covered on sputum cultures. Detection of Mycobac-
other aetiology. This corresponds to a diagnostic
terium tuberculosis-specific T-cells in the bronchoal-
sensitivity of 90% (95%CI 56–100) and specificity of
veolar lavage using enzyme-linked immunospot as-
100% (95%CI 59–100). This pilot study demonstrates
say is a promising tool for the diagnosis of active
proof-ofprinciple for a new T-cell-based diagnostic
pulmonary tuberculosis in immunocompromised
test for TBM which is rapid, sensitive and specific.
patients with negative acid-fast bacilli smears.
Collaboration: Ernst M, Div. of Immune Cell-Analytics, Research Center Borstel; Nau R, Dept. of Neurology, University Hospital Göttingen; Lalvani A,
Thomas MM, Imperial College, London, UK.
Use of a T-cell interferon-γγ release assay for the
diagnosis of tuberculous pleurisy
Jafari C, Greinert U, Lange C.
The diagnosis of pleural tuberculosis (plTB) by the
Fig. 80. Computed tomography image of the chest of a patient with
AFB-smear negative tuberculosis and non specific intersti-
tial pneumonitis.
analysis of pleural effusions (PEs) with standard diagnostic tools is difficult. In routine clinical practice,
the present authors evaluated the performance of
Collaboration: Ernst M, Div. of Immune Cell-Analyt-
a commercially available Mycobacterium tubercu-
ics, Research Center Borstel; Lotz W.
losis (MTB)-specific enzyme-linked immunospot assay on peripheral blood mononuclear cells (PBMCs) and pleural effusion mononuclear cells
(PEMCs) in patients with suspect plTB. The T-SPOT.TB
test (Oxford Immunotec Ltd, Abingdon, UK) was per-
Wissenschaftlicher Jahresbericht 2007-2008
193
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
formed on PBMCs and PEMCs in 20 patients with a
aimed to ascertain in routine clinical practice the ac-
clinical and radiological suspect of plTB and in 21
curacy of a novel assay using selected peptides en-
control subjects with a diagnosis of PE of nontuber-
coded in the mycobacterial genomic region of dif-
culous origin at four centres participating in the Eu-
ference (RD) 1 for the diagnosis of active tuberculo-
ropean Tuberculosis Network. In total, 18 (90%) out of
sis in comparison with tuberculin skin test (TST), Quan-
20 patients with plTB tested T-SPOT.TB-positive on PB-
tiFERON-TB GOLD In-Tube (Cellestis Ltd., Carnegie,
MCs and 19 (95%) out of 20 on PEMCs. Among con-
Australia) and T-SPOT.TB (Oxfordimmunotec, Abing-
trols, T-SPOT.TB was positive in seven out of 21 (33%)
don, UK). 425 individuals from 6 different European
patients when performed on PBMCs (these patients
centres were prospectively enrolled. We found that
were assumed to be latently infected with MTB) and
sensitivity of the novel test, TST, QuantiFERON-TB
five (23%) out of 21 when performed on PEMCs. Sen-
GOLD In-Tube and T-SPOT.TB was respectively 73.1%,
sitivity and specificity of T-SPOT.TB for the diagnosis
85.3%, 78.1%, and 85.2%; specificity was respectively
of active plTB when performed on PEMCs were 95
70.6%, 48.0%, 61.9% and 44.3%; positive likelihood ra-
and 76%, respectively. Enumerating Mycobacterium
tios were respectively 2.48, 1.64, 2.05, and 1.53; ne-
tuberculosis-specific T-cells in pleural effusion mo-
gative likelihood ratios were respectively 0.38, 0.31,
nonuclear cells by ELISPOT is feasible in routine cli-
0.35, 0.33. Sensitivity of TST combined with the novel
nical practice and may be useful for a rapid and ac-
test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB in-
curate diagnosis of pleural tuberculosis.
creased up to 92.4%, 97.7% and 97.1%, respectively.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
The likelihood ratios of combined negative results of
TST with, respectively, the novel test, QuantiFERON-TB
GOLD In-Tube and T-SPOT.TB were 0.19, 0.07 and 0.10.
The assay based on RD1 selected peptides has similar accuracy for active tuberculosis compared with
TST and commercial IGRAs. Then, independently of
the spectrum of antigens used in the assays to elicit
mycobacterial specific immune responses, the novel
test, IGRAs, and the TST do not allow an accurate
Fig. 81. Top view of a single well from a 96 well microtiter plate.
Spots represent single cells among 250 000 mononuclear cells that
have produced interferon-gamma in response to contact with peptides of the „early-secretory-antigenic-target-6, which is incoded in
the RD-1 region of M. tuberculosis.
identification of active tuberculosis in clinical practice. However, the combined use of the novel assay
or commercial IGRAs with TST may allow exclusion
of tuberculosis.
Collaboration: Ernst M, Div. of Immune Cell-Analytics, Research Center Borstel; Losi M, Modena Italy,
Collaboration: Ernst M, Div. of Immune Cell-Analyt-
and members of the TBNET.
ics, Research Center Borstel; Goletti D, National Institute for Infectious Diseases, Rome, Italy, and members of the TBNET.
Accuracy of immunodiagnostic tests or active tuberculosis. Results from a multicenter TBNET-study
Lange C.
Relationship between tuberculin skin test reactivity, memory CD4 subset and circulating FoxP3 ex-
The clinical application of IFN-γ release assays
pressing cells in HIV-infected persons
(IGRAs) has recently improved the diagnosis of latent
Sarrazin H, Lange C.
tuberculosis infection. In a multicenter study of the Tuberculosis Network European Trialsgroup (TBNET) we
194
Lack of reactivity to the tuberculin skin test (TST) is
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical Infectious Diseases and Clinical Studies
widely observed in advanced HIV-1 infection. The
Research Reports
Supported in part by: HW & J Hector Foundation.
TST reaction site and PPD uninfiltrated skin were
biopsied in 15 HIV-1 infected and 23 uninfected persons without active tuberculosis from a community
Distinct, specific IL-17- and IL-22-producing CD4+
with a very high tuberculosis incidence. 8µm histo-
T cell subsets contribute to the human anti-my-
logical sections were immunohistochemically
cobacterial immune response
stained for CD4, CD8, CD28, CD45RA, CD45RO,
Kalsdorf B.
CD62L, CD1a, HLA-DR, granulyzin, IFN-γ and FoxP3
and analysed by single cell in situ digital imaging.
We investigated whether the proinflammatory T cell cy-
Peripheral blood mononuclear cells were analysed
tokines IL-17 and IL-22 are induced by human my-
by FACS. In TST reactive skin test biopsies, HIV-1 in-
cobacterial infection. Remarkably, >20% of specific
fected persons showed less CD4+ T cells at the TST
cytokine-producing CD4(+) T cells in peripheral blood
site (p=0.036), but more HLA-DR+ T cells (p=0.037)
of healthy, mycobacteria-exposed adults expressed IL-
compared to HIV-1 uninfected. In the HIV-1 infected
17 or IL-22. Specific IL-17- and IL-22-producing CD4(+) T
group, the total number of cells (p=0.008) and num-
cells were distinct from each other and from Th1 cyto-
bers of CD45RO+ memory T cells (p=0.003) were
kine-producing cells. These cells had phenotypic cha-
significantly higher in TST-reactive persons than in
racteristics of long-lived central memory cells. In pati-
TST-unreactive. In HIV-1 infected persons, the TST in-
ents with tuberculosis disease, peripheral blood fre-
duration inversely correlated with numbers of Fo-
quencies of these cells were reduced, whereas bron-
xP3+ T cells in the blood (p = 0.026) but was unre-
choalveolar lavage fluid contained higher levels of
lated to the circulating CD4+ T cell number. In HIV-
IL-22 protein compared with healthy controls. IL-17 was
1 infection, the TST depends upon memory T cells
not detected in this fluid, which may be due to sup-
and relates better to numbers of circulating FoxP3+
pression by Th1 cytokines, as PBMC IL-17 production
CD4+ T cells than total CD4+ T cells.
was inhibited by IFN-gamma in vitro. However, Th1
cytokines had no effect on IL-22 production in vitro. Our
results imply that the magnitude and complexity of the
anti-mycobacterial immune response have historically
been underestimated. IL-17- and IL-22-producing
CD4(+) T cells may play important roles in the human
immune response to mycobacteria.
Collaboration: Scriba T, Hankom W and colleagues,
South African Tuberculosis vaccine Initiative, Wilkinson RJ, Institute of Infectious Diseases and Molecular Medicine, Cape Town, Rep. South Africa.
Fig. 82. Example of a microscopic view of a skin sample of tuberculin skin test reactive skin, obtained from an HIV-1 infected person and immunohistochemically stained with monoclonal antibodies against CD45 RO (original magnification 220x).
LTBI: latent tuberculosis infection or lasting immune response to Mycobacterium tuberculosis. A
Collaboration: Wilkinson KA, Rangaka MX, , van
TBNET consensus statement
Veen K, Wilkinson RJ, Institut of Infectious Diseases
Lange C.
and Molecular Medicine, Cape Town, Rep. South
Africa; Andersson J, Radler L, Center for Infectious
Tuberculosis control relies on the identification and
Medicine, Karolinska Instutute, Stockholm, Sweden.
preventive treatment of individuals who are latent-
Wissenschaftlicher Jahresbericht 2007-2008
195
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
ly infected with Mycobacterium tuberculosis. Howe-
tries of the World Health Organization European re-
ver, direct identification of latent tuberculosis infec-
gion using a standard questionnaire. Contacts are
tion is not possible. The diagnostic tests used to iden-
universally screened for active tuberculosis and la-
tify individuals latently infected with M. tuberculosis,
tent tuberculosis infection (LTBI). Most countries
the in vivo tuberculin skin test and the ex vivo inter-
(.70%) screen those with HIV infection, prisoners and
feron-gamma release assays (IGRA), are designed
in-patient contacts. Screening of immigrants is rela-
to identify an adaptive immune response against, but
ted to their contribution to national rates of tuber-
not necessarily a latent infection with M. tuberculosis.
culosis. Only 25 (50%) out of 50 advise a request for
The proportion of individuals who truly remain infec-
symptoms in their guidelines. A total of 36 (72%) out
ted with M. tuberculosis after tuberculin skin test or
of 50 countries recommend sputum examination for
IGRA conversion is unknown. It is also uncertain how
those with a persistent cough; 13 countries do not,
long adaptive immune responses towards mycob-
even if the chest radiograph suggests tuberculosis.
acterial antigens persist in the absence of live my-
Nearly all countries (49 out of 50) use tuberculin skin
cobacteria. Clinical management and public health
testing (TST); 27 (54%) out of 50 countries also per-
care policies for preventive chemotherapy against tu-
form chest radiography irrespective of the TST result.
berculosis could be improved, if we were to gain a
Interpretation of the TST varies widely. All countries
better understanding on M. tuberculosis latency and
use 6–9 months of isoniazid for treatment of LTBI,
reactivation. This statement by the TBNET summarizes
with an estimated median (range) uptake of 55%
knowledge and limitations of the currently available
(5–92.5%). Symptoms and sputum examination
tests used in adults and children for the diagnosis of
could be used more widely when screening for ac-
latent tuberculosis infection. We conclude that the
tive tuberculosis. Treatment of latent tuberculosis in-
main issue about testing is to restrict it to those who
fection might be better focused by targeted use of
are known to be at higher risk of developing tuber-
interferon-gamma release assays.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
culosis and who are willing to accept preventive chemotherapy.
Collaboration: Bothamley G ,London, UK; Ditiu L,
WHO, Copenhagen, Denmark, Migliori GB, Tradate,
Collaboration: Ehlers S, Div. of Molecular Infection
Italy, members of the TBNET.
Biology, Hölscher C, Junior Research Group, Molecular Infection Biology, Research Center Borstel;
Mack U, Oslo, Norway and members of the TBNET.
Epidemiology and clinical management of XDRTB: A systematic review by TBNET
Lange C.
Active case finding of tuberculosis in Europe. A TBNET (Tuberculosis Network European Trialsgroup)
Extensively drug-resistant tuberculosis (XDR-TB) is
survey
present in all regions and poses serious challenges
Lange C.
for public health and clinical management. Laboratory diagnosis is difficult and little evidence exists
Tuberculosis control depends on successful case fin-
to guide clinicians in treating people with XDR-TB ef-
ding and treatment of individuals infected with My-
fectively. To summarize the available data on dia-
cobacterium tuberculosis. Passive case finding is wi-
gnosis and treatment, we performed a systematic
dely practised. The present study aims to ascertain
review on 13 recent studies of the epidemiology
the consensus and possible improvements in active
and clinical management of XDR-TB. Studies that
case finding across Europe. Recommendations from
met inclusion criteria were reviewed to assess me-
national guidelines were collected from 50 coun-
thodology, treatment regimens, and reatment out-
196
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
comes. Meta-analysis of currently available data is
sed, 361 (7.9%) were MDR and 64 (1.4%) were XDR.
not possible because of inconsistent definitions and
XDR-TB cases had a relative risk (RR) of 1.58 to have
methodologies. Data show that XDR-TB can be suc-
an unfavourable outcome compared with MDR-TB
cessfully treated in up to 65% of patients, particularly
cases resistant to all first-line drugs (isoniazid, ri-
those who are not co-infected with HIV. However,
fampicin ethambutol, streptomycin and, when test-
treatment duration is longer and outcomes are in
ed, pyrazinamide), and an RR of 2.61 compared
general poorer than for non-XDR-TB patients. To
with ‘‘other’’ MDR-TB cases (those susceptible to at
strengthen the evidence for XDR-TB diagnosis, tre-
least one first-line anti-TB drug among ethambutol,
atment and prevention, future studies should: i) be
pyrazinamide and streptomycin, regardless of re-
prospective in design, ii) adopt standardized, in-
sistance to the second-line drugs not defining XDR-
ternationally accepted definitions, iii) use quality-as-
TB). The emergence of extensively drug-resistant tu-
sured laboratory testing for all first-and second-line
berculosis confirms that problems in tuberculosis
drugs, and iv) collect data on an agreed-upon set
management are still present in Europe. While wait-
of standard variables allowing for comparisons
ing for new tools which will facilitate management
across studies. Early diagnosis and aggressive ma-
of extensively drug-resistant tuberculosis, accessibi-
nagement of XDR-TB provide the best chance of po-
lity to quality diagnostic and treatment services
sitive outcome, but prevention is still paramount.
should be urgently ensured and adequate public
health policies should be rapidly implemented to
Collaboration: Ruesch-Gerdes S, National Refer-
prevent further development of drug resistance.
ence Center for Mycobacteria, Research Center
Borstel; Sotgiu G, Sassari (Italy), Migliori GB Tradate
Collaboration: Migliori GB, Tradate, Italy, and mem-
(Italy) and members of the TBNET.
bers of the TBNET.
Clinical and operational value of the XDR-TB def-
Multi-drug resistant and extensively drug resistant
inition
tuberculosis, Germany
Lange C.
Eker B, Lange C.
