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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 (Vκ, Vλ) chain genes were analysed by single cell polymerase chain reaction and direct sequencing. Sixteen immunoglobulin VH-Vκ or VH-Vλ 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. [ABSTRACT FROM AUTHOR]

Objectives. Results from open-label trials suggest that methotrexate (MTX) and leflunomide (LEF) are effective for maintenance of remission in Wegener's granulomatosis (WG), but data from randomized controlled clinical trails are not yet available. [ABSTRACT FROM PUBLISHER]

SUMMARY Although HLA class I expression is diminished in patients with defects in the transporter associated with antigen presentation (TAP), recurrent Gram-negative bacterial lung infections are found from childhood onwards. As MHC class II-mediated responses are normal, other mechanisms that contribute to susceptibility to infections are presumed. The bactericidal/permeability-increasing protein (BPI) is a potent neutrophil antibiotic that neutralizes endotoxin efficiently. As antineutrophil cytoplasmic autoantibodies (ANCA) against BPI were found in the majority of cystic fibrosis patients and correlate with disease severity we examined the prevalence of BPI–ANCA and their contribution to susceptibility to bacterial infections in six TAP-deficient patients. Although only two patients showed ANCA in indirect immunofluorescence, BPI–ANCA occurred in five of six patients in ELISA. Purified IgG from BPI–ANCA-positive sera (five of six) inhibited the antimicrobial function of BPI in vitro . Epitope mapping revealed binding sites not only on the C-terminal but also on the antibiotic N-terminal portion of BPI, indicating that short linear BPI peptide fragments may be long-lived enough to become immunogens. In conclusion, BPI–ANCA are associated strongly with TAP deficiency. Inhibition of the antimicrobial BPI function by BPI–ANCA demonstrates a possible mechanism of how autoantibodies may contribute to increased susceptibility for pulmonary Gram-negative bacterial infections by diminished bacterial clearance. [ABSTRACT FROM AUTHOR]

SUMMARY Wegener’s granulomatosis (WG) is characterized by a predominance of the type 1 T-helper cell (Th1) response. We have studied monocytic cytokine expression in untreated patients and in patients who did not respond to prior methotrexate or trimethoprim-sulphamethoxazole therapy, i.e. patients with active disease. Intracytoplasmic IL-12 and TNF-α expression was significantly increased in WG compared with healthy controls. IL-8 expression was not increased. Two and 12 weeks of daily standard oral cyclophosphamide and corticosteroid (CYC + GC) treatment induced a stable remission of the disease. Elevated IL-12 and TNF-α expression of monocytes was normalized. The active metabolite of CYC was shown to down-regulate IL-12 mRNA in vitro . Monocytic cytokines, especially IL-12, may have a role in the early determination and skewing of the immunoregulatory response towards a Th1 profile. It appears that CYC + GC exerts its effect by normalizing the Th1-driving cytokine pattern, and CYC may maintain this mode of action. Normalization of the skewed cytokine pattern may be a prerequisite and an indicator of inducing a remission in WG. [ABSTRACT FROM AUTHOR]

CTLA-4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA-4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Furthermore, CTLA-4 has a role in inducing a Th1 response and suppressing Th2 cytokines, an effect which is antagonized by CD28. Many autoimmune diseases are characterized by an overwhelming production of Th1 cytokines. Recently, the predominance of the Th1 cytokine pattern has been directly observed in the granulomatous inflammation of patients with Wegener's granulomatosis. The balance between CD28 and CTLA-4 expression by T lymphocytes could be a factor in the pathogenesis of autoimmune diseases. Down regulation of CD28 predominantly on CD8+ T cells has been described in Wegner's granulomatosis; however, analysis of CTLA-4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA-4 on PBMC in patients with Wegener's granulomatosis (n = 25) in comparison with healthy controls (n = 19). Expression levels of CTLA-4 were significantly increased selectively on CD4+ and possibly also on CD4-/CD8- T cells in Wegener's granulomatosis. High CTLA-4 expression by T lymphocytes was associated with more severe disease. In contrast, after stimulation with the mitogen PHA, CTLA-4 levels were strongly increased on T cells from controls but in T cells from Wegener's granulomatosis patients this response was severely impaired. Interestingly, while CTLA-4 was seen exclusively on T cells in control individuals, about half of the Wegener's patients showed CTLA-4 expression by a fraction of peripheral B lymphocytes. CTLA-4 positive B cells in the periphery were associated with less acute disease. [ABSTRACT FROM AUTHOR]

Amphipathic variable-region heavy chain 11-mer peptides from monoclonal human IgM antiproteinase-3 antibodies were studied for peripheral blood lymphocyte stimulation in 21 patients with Wegener's granulomatosis (WG) or microscopic polyangiitis (MPA), connective tissue disease controls and normal control subjects. Positive T-cell activation was observed in most experiments with WG patients' lymphocytes