Prognosis of vasculitis associated myelodysplasia.

Systemic and immune manifestations have been reported in patients with MDS. The correlation between immunological abnormalities and prognosis in myelodysplastic syndrome patients remains controversial. Most of the authors agree that the median survival in myelodysplastic syndrome is not related to the presence of systemic and immune manifestations, but only with the existence of a systemic vasculitis.

Autologous haematopoietic stem cell transplantation for systemic lupus erythematosus: data from the European Group for Blood and Marrow Transplantation registry.

OBJECTIVES: Patients with systemic lupus erythematosus (SLE) refractory to conventional immunosuppression suffer substantial morbidity and mortality due to active disease and treatment toxicity. Immunoablation followed by autologous stem cell transplantation (ASCT) is a novel therapeutic strategy that potentially offers new hope to these patients. METHODS: This retrospective survey reviews the efficacy and safety of ASCT in 28 SLE patients from eight centres reported to the European Group for Blood and Marrow Transplantation (EBMT) registry between 2001 and 2008. RESULTS: Median disease duration before ASCT was 52 (nine to 396) months, 25/28 SLE patients (89%) were female, age 29 (16-48) years. At the time of ASCT, eight (one to 11) American College of Rheumatology (ACR) diagnostic criteria for SLE were present and 17 (60%) patients had nephritis. Peripheral blood stem cells were mobilized with cyclophosphamide and granulocyte-colony stimulating factor in 93% of patients, and ex vivo CD34 stem cell selection was performed in 36%. Conditioning regimens were employed with either low (n = 10) or intermediate (18) intensities. With a median follow-up of 38 (one to 110) months after ASCT, the five-year overall survival was 81 ± 8%, disease-free survival was 29 ± 9%, relapse incidence (RI) was 56 ± 11% and non-relapse mortality was 15 ± 7%. Graft manipulation by CD34+ selection was associated with a lower RI (p = 0.001) on univariate analysis. There were five deaths within two years after ASCT: three caused by infection, one by secondary autoimmune disease and one by progressive SLE. CONCLUSIONS: Our data further support the concept of immunoablation and ASCT to re-induce long-term clinical and serologic remissions in refractory SLE patients even in the absence of maintenance therapy. This study also suggests a beneficial effect of ex vivo graft manipulation on prevention of relapses post-transplantation in SLE.

[Mesenchymal stem cells and rheumatism. State of the art]. / Mesenchymale Stammzellen und Rheuma. "State of the art".

Mesenchymal stem cells (MSC) are multipotent. They are able to regenerate tissue damage and, in parallel, inhibit inflammation and fibrosis. As they are non-immunogenic, MSCs can be safely transplanted using autologous and allogeneic procedures: an option for refractory connective tissue diseases and osteoarthritis.

Association of rheumatoid arthritis with Mdm2 SNP309 and genetic evidence for an allele-specific interaction between MDM2 and p53 P72R variants: a case control study.

OBJECTIVE: This study examines two common, functional, single nucleotide polymorphisms (SNP) in the genes coding the human homolog of murine-double-minute-2 (MDM2) and p53 in patients with rheumatoid arthritis (RA) based on the hypothesis that p53 may be an important negative regulator of the pro-inflammatory transcription factor nuclear factor kappa b (NFKappaB). METHODS: Genomic DNA was obtained from 221 patients with RA who fulfilled at least 4 ACR criteria and from 521 healthy controls. Mdm2 SNP309 and p53 P72R were genotyped by polymerase chain reaction and restriction enzyme analysis. RESULTS: In RA patients the frequencies of the mdm2 SNP309 G allele and both G-containing genotypes were significantly reduced (G allele: OR: 0.75, 95% CI: 0.59-0.95, p=0.016; genotype TG: OR: 0.71, 95% CI: 0.50-1.00; genotype GG: OR. 0.58, 95% CI: 0.34-0.99; both: p=0.049). Concerning p53 P72R, no differences in allele or genotype frequencies were detected. A combined analysis of both polymorphisms revealed a significant interaction between them (p=0.046). In individuals carrying >1 p53 72R allele, MDM2 had a protective effect, whereas in individuals homozygous for p53 72P, MDM2 had the opposite effect. CONCLUSION: The function of MDM2 depends on the p53 P72R genotype, resulting in either an increased or reduced risk for RA. We suggest that in most cases MDM2 stabilizes the conformation of p53, whereas in p53 PP-positive subjects MDM2 supports the degradation of p53.