Currently, no information is available on the effect
We evaluated risk factors and treatment outcomes
of resistance/susceptibility to first-line drugs diffe-
associated with multidrug-resistant (MDR)- and ex-
rent from isoniazid and rifampicin in determining
tensively drug-resistant (XDR)-tuberculosis (TB) in
the outcome of extensively drug-resistant tuberculo-
Germany in 2004-2006. In 184/4,557 (4%) of culture-
sis (XDR-TB) patients, and whether being XDR-TB is
positive TB cases, M. tuberculosis isolates were
a more accurate indicator of poor clinical outcome
MDR. 148/184 (80%) patients with MDR-TB were
than being resistant to all first-line anti-tuberculosis
born in countries of the former Soviet Union. 7/184
(TB) drugs. To investigate this issue, a large series
(4%) met XDR-TB criteria. In patients with XDR-TB hos-
of multidrug-resistant TB (MDR-TB) and XDR-TB cases
pitalization was longer (mean±SD 202±130 vs.
diagnosed in Estonia, Germany, Italy and the Rus-
123±81 days; p=0.015) and resistance to all first li-
sian Federation during the period 1999–2006 were
ne drugs was more frequent (36% vs. 86%; p=0.013)
analysed. Drug-susceptibility testing for first- and se-
than in patients with MDR-TB. 74/184 (40%) patients
cond-line anti-TB drugs, quality assurance and tre-
received treatment with linezolid. Treatment suc-
atment delivery was performed according to World
cess rates ranged from 59.2% for the entire cohort
Health Organization recommendations in all study
(59.3% for MDR-TB and 57.1 % for XDR-TB) to 87.2%
sites. Out of 4,583 culture-positive TB cases analy-
for those with a definitive outcome (n=125; 87.5% for
Wissenschaftlicher Jahresbericht 2007-2008
197
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
MDR-TB and 80% for XDR-TB). Extensive antibiotic
To investigate this issue, a large series of MDR- and
susceptibility testing and availability of second- and
XDR-TB cases diagnosed in Estonia, Germany, Italy
third-line drugs under inpatient management con-
and the Russian Federation (Archangels Oblast)
ditions allow achieving relatively high treatment suc-
between 1999 and 2006 were analysed. All study
cess rates in MDR- and XDR-TB.
sites performed drug susceptibility testing for first-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
and second-line anti-TB drugs, laboratory quality assurance and treatment delivery according to World
Health Organization recommendations. Out of
4,583 culture-confirmed cases, 240 MDR- and 48
XDR-TB cases had a definitive outcome recorded
(treatment success, death, failure). Among MDRand XDR-TB cases, capreomycin resistance yielded
a higher proportion of failure and death than capreomycin-susceptible cases. Resistance to capreomycin was independently associated with unfavourable outcome (logistic regression analysis:
odds ratio 3.51). In the treatment of patients with
multidrug-resistant and extensively drug-resistant tuberculosis, resistance to the injectable drug capreomycin was an independent predictor for therapy failure in this cohort. As Mycobacterium tuberculosis drug resistance is increasing worldwide, there
is an urgent need for novel interventions in the fight
against tuberculosis.
Collaboration: Migliori GB, Tradate, Italy, and memFig. 83a-c. Contact screening with a new blood test to investigate
a tuberculosis outbreak in a school.
bers of the TBNET.
Collaboration: Ruesch-Gerdes S, National Refer-
Fluoroquinolones: are they essential to treat
ence Center for Mycobacteria, Research Center
MDR-TB?
Borstel;
Lange C.
27
collaborating
hospitals
in
Ger-
many,members of the TBNET.
Several publications have already demonstrated
that resistance to fluoroquinolones (FQ) is indeResistance to second-line injectables and treat-
pendently associated with poor outcome and/or
ment outcomes in multidrug-resistant and exten-
that the possibility of including FQ in regimens im-
sively drug-resistant tuberculosis cases
proves treatment outcomes of multidrug-resistant
Lange C.
(MDR)-TB cases. This happened before the (recent)
description of XDR-TB. We do not know how many
No information is currently available on the influ-
of the patients with MDR-TB strains were, in fact, in-
ence of injectable second-line drugs on treatment
fected with XDR Mycobacterium tuberculosis. We
outcomes of multidrug-resistant (MDR) and exten-
wanted to establish the role of the different XDR-de-
sively drug-resistant (XDR) tuberculosis (TB) patients.
fining components (e.g. isoniazid and rifampicin,
198
Wissenschaftlicher Jahresbericht 2007-2008
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
FQ and injectable second-line drugs) in determining
1.14–4.89; p,0.02). The findings of our analysis sug-
poor treatment outcomes. Our group has shown for
gest that FQ contribute to increase the risk of death
the first time that XDR-TB cases in Italy and Germany
and failure, being a key XDR-defining variable. In
have a five-fold increase in the risk of death (relati-
conclusion, apart from linezolid, fluoroquinolones
ve risk (RR) 5.45; 95% confidence interval (CI) 1.95–
represent the only ‘‘new’’ class of active drugs cur-
15.27; p,0.01), require longer hospitalisation than
rently available to treat drug-resistant tuberculosis.
MDR-TB cases (241.2¡177.0 versus 99.1¡85.9 days;
They are effective and relatively well tolerated. Fur-
p,0.001), have a longer treatment duration
thermore, fluoroquinolones have the potential to al-
(30.3¡29.4 versus 15.0¡23.8 months; p,0.05) and, for
low a reduction in the (still long) short-course che-
the few cases who converted, need a longer time
motherapy regimens. Unfortunately, rapid selection
to smear/culture conversion (p,0.01). The findings of
of drug resistance mutants to fluoroquinolones is a
a second study, which included additional cases
well-known phenomenon. Prevention of develop-
from Estonia and the Russian Federation, demon-
ment of further drug resistance is imperative until
strated that XDR-TB cases had an RR of 1.58 to
new drugs become available in the treatment
achieve death or failure compared with MDR-TB ca-
arena.
ses resistant to all first-line drugs (95% CI 1.14–2.20;
p,0.05) and an RR of 2.61 (95% CI 1.45–4.69; p,0.001)
Collaboration: Migliori GB, Tradate, Italy, and mem-
compared with MDR-TB cases in which susceptibili-
bers of the TBNET.
ty to at least one first-line drug still existed. These
data support the observation that the loss of first-line drugs different from rifampicin and isoniazid has
Clinical presentation and diagnosis of tubercu-
a role in worsening prognosis of MDR-TB cases. In
losis
order to better understand the role of FQ in deter-
Kalsdorf B, Strassburg A, Greinert U, Lange C.
mining poor treatment outcomes in MDR-TB cases,
we re-analysed data from the four-country study to
Recently, major advances have been accomplished
assess whether there is any difference in death or
in the diagnosis of active tuberculosis.
mortality in MDR-TB cases resistant or susceptible to
FQ. The overall sample included 425 MDR-TB cases
(361 MDR, 64 XDR). A total of 87 (20%) were resistant
to FQ, 23 (26%) being MDR and 64 (74%) XDR. Although the proportion of MDR-TB cases resistant to
FQ was similar in the three countries reporting FQ
resistance (i.e. 18, 24 and 24% in Italy, Germany and
Estonia, respectively), the proportion of XDR-TB cases among FQ-resistant cases was largely different
(50, 27 and 88% in Italy, Germany and Estonia, respectively). FQ-resistant MDR-TB cases yielded a higFig. 84. Examining the induration in the tuberculin skin test.
her proportion of deaths than non-FQ-resistant cases (20 versus 12%; p50.020), as well as a higher
A comprehensive diagnostic approach of a patient
proportion of treatment failures (19 versus 9%;
with possible tuberculosis includes a detailed me-
p50.038). At the multiple regression analysis, the
dical history and clinical examination as well as the
presence of XDR-TB is the only independent risk fac-
results of radiological, microbiological, immunolo-
tor for both death (odds ratio (OR) 2.07; 95% CI
gical, molecular-biological and histological me-
1.05–4.05; p,0.034) and failure (OR 2.37; 95% CI
thods. In concert, these results enable the clinician
Wissenschaftlicher Jahresbericht 2007-2008
199
Division of Clinical Infectious Diseases and Clinical Studies
Research Reports
to develop rapidly a decision with a high probabi-
Among the important novel developments ranks the
lity of a diagnosis or exclusion of active tuberculo-
analysis of serum procalcitonin for a better identifi-
sis. Therapeutic decisions can thus be made early,
cation and treatment monitoring of bacterial pneu-
even though corrections in these decisions need to
monias compared to conventional tests and a sim-
be considered depending on the results of Mycob-
ple scoring system, like the CRB-65/CURB score, for
acterium tuberculosis -cultures and sensitivity
a rapid risk stratification. A rational diagnostic ap-
testing.
proach is necessary to identify causative microor-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
ganisms of pulmonary infectious diseases depenCollaboration: Lotz J, Medical University Hannover.
ding on the severity of the illness, the exposition and
predisposition of the patient. In addition to the classical microbiological methods, rapid test systems
Diagnosis of respiratory infectious diseases
for the identification of microorganisms are beco-
Strassburg A, Lange C.
ming increasingly important.
Lower respiratory tract infections rank among the
Collaboration: Zabel P, Div. of Clin. Immunophar-
most important illnesses in medicine. However, the
macology, Research Center Borstel; Magnussen H,
identification of a causative microbiological agent
Hospital Großhansdorf, University of Lübeck, Rupp
is often difficult.
J, University of Lübeck, Herth, FJF, Thorax Clinic Heidelberg, University of Heidelberg.
Theses
Dissertation
Döscher, Gabriele
Evaluation eines neuen Testverfahrens zur Diagnose einer Infektion mit Mycobacterium tuberculosis.
Medizinische Fakultät
Universität zu Lübeck, 2007.
Fig. 85. Examination of sputum in patients with pneumonia: a)
suameous epithelial cells representing an insufficient specimen
from the upper respiratory tract, b) polymorphonuclear granulocytes and Gram-positive diplococci in a patient with pneumococal
pneumonia.
Pulmonary infections must be differentiated from
non-infectious causes of pulmonary diseases with similar symptoms and infiltrates on chest imaging.
200
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Preise • Awards
Ernennungen • Appointments
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Wissenschaftlicher Jahresbericht 2007-2008
228
Preise • Awards
Ernennungen • Appointments
Ernennungen • Auszeichnungen
Appointments • Awards
Wissenschaftlicher Jahresbericht 2007-2008
229
Preise 2007
Awards 2007
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Preise 2007
Dipl.-Biol. Lena Heinbockel
Diplompreis 2007 des Vereins zur Erforschung in-
Dr. rer. nat. Steffen Bade
fektiologischer und allergischer Prozesse.
Posterpreis für den besten freien Vortrag
2. Gemeinsamer Deutscher Allergiekongress des
Ärzteverbands Deutscher Allergologen e.V., der
Dr. med. Barbara Kalsdorf
Deutschen Gesellschaft für Allergologie und Kli-
CFAR Travel Scholarship
nische Immunologie und der Gesellschaft für Pä-
Tuberculosis Research Unit, Case Western Re-
diatrische Allergologie und Umweltmedizin.
serve University, Cleveland, USA.
Lübeck, September 2007.
PD Dr. rer. nat. Frank Petersen
Dr. rer. nat. Wolf-Meinhard Becker,
Science Prize of the Signal Transduction Society
Prof. Dr. rer. nat. Arnd Petersen
(Acris Award)
Forschungspreis der Stiftung Kanert für Allergie-
for the Contribution “CXCL4 (Platelet factor 4) dif-
forschung.
ferentially regulates respiratory burst, survival,
and differentiation of human monocytes by using
distinct signaling pathways”.
Weimar, November 2007.
Dr. rer. nat. Dominik Rückerl
Promotionspreis des Kreises Segeberg 2007.
Dr. med. Alan Strassburg
ERS/ELF Long Term Fellowship,
Dr. rer. nat. Jenny Debarry
Tuberculosis Immunology Unit, Imperial College,
Promotionspreis des Krei-
London.
ses Segeberg 2007.
Michael Weidhase
Landesbester in der
Ausbildung zum/r Biolaboranten/in, SchleswigHolstein.
Nina-Christin Frank
Landesbeste in der Ausbildung zum/r Tierpfleger/in,
Schleswig-Holstein.
230
Wissenschaftlicher Jahresbericht 2007-2008
Preise 2008
Preise 2008
Awards 2008
PD Dr. rer. nat Norbert Reiling
STS Science Award 2008
Dr. rer. nat. Steffen Bade
Signal Transduction Society, Weimar 2008
3. Gemeinsamer Deutscher Allergiekongress des
Ärzteverbands Deutscher Allergologen, der Deutschen Gesellschaft für Allergologie und Klinische
Riecken S, Stratmann C, Fölster-Holst R, Petersen
Immunologie und der Gesellschaft für Pädiatri-
A, Becker W.-M.
sche Allergologie und Umweltmedizin
3. Gemeinsamer Deutscher Allergiekongress des
Posterpreis für den besten freien Vortrag, Sep-
Ärzteverbands Deutscher Allergologen, der Deut-
tember 2008, Erfurt.
schen Gesellschaft für Allergologie und Klinische
Immunologie und der Gesellschaft für Pädiatrische Allergologie und Umweltmedizin
Dr. rer. nat. Erwin Duitman
Posterpreis, September 2008.
OEGAI Joint annual Meeting of Immunology
Posterpreis,
September 2008.
Kleinheinz A, Lepp U, Petersen A, Becker W.-M.,
Hausen B.M.
3. Gemeinsamer Deutscher Allergiekongress des
Ärzteverbands Deutscher Allergologen, der Deutschen Gesellschaft für Allergologie und Klinische
Dipl. Biol. Katharina Scholz
Immunologie und der Gesellschaft für Pädiatri-
Diplompreis 2008 des Ver-
sche Allergologie und Umweltmedizin
eins zur Erforschung infek-
Posterpreis, September 2008.
tiologischer und allergischer Prozesse.
Maria Lammers
Landesbeste in der Ausbildung zum/r Biolaboranten/in, Schleswig-Holstein.
Dr. rer. nat. Kay Vogel
Promotionspreis des Kreises Segeberg 2008.
Dr. rer. nat. Christina Moulakakis
Promotionspreis des Kreises Segeberg 2008.
und American Thoracic Society Travel Award,
September 2008.
Wissenschaftlicher Jahresbericht 2007-2008
231
Ehrungen /Ernennungen 2007/2008
Berufungen 2007/2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Ehrungen 2008
Berufungen 2007
Prof. Dr. med. Hans-Dieter Flad, Emeritus
Prof. Dr. med. Stefan Ehlers
Medal of Professor Ludwik Hirszfeld
Ruf auf die W3-Professur
XIIIth Congress of Polish Society of Experimental
„Molekulare Entzündungs-
and Clinical Immunology
medizin“
Mai, 2008.
Christian-Albrechts-Universität zu Kiel
Oktober 2007
Ruf angenommen.
Berufungen 2008
PD Dr. rer. nat. Thomas
Gutsmann
W3-Bionanomechanik
Universität Ulm
September 2008
Ruf abgelehnt
Ernennungen 2007
Prof. Dr. rer. nat. Otto Holst
wurde zum Präsident der European Carbohydrate Organisation ernannt.
232
Wissenschaftlicher Jahresbericht 2007-2008
Wissenschaftliche Veranstaltungen
Colloquia and Symposia
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Wissenschaftlicher Jahresbericht 2007-2008
234
Wissenschaftliche Veranstaltungen
Colloquia and Symposia
Wissenschaftliche Veranstaltungen
Colloquia and Symposia
Wissenschaftlicher Jahresbericht 2007-2008
235
Seminare 2007
Colloquia 2007
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Zentrumsseminare 2007
Prof. Dr. Jabbar Ahmed, Forschungszentrum Bor-
Aik Bossing, M.D., Department of Pulmonary Dis-
stel, LG Vet. Infektiologie und Immunologie,
eases and Tuberculosis, Diakonessenhuis Utrecht,
12.06.2007
Niederlande, 23.10.2007
Theileria-transformierte Leukozyten als Modellsy-
Use of the ELISPOT technique and Cross-spot tech-
stem für Untersuchungen von Proliferation und
nique to establish the diagnosis of tuberculosis.
Apoptose.
Prof. Dr. Helmut Brade, Forschungszentrum Borstel,
Dr. Dirk Albrecht, Forschungszentrum Borstel, Me-
LG Medizinische und Biochemische Mikrobiologie,
dizinische Klinik, 24.05.2007
09.01.2007
Update zur nicht-invasiven Beatmung.
Monoklonale Antikörper gegen Kohlenhydrate –
Grundlagen und Anwendung.
