Zeitschrift für Rheumatologie – Supplement 1 - Deutsche ...

teins along with lineage, activation and migration markers, cytokine
and complement receptors. Finally we present a prototype of a database
necessary for data storage and data analysis.
In summary, the approach presented provides a powerful platform for
validation of candidate genes at the protein level and their monitoring
for clinical applications.
POFER-21
Genetic Association of Progressive Systemic Sclerosis (SSc) with
PTPN22 Polymorphisms
Kirsten H. 1 *, Ahnert P. 1 *, Dümmler J. 2 , Hunzelmann N. 3 , Vaith P. 4 , Melchers I. 2
1 2 Universität Leipzig, IKIT/BBZ, Klinische Forschergruppe für Rheumatologie,
Universitätsklinikum Freiburg,
3 Universitätshautklinik Köln,
4 Rheumatologie und Klin. Immunologie, Universitätsklinikum Freiburg
*contributed equally
Recently, associations between type I diabetes, rheumatoid arthritis
as well as several other autoimmune diseases (AIDs) and the PTPN22
single nucleotide polymorphism (SNP) 1858C→T were discovered.
PTPN22 (encoding protein tyrosine phosphatase, non-receptor type
22) located on chromosome 1p13 has 21 exons spanning 58 kb. Th e
variant 1858C→T in codon 620 results in the exchange of Arg to Trp
(R620W). PTPN22 (also known as Lyp or Pep) is expressed primarily
in lymphoid tissue, and most probably involved in the negative regulation
of T cell activation via interaction with the protein tyrosine kinase
Csk. It was suggested that the mutation 620W may interfere with the
interaction between Lyp and Csk. However, functional data comparing
homozygous and heterozygous 620W carriers are not yet available.
We collected DNA from 177 patients with SSc and 184 healthy blood
donors (HD). Samples were analyzed for 13 SNPs covering PTPN22,
including 1858C→T (rs2476601). SNPs were selected to represent the
most common haplotypes. Th e analysis was performed by PCR, single
base extension and MALDI-TOF mass spectrometry.
Among HD, the allele frequency of 1858T was 9.0 %, similar to published
data. Genotype frequency of 1858T/T was 0.5 %. Among SSc patients,
the allele frequency of 1858T was 12.4 %, genotype frequency of
1858T/T was 2.8 %. Th e diff erence in allele frequencies did not reach
statistical signifi cance, however, the diff erence in genotype frequencies
was signifi cant (p = 0.01, genotype relative risk test, Lathrop. Tissue
Antigens. 1983; 22:160–166). Data concerning subgroups of patients,
additional polymorphisms and the other 4 major haplotypes of
PTPN22, together accounting for about 98.5 % of detectable variants,
will also be discussed.
So far, associations observed in AIDs with PTPN22 always only concerned
a subpopulation of patients since only a subset of the patients
carried the disease associated variant. For PTPN22, maximally 20 % of
AID patients were shown to carry the allele 1858T and even less carried
the homozygous 1858T/T genotype. Th erefore, only a small subpopulation
of patients may be infl uenced by functional eff ects due to PTPN22.
In SSc patients, this subpopulation may be quite small, but it does exist.
Supported by grants of the BMBF (“German Network for Systemic Scleroderma”
to IM and NH, “University and Science” to PA), the Sächsische
Aufb aubank (PA) and the EFRE (PA).