The p53 G72C and MDM2 T309G single nucleotide polymorphisms in patients with Wegener's granulomatosis.

OBJECTIVE: Wegener's granulomatosis (WG) is a rare disease with unknown aetiology, but there is evidence for a complex genetic background. The tumor suppressor p53 and its most important negative regulator, MDM2, are positioned in the centre of a pathway that eliminates damaged cells through apoptosis. Furthermore, p53 is one of the most important negative regulators of the pro-inflammatory transcription factor nuclear factor kappa b (NFkappaB). In this respect the investigation of polymorphisms in the p53-network could be a promising approach contributing susceptibility of WG and its course of disease. METHODS: A case control study with 132 patients with WG and 512 healthy blood donors was conducted to evaluate an association of p53-SNP G72C or MDM2-SNP T309G with WG. SNPs were genotyped by polymerase chain reaction (PCR) and subsequent differential enzymatic restriction. All patients showed the clinical pathological findings of WG according to the ACR classification criteria of 1990. RESULTS: The p53 G72C and MDM2 T309G polymorphisms did not show any difference between WG patients and controls. The subgroup analysis of gender differences and earlier onset of WG (younger than median age of 51 years at diagnosis) did not show any differences in allelic or genotype frequencies of p53 G72C or MDM2 T309G SNP between WG patients and the control group. CONCLUSIONS: Our study showed no association between the p53 SNP G72C and the MDM2 SNP T309G with susceptibility or course of disease in patients with WG. The data presented do not suggest that alterations in the p53-network play a key role in the pathogenesis of WG.

A novel system to test for specificity of B cell receptors from tissue of Wegener's granulomatosis patients.

OBJECTIVE: Wegener's granulomatosis (WG) is characterized by granulomatous inflammation of the respiratory tract and Anti Neutrophil Cytoplasmic Antibody (ANCA)-associated systemic vasculitis. The pathognomonic ANCA in WG is typically directed against proteinase 3 (PR3). Germinal centre-like clusters of lymphocytes were seen in granulomata of WG patients suggesting an antigen-driven maturation of B lymphocytes potentially leading to ANCA formation. The goal of this study was to develop a system to determine the specificity of B cells found in WG granulomata via the generation of fab fragments as antibody analogues. These fab fragments have the identical antigen binding site like the B-cell receptor from which the DNA was derived. METHODS: Single B cells were isolated from B cell clusters within the granuloma of a WG patient by laser-assisted microdissection. Their immunoglobulin genes (VH/Vkappa, VH/Vlambda) were characterized by seminested single cell PCR and cloned into a phagemid vector in order to produce fab fragments. The fabs were characterized by protein gel electrophoresis and western blot. RESULTS: The immunoglobulin genes from lymphocyte infiltrates of WG granulomata reveal antigen-driven selection. On the basis of two individual couples of mutated VH/Vlambda PCR products functional fabs were generated that represent the B cell receptors of WG tissue-derived single B cells. CONCLUSION: This is the first in vitro model to test for specificity of B cell receptors from WG granulomata. With respect to ANCA origin in WG this system provides a tool to elucidate the structure-function relationship of apparently antigen-driven maturation of B cells within Wegener's granuloma.

Single cell analysis of B lymphocytes from Wegener's granulomatosis: B cell receptors display affinity maturation within the granulomatous lesions.

Increased amounts of anti-neutrophil cytoplasm antibody (ANCA) directed against proteinase 3 (PR3) are a diagnostic and pathogenic hallmark of full-blown Wegener's granulomatosis (WG). Aggregates of B lymphocytes proximal to PR3+ cells as well as plasma cells have been described as substantial components of Wegener's granuloma and could participate in forming tertiary lymphoid structures, which might promote autoantibody formation. Our aim was a molecular analysis of single B cells in order to develop a methodological approach that allows examination of potential ANCA formation in the tissue. Single B cells from cryo-conserved endonasal biopsies of three WG patients were isolated, using laser-assisted microdissection. Subsequently, their immunoglobulin variable heavy (VH) and light (Vkappa, Vlambda) chain genes were analysed by single cell polymerase chain reaction and direct sequencing. Sixteen immunoglobulin VH-Vkappa or VH-Vlambda chain gene couples were characterized. Twelve of these immunoglobulin gene couples resembled memory B cells. Two offsprings of one B cell were detected, indicating clonal expansion. VH genes representing 39 single B cells of WG tissues displayed significantly more mutations when compared with VH genes from peripheral blood of a healthy donor. The findings confirm and extend our previous results, arguing for an initial selection and affinity maturation of B cells within Wegener's granuloma. Further, the methodology provides the initial basis for the recombinant generation of antibodies derived from tissue cells.