Dr. Sabine Anders, Forschungszentrum Borstel, Medizinische Klinik, 08.02.2007
PD Dr. Sven Brandau, Forschungszentrum Borstel,
Vertrauen ist gut, Kontrolle ist besser. Controlling
LG Immuntherapie, 13.02.2007
und Qualitätsmanagement im Krankenhaus.
Interaktion von malignem Gewebe und Wirtsimmunität.
Dipl. Biol. Ellen Andresen, Forschungszentrum Borstel, LG Klinische Infektiologie und Angeborene Im-
Prof. Dr. Klaus Brandenburg/Dipl.-Phys. Jörg Ho-
munologie, 05.04.2007
we, Forschungszentrum Borstel, LG Biophysik,
Die Rolle antimikrobieller Peptide bei der COPD.
06.02.2007
Einsatz von Synchrotronstrahlung zur Strukturauf-
Dr. Wolf-Meinhard Becker, Forschungszentrum Bor-
klärung von Biomolekülen am Hamburger HASY-
stel, LG Molekulare und Klinische Allergologie,
LAB. Wie findet PETRA die Struktur der Moleküle?
10.04.2007
Prof. Dr. Thomas Brüning, Berufsgenossenschaftli-
Erdnussallergie.
ches Forschungsinstitut für Arbeitsmedizin (BGFA),
Dr. Christoph Beisswenger, Department of Micro-
Institut für Ruhr-Universität Bochum, 23.01.2007
biology and Pediatrics, University of Pennsylvania
Die besondere Bedeutung des Biomonitorings in
School of Medicine, Philadelphia, Pennsylvania,
der arbeitsmedizinischen Vorsorge.
USA, 04.09.2007
Transepithelial migration of invasive bacterial
Prof. Dr. Petra Dersch, Institut für Mikrobiologie, TU
pathogens.
Braunschweig, 24.04.2007
Enteropathogenic Yersiniae: how to enter and ex-
Prof. Dr. Rikard Blunck, Université de Montréal, GÉ-
ploit human cells.
PROM, Canada, 10.07.2007
Fluorescence spectroscopy of proteins in planar
Dr. Daniel Drömann, Forschungszentrum Borstel,
lipid bilayer.
LG Klinische Immunpharmakologie, 08.05.2007
Infektionen im humanen Lungenmodell.
Prof. Dr. Jan Born, Inst. f. Neuroendokrinologie, Universitätsklinikum Schleswig-Holstein, Campus Lü-
Dr. med. Frank Eberhardt, Forschungszentrum Bor-
beck, 18.01.2007
stel, Medizinische Klinik, 30.08.2007
Schlaf und Gedächtnisbildung.
Therapie des NSCLC (Update vom ASCO 2007).
236
Wissenschaftlicher Jahresbericht 2007-2008
Seminare 2007
Colloquia 2007
Dr. Niko Föger, Institut für Immunologie, Otto-von-
PD Dr. Thomas Jacobs, Bernhard-Nocht-Institut für
Guericke Universität, Magdeburg, 05.06.2007
Tropenmedizin, Hamburg, 16.10.2007
Control of T-lymphocyte trafficking and cellular
Regulation of T-cells during experimental malaria:
homeostasis by the actin cytoskeleton.
Protection versus Pathology.
PD Dr. Andreas Frey, Forschungszentrum Borstel,
Dr. Pernille Jensen, Imagnia AG, Malmö, Schwe-
Medizinische Klinik, 09.10.2007
den, 30.03.2007
Peptid-basierte Targetingsysteme für die molekula-
Signal enhancement of >10.000 times with hyper-
re Bildgebung und Zelltyp-spezifische Antigenauf-
polarized NMR for real time measurements of in vit-
nahme.
ro and in vivo metabolism.
PD Dr. Karoline Gaede, Forschungszentrum Borstel,
Dr. Stefan Kluge, Klinik für Intensivmedizin, Univer-
LG Immunpharmakologie, 03.05.2007
sitätsklinikum Eppendorf, Hamburg, 01.02.2007
Chronische Berylliose – Risikofaktoren.
Pulmonale Mykosen.
Dr. med. Volker Geist, Segeberger Kliniken, Bad Se-
Dr. Roland Lang, Technische Universität München,
geberg, 06.09.2007
22.05.2007
pAVK: Aktuelle Diagnostik und Therapie.
Balancing macrophage activation: from IL-10 to
DUSP1.
Delia Goletti M.D. Ph.D., National Institute for Infectious Diseases, Rom, Italien, 02.10.2007
PD Dr. Gerd Leimenstoll, Klinik für Allgemeine In-
Immune diagnostic assays for the diagnosis of ac-
nere Medizin, Universitätsklinikum Schleswig Hol-
tive and latent tuberculosis infection.
stein, Campus Kiel, 22.02.2007
Management der Niereninsuffizienz.
Prof. Dr. Ernst Hempelmann, Witwatersrand University, Johannesburg, Südafrika, 25.09.2007
Prof. Dr. Helgo Magnussen, Krankenhaus Großhan-
Malariaequipment: Die Achillesferse der Malaria-
sdorf, 22.03.2007
parasiten.
Phänotypisierung der COPD.
Prof. Dr. Otto Holst, Forschungszentrum Borstel, LG
Dr. Lauren Mashburn Warren, NMS Building, Uni-
Strukturbiochemie, 11.12.2007
versity of Texas, Austin, USA, 19.06.2007
Allergy-protective cowsheds: a dirty story?
Quorum sensing and insights into membrane vesicle formation.
Prof. Dr. Christian G. Hübner, Institut für Physik, Universität zu Lübeck, 03.07.2007
Dr. Sebastian Meier, Institute of Molecular Biology,
Intrinsic motions along an enzymatic reaction tra-
University of Copenhagen, Denmark, 30.03.2007
jectory.
Conformational landscapes of proteins: collective
motions, unfolded states and fold evolution.
Dr. Tamitake Itoh, National Institute of Advanced Industrial Science and Technology, Health Technolo-
Dr. Markus Mohrs, Trudeau Institute, Saranac Lake,
gy Research Center, Bio-Nano Analysis Team, Taka-
USA, 26.06.2007
matsu, Japan, 01.10.2007
Interrogating immune responses with cytokine re-
Surface-Enhanced Resonance Raman Scattering (SERS)
porter mice.
as an imaging tool for single molecular biomaterials.
Wissenschaftlicher Jahresbericht 2007-2008
237
Seminare 2007
Colloquia 2007
Prof. Dr. Sergei Nedospasov, Engelhardt Institute
Prof. Dr. Udo Schumacher, Universitätsklinikum Ep-
of Molecular Biology, Moscow und Deutsches Rheu-
pendorf, Hamburg, 13.03.2007
maforschungszentrum Berlin, 14.06.2007
Klinisch relevante Tiermodelle zur Tumormetasta-
Distinct physiological functions of TNF may have dif-
sierung.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
ferent physiological thresholds and are associated
with production by distinct cell types.
Dr. S. Schwarting, Dt. Gesellschaft Zahnärztliche
Schlafmedizin, 19.04.2007
Prof. Dr. Joost Oppenheim, National Cancer Insti-
Zahnschienen gegen Schnarchen und Schlafapnoe
tute, Laboratory of Molecular Immunregulation,
– Wirkungsweise, Leitlinien, klinische Anwendung.
NIH, Bethesda, USA, 19.03.2007
Alarmins are a subset of danger signals.
Dr. Silvano Sozzani, Section of General Pathology and
Immunology, University of Brescia, Italien, 16.01.2007
Dr. Tobias Pukrop, Abt. Hämatologie/Onkologie,
Role of LCCR in pulmonary dendritic cell migration.
Universität Göttingen, 30.01.2007
Wnt5a signaling in tumor progression.
Dr. med. Hendrik Treede, Universitätsklinikum Eppendorf, Hamburg, 13.09.2007
Prof. Dr. Hans-Reimer Rodewald, Institut für Immu-
Lungentransplantation: Indikation, Technik und Prognose.
nologie, Universitätskrankenhaus Ulm, 18.09.2007
Mechanism of a Mast Cell Protease-Mediated In-
PD Dr. Thomas Tschernig, Functional and Applied
nate Response.
Anatomy, Medical School of Hannover, MHH,
06.03.2007
Dr. Philip Rosenstiel, Institut für Klinische Mole-
MALP-2 stimulates the lung immune system.
kuarbiologie, Christian-Albrechts-Universität, Kiel,
20.02.2007
Dr. Cornelis M. van Drunen, Head ENT Research
Defence mechanisms at epithelial interfaces: les-
Laboratory, Academic Medical Center, Amsterdam,
sons from complex genetics.
06.11.2007
Primary nasal epithelium of house dust mite aller-
Dr. Jan Rupp, Medizinische Klinik III, Universitätsklinikum
Schleswig-Holstein,
Campus
gic individuals displays an activated state.
Lübeck,
07.06.2007
PD Dr. Thomas Vogel, Institut für experimentelle
Chlamydien und Mycoplasmen Pneumonien.
Dermatologie, Universität Münster, 18.12.2007
MRP8 and MRP14 are novel endogenous ligands of
PD Dr. Jan Rupp, Institut für Medizinische Mikro-
Toll- like receptor 4 and promote lethal endotoxin -
biologie und Hygiene, Universitätsklinikum Schles-
induced shock.
wig-Holstein, Campus Lübeck, 20.11.2007
C. pneumoniae interferes with immune regulatory
Prof. Dr. Jürgen Westermann, Institut für Anatomie,
mechanisms of antigen-presenting cells – is there a
Universität zu Lübeck, 27.03.2007
functional role in the pathogenesis of asthma?
CD4 memory T-cells on trial: Immunological memory without a memory T-cell.
Prof. Dr. Axel Scheidig, ZBM, Christian-AlbrechtsUniversität zu Kiel, 27.02.2007
Prof. Dr. Barbara Wollenberg, HNO-Klinik, Univer-
Insight into enzymatic reaction mechanisms by pro-
sitätsklinikum Schleswig-Holstein, Campus Lübeck,
tein crystallography.
28.06.2007
Akute und chronische Sinusitiden.
238
Wissenschaftlicher Jahresbericht 2007-2008
Seminare 2008
Colloquia 2008
Zentrumsseminare 2008
Dr. Kerstin Andrae Marobela, University of
Functional genetics of multiple sclerosis using hu-
Botswana, Gaborone, Botwana, 30.06.08
manised mice.
Traditional medical knowledge and modern diseases.
Prof. Dr. M. Gavrovi -Jankulovi , Faculty of Chemistry, University of Belgrade, 18.11.08
Prof. Dr. Ben Appelmelk, Dept. of Medical Micro-
Modulation of T cell proliferation by mannose spe-
biology, Vrije Universiteit Medical Center, Amster-
cific banana lectin.
dam, 21.10.08
DC-SIGN ligands on Mycobacterium tuberculosis
Prof. Dr. Michael Glocker, Proteom-Zentrum Ro-
and Heliobacter pylori and their interaction with the
stock, Institut für Immunologie, Uni Rostock, 13.02.08
host.
Peptidsignaturen beim Mamma-Carcinom: „Labelfree“ – Quantifizierung durch LC-MS im Vergleich zu
PD Dr. Iris Bittmann, Pathologisches Institut, Diako-
2D Page – MALDI-TOF Peptide Mass Fingerprinting.
niekrankenhaus Rotenburg (Wümme), 11.11.08
Muster des immunologisch getriggerten Lungen-
Dr. Andrea Gómez-Zavaglia, Universidad de
schadens.
Buenos Aires, Argentinien, 22.01.08
Characterization of S-layer proteins from Lacto-
Prof. Dr. Heimo Breiteneder, Institut für Pathophy-
bacillus by FTIR spectroscopy.
siologie, Zentrum für Physiologie, Pathophysiologie
und Immunologie (CEPPI), Medizinische Universität
Prof. Dr. Mathias Hornef, Institut für Med. Mikrobi-
zu Wien, 04.03.08
ologie, Med. Hochschule Hannover, 05.02.08
Die Evolution der Allergenität.
Innate recognition and response by the intestinal
epithelium.
Prof. Dr. Philip Calder, Professor of Nutritional Immunology, University of Southhampton, England,
Dr. Birgit Jung und Dr. Detlef Stiller, Boehringer In-
28.10.08
gelheim Pharma, Biberach an der Riss, 21.04.08
Polyunsaturated fatty acids and inflammation: from
Tiermodell in der COPD.
the bench to bedside.
Moderne Bildgebung in der Atemwegsforschung.
Prof. Dr. André Fischer, Laboratory of Aging and
Dr. Axel Kallies, The Walter and Eliza Hall Institute
Cognitive Diseases, EURYI Research Group, Max
of Medical Research, Parkville Victoria, Australia,
Planck Society, Göttingen, 16.09.08
29.04.08
Epigenetic strategies to treat neurological diseases.
Differentiation of mature effector lymphocytes depends on Blimp1.
Prof. Dr. Wolfgang Fischer, Max Planck Institut für
Biophysikalische Chemie, Göttingen, 23.09.08
Dr. Peter König, Institut für Anatomie, Universität Lü-
Molecular analysis of histone modifications and
beck, 11.03.08
chromatin domains.
Mechanismen und Regulation des zilienvermittelten
Partikeltransports in den Atemwegen.
Dr. Manuel Friese, Institut für Neuroimmunologie
und Klinische Multiple Sklerose Forschung, UKE,
Prof. Dr. Paul Kosma, Dept. of Chemistry, Universi-
07.10.08
ty of Natural Resources and Applied Life Sciences,
Wissenschaftlicher Jahresbericht 2007-2008
239
Seminare 2008
Colloquia 2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Wien, 12.02.08
ical Sciences, Royal Institute of Medical Sciences,
Chemical synthesis of nucleotide-activated hep-
Royal Free and University College Medical School,
toses and 4-aminoarabinose LPS epitopes.
London, 01.04.08
Geographical variation in the priming of the im-
Prof. Dr. Guido Kroemer, INSERM, Villejuif, Frankre-
mune system: implications for the design of vac-
ich, 25.11.08
cines for tuberculosis.
Immunogenic cell death in anti-cancer chemotherDr. Günter Roth, Interfakultäres Institut für Zellbio-
apy.
logie, Eberhard-Karls-Universität, Tübingen, 20.05.08
Dr. Sabine Küsters, Director of European Opera-
Softlithography and printing of microarrays.
tions, Rules-Based Medicine Inc., Krefeld, Germany/Austin, Texas, USA, 18.06.08
Prof. Dr. Ruth Schmitz-Streit, Institut für Allgemeine
High content immunoassays for biomarker profiling
Mikrobiologie, Christian-Albrechts-Universität, Kiel,
across species.
06.05.08
From nitrogen regulation in prokarya to gene min-
Prof. Dr. Roland Martin, Institut für Neuroimmuno-
ing of novel biocatalysts.
logie und Klinische Multiple Sklerose Forschung,
UKE, 27.05.08
Annette Schön, Brüsselbüro der Leibniz-Gemein-
The role of T cells in multiple sclerosis.
schaft, 26.02.08
Einführung in die EU-Forschungsförderung und Aus-
Dr. Diana Mittag, Department of Immunology,
blick auf die nächste Ausschreibung zum Thema 1
Monash University, Melbourne, 03.06.08
Gesundheit.
Hitchhikers on ryegrass pollen shaping the T cell reProf. Dr. Yoichi Shinkai, Research Center for Infect.
sponse – a T cell’s guide to the allergy.
Diseases, Institute for Virus research, Kyoto UniverProf. Dr. Gunnar Pejler, Dept. of Anatomy, Physiol-
sity, Japan, 19.05.08
ogy and Biochemistry, Swedish University of Agri-
Function and regulation of histone lysine methyla-
cultural Sciences, Uppsala, 15.04.08
tion.