POFER-22
Cyclophosphamide reduces cellular infi ltrates in the infl amed
kidneys rather than aff ecting lymphoproliferation or auto-antibody
levels in a murine model of lupusnephritis
Humrich JY. 1 , Schürer S. 2 , Wittenburg G. 2 , Enghard P. 1 , Undeutsch R. 1 ,
Berek C. 2 , Riemekasten G. 1
1 Rheumatologie und Klinische Immunologie, Charité Berlin, 2 DRFZ Berlin
Cyclophosphamide is commonly used as a standard therapy for the
treatment of lupusnephritis. Based on its DNA-alkylating properties it
is assumed that the benefi cial eff ect of cyclophospamide is due to the
inhibition of proliferation of autoreactive T and B cells in secondary
lymphoid organs, which results in a decreased generation of nephritogenic
auto-antibodies. We used the (NZBxNZW) F1 lupus model to
evaluate the eff ects of a cyclophospamide pulse-therapy on kidney infl
ammation and cellular activity in secondary lymphoid organs. Mice
were treated daily with 1mg of cyclophosphamide or as control with
PBS for the duration of one week. Sera were collected before and aft er
treatment, and the proteinuria was simultaneously determined. Th ree
weeks and fi ve weeks aft er therapy kidneys and spleens were isolated
from fi ve mice in each group. Th e phenotype and activation status of
splenic CD4+ T cells and B cell subpopulation were examined by fl ow
cytometry. Kidneys and one part of the spleens were prepared for immunohistological
analyses. Sera were screened for anti-ds-DNA antibody
levels by ELISA. Treatment with cyclophosphamide signifi cantly
reduced proteinuria almost to normal levels and signifi cantly prolonged
the survival of the treated group. Immunohistological analysis of
kidney sections showed a marked reduction of the cellular infi ltrate
in parallel to the reduction of proteinuria. In contrast, we found only
moderate eff ects on the phenotype of splenic CD4+ T cell with a slight
decrease of CD69+ cells and an increase of CD62L+ cells, while no
changes in the frequencies of splenic CD138+ plasmablasts, CD23+ follicular
B cells, CD21+ marginal zone B cells or PNA++ germinal center
B cells could be observed. Complementary to this histological analysis
of the spleens showed only moderate eff ects on the architecture and the
size of the lymphoid follicles. Most interestingly we could not detect a
reduction of the serum anti-ds-DNA antibody levels aft er treatment.
Th us we conclude that cyclophophamide interacts directly with kidney
infi ltrating cells at the site of infl ammation rather than aff ecting proliferation
of autoreactive T and B cells in secondary lymphoid organs.
Furthermore we suggest that auto-antibodies and humoral immunity
might not be such relevant for lupusnephritis, since improvement of
nephritis was achieved despite the presence of high levels of anti-ds-
DNA antibodies in the sera aft er treatment with cyclophosphamide.
POFER-23
Das Wegener Autoantigen Proteinase 3 (PR3) in Organo- und
Pathogenese
Relle M., Galle PR., Schwarting A.
I. Medizinische Klinik und Poliklinik, Uniklinikum Mainz, Mainz
Zielsetzung: Die Proteinase 3 (PR3) ist eine neutrale Serin-Protease
Neutrophiler Granulozyten, Mastzellen und Monozyten. Sie ist auch
als Myeloblastin, ein Wachstumsfaktor myeloider Zellen, bekannt.
Rahmen der Wegenerschen Granulomatose ist die PR3 das Hauptzielantigen
antineutrophiler cytoplasmatischer Autoantikörper (c-
ANCA). Nach wie vor kontrovers diskutiert wird die Expression der
PR3 in non-myeloiden Zellen, obwohl sie zweifelsfrei im Endothel, in
Nierenzellen und in epithelialen Tumorzell-Linien nachgewiesen werden
konnte.
Methoden: Da das Expressionsprofi l der PR3 im Bezug auf die Pathophysiologie
von Autoimmunerkrankungen, wie z. B. dem Morbus Wegener
von essentieller Bedeutung ist, wurden Dot Blot- und Northern
Blot-Analysen durchgeführt, um PR3-Transkripte in verschiedenen
Organen, Tumoren und Tumorzell-Linien zu detektieren. Ferner wurden
PR3-spezifi sche Primer eingesetzt, um PR3-Transkripte sowohl in
Zeitschrift für Rheumatologie · Supplement 1 · 2006 | S81