[B lymphocyte differentiation in granulomatous tissues of the lung and the nasal mucosa in Wegener's granulomatosis: origin of anti-neutrophil cytoplasmic antibody formation?]. / B-Lymphozyten-Differenzierung in granulomatösen Geweben der Lunge und der Nasenschleimhäute beim Morbus Wegener: Ursprungsort der ANCA-Bildung?

Wegener's granulomatosis (WG) starts with granulomatous inflammation of the respiratory tract before it converts into a potentially organ and life threatening systemic vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA). The site of formation of the highly specific ANCA directed against "Wegener's autoantigen" proteinase 3 (PR3) is still unknown. Previously, we have shown that follicle-like B lymphocytic infiltrates in the vicinity to PR3 expressing cells in WG-granulomata. We characterized the immunoglobulin-VH repertoire in lung and nasal granulomata (paraffin embedded) from four WG patients. A total of 115 individual VH genes were characterized and compared to 84 VH genes from the peripheral blood of a healthy donor. We found an increased frequency of mutations with a bias to amino acid exchanges within the antigen binding sites (CDR) 1 and 2 in WG tissue. A large number of mutations led to negatively charged amino acids and may increase affinity to the positively charged PR3. Furthermore, the occurrence of differently mutated members of one B cell clone indicates clonal expansion and intraclonal diversification by an antigen, e.g. PR3. Several WG tissue derived genes displayed similarities to published sequences from peripheral PR3 ANCA producing B cells. Thus, granulomata of the lower and upper respiratory tract contain follicle-like B cell clusters with a selected VH repertoire infiltrate in WG. WG granulomata could be the place of autoantigen presentation and formation of high-affinity ANCA within neoformed ectopic or tertiary lymphoid-like tissue areas.

B lymphocyte maturation in Wegener's granulomatosis: a comparative analysis of VH genes from endonasal lesions.

BACKGROUND: Anti-neutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) are highly specific for Wegener's granulomatosis (WG). Evidence for a pivotal role of PR3-ANCA in the induction of vasculitis has been demonstrated. B cell clusters have been observed within endonasal biopsy specimens. OBJECTIVES: To determine whether B cell selection and maturation take place in granulomatous lesions of WG. METHODS: Granulomatous lesions and the immunoglobulin (VH) gene repertoire from nasal tissue of six WG patients-two active and two smouldering localised WG (ANCA negative, restricted to respiratory tract), plus one active and one smouldering PR3-ANCA positive generalised WG-were characterised by immunohistochemistry, polymerase chain reaction, cloning, DNA sequencing and database comparison. RESULTS: B lymphocyte-rich, follicle-like areas were observed proximal to PR3 positive cells and plasma cells in granulomatous lesions; 184 VH genes from these granulomatous lesions were compared with 84 VH genes from peripheral blood of a healthy donor. The mutational pattern of VH genes from active WG resembled memory B cells. Structural homologies of VH genes from granulomatous lesions to PR3-ANCA encoding genes were detected. Significantly more genes (55%, 45%, and 53%, respectively) from active WG compared with the healthy repertoire carried mutations to negatively charged amino acids within the binding site coding regions, favouring affinity to the positively charged PR3. CONCLUSIONS: Selection and affinity maturation of potentially PR3-ANCA producing autoreactive B cells may start in granulomatous lesions, thereby contributing to disease progression from ANCA negative localised to PR3-ANCA positive generalised WG.

Lack of efficacy of rituximab in Wegener's granulomatosis with refractory granulomatous manifestations.