The role of mast cell chymase in inflammatory disDr. Alice Sijts, Dept. of Infectious Diseases and Im-
ease.
munology, Faculty of Veterinary Medicine, UniversiProf. Dr. Francesco Peri, Dept. of Biotechnology
ty of Utrecht, 01.07.08
and Biosciences, University of Milano-Bicocca, Mi-
How the antigen processing pathway regulates
lano, 04.11.08
CD8 T cell responses to self and foreign antigens.
Sugar-derived bioactive compounds: lipid A antagPD Dr. Christian Taube, 3. Med. Klinik, Univer-
onist and Ras oncoprotein inhibitors.
sitätsklinikum Mainz, 14.10.08
Prof. Dr. Satish Raina, Amrita Institute of Medical
Regulation of allergic airway inflammation.
Science, Cochin, Indien, 15.01.08
Signal transduction pathway in E. coli in response
Prof. Dr. Diethard Tautz, Abteilung Evolutionsgene-
to outer membrane alterations.
tik, Max-Planck-Institut für Evolutionsbiologie, Plön,
29.01.08
Prof. Graham Rook, Centre for Infectious Diseases
Tracing the genetics of adaptations in the house
and International Health, Windeyer Institute of Med-
mouse.
240
Wissenschaftlicher Jahresbericht 2007-2008
Seminare 2008
Colloquia 2008
Dr. Zhao–Qi Wang, Leibniz-Institut für Altersforschung – Fritz Lipmann Institut e.V. Jena, 19.02.08
DNA repair defects in human diseases: lessons from
mouse models.
Prof. Dr. Rainer Zawatzky, DKFZ Heidelberg,
17.06.08
The growing family of Interferon-induced genes: recent studies on a new member
Wissenschaftlicher Jahresbericht 2007-2008
241
Weiterbildungseminare 2007
Colloquia 2007
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Weiterbildungsseminare der Klinik 2007
Dr. Sven Hirschfeld Araujo, BUKH Boberg, Querschnittsgelähmten-Zentrum, 22.11.2007
Zwerchfellschrittmacher.
Dr. Frank Eberhardt, Medizinische Klinik Borstel,
30.08.2007
Therapie des NSCLC.
Dr. Volker Geist, Segeberger Kliniken, 6.09.2007
pAVK: Aktuelle Diagnostik und Therapie.
PD Dr. Hans-Peter Hauber, Medizinische Klinik
Borstel, 4.10.2007
Update GINA Guidelines.
Klinisch-Pathologische Konferenz, 27.09.2007
Klinisch-Pathologische Konferenz, 1.11.2007
Klinisch-Pathologische Konferenz, 29.11.2007
Dr. Helgo Magnussen, KH Großhansdorf,
25.10.2007
Phänotypisierung der COPD.
Dr. Hendrik Treede, UKE, Hamburg, 13.09.2007
Lungentransplantation: Indikation, Technik und
Prognose.
PD Dr. Gunther Wiest, Asklepios Klinik Harburg,
8.11.2007
Pulmonale Hypertonie: State of the art.
242
Wissenschaftlicher Jahresbericht 2007-2008
Weiterbildungseminare 2008
Colloquia 2008
Weiterbildungsseminare der Klinik 2008
Dr. Alexander Balling, Psychosomatische Klinik,
Klinisch-Pathologische Konferenz, 23.10.2008
Bad Bramstedt, 19.06.2008
Morbide Adipositas: Behandlungsoptionen aus
Klinisch-Pathologische Konferenz, 27.11.2008
psychosomatischer Sicht.
Klinisch-Pathologische Konferenz, 18.12.2008
Dr. Hans-Jörg Baumann, II. Med. Klinik, UKE,
Hamburg, 3.04.2008
Dr. Thomas Köhnlein, Abtl. Pneumologie, MHH,
Die Buchstabensuppe der Beatmungsmodi.
4.12.2008
Alpha 1-Antitrypsin and COPD.
Dr. Peter Buggisch, I. Medizinische Klinik, UKE,
Hamburg, 15.05.2008
PD Dr. Christoph Lange, Medizinische Klinik Bor-
Therapie der akuten und chronischen viralen He-
stel, 5.06.2008
patitis.
Exazerbation der COPD.
PD Dr. Fabian Fehlauer, Strahlenzentrum Ham-
Dr. Michael Melullis, Klinik f. Chirurgie, UKSH, Lü-
burg Nord, 17.01.2008
beck, 7.02.2008
Strahlentherapie beim Bronchialkarzinom.
Adipositaschirurgie: Indikation, Technik und Prognose.
Dr. Volker Geist, Segeberger Kliniken, 24.01.2008
pAVK: Aktuelle Diagnostik und Therapie.
PD Dr. Andreas Meyer, Krankenhaus Mariahilf,
Mönchengladbach, 21.02.2008
PD Dr. Hans-Peter Hauber, Medizinische Klinik
Nicht-medikamentöse Therapie von COPD und
Borstel, 17.04.2008
Asthma.
Update pulmonale Hypertonie.
Dr. Ernst Müller, Medizinische Klinik Borstel,
PD Dr. Hans-Peter Hauber, Medizinische Klinik
6.11.2008
Borstel, 18.09.2008
Nicht-kleinzelliges Bronchialkarzinom – State of the art.
Neues vom ATS 2008.
Dr. Thomas Nauert, LGASH, Kiel, 20.03.2008
Klinisch-Pathologische Konferenz, 31.01.2008.
Asbestbedingte Berufskrankheiten.
Klinisch-Pathologische Konferenz, 28.02.2008.
Dr. Adriana Sorete Arbore, Lasi, Rumänien,
6.03.2008
Klinisch-Pathologische Konferenz, 27.03.2008.
Tuberculosis in Romania.
Klinisch-Pathologische Konferenz, 24.04.2008.
Dr. Ulrich van Laak, Medizinische Klinik Borstel,
20.11.2008
Klinisch-Pathologische Konferenz, 29.05.2008.
Apnoetauchen.
Klinisch-Pathologische Konferenz, 27.06.2008.
Prof. Dr. Robert J. Wilkinson, University Cape
Town, South Africa, 8.07.2008
Klinisch-Pathologische Konferenz, 25.09.2008.
HIV infection and XDR tuberculosis.
Wissenschaftlicher Jahresbericht 2007-2008
243
Symposien und Workshops 2007
Symposia and Workshops 2007
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Symposien und Workshops 2007
Meeting des Schistosomen-Ei-Konsortiums: Notting-
SIICA 5th National Conference for the session
ham – Cambridge – Leiden – Gießen – Borstel
Cytokines and Inflammatory Mediators.
Organisation: PD Dr. Helmut Haas, Dr. Gabriele
Gruppo Regionale Immunologia, Immunologia
Schramm
Clinica e Allergologia (GRIICA-FVG)
Forschungszentrum Borstel
Vorsitz: Prof. Dr. Dr. Silvia Bulfone-Paus
27.-29. März 2007
Triest, Italien
6.-9. Juni 2007
EU-FP-6-ICTTD-3 Workshop „Attenuated Vaccines
for Animal Diseases“
NGFN2-Abschlusssymposium „Umweltnetz”
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr.
Co-Organisation: Prof. Dr. Stefan Ehlers
Jabbar Ahmed
Christian-Albrechts-Universität zu Kiel
Forschungszentrum Borstel
Juli 2007
16.-19. April 2007
EU-FP-6-ASEMDialog Meeting „The EU, China and
EU-FP-6-ICTTD-3 Workshop „Isolation, cultivation
South East Asia Dialog for the Development of Re-
and monitoring techniques for the production
search Areas in Animal Health of Mutual Interest“
and application of attenuated vaccines“
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr. Jab-
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr.
bar Ahmed
Jabbar Ahmed
Beijing, China
Forschungszentrum Borstel
13.-15. August 2007
19.-24. April 2007
14th European Carbohydrate Symposium
Arbeitskreis Infektiologie in Schleswig-Holstein
Organisation: Forschungszentrum Borstel et al.
Organisation: PD Dr. Christoph Lange
Tagungspräsident: Prof. Dr. Otto Holst, FZB
Kiel
Lübeck
25. April
2.-7. September 2007
EU-FP-6-INCOME Meeting „Harmonisation and Dis-
Workshop „Regulation of Cell Survival”
tribution of Pathogen Detection and Differentiation
37. Annual Meeting of the German Society for Im-
Tools“
munology
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr. Jab-
Vorsitz: Prof. Dr. Dr. Silvia Bulfone-Paus
bar Ahmed
Heidelberg
Losbon, Portugal
6.-7. September 2007
15.-18. Mai 2007
TBNET Meeting
20. Allergiekolloquium
Co-Organisation: PD Dr. Christoph Lange
„Aktuelles aus der Allergologie“
Stockholm, Schweden
Organisation: Prof. Dr. Peter Zabel et al.
14.-15.September 2007
Forschungszentrum Borstel
Mai 2007
244
Wissenschaftlicher Jahresbericht 2007-2008
Symposien und Workshops 2007
Symposia and Workshops 2007
EU-FP-6-ICTTD-3 „Asian Component Satellite Meeting“
Borsteler Bronchoskopiekurs
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr. Jab-
Organisation: PD Dr. Christoph Lange, Dr. Ernst
bar Ahmed
Müller
Sansibar, Afrika
Forschungszentrum Borstel
26. September 2007
3. November 2007
2. Gemeinsamer Deutscher Allergiekongress
30. Arbeitstagung der Norddeutschen Immuno-
der Deutschen Gesellschaft für Allergologie und
logen
klinische Immunologie, des Ärzteverbandes
„Biology of Inflammation“
Deutscher Allergologen und der Gesellschaft für
Organisation: Prof. Dr. Dr. Silvia Bulfone-Paus et
Pädiatrische Allergologie und Umweltmedizin
al.
Co-Organisation: Forschungszentrum Borstel
Forschungszentrum Borstel
Tagungspräsident: Dr. Wolf-Meinhard Becker, FZB
16. November 2007
Lübeck
26.-29. September 2007
EU-FP-6-INCOME “Transboundary animal diseases
& tick and tick-borne diseases”
Scanning Probe Microscopies and Organic Ma-
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr. Jab-
terials XVI
bar Ahmed
Organisation: PD Dr. Thomas Gutsmann
RAZI Vaccine & Serum Research Institute, Hessa-
Hamburg
rak, Karaj, Iran
September 2007
18.-22. November 2007
Treffen von deutschen (Glyko)Lipidforschern
Arbeitskreis Infektiologie in Schleswig-Holstein
Organisation: PD Dr. Thomas Gutsmann
Organisation: PD Dr. Christoph Lange
Hamburg
Kiel
September 2007
27. November 2007
Pneumocampus „Pneumologische Infektiologie“
Treffen der Norddeutschen Biophysiker
Organisation: PD Dr. Christoph Lange
Organisation: PD Dr. Thomas Gutsmann, PD Dr.
Lübeck
Andra Schromm
12.-13.Oktober 2007
Forschungszentrum Borstel
30. November 2007
SFB/TR22 Meeting Schloss Rauischholzhausen
Steering Committee: Prof. Dr. Dr. Silvia Bulfone-Paus
Statusseminar des Sonderforschungsbereichs
Marburg
415 „Spezifität und Pathophysiologie von Signal-
24.-26. Oktober 2007
transduktionswegen“
Co-Organisation: Prof. Dr. Dr. Silvia Bulfone-Paus
EU-FP-6-ConFluTech „Avian Influenza-Technology
Forschungszentrum Borstel
Transfer & Training“
30. November - 1. Dezember 2007
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr. Jabbar Ahmed
Bucharest, Romania
29.-31. Oktober 2007
Wissenschaftlicher Jahresbericht 2007-2008
245
Symposien und Workshops 2008
Symposia and Workshops 2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Symposien und Workshops 2008
12. Symposium “Infektion und Immunabwehr”
Forschungszentrum Borstel
der gemeinsamen Fachgruppe “Infektionsimmu-
25.-26. April 2008
nologie” der DGHM und DGfI
Organisator: Prof. Dr. Christoph Hölscher
Workshop “Technical workshop on epidemiologi-
Burg Rothenfels
cal tools”
7.-9. März 2008
Organisation, Durchführung: Prof. Dr. Ulrike Seitzer,
Prof. Dr. Jabbar Ahmed
Symposium der European Respiratory Society
Duhok, Irak
“State of the Art in Tuberculosis 2008
30. April–9. Mai 2008
und
Treffen der Arbeitsgruppe Tuberkulosebera-
Workshop „Technical workshop on molecular di-
tungsstellen des ÖGD Schleswig-Holstein
agnostic tools”
Organisation: PD Dr. Christoph Lange, PD Dr.
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr.
Stefan Niemann, Prof. Dr. Peter Zabel
Jabbar Ahmed
Forschungszentrum Borstel
Berlin
9. April 2008
5.-9. Mai 2008
49. Kongress der Deutschen Gesellschaft für
Workshop „Tierschutz und Versuchstiere in der
Pneumologie und Beatmungsmedizin e.V.
Forschung“
Tagungspräsident: Prof. Dr. Peter Zabel et al.
Organisation: Dr. Ilka Monath
Lübeck
Forschungszentrum Borstel
9.-12. April 2008
8. Mai 2008
EU-funded Collaborative Networks Income and
Workshop „Real Time PCR“
Lab-On-Site
Organisation: PD Dr. Stefan Niemann
“Technology Transfer of Molecular Diagnostic
Forschungszentrum Borstel
Tools”
14.-16. Mai 2008
Organisation, Durchführung: Prof. Dr. Ulrike Seitzer,
Prof. Dr. Jabbar Ahmed
Central European Workshop on “Application of
Lanzhou, China
Molecular genetic methods in veterinary diagnos-
14.-18. April 2008
tic virology”
Organisation: Prof. Dr. Jabbar Ahmed
21. Allergie-Kolloquium
Kosice, Slovak Republic
„Update Allergologie“
27.-29. Mai 2008
Organisation: Prof. Dr. Peter Zabel et al.
Forschungszentrum Borstel
EU-FP 6 INCOME Workshop „Bioinformatics“
19. April 2008
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr.
Jabbar Ahmed
SFB/TR 22 „Allergische Immunantwort der Lunge“
Forschungszentrum Borstel
Retreat
8.-14. Juni 2008
Organisation: Prof. Dr. Dr. Silvia Bulfone-Paus,
Prof. Dr. Harald Renz (Univ. Marburg)
246
Wissenschaftlicher Jahresbericht 2007-2008
Symposien und Workshops 2008
Symposia and Workshops 2008
Workshop, Arbeitskreis „Vakzine“ der Deutschen
5. Borsteler Herbstseminar für Pathologie und
Gesellschaft für Immunologie
Biomedizin
Organisation: PD Dr. Andreas Frey et al.
Organisation: Prof. Dr. Dr. Ekkehard Vollmer, PD
Forschungszentrum Borstel
Dr. Torsten Goldmann, Holger Schultz et al.
12. Juni 2008
Forschungszentrum Borstel
12.-13. September 2008
EU-FP 6 INCOME Workshop „Host-Pathogen-Interaction“
Regional Training Course on “Rapid Diagnosis of
Organisation: Prof. Dr. Ulrike Seitzer, Prof. Dr.
Avian Influenza (Bird Flu)”
Jabbar Ahmed
Organisation: Prof. Dr. Jabbar Ahmed
Forschungszentrum Borstel
Vladimir, Russian Federation
18. Juni 2008
15.-26. September 2008
Symposium der „Akademie der Wissenschaften
SFB 415 „„Spezifität und Pathophysiologie von
in Hamburg“
Signaltransduktionswegen”
Organisation: Dr. Bettina Brand
Status Quo Meeting
Forschungszentrum Borstel
Organisation: Prof. Dr. Dr. Silvia Bulfone-Paus,
21. Juni 2008
Prof. Dr. Dietrich Kablitz (Univ. Kiel) et al.
Forschungszentrum Borstel
Workshop „Moderne Wirkstoffforschung“
19.-20. September 2008
Organisation: Dr. Hinrich Habeck, Ascenion;
ScreeningPort GmbH Hamburg
Workshop Fachgruppen “Microbial Systematics
Forschungszentrum Borstel
and Infection Epidemiology”
23. Juni 2008
60. Jahrestagung der Deutschen Gesellschaft für
Hygiene und Mikrobiologie
Conference of the Cluster of Excellence “Inflam-
Organisation: PD Dr. Stefan Niemann
mation at Interfaces”
Dresden
Organisation: PD Dr. Frank Petersen
21.-24. September 2008
Universität Lübeck
27.-28. Juni 2008
Ticks and Tick-borne Diseases VI
Scientific Committee: Prof. Dr. Jabbar Ahmed
29th Annual Congress of the European Society of
Buenos Aires, Argentinien
Mycobacteriology
22.-26. September 2008
Steering Committee: PD Dr. Stefan Niemann
Plovdiv, Bulgarien
13. Forum pneumologicum
6.-9. Juli 2008
„3 Jahre Zentrum für Klinische Infektiologie“
Organisation: Prof. Dr. Peter Zabel et al.
Research in Swine Viral Diseases
Forschungszentrum Borstel
Organisation: Prof. Dr. Jabbar Ahmed
11. Oktober 2008
Shanghai
7.-9. Juli 2008
Wissenschaftlicher Jahresbericht 2007-2008
247
Symposien und Workshops 2008
Symposia and Workshops 2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
BMBF Verbund „Lungentuberkulose – Wirts- und
National Training Course on Avian Influenza Diag-
Erregerdeterminanten für Resistenz und Krank-
nostics
heitsausprägung”
Organisation: Prof. Dr. Jabbar Ahmed
Statusseminar
Bucharest, Romania
Organisation: Prof. Dr. Stefan Ehlers, PD Dr. Ka-
8.-9. Dezember 2008
roline Gaede
Forschungszentrum Borstel
National Training Course on Avian Influenza Diag-
30.-31. Oktober 2008
nostics
Organisation: Prof. Dr. Jabbar Ahmed
31. Arbeitstagung der Norddeutschen Immunolo-
Tbilisi, Georgien
gen
15.-17. Dezember 2008
„Frontiers in Immunoregulation“
Organisation: Prof. Dr. Dr. Silvia Bulfone-Paus et al.
National Training Course on Avian Influenza Diag-
Forschungszentrum Borstel
nostics
14. November 2008
Organisation: Prof. Dr. Jabbar Ahmed
Istanbul, Turkey
National Avian Influenza workshop for local exper-
15.-19. Dezember 2008
tise’s
Organisation: Prof. Dr. Jabbar Ahmed
Symposium: Deutsch-Ukrainische Partnerschaftsi-
Athens, Griechenland
nitiative
17.-19. November 2008
Organisation und Durchführung: Dr. Sabine
Rüsch-Gerdes, PD Dr. Elivra Richter
Workshop „Spiroergometrie“
Forschungszentrum Borstel
Organisation: PD Dr. Hans-Peter Hauber et al.
18. Dezember 2008
Forschungszentrum Borstel
22. November 2008
GABRIEL – Workshop
Organisation: Prof. Dr. Otto Holst et al.
Forschungszentrum Borstel
27.-28. November 2008
SFB/TR 22 – Begutachtung
Organisation: SFB/TR 22
Philipps-Universität Marburg
1.-2. Dezember 2008
World Vaccine Congress 2008
Scientific Advisory Board: Prof. Dr. Jabbar Ahmed
Foshan, PRChina
1.-5. Dezember 2008
248
Wissenschaftlicher Jahresbericht 2007-2008
Lehre • Ausbildung
Teaching • Training
Fo r s c h u n g s z e n t r u m
Bo r s t e l
250
Wissenschaftlicher Jahresbericht 2007-2008
Lehre • Ausbildung
Teaching • Training
Lehre • Ausbildung
Teaching • Training
Wissenschaftlicher Jahresbericht 2007-2008
251
Lehre 2007 - 2008
Teaching 2007 - 2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Lehre
Bulfone-Paus S, Brandt E, Gerdes J, Heine H, Petersen F, Seitzer U, Hölscher C, Reiling N.
A
Ausgewählte Themen der Immunologie und Zell-
Ahmed JS.
biologie
Anleitung zum selbstständigen wissenschaftlichen
Forschungszentrum Borstel WS 2006/2007, SS 2007,
Arbeiten
WS 2007/2008, SS 2008
Fachbereich Veterinärmedizin, FU Berlin, WS
2006/2007, SS 2007, WS 2007/2008, SS 2008
Bulfone-Paus S, Ulmer AJ, Petersen F, Ernst M,
Haas H, Seitzer U, Reiling N, Heine H, Scholzen
Andrä J.
T, Petersen A, Schromm AB, Kasper B, Lee K-H.
Vorlesung: Biochemie und Biophysik biologischer
Neue Entwicklungen in der Immunologie für Natur-
Membranen
wissenschaftler und Mediziner
Fachbereich Chemie, Universität Hamburg, WS
Mathematisch-Naturwissenschaftliche Fakultät
2006/2007, SS 2007, WS2007/2008, SS 2008
Christian-Albrechts-Universität zu Kiel, WS
2006/2007, SS 2007, WS 2007/2008, SS 2008
Vorlesung, Biochemisch Analytik
Fachbereich Chemie, Universität Hamburg, WS
Bulfone-Paus S, Brandt E, Gerdes J, Ernst M, Pe-
2006/2007, SS 2007, WS 2007/2008, SS 2008
tersen F, Seitzer U.
Anleitung zum selbstständigen wissenschaftlichen
B
Arbeiten
Brade H, Ehlers S, Holst O, Zähringer U, Hölscher
Universität zu Lübeck WS 2006/2007, SS 2007, WS
C, Reiling N, Müller-Loennies S.
2007/2008, SS 2008
Immunchemisches Seminar
Universität zu Lübeck, WS 2006/07, SS 2007, WS
Bulfone-Paus S, Heine H, Ulmer AJ, Schromm AB,
2007/08, SS 2008
Petersen F mit wissenschaftlichen Mitarbeitern.
Anleitung zum selbstständigen wissenschaftlichen
Brade H, Holst O, Zähringer U, Müller-Loennies S,
Arbeiten auf dem Gebiet der Immunologie und
Schromm AB.
Zellbiologie
Anleitung zum wissenschaftlichen Arbeiten
Mathematisch-Naturwissenschaftliche Fakultät
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
Christian-Albrechts-Universität zu Kiel, WS
2007/2008, SS 2008
2006/2007, SS 2007, WS 2007/2008, SS 2008
Brandenburg K.
Bulfone-Paus S, Ulmer A, Ernst M, Brandt E, Hei-
Vorlesung, Physikalische Grundlagen des Lebens
ne H, Petersen F, Seitzer U, Orinska Z, Scholzen
Mathematisch-Naturwissenschaftliche Fakultät
T, Reiling N, Kasper B, Petersen A, Hölscher C.
Christian-Albrechts-Universität zu Kiel, WS
Praktikum Immunologie für Naturwissenschaftler
2006/2007
und Mediziner
am Forschungszentrum Borstel
Vorlesung, Biophysik elektromagnetischer Strahlung
Mathematisch-Naturwissenschaftliche Fakultät
Mathematisch-Naturwissenschaftliche Fakultät
Christian-Albrechts-Universität zu Kiel, SS 2007, SS
Christian-Albrechts-Universität zu Kiel, SS 2007
2008
Praktikum Kernphysik und Radiochemie
Fachhochschule Lübeck, WS 2006/2007
252
Wissenschaftlicher Jahresbericht 2007-2008
Lehre 2007 - 2008
Teaching 2007 - 2008
E
Ehlers, Reiling N, Hölscher C, Niemann S.
Gutsmann T, Andrä J, Brandenburg K.
Infection Immunology
Christian-Albrechts-Universität zu Kiel, WS
Masterstudiengang Molecular Life Sciences
2006/2007, SS 2007, WS 2007/2008, SS 2008
Anleitung zum wissenschaftlichen Arbeiten
Universität zu Lübeck, WS 2006/2007, WS
2007/2008
Gutsmann T.
Vorlesung: Grundlagen der Biophysik
Anleitung zum wissenschaftlichen Arbeiten
Christian-Albrechts-Universität zu Kiel, WS
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
2006/2007, WS 2007/2008
2007/2008, SS 2008,
Vorlesung: Membranbiophysik
Ehlers S, Hölscher C, Reiling N, Laskay T,
Christian-Albrechts-Universität zu Kiel, SS 2007, SS
Schromm AB, Haas H, Niemann S.
2008
Seminar, Molekulare Infektiologie und Immunologie
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
Fortgeschrittenen Praktikum Iii, Biophysik
2007/2008, SS 2008
Christian Albrechts-Universität zu Kiel, WS
2006/2007, WS 2007/2008
Ehlers S, Reiling N, Schramm G, Niemann S,
G
Richter E, Hillemann D, Müller-Loennies S, Goldmann T, Seitzer U.
Haas H, Petersen A, Schramm G, Becker WM,
FP II Praktikum Mikrobiologie für Studenten der
Schocker F, Frey A.
Biochemie/Molekularbiologie
Aktuelle biochemische, molekulare und zelluläre
Christian Albrechts-Universität zu Kiel, WS
Aspekte der Allergieforschung
2006/2007, WS 2007/2008
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
2007/2008, SS 2008
F
Frey A.
Hauber, H-P.
Blockpraktikum Strukturbiologie: Herstellung von
Einführung in die nicht-invasive Beatmung
Biokonjugaten und ihre Anwendung in Analytik
Universität zu Lübeck, WS 2007/2008, SS 2008
und Wirkstoff-Targeting.
Masterstudiengang Molecular Life Sciences
Heine H., Schromm AB, Bulfone-Paus S, Stamme
Universität zu Lübeck, WS 2006/2007, WS
C, Brade H, Zähringer U, Lindner B, Ernst M,
2007/2008
Baumann R, Reiling N, Vollmer E, Gutsmann T,
Petersen F,
G
Gaede K, Goldmann T, Niemann S.
Mentoring program for PhD and MD students
Forschungszentrum Borstel WS 2006/2007, SS 2007
Molekularbiologische Methoden für Mediziner
Universität zu Lübeck, WS 2006/2007, WS
Hölscher C.
2007/2008
Praktikum, Transgene Expression in Mäusen
Universität zu Lübeck, WS 2006/2007, WS
Gutsmann T, Andrä J.
2007/2008
Vorlesung, Grundlagen der Membranphysik
Universität zu Lübeck, SS 2007, SS 2008
Hölscher C, Niemann S.
Praktikum, Molekulare Infektiologie und Immunologie
Wissenschaftlicher Jahresbericht 2007-2008
253
Lehre 2007 - 2008
Teaching 2007 - 2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
Vorlesung, Physik der Sinne
2007/2008, SS 2008
Mathematisch-Naturwissenschaftliche Fakultät
Christian-Albrechts-Universität zu Kiel, WS
Hölscher C, Reiling N, Ehlers S, Niemann S, Holst
2006/2007, WS 2007/2008
O, Lange C, Goldmann T.
Seminar, Aktuelle Ergebnisse der Tuberkuloseforschung
Vorlesung, Einführung in die moderne Massen-
Forschungszentrum Borstel, WS 2006/2007, SS
spektrometrie
2007, WS 2007/2008, SS 2008
Mathematisch-Naturwissenschaftliche Fakultät
Christian-Albrechts-Universität zu Kiel, SS 2007, SS
2008
Holst O.
Vorlesung: Microbial toxins
Universität Katowice, Polen, WS 2007/2008, SS
Lindner B, Peters T, Mesters JR.
2008
Einführung in die Strukturanalytik
Studiengang Molecular Life Sciences
Holst O, Ehlers S, Mamat U, Goldmann T, Seitzer
Universität zu Lübeck, SS 2007, SS 2008
U, Zähringer U, Reiling N, Niemann S, Hillemann
D, Brade H, Vollmer E, Richter E, Schultz H.
Lindner B, Zähringer U.
Mentorenprogramm des Studiengangs Molecular
Wahlpflichtveranstaltung Massenspektrometrie
Life Science, Vorlesungen, Seminare
Bachelor Studiengang Molecular Life Sciences
Universität zu Lübeck, WS 2006/2007; SS 2007, WS
Universität zu Lübeck, WS 2005/2006
2007/2008, SS 2008
M
L
Mamat U, Dalski A, Zühlke C.
Lange C.Seminar Querschnittsbereich Infektiolo-
Vorlesung, Praktikum, Genetik
gie/Immunologie
Bachelor Studiengang Molecular Life Sciences
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
Universität zu Lübeck, SS 2007, SS 2008
2007/2008, SS 2008
P
Seminar Themen aus der Inneren Medizin
Petersen A.
Universität zu Lübeck, WS 2006/2007, SS 2007, Ws
Blockpraktikum Strukturbiologie: Untersuchungen
2007/2008, SS 2008
der RNase-Aktivität des Erdnussallergens Ara h8.
Masterstudiengang Molecular Life Sciences, Uni-
Laskay T, Rupp J, Knobloch J, Holst O, Ohgke H,
versität zu Lübeck, WS 2007/2008
Niemann S.
Vorlesung, Praktikum Mikrobiologie
Petersen A, Flesch BK.
Studiengang Molecular Life Sciences
Elektrophoretisches Praktikum für Naturwissen-
Universität zu Lübeck, WS 2006/2007, WS
schaftler und Mediziner
2007/2008
Universität zu Lübeck und
Christian-Albrechts-Universität zu Kiel, SS 2007, SS
2008
Lindner B.
Physikalisches Praktikum Teil III: Biophysikalische
R
Arbeitsmethoden
am Forschungszentrum Borstel, WS 2006/2007, WS
Reiling N.
2007/2008
Vorlesung: Infektionsimmunologie der Tuberkulose
254
Wissenschaftlicher Jahresbericht 2007-2008
Lehre 2007 - 2008
Universität zu Lübeck, WS 2007/2008
Teaching 2007 - 2008
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
2007/2008, SS 2008
S
Stamme C.
Zabel P, Lange C.
Anleitung zum wissenschaftlichen Arbeiten
Praktische Ausbildung in der Inneren Medizin
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
2007/2008, SS 2008
2007/2008, SS 2008
Repetitorium Zellbiologie
Zabel P, Schunkert H, Fehm L, Gross W. et al.
Universität zu Lübeck / Forschungszentrum Borstel,
Vorlesung, Innere Medizin
WS 2006/2007, SS 2007, WS 2007/2008, SS 2008
Universität zu Lübeck, SS 2007, SS 2008
V
Vorlesung, Pathophysiologie der Inneren Medizin
Vollmer E, Goldmann T, Schultz H.
Universität zu Lübeck, WS 2006/2007, WS
Anleitung zum wissenschaftlichen Arbeiten
2007/2008
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
2007/2008, SS 2008
Blockpraktikum der Inneren Medizin
Universität zu Lübeck, SS 2007, WS 2007/2008, SS
Seminar, Ausgewählte Kapitel zur Tumorpatholo-
2008
gie
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
2007/2008, SS 2008
Vollmer E, Welker L, Schultz H.
Klinisch-pathologische Konferenz
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
2007/2008, SS 2008
Vollmer E, Goldmann T, Niemann S, Drömann D,
Schultz H.
Komplexe Experimentalmodelle in der Pneumologie
Universität zu Lübeck, WS 2006/2007, SS 2007, WS
2007/2008, SS2008
Z
Zabel P.
Vorlesung, Atemwegserkrankungen im Problemorientierten Lernen (POL)
Universität zu Lübeck, SS 2007, WS 2007/2008, SS
2008
Zabel P, Petersen A, Haas H, Lange C, Frey A,
Niemann S, Gaede KI, Hauber H-P.
Anleitung zum wissenschaftlichen Arbeiten
Wissenschaftlicher Jahresbericht 2007-2008
255
Ausbildung 2007 - 2008
Training 2007 - 2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Ausbildung
LEONARDO-DA-VINICI Programm
Romina Pritzkow
In 2007 und 2008 haben 26 Auszubildenden ihre
berufliche Ausbildung erfolgreich abgeschlossen.
„Wege entstehen dadurch, dass man sie geht
und Chancen multiplizieren sich, wenn man sie er-
Biolaboranten, 2007: Björn Adomeit, Jessica Dob-
greift”
schanski, David Gräser, Stefanie Heller, Marie-Luise Helms, Tjard Jörß, Kerstin Kopp, Stefan Kruse,
Mit diesen Lebensweisheiten kann man wohl zu-
Nadine Ruske, Ulrike Stein, Michael Weidhase,
sammenfassend das ausdrücken, was uns 13 Aus-
Doreen Weigel, Chrystin Wildgrube
zubildenden für 4 Wochen geboten wurde. Wir
durften teilnehmen am Leonardo - da - Vinci - Pro-
Biolaboranten, 2008: Simon Berg, Franziska Da-
jekt und 28 - 31 Tage lang, in einem EU - Land un-
duna, Lisa Dost, Luisa Freese, Franziska Hillers,
serer Wahl, Kultur, Sprache und Arbeit kennen ler-
Maria Lammers, Linda Lang, Corinna Studt, So-
nen. Ganz auf uns alleine gestellt, übten wir uns
phie Weber, Birte Wegner, Kathleen Wilke
in der englischen - und der jeweiligen Muttersprache, erfuhren neue Arbeitstechniken und er-
Tierpfleger, 2007: Nina Frank
lebten Kultur pur. Dieser Monat gab uns die Möglichkeit Einblicke in andere Länder zu gewinnen,
Tierpfleger, 2008: Tina Lipski
Erfahrungen sammeln zu können und Zukunftsperspektiven zu entwickeln.
Ebenso lehrte uns der Aufenthalt Verantwortung
LEONARDO-DA-VINICI Programm
zu übernehmen und Selbstbewusstsein zu erlan-
Das LEONARDO DA VINCI Programm der Eu-
gen. Ganz besonderer Dank gilt der Ausbil-
ropäischen Union ist im Bereich der beruflichen
dungsleiterin vom FZ - Borstel, Fr. Dr. Zähringer,
Aus- und Weiterbildung einzigartig. Es unterstützt
die uns die Türen zur großen weiten Welt ein Stück
die transnationale Zusammenarbeit zwischen
weiter öffnete.
den Akteuren in diesem Bereich, indem es Auslandsaufenthalte zum beruflichen Lernen fördert
Danke – Thanks – Gracias – Hvala - Tack
und in europäischen Partnerschaften innovative
Lehr- und Lernmaterialien oder Zusatzqualifikationen entwickelt. Die Teilnahme des FZB an diesem Programm wurde 2004 durch die Leiterin der
Ausbildung, Dr. Susanne Zähringer, initiiert. Für
die Originalität der Antragstellung, Organisation
und Zusammenarbeit mit den europäischen Partner wurde Dr. Zähringer in den letzten drei Jahren jeweils mit dem Qualitätsurteil „best practice“
ausgezeichnet – das allein spricht für sich!
Hier einige Originalkommentare und Berichte der
Auszubildenden mit eigenem Fotomaterial:
256
Alexander Börnhöft, Thore Masekowitz
Universita degli Studi Napoli Federicoll, Neapel, Italien
„... die Italiener sind nett und alles ist so crazy ...“
Wissenschaftlicher Jahresbericht 2007-2008
Ausbildung 2007 - 2008
Training 2007 - 2008
Katharina Langer, Ann-Kathrin Jarms
University Clinic for Respiratory and Allergic Diseases Golnik,
Bled, Slowenien
„ ... klasse war die Gastfreundschaft der Slowenen – das geilste war das Paragliden ... ”
Jan Demmer, Kai Treichel
Instituto Zooprofilattico Sperimentale „A. Mirri“ della Sicilia, Italien
„ ... afrikanische Kultur & Land sind sehr interessant ...“
Bericht über den Slowenien-Aufenthalt im Rahmen des Leonardo-Projektes 2008
Katharina Langer
Im Rahmen des Leonardo-Projektes traten AnnKathrin Jarms und ich am 26.09.2008 unsere Reise nach Slowenien an. Gegen Abend des
27.09.2008 erreichten wir unser Apartment in BoFranziska Ganzert, Philip Wildgrube
University Lund, Schweden
„ ... Land, Leute und Arbeit waren einfach toll ... “
desce/Bled, hier bezogen wir ein 2-Zimmer Apartment mit Einbauküche und Bad in einem nett angelegten Bauernhaus.
Am 29.09.2008 traten wir unseren ersten ArbeitsRomina Pritzkow, Melanie Röpcke, Svenja Schulz, Özge Kök
Facultad de Ciencias de Quimicas de Moncloa, Madrid,
Spanien
„ ... wunderschöne Sehenswürdigkeiten & interessante neue Arbeitstechniken erlernt ...”
tag in der „University Clinic of Respiratory and Allergic Diseases Golnik“ an. Unsere erste Arbeitswoche sollten wir in der Immunologie verbringen.
Die immunologische Abteilung steht seit 2002 un-
Wissenschaftlicher Jahresbericht 2007-2008
257
Ausbildung 2007 - 2008
Training 2007 - 2008
ter der Leitung von Dr. Peter Korosec, seine 5 Mit-
Die Identifikation dieser Mykobakterienstämme
arbeiter entwickeln standardisierbare immuno-
soll später epidemiologischen Forschungen zur
logische und genetisch diagnostische Tests für
Verbreitung der verschiedenen Mykobakterien-
Lungenerkrankungen und Allergien. Darüberhin-
stämme in Slowenien dienen.
Fo r s c h u n g s z e n t r u m
Bo r s t e l
aus werden hier Hypersensitivitäts-, immunologische, serologische und genetische Tests sowie
Neben all der interessanten Arbeit blieb uns im-
Tests zu Autoimmunerkrankungen durchgeführt.
mer genügend Zeit, Slowenien kennen zu lernen.
In dieser ersten Arbeitswoche war es unsere Auf-
So hatten wir zum Beispiel die Gelegenheit, ein
gabe, aus dem Vollblut von Patientenproben
Wochenende in dem Apartment einer Arbeitskol-
DNA zu Isolieren und die Konzentration der iso-
legin in dem Hafenstädtchen Piran zu verbringen,
lierten DNA fotometrisch zu messen. Hierbei fiel
welches sich auf einer Landzunge in den Golf von
auf, dass in sehr großem Maße auf die Sterilität
Venedig erstreckt. Nachdem wir in den ersten
geachtet wurde: So wurde z.B., entgegen unserer
Wochen in Bled u. Umgebung. die schneebe-
bisherigen Erfahrung, unter einer Cleanbench ge-
deckten Kuppen 2000m hoher Berge bestaunt hat-
arbeitet. Die Arbeit in dieser Abteilung war für
ten, bot sich uns nun, nur 200km südwestlich, ein
mich insofern sehr interessant, als dass mich an
absolutes Kontrastprogramm: Mediterranes Kli-
dem Leonardo-Projekt vor allem interessierte, wie
ma mit Palmen, Olivenhainen und natürlich
bereits bekannte Methoden in Laboratorien an-
Strand- das alles bei ca. 28°C und Meerwasser
derer Länder durchgeführt werden und dass es
mit toller Badetemperatur.
hier tatsächlich große Unterschiede zu vermerken
gibt.
Ein weiteres Highlight unseres Aufenthaltes war
das Paragliden. Wie für so vieles hatte auch hier
Am 09.10.2008 wechselten wir dann aus der Im-
Manca Zolnir ihre Kontakte spielen lassen und ei-
munologie in die Mykobakteriologie. Leiterin die-
nen Tandemflug mit einem Paragliding-Piloten or-
ser Abteilung ist seit 1995 Dr. Manca Zolnir-Dovc,
ganisiert. Ich denke wer einmal frei wie ein Vogel
für sie sind 6 Mitarbeiter tätig. Die Hauptaufga-
über das traumhafte Panorama der julischen Al-
ben dieses Labors liegen in der direkten mikro-
pen geflogen ist, der wird diesen Eindruck sein
skopischen Untersuchung von Patientenproben,
Leben lang nicht mehr vergessen.
bakteriologischen Kulturen und der Identifikation
von Mykobakterien. All diese Untersuchungen
dienen dazu, dem jeweiligen Patienten die richtige Diagnose zu stellen und geeignete Behandlungen zu entwickeln. Neben der Untersuchung
von Patientenproben werden auch Forschungsprojekte, zum Beispiel im Bereich „molecular genotyping and molecular epidemiology of tubercle
bacilli“, in dieser Abteilung durchgeführt. Unsere
Aufgabe in diesem Labor war es zunächst, PCRs
von isolierter DNA (aus Patientenproben aus dem
Jahr 2007) durchzuführen. Diese amplifizierten
Natürlich haben wir auch nahezu das komplette
DNA-Proben wurden später von uns für eine Me-
Programm touristischer Sehenswürdigkeiten Slo-
thode namens Spoligotyping verwendet, mit wel-
weniens „abgearbeitet“, wie z.B. die Tropfstein-
cher verschiedene klinisch bedeutsame Myko-
grotte „Postojnska Jama“, den Markt und die Alt-
bakterienstämme identifiziert werden können.
stadt von Ljubljana, eine Wanderung auf den
258
Wissenschaftlicher Jahresbericht 2007-2008
Ausbildung 2007 - 2008
Training 2007 - 2008
1500m hohen Gipfel des „Kriska Gora“, eine Wanderung entlang des Bohinj-Sees bis hin zum „Slap
Savice“ (einem beeindruckenden Wasserfall) sowie eine Fahrt mit der Seilbahn auf den „Vogel“
auf ca. 1600m Höhe mit beeindruckendem Blick
auf den Bohinj See.
Zusammenfassend muss ich sagen, dass dieser
Monat in Slowenien mit Sicherheit die tollste Auslandserfahrung war, die ich bisher gemacht habe. Auch, weil Slowenien ein Land ist, von dem
ich bisher eine völlig falsche Vorstellung hatte,
bin ich froh, gerade dieses Land gewählt zu haben und kann Slowenien und v.a. die Klinik Golnik jedem weiteren Auszubildenden, der in den
Genuss der Leonardo-Teilnahme kommt, nur
wärmstens empfehlen. Ich habe bisher nirgendwo in Europa eine solche Gastfreundschaft empfunden wie dort und bisher in keinem anderen
Land so tiefe Einblicke in den Alltag der dort lebenden Menschen nehmen können. Alle Kolleginnen und Kollegen waren uns gegenüber sehr
aufgeschlossen und interessiert, auch sprachlich
gesehen gab es keine Schwierigkeiten, da fast alle über sehr gute Englischkenntnisse verfügten.
Besonders hervorzuheben ist in diesem Zusammenhang erneut Manca Zolnir, die sich rührend
um unser Wohlergehen kümmerte und uns sowohl
in beruflichen wie auch die Freizeit betreffenden
Fragen mit Rat und Tat zur Seite stand und viel
Zeit für uns opferte.
Ich hoffe, dass in den nächsten Jahren viele weitere Azubis ihre evtl. vorhandene Scheu gegenüber Slowenien ablegen und im Rahmen des Leonardo-Programms dorthin reisen werden. Ich, für
meinen Teil, war absolut begeistert und froh,
dass ich diese Gelegenheit genutzt habe.
Wissenschaftlicher Jahresbericht 2007-2008
259
Chronik • Neue Entwicklungen • Haushaltsplan
Chronicle • New Developments • Budget
Fo r s c h u n g s z e n t r u m
Bo r s t e l
260
Wissenschaftlicher Jahresbericht 2007-2008
Chronik • Neue Entwicklungen • Haushaltsplan
Chronicle • New Developments • Budget
Chronik • Neue Entwicklungen • Haushaltsplan
Chronicle • New Developments • Budget
Wissenschaftlicher Jahresbericht 2007-2008
261
Chronik 2007
Chronicle 2007
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Chronik 2007
18. Juni 2007
Sitzung des Kuratoriums und der Stiftungsver-
13. Februar 2007
sammlung des Forschungszentrums Borstel.
Besuch von Prof. Dr. Bernd Rohwer, Geschäftsführer der Industrie- und Handelskammer SchleswigHolstein, anlässlich einer Bereisung des Kreises
21. Juni 2007
Segeberg.
Besuch des Ministerpräsidenten, Peter Harry Carstensen, mit diplomatischen Korps anlässlich der
Kieler Woche und der damit verbundenen Reise
8. März 2007
ins Land.
Besuch des FDP-Kreisverbandes Segeberg.
15. Juni 2007
„… wer den Mythos von Borstel, das Geheimnis
dieses wunderbaren und im internationalen Wettbewerb so herausragend erfolgreichen Forschungszentrums für Medizin und Biowissenschaften ergründen will, der muss in das Herrenhaus, die Parkallee 1, eintreten, so wie Sie alle es
getan haben, sich niederlassen – und dann
schauen, hören, tasten und spüren. Sie werden
sehen, dass die Schönheit des Gebäudes zeitlos
28. Juni / 26. Juli / 30. August 2007
ist, so zeitlos, wie die Schönheit der Wissenschaft.
800 Jahrfeier der Gemeinde Sülfeld. Anlässlich
Das Klima dieses Hauses inspiriert die Fantasie,
dieses Jubiläums veranstaltete das FZB eine an
generiert neue Ideen und befördert die für die
die Öffentlichkeit gerichtete Veranstaltungsreihe
Wissenschaft alles entscheidende Kreativität. Das
zum Thema „Gesundheit der Lunge“.
Herrenhaus verkörpert mithin den genius loci Borstels …“. Zitat aus der Rede von Prof. Ernst Th.
Rietschel, Präsident der Leibniz-Gemeinschaft,
19. Juli 2007
anlässlich des Festaktes zur Eröffnung des Wis-
Besuch des Lions Club Henstedt-Ulzburg mit einer
sens- und Kommunikationszentrums im Herren-
Besichtigung des Herrenhauses und Diskussion
haus Borstel.
zum Thema „Allergie“.
27. Juli – 9. September 2007
Joost J. Oppenheim, Chief of the Laboratory of
Molecular Immunoregulation at NCI, NIH has visited the Research Center Borstel on his sabbatical leave.
Dr. Oppenheim was a pioneer in identifying cytokines and chemokines such as IL-1, IL-8 and
MCP-1, and in characterizing their function. This
earned him the nickname “Father of Cytokines”.
262
Wissenschaftlicher Jahresbericht 2007-2008
Chronik 2007
Chronicle 2007
Dr. Oppenheim has contributed to the field with
regionalen Wirtschaft und der IHK zum Thema
well over 550 papers (includes books, chapters,
Forschung/Entwicklung/Innovationsförderung.
reprints and reviews).
12. Dezember 2007
Erste Weihnachtsfeier des FZB nach der Restaurierung des Herrenhauses und der Eröffnung des
Wissens- und Kommunikationszentrums. Für ein
stimmungsvolles Programm und die wunderschöne Dekoration des Hauses sorgten Ulrike Schroer
und Grudrun Lehwark-Yvetot.
29. August 2007
Kühl war es auf dem Sommerfest 2007! Doch auch
das Wetter konnte den Borstelern die gute Stimmung nicht vermiesen. Die zweite Borstel Band
„ex vivo“ heizte richtig ein, genauso wir die härtesten Griller Norddeutschlands und alle weiteren Köstlichkeiten aus der inzwischen weltweit bekannten Küche des Zentrums.
7. November 2007
Besuch der Staatssekretärin Karin Wiedemann,
Ministerium für Wissenschaft, Wirtschaft und Verkehr.