OBJECTIVE: To investigate the safety and efficacy of rituximab (RTX) in patients with refractory Wegener's granulomatosis (WG). PATIENTS AND METHODS: Eight consecutive patients with active refractory WG were included. In all patients disease activity had persisted despite standard treatment with cyclophosphamide and prednisolone, as well as tumour necrosis factor alpha blockade 3 months before inclusion in the study. Patients had particular granulomatous manifestations like retro-orbital granulomata (n=5), nodules of the lungs (n=1), and subglottic stenosis (n=2). RTX was given intravenously every 4th week in combination with the standard treatment in five patients and with methotrexate in two others. Disease extent and activity were monitored clinically by interdisciplinary care, immunodiagnostics (ANCA serology, B cells by flow cytometry), and magnetic resonance imaging. RESULTS: Beneficial response and a reduction in disease activity were seen in three patients, two of whom went into complete remission. In three other patients, disease activity remained unchanged while the disease progressed in the remaining two patients. In all patients peripheral blood B cells fell to zero during treatment with RTX. cANCA titres remained unchanged in all except one patient. CONCLUSION: In this pilot study, B lymphocyte depletion was not associated with a change of the ANCA titres or obvious clinical improvement of refractory granulomatous disease in patients with WG. Further studies are needed to evaluate the role of RTX in WG.

Is PR3-ANCA formation initiated in Wegener's granulomatosis lesions? Granulomas as potential lymphoid tissue maintaining autoantibody production.

In Wegener's granulomatosis (WG), antiproteinase 3 (PR3) autoantibodies (PR3-ANCA) are crucial in the development of generalized vasculitis. Wegener's pathognomonic lesion, a granulomatous inflammation of the upper and lower respiratory tract, contains abundant lymphocytes and macrophages. Lymphocyte clusters in germinal center-like formation within the granulomatous lesion are frequently observed, which suggests antigen-driven B cell maturation. Wegener's autoantigen PR3, the target for autoreactive B and T cells, is expressed in granulomatous lesions. Disease progression in WG is accompanied by a profound generalized alteration of T cell differentiation with an increase of effector memory T cells (CD4(+)CD28(-)). The cytokine profile suggests an aberrant Th1-type response either to an environmental trigger and/or the autoantigen PR3 itself. Staphylococcus aureus, a risk factor for disease exacerbation, is widely present in the upper airways in WG. The Ig gene repertoire from WG lesions indicates a predominance of VH3+ B cells with affinity to PR3 as well as to the S. aureus B cell superantigen SPA. Hence, within the WG lesion, S. aureus might support the maturation of PR3-affinity B cells that enter a germinal center reaction in contact with PR3 and T cells and expand, leading to PR3-ANCA production. Thus, granulomatous lesions could represent a potential lymphoid tissue-maintaining autoantibody production rather than a simple, random leukocyte accumulation in WG.

Rituximab induces remission in refractory HCV associated cryoglobulinaemic vasculitis.

OBJECTIVES: To report the successful induction of remission with the monoclonal anti-CD20 antibody rituximab in a patient with hepatitis C virus (HCV) associated cryoglobulinaemic vasculitis and a non-Hodgkin's lymphoma (NHL) resistant to previously advocated conventional treatments. CASE REPORT: The patient was a 45 year old woman with HCV associated cryoglobulinaemic vasculitis, with purpura, arthralgia, constitutional symptoms, and a polyneuropathy. A malignant NHL was found as underlying lymphoproliferative disease. At this stage the disease was refractory to interferon alpha2b and ribavirin and to subsequent immunosuppressive treatment with cyclophosphamide. Six rituximab infusions targeting the CD20 antigen on cells of the B cell lineage induced remission of the vasculitis. Bone marrow biopsy disclosed absence of the NHL. Remission has subsequently been maintained and HCV eliminated with the new pegylated interferon alpha2b and ribavirin for nearly one year. CONCLUSIONS: Transition of the underlying "benign" lymphoproliferative disease to a malignant lymphoma may result in difficult to treat HCV associated cryoglobulinaemic vasculitis. Rituximab offers a new possibility for inducing remission in refractory HCV associated cryoglobulinaemic vasculitis and the lymphoproliferative disorder. After remission, HCV may subsequently be eliminated with pegylated interferon alpha2b and ribavirin.

Effectiveness of TNF-alpha blockade with infliximab in refractory Wegener's granulomatosis.