15. November 2007
Besuch der IHK Nord zum Thema „Forschung erforschen“.
Wirtschaftgespräche im Kreis Segeberg, Informations- und Meinungsaustausch zwischen der
Wissenschaftlicher Jahresbericht 2007-2008
263
Chronik 2008
Chronicle 2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Chronik 2008
der Abteilung „Molekulare Infektiologie“ willkommen.
22. Mai 2008
Veranstaltungsreihe „Forschung erforschen!“ Zu
Gast waren Teilnehmer der Innovationstour der
Metropol-, Industrie- und Handelskammern, Norddeutschland.
4. Juni 2008
Sitzung des Beirats der Region Südwest, Projektgesellschaft Norderelbe.
12. Juni 2008
Besuch des Lions Club Henstedt-Ulzburg, der das
17. Oktober 2008
Schullabor am FZB durch Spenden finanziell un-
Verleihung des Professor-Otto-Roth Preises, des
terstützt. Durch diese Initiative hat der Lions Club
Heinrich-Dräger-Wissenschaftspreises und des Fa-
weiteren Schulen die Teilnahme am Projekt „For-
kultätspreises der Gesellschaft der Freunde und
schung und Schule“ ermöglicht.
Förderer der Universität Lübeck im Herrenhaus
Borstel.
14. November 2008
Verleihung des Nils-Ilja-Richter Preises der Deutschen Gesellschaft für Autoimmun-Erkrankungen
im Rahmen der 31. Arbeitstagung der Norddeutschen Immunologen in Anwesenheit von Minister
Dr. Werner Marnette.
9. Dezember 2008
Die traditionelle Weihnachtsfeier, wie immer im
prächtig geschmückten Herrenhaus, konnte eine
28. August 2008
Amtseinführung des neuen Direktors, Prof. Dr.
Ulrich Schaible. Geladene Gäste aus Universitäten und außeruniversitären Einrichtungen sowie
Premiere verzeichnen. Der Borstel Chor hatte mit
den zuständigen Ministerien hießen den Direktor
einer Reihe von internationalen Weihnachtslie-
264
Wissenschaftlicher Jahresbericht 2007-2008
Chronik 2008
Chronicle 2008
dern seinen ersten Auftritt und erhielt dafür einen
Riesenapplaus. Ansprachen, Musik, Preisverleihungen, kulinarische Köstlichkeiten und „Spitzen“
in Wort und Ton der Comedian Scientists sorgten
für einen mehr als gelungenen und stimmungsvollen Abend.
18. Dezember 2008
Aufsichtsratssitzung der Norgenta, Norddeutsche
Life Science Agentur.
Wissenschaftlicher Jahresbericht 2007-2008
265
Neue Entwicklungen
New Developments
Neue Entwicklungen
bioimaging, flow cytometric sorting capacity, BSL3 fa-
Fo r s c h u n g s z e n t r u m
Bo r s t e l
cility upgrade). Two newly created structural W3 pro-
Exzellenz-Cluster „Entzündung
an Grenzflächen“
fessorships affiliated with the universities of Lübeck
and Kiel (Experimental Pneumology, Heinz Fehrenbach; Molecular Inflammation Medicine, Stefan
A proud founding pillar of the Cluster
of Excellence in Inflammation
Research
Ehlers) and one Kiel-affiliated W1 junior professor-
Stefan Ehlers, Member of the Executive Group
frastructures and personnel in Borstel through Clus-
ship (Mouse Model Systems for Microbe-Induced Inflammation, Guntram Grassl) receive support for in-
ter funding. In addition, several independent young
On October 19, 2007, the Cluster of Excellence “In-
researcher and postdoctoral positions are funded
flammation at Interfaces” was approved for funding
via the molecular research pipelines on collagen VII,
through 2012. The Cluster is a research consortium
gp130 and NOD-like receptors and thus form an in-
comprising five faculties of the universities of Kiel and
tegral part of the interdisciplinary Cluster effort. Al-
Lübeck, the Max-Planck-Institute for Evolution Biology
together, approx. 20% of all Cluster funds (total of ⇔
in Plön and the Research Center Borstel. Research fo-
30 million over 5 years) have been acquired by Bors-
cuses on human chronic inflammatory barrier disor-
tel researchers.
ders, such as Crohn disease, psoriasis and sarcoidosis. The aim of the Cluster is to re-structure the
The Research Center Borstel is represented in the
scientific landscape in Schleswig-Holstein into a high-
Cluster Steering Committee by Silvia Bulfone-Paus
ly matrixed field of interested scientists who, based
and Ulrich Zähringer, and within the Cluster Executive
on new genetic input concerning disease etiology,
Group by Stefan Ehlers. Borstel will also participate
predict structure information and describe multiple in-
in a PhD program “Inflammation Research” to be
tertwined pathways and pathophysiologies in mod-
conducted by the newly recruited young professors
el systems of barrier diseases in a highly parallel
of the Cluster. Members of all three departments
fashion. In its first year of existence, the Cluster has
profit from the Cluster via its international visibility
filled 3 W3 structural professorships as well as 12 W2
(e.g. monthly Cluster Lectures with high profile re-
and 3 W1 strategic researcher positions at the uni-
searchers), access to high-throughput and high-tech
versities of Kiel and Lübeck, and has recruited 12 in-
platforms (genome wide sequencing, DESY PETRA III
dependent young scientists at the postdoctoral level
beamline, population representative biobank pop-
in the research pipelines focused around NOD-like re-
gen), and free online access to the Elsevier Freedom
ceptors, gp130 cytokines, and collagen VII. An infra-
Collection and Nature Journal Series. Most impor-
structural milestone will be the inauguration of the
tantly, however, the successful application as an in-
Comprehensive Center of Inflammation Medicine in
tegral founding member of the Cluster is another
April 2009 where patients will be diagnosed and
proof of excellence for the disease-oriented molecu-
treated based on an interdisciplinary clinical confer-
lar research that is the centerpiece of the Research
ence and where therapy and prevention studies will
Concept in Borstel.
be initiated with new anti-inflammatory agents and
molecular nutrition. The Research Center Borstel provides access to its technological platforms (e.g.
Literarisches zum Cluster
biosafety level 2 and 3 facilities, advanced mass
spectrometry, unique M. tuberculosis strain collec-
Ein imaginäres Gespräch
tions, certified infectious disease study center) and is
Hans-Dieter Flad*
rewarded by several infrastructural investments (e.g.
Am 6. Dezember 1769 fand am Hof des Königs
266
Wissenschaftlicher Jahresbericht 2007-2008
Neue Entwicklungen
New Developments
Christian VII. von Dänemark ein Gespräch zwi-
gnose gestellt habe („febris continua epidemica
schen Johann Hartwig Ernst Graf von Bernstorff,
maligna petechialis“) und, entgegen dem Rat an-
dem allmächtigen Staatsminister und Handels-
derer Ärzte, als Therapie „Lüftung der Kranken-
minister, und Johann Friedrich Struensee, Leib-
stuben, nicht zu warm zudecken, nicht zu stark hei-
arzt des Königs, Physikus der Herrschaft von Pin-
zen, kühlende Waschungen mit Wasser-verdünn-
neberg und Altona und Landphysikus der Graf-
tem Weinessig“ empfohlen habe. Damit eng ver-
schaft Rantzau, statt.
bunden sind auch Maßnahmen, die den
Sanitätsdienst in unserem Lande betreffen.
Struensee: Exzellenz, ich beabsichtige, einen Ver-
Bernstorff: Struensee, ich bin mit Ihrem Vorschlag
bund der medizinischen Diagnostik und der Pati-
einverstanden. Was das Gebiet betrifft so sollte
entenversorgung in Dänemark und Norddeutsch-
dieses „Exzellenzcluster“, wie Sie das nennen,
land zu gründen, ein sogenanntes Medizinisches
ganz Norddeutschland einschließlich meinen
Exzellenzcluster für das ganze Land.
Stammsitz Borstel und weiter Hamburg und Alto-
Bernstorff: Das klingt interessant, zumal es auch
na umfassen.
meinen Titel beinhaltet. Als Staatsminister, der für
Struensee: Sehr richtig, Exzellenz.
den Handel zuständig ist, habe ich natürlich ein
Interesse daran, dass die Bevölkerung gesund ist
Am 15. September 1770 wurde Bernstorff aus po-
und vor Krankheiten geschützt werden kann.
litischen Gründen unter Mithilfe von Struensee
Aber: Was haben Sie vor? Sind Sie überhaupt für
von König Christian dem VII. entlassen. Bernstorff
ein solches Vorhaben qualifiziert?
bemerkte hierzu: „Was ich erwartet habe, ist ge-
Struensee: Ich denke schon, Exzellenz. Zunächst
schehen. Ich habe eine Schlacht verloren. Die Op-
einmal muss die Verhütung von Krankheiten, die
position ist zu stark gewesen. … Ich bin es nun zu-
Prophylaxe, verbessert werden. Ich bin, wie Sie
frieden, ich ziehe bald nach Borstel, wo ich es gut
wissen, ein Anhänger des Contagiums, d. h. der
habe und gern sein mag.“ Aber in Borstel kann
Übertragbarkeit von Krankheiten durch kleine Er-
er nicht bleiben, weil seine Schwiegermutter we-
reger, die, wie Leibniz propagiert, mit dem Mi-
gen der Schmach der Entlassung ihn nicht auf-
kroskop erkannt werden können. Krankheiten ent-
nehmen will. Struensee wird am 17. Januar 1772
stehen nicht, wie die Humoralpathologie be-
verhaftet und am 24. April zum Tode verurteilt. Sei-
hauptet, als Folge einer Säfteverderbnis durch
nen Feinden war es gelungen, ihm ein Verhältnis
Ausdünstungen aus dem Boden. So habe ich be-
zu Königin Caroline Mathilde nachzusagen. Seine
obachtet, dass die Exkremente von Ruhr- und
Reformen des Sanitätswesens und seine seu-
Faulfieber-Kranken im Fleetwasser von Hamburg
chenprophylaktischen Verdienste im Sinne einer
ansteckend sind und Gesunde krankmachen,
Exzellenzinitiative fielen der Vergangenheit zum
während das Trinkwasser von Altona von besse-
Opfer. Erst 235 Jahre später konnte seine Idee ei-
rer Qualität ist und weniger neue Krankheitsfälle
nes Exzellenzclusters in Norddeutschland inklusi-
hervorruft. Weiterhin habe ich gute Erfahrung mit
ve Borstel verwirklicht werden. Die wissenschaft-
der Pockenimpfung. Ich sage immer: „So wie die
lichen Grundlagen waren gelegt.
Eigenschaft des Magneten durch Berührung aus
vielen Eisenstücken übergehen kann, ebenso
Die Mächtigen interagierten und kooperierten
kann das Blatterngift von einem Kranken auf vie-
miteinander. Ehefrauen und Schwiegermütter wur-
le Gesunde, welche noch keine Blattern haben,
den in das neue Konzept mit einbezogen.
übertragen werden“. Auch dürfte Ihnen, Exzel-
(Literatur beim Verfasser)
lenz, bekannt sein, dass ich 1759 im Kreis Pinne-
*Prof. Dr. med. Hans-Dieter Flad war 20 Jahre als Direktor der
berg bei Fälle von Fleckfieber die richtige Dia-
Abteilung „Immunologie und Zellbiologie“ am FZB tätig.
Wissenschaftlicher Jahresbericht 2007-2008
267
Neue Entwicklungen
New Developments
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Neues Gesicht am FZB
Lötzer besonders wichtig, die Wissenschaftler dabei zu unterstützen, ihre Themenvorschläge bei
EU-Forschungsreferat
der EU-Kommission bekannt zu machen und star-
Seit Oktober 2008 ist das EU-Forschungsreferat
ke internationale Konsortien zu bilden. Die För-
neu besetzt. Dr. rer. nat. Katharina Lötzer wech-
derung von Forschungsprojekten durch die Eu-
selte an das FZB, um die Wissenschaftler in allen
ropäische Union ist nicht nur ein wichtiger Bau-
Fragen der EU-Forschungsförderung zu unterstüt-
stein zur Finanzierung relevanter Forschungsvor-
zen. Frau Lötzer war zu-
haben, sondern festigt auch die internationale
vor als Wissenschaftlerin
Akzeptanz des FZB und seiner Wissenschaftler.
am Institut für Vaskuläre
Medizin der FriedrichSchiller-Universität Jena
Scientific Reinforcement
im Bereich der Atherosklerose-Forschung tätig.
Department Molecular Infection Biology
Nach Abschluss ihrer
In August 2008, Prof. Dr. rer. nat. Ulrich E. Schaible
Promotion im Fach Zell-
succeeded Ernst Rietschel in leading the Depart-
biologie leitete sie die
ment Immunochemistry and Biochemical Microbiol-
Arbeitsgruppe Endothelzellbiologie und sammel-
ogy renamed as Department of Molecular Infection
te während dieser Tätigkeit umfangreiche Erfah-
Biology. In addition he is holding the chair for Bio-
rungen in Forschungsförderung und Projektma-
chemical Mircobiology at the University of Lübeck.
nagement. Erste Berührungen mit der Leibniz-Ge-
Parts of his research team are still working at the
meinschaft ergaben sich bereits während ihres
London School of Hygiene & Tropical Medicine, In-
Studiums der Ökotrophologie an der Christian-
fectious and Tropical Diseases where he had a
Albrechts-Universität zu Kiel bei einem einjähri-
chair in immunology (2006-2008) and is now ap-
gen Forschungsaufenthalt am Deutschen Institut
pointed as a Honorary Professor.
für Ernährungsforschung in Potsdam-Rehbrücke.
Durch ihren Wechsel nach Borstel kann Frau Lötzer ihre Interessen im Bereich Wissenschaft, Wirtschaft und Organisation verbinden und für das
FZB einsetzen. Sie hält engen Kontakt zu den Nationalen Kontaktstellen und dem Büro der LeibnizGemeinschaft in Brüssel, um die Borsteler Wissenschaftler und Entscheidungsträger stets auf
dem aktuellen Stand halten zu können. Im Januar 2009 wird sie daher im Brüsseler Büro hospitieren, um vor Ort mit spezifischen Angelegenheiten der EU-Forschungsförderung bekannt zu
werden. Hierzu gehört insbesondere das Knüpfen
von Kontakten, etwa zu Vertretungen der Länder
und Organisationen sowie zur Europäischen Kommission. Denn neben der Informationsvermittlung
The Department of Molecular Infection Biology
sowie der Unterstützung der Wissenschaftler von
(MIB) combines research on the many facets of
Projektidee und Antragstellung bis Vertragsver-
bacterial infections to study them in their entirety.
handlungen und Projektmanagement ist es Frau
The research agenda covers from basic molecular
268
Wissenschaftlicher Jahresbericht 2007-2008
Neue Entwicklungen
New Developments
analysis of bacterial structures and their genetic
basis, studies on pathogen and host interactions
and the resulting immune responses as well as
translational approaches in collaboration with the
Clinical Department. The prime foci of MIB are on
pulmonary pathogens, in particular those, which
cause Tuberculosis and Cystic Fibrosis, but also on
Chlamydia and other Gram-negative ones including environmental bacteria and their influence on
lung immunity such as allergic reactions. Tuberculosis research comprises studies on host genetics,
host cell/pathogen interactions and anti-tuberculo-
tion and structural changes leading to a disease-
sis immunity. Factors determining virulence and
specific patho-physiology. The integrative research
pathogenicity - primarily in infections with Gram-
concept is based on the in vivo study of animal
negative bacteria - are studied with respect to their
models and comprises the seek for specific molec-
structural, genetic and functional characteristics
ular mechanisms linked to the development of lung
with a focus on immune modulation by bacterial
(dys)function and pathology. The central aim is the
compounds and membrane biophysics of receptor-
identification of novel therapeutic targets.
ligand interactions.