OBJECTIVE: To study the effect of the chimeric monoclonal anti-tumour necrosis factor alpha (TNF-alpha) antibody infliximab in the induction of remission in six patients refractory to standard treatment with cyclophosphamide and corticosteroids. In four patients, other measures for treating refractory Wegener's granulomatosis (WG) that have been advocated previously, i.e. intensified cyclophosphamide therapy and additional intravenous immunoglobulin, were ineffective. METHODS: Patients received infliximab (3 mg/kg in two patients and 5 mg/kg in four patients) with a 2-week interval after the first administration and 4-week intervals between infusions until remission, in addition to cyclophosphamide and corticosteroids. Vasculitis activity was assessed with the Birmingham Vasculitis Activity Score (BVAS). A standardized interdisciplinary approach was used for the follow-up of specific organ involvement. RESULTS: Remission was induced in five patients and corticosteroid doses could be tapered. Acute-phase responses (e.g. C-reactive protein) normalized. Titres of c-ANCA (cytoplasmic pattern antineutrophil cytoplasmic antibodies) were no longer detectable. The BVAS was reduced to zero. The higher dose of infliximab (5 mg/kg) seemed more effective in inducing remission. One patient was withdrawn because of suspected systemic infection. Five patients remained in remission for 6-24 months of follow-up. CONCLUSION: The data suggest that infliximab may provide an effective and more specific therapeutic option in the treatment of active WG refractory to standard treatment.

[Induction of remission with infliximab in therapy-refractory Wegener's granulomatosis - Follow-up of six patients]. / Remissionsinduktion mit Infliximab bei therapierefraktärer Wegenerscher Granulomatose - Verlaufsbeobachtung1.

UNLABELLED: BACKGROUND AND QUESTION: The prognosis of patients with Wegener's granulomatosis (WG) refractory to standard treatment for the induction of remission (cyclophosphamide and steroids) has been poor so far. We report on the results of the follow-up of six patients refractory to the standard regimen (Fauci's scheme) with progressive, imminent visual loss, pulmonary and renal involvement, respectively. How long can remissions be successfully maintained with anti-TNF-alpha-antibody infliximab? What side effects occur? PATIENTS AND METHODS: Patients received infliximab (3 mg/kg) in addition to standard therapy with cyclophosphamide and steroids. Intervals between the first two infliximab infusions were 2 weeks, thereafter 4 weeks. Based on the impression of higher efficacy patients received 5 mg/kg infliximab for subsequent infusions. RESULTS: Remission was induced after 4-6 infliximab infusions in five patients. Remission has been maintained in four patients for 16-26 months. After 12 months a pulmonary relapse occurred in one patient, who received azathioprine for the maintenance of remission. Infliximab was stopped in another patient because of a suspected infection. In the light of high cumulative cyclophosphamide doses (100 g/275 g) and cyclophosphamide induced hemorrhagic cystitis, infliximab was added to azathioprine in the two patients with a pulmonary relapse and protrusion of the eye with imminent visual loss, respectively. Remission was induced in both patients. A carcinoid of the bronchus was diagnosed in one patient after 12 months in remission. CONCLUSION: Infliximab means a new therapeutic option and offers better perspectives for a patient group with previously bad prognosis.

B lymphocyte involvement in ankylosing spondylitis: the heavy chain variable segment gene repertoire of B lymphocytes from germinal center-like foci in the synovial membrane indicates antigen selection.

The synovial membrane (SM) of affected joints in ankylosing spondylitis (AS) is infiltrated by germinal center-like aggregates (foci) of lymphocytes similar to rheumatoid arthritis (RA). We characterized the rearranged heavy chain variable segment (VH) genes in the SM for gene usage and the mutational pattern to elucidate the B lymphocyte involvement in AS. Cryosections from an AS-derived SM were stained for B and T lymphocytes. B cells were isolated from different areas of a focus. The rearranged VH genes were amplified by semi-nested polymerase chain reaction (PCR) using oligonucleotides specific for the six different VH families and heavy chain joining segments (JHs). PCR products were cloned and sequenced.Fifty-nine of 70 different heavy chain gene rearrangements were potentially functional. Most of the rearranged genes were mutated (range, 1-15%). Thirty of 70 products had a mutational pattern typical for antigen selection. Most of the rearranged VH genes belonged to the VH3 family (54%), consistent with data from healthy donors and patients with RA, while VH4 genes, in contrast to RA, were identified less frequently (10%) and VH5 genes were over-represented (11%). In contrast to RA, neither VH6 genes nor the autoimmunity-prone VH4-34 were seen, whereas another autoimmunity-prone gene, V3-23, was predominantly used (11%). One VH1-derived and one VH3-derived B cell clone were expanded. CDR3 were shorter and more variable in length than in RA. Comparable with RA and reactive arthritis, there is a biased repertoire of selected VH genes, whereas the panel of represented genes is different and less clonal expansion was observed.