Severe asthma displays a complex clinical phenotype with chronic impairment of lung function periodically necessitating intensive care hospitalization
and mechanical ventilation. Recent data suggest
that chronic airway inflammation is different from
the classical T helper 2 cell driven airway
eosinophilia. It involves influx of neutrophils as well
as a contribution of T helper 17 cells. However, the
factors leading to the altered inflammatory response and ultimately to progression and aggravation of the asthmatic phenotype remain elusive.
Work in the division utilizes several animal models
together with extensive lung function assessment
and state-of-the-art stereology in order to elucidate
Experimental Pneumology
the role of neutrophils and T helper 17 cells.
In July 2008, Prof. Dr. rer. nat. Heinz Fehrenbach
joined the Research Center Borstel to establish the
Pulmonary emphysema is characterized by the
section “Experimental Pneumology” which compris-
loss of alveoli, which is considered to be irreversible
es additional three research groups. Furthermore,
with no curative therapy being available. Although
he is holding the chair in Experimental Pneumology
cigarette smoking is the most prominent cause of
(W3) at the University of Lübeck.
the disease, systemic effects are implicated in its
pathogenesis. Increased interest in regenerative
His division “Inflammation and Regeneration” will
therapeutic approaches has been stimulated by re-
focus on the genuine patho-physiological process-
cent studies indicating that the lung possesses an
es and the specific cell types, which link inflamma-
endogenous regenerative capacity, which may be
Wissenschaftlicher Jahresbericht 2007-2008
269
Neue Entwicklungen
New Developments
influenced by systemic factors. Several growth fac-
chemokines, and receptors is evaluated. First ex-
tors (HGF, KGF, VEGF, PDGF, TGFb) exerting cell
periments have shown that very low tidal volumes
type-specific effects are candidate key mediators in
induce pulmonary inflammation. Most previous
alveolar regeneration. Work in the division will fo-
studies used animals with healthy lungs. It is not
cus on the identification of the relative contribution
clear whether so called protective ventilation strate-
of these known as well as of still unknown pul-
gies can be used in injured lungs. Therefore differ-
monary and systemic pathways underlying such en-
ent ventilation strategies are applied to mice with
dogenous and potentially inducible regenerative
injured lungs (bleomycin-induced fibrosis, elastase-
processes, which may lead to the restoration of a
induced emphysema; 2 hit model).
Fo r s c h u n g s z e n t r u m
Bo r s t e l
proper lung structure and function.
Work on granulomatous and fibrotic interstitial lung
disease focuses on the mechanisms of disease susPathophysiology of Inflammation
ceptibility. Using genome wide linkage analysis
Head: PD Dr. Hans-Peter Hauber
gene polymorphisms are identified that are linked
with disease susceptibility.
The group „Pathophysiology of Inflammation“ is interested in pathomechanisms of acute and chronic
pulmonary inflammation and the resultant functional impairment. Work mainly focuses on COPD,
bronchial asthma, pneumonia, and chronic granulomatous diseases (eg. Sarcoidosis, berylliosis).
Moreover, ventilator-induced injury and ARDS are
main topics of the group.
One main area of research is the investigation of
the mechanisms that lead to mucus hypersecretion
in chronic inflammatory lung disease. Using a mod-
Barrier Integrity
el of explanted human mucosal tissue of the upper
The Team: Ingmar Lautenschläger (head), Dr.
airways stimulation of the mucosa with bacterial
Heike Dombrowsky (scientific staff), Jürgen Sarau
products (LPS, PAM3), living bacteria (Chlamydia,
(technical staff).
Pseudomonas aeruginosa), and Th2 type cytokines
(IL-4, IL-9, IL-13) to mimik bacterial and allergic in-
As a barrier organ, the intestine has the delicate
flammation ex vivo can be achieved. By evaluating
task of distinguishing between “friend and foe” on
mechanisms of mucus hypersecretion new thera-
a molecular basis, incorporating the former and
peutic targets can be defined.
tagging them harmless while warding off the latter.
Failure of this barrier function may result in severe
Another main area of research is the investigation
illness. In close cooperation with the Department of
of the pathophysiological mechanisms that are in-
Anaesthesiology and Intensive Care Medicine at
volved in the ventilator-induced lung injury. Several
the University Medical Center Schleswig-Holstein,
studies have demonstrated that both very high and
Campus Kiel, and the Institute of Pharmacology and
very low pressure can induce lung injury and may
Toxicology at the RWTH Aachen University, the Di-
affect the whole organism. Using a model of venti-
vision of Barrier Integrity focuses in particular on elu-
lated mice the effect of different ventilation strate-
cidating mechanisms of interface disorders and im-
gies on gene and protein expression of cytokines,
mune responses in this organ.
270
Wissenschaftlicher Jahresbericht 2007-2008
Neue Entwicklungen
New Developments
Acute inflammatory diseases such as sepsis and
at the interface between M. tuberculosis and its host
multiple organ failure are accompanied by loss of
cell, the macrophage. The detailed characterization
endothelial and epithelial barrier functions with sub-
of the interaction between mycobacteria and
sequent edema formation. The impact of the intes-
macrophages is the major focus of the division. We
tine as an immunologically highly active surface or-
use a variety of biochemical, immunological as well
gan on this pathology is not well understood. Using
as cell- and microbiological approaches to monitor
a unique and complex isolated perfused small in-
the infection in “real” cells, such as primary human
testine model with real time access to all compart-
and murine macrophages. We study the functional
ments (vasculature, lumen, lymphatics and intestin-
relevance of a distinct mediator or signaling path-
al tissue), we will study the molecular mechanisms
way analyzed in vitro experiments and if possible
of intestinal edema formation caused by bacterial
also in an in vivo infection situation. To come as
components such as LPS and inflammatory media-
close as possible to the natural infection situation
tors. Comprehension of these mechanisms will en-
we challenge immunocompetent and selectively
able us to test novel therapeutic approaches, e.g.
gene-deficient mice with M. tuberculosis via the
immunonutrition or proteasome inhibition.
aerosol route and analyze the course of infection by
monitoring macrophage activation, bacterial bur-
Allergic diseases, including food allergies, have
den, as well as granuloma development.
doubled in number within the past 25 years, affectaccess:
Defining
the
M.
tuberculo-
ing now approximately 10-15% of the population in
New
industrialized countries. Failure of desensitization to-
sis/macrophage interface
wards food ingredients is noted with increased fre-
M. tuberculosis actively blocks phagosome/lysosome
quency resulting in impairment of everyday life and
fusion and is able to survive and even replicate in
even in life threatening events. In order to close this
macrophages. Components of the mycobacterial cell
therapeutic gap, we will, in collaboration with the
wall, e.g. glycolipids and lipoproteins, modulate cel-
Division of Mucosal Immunology and Diagnostics,
lular functions so that the cell is not capable of erad-
focus on the intestinal antigen absorption, process-
icating the invading microorganism. However the
ing and translocation, and examine food allergens
question which signaling pathways are initiated in
in the abovementioned isolated perfused intestine
which compartment and at what time during M. tu-
model. Of particular interest is the isolation and
berculosis infection is far from being completely un-
characterisation of allergen carrying components
derstood. To tackle this issue we have developed a
as well as factors modulating the immunogenicity of
novel immunomagnetic technique which allows a
allergens with the long-term goal of being able to
rapid isolation of phagosomes from mycobacteria-in-
induce immunological tolerance in allergic patients.
fected primary macrophages. This enables us to perform a detailed biochemical characterization of the
molecular processes occurring at the (intracellular)
Microbial Interface Biology
interface between mycobacteria and macrophages.
Head: PD Dr. Norbert Reiling
Novel responses: Deciphering the role of
“To get it, all you have to do is breathe”- Tubercu-
Wnt/Frizzled signaling in mycobacterial infections
losis is acquired through inhalation of Mycobac-
The contact between mycobacterial structures and
terium tuberculosis. In the lung the bacteria get in-
the macrophage elicits a variety of immunological
to contact with alveolar macrophages. Whether
responses: Depending on the mycobacterial strain,
these cells are capable of killing the bacteria de-
the cells produce a wide array of pro- and anti-in-
pends on a number of processes which take place
flammatory mediators. We have identified several
Wissenschaftlicher Jahresbericht 2007-2008
271
Neue Entwicklungen
New Developments
Fo r s c h u n g s z e n t r u m
Bo r s t e l
members
of
the
ics of bacterial recognition, uptake and phagoso-
Wnt/Frizzled signal-
mal processing by antigen presenting cells. In this
ing cascade to be
context the formation of receptor complexes, mem-
involved in the reg-
brane and intracellular trafficking as well as signal
ulation of antimy-
transduction pathways will be studied in detail. To
cobacterial effector
this end it is planned to utilize fluorescence-based
functions
the
techniques such as FRAP (fluorescence recovery af-
host. Both Toll and
ter photobleaching), FRET (fluorescence resonance
WNT
energy transfer) and the photoactivation of fluores-
of
signaling
pathways are evolutionarily
cent protein tags.
highly
conserved and have only recently been found to intersect in Drosophila. A major focus of the group is
the in-depth analysis of the novel functions of this
signaling network, already well known for their roles
in embryogenesis and cancer, in inflammation and
infection.
Service Unit Fluorescence Cytometry
Head: Dr. Thomas Scholzen
The service unit Fluorescence Cytometry offers scientists at the FZB and associated universities access
to modern fluorescence-based techniques. These include flow cytometry, which allows to analyze large
numbers of cells in a quantitative manner and, by
utilizing FACS sorting, to obtain highly purified populations of living cells for subsequent experiments.
However, to get additional information about the
spatial distribution of components and the chronological sequence of events, complementary methods must be applied. In this regard high resolution
live cell imaging is a key technique for the spatialtemporal analysis of complex processes. For this
purpose the service unit provides state-of-the-art
confocal and wide-field fluorescence microscopes.
The Fluorescence Cytometry is scientifically associated with the division of Innate Immunity (Head PD.
Dr. Holger Heine), working on allergy protection
based on the immunomodulatory effects evoked by
certain bacterial strains. The Fluorescence Cytometry unit contributes to this project by applying live
cell imaging techniques to investigate the dynam-
272
Wissenschaftlicher Jahresbericht 2007-2008
Haushaltsplan
Budget
Haushaltsplan
Institutioneller Hauhalt 2007: 16,96 Mio. Euro
Eingeworbene Drittmittel 2007: 6,0 Mio. Euro
Institutioneller Hauhalt 2008: 15,36 Mio Euro
Eingeworbene Drittmittel 2008: 6,8 Mio. Euro
Wissenschaftlicher Jahresbericht 2007-2008
273
Personenregister
Register of Persons
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Personenregister
Ahmed JS; 109, 118
Albrecht S; 47
Alexander C; 50-56
Ali AM; 113
Aly S; 64-65
Andrä J; 52-56
Andresen E; 125
Bade S; 184-187, 189,
230, 231
Bakheit MA; 113
Barths D; 181-182
Becker WM; 175-179,
230, 231
Behrends J; 66, 70
Billur M; 106-107
Böhling A; 55
Brade H; 32-35
Brade L; 32, 34
Brandenburg K; 52-55,
59
Brandt E; 102-105
Brandt K; 89
Brown J; 106
Budagian V; 88-89
Bulanova E; 88-89, 90
Bulfone-Paus S; 6-9,
80-91
Bullwinkel J; 106-107
Debarry J; 126-129, 230
Drömann D; 146
Duitman E; 88, 90, 231
Eberhardt F; 145
Ehlers S; 62-68, 232, 266
EkerB; 197
Ernst M; 99-101
Farhat K; 92-95
Fattakhova G; 97
Fehrenbach H; 269
Feuerriegel S; 169
Flad HD; 232, 266-267
Frey A; 184-190
Fujimoto N; 189, 190
Funk C; 46
Gaede K; 147-148
274
Schwartzkopff F; 100
Seitzer U; 109-118
Singh PB; 106-107
Sodenkamp J; 71
Mamat U; 36
Stamme C; 60, 74-76
Miranda J; 118
Stein K; 126-129
Mirghomizadeh F; 118
Steinhäuser C, 67
Mittelstädt J; 122
Moulakakis C; 60, 74-75, Stellmacher F; 149, 155
Strassburg A; 99, 100,
231
Müller-Loennies S; 32-35 191-193, 199-200, 230
Galle J; 155
Gerber S; 114
Gerdes J; 106-108
GhulamO; 103
Goldmann T; 149-156,
182
Grage-Griebnow E; 67
Greinert U; 99, 191-192,
193, 199
Gutsmann T; 52-58, 232
Liu Z; 113, 116-117
Loetzer K; 268
Haas H; 180-183
Haller D; 109
Hammer M; 53
Hanuszkiewicz A; 36-38,
40, 42-43
Hauber HP; 143-145, 270
Heine H; 125-129
Heinemann S; 144
Heitmann L; 60-70
Hessmann M; 70
Hillemann D; 159,
161-162
Hoerster R; 191
Hölscher C, 69-73
Holst O; 36-44, 232
Homolka S; 170, 172-173
Howe J; 52-53, 56-57, 59
Hübner G; 48
Ulmer AJ; 92-96
Nguyen XH; 97
Niemann S; 162, 164-171
Vogel K; 41, 231
Vollmer E; 149-156
Orinska Z; 88-89, 90
Jafari C; 99-100, 191-194
Kacynski Z; 38
Kähler D; 149, 154
Kalsdorf B; 99, 191-192,
195, 199, 230
Kasper B; 119-121
Keese S; 60
Keller C; 62, 63-64
Kirsten D; 99
Kondakova A; 49
Kubica T; 170-172
Lang DS; 149-152
Lange C; 99, 191-200
Lautenschläger I; 270
Lee KH; 97-98
Leonhardt L; 121, 123
Lindner B; 47-50
Petersen A; 175-179, 231
Petersen F; 119-124, 230
Plinke C; 169
Reiling N; 62, 65, 66-68,
271
Renneker S; 114
Reuter F; 184-186
Richter E; 159-163
Riecken S; 177, 178, 231
Riekenberg S; 92-94
Röckendorf N; 186-190
Romey R; 107
Röschmann K; 94-96
Rückerl D; 70, 230
Rückert R; 88,89
Rüsch-Gerdes S; 157163, 167-171
Walter K; 62-64, 66
Warmbold C; 95
Wehry K; 187, 189-190
Wittkowski M; 38
Wölbeling F; 172
Woller G; 121
Yu X; 124
Zabel P; 6-9, 132-148
Zähringer U; 45-51
Salih DA; 110, 111, 113,
116-117
Sarrazin H; 194
Schaible UE; 6-9, 16-30,
268
Schiemann F; 102-103
Schneider I; 108
Schocker F; 178
Scholzen T; 107, 272
Schramm G; 180-182
Schromm AB; 59-61
Schulmistrat J; 102, 104
Schultz H; 149-156
Schurek B; 98
Wissenschaftlicher Jahresbericht 2007-2008
Kiel
B 404/A 21
A7
Bad Bramstedt
Bad Segeberg
B4
Bad Oldesloe
B 433
B 75
Kaltenkirchen
Borstel
B 432
A1
Hamburg
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Parkallee 1-40
23845 Borstel
Tel. +49 (0)4537.188 0
[email protected]
www.fz-borstel.de
B 208
Lübeck
Thomas Gutsmann, Leiter der Biophysik
Thomas Gutsmann, Head of the Division of Biophysics
Maria Lammers, Landesbeste in der Ausbildung zur Biolaborantin, Schleswig- Holstein
Maria Lammers, the best trainee (lab
assistant in biology) in Schleswig-Holstein
Kay Vogel, Promotionspreis des
Kreises Segeberg, 2008
Kay Vogel, laureate of the "Promotionspreis
Kreis Segeberg", best graduation 2008
Fo r s c h u n g s z e n t r u m
Bo r s t e l
Parkallee 1-40
23845 Borstel
[email protected]
www.fz-borstel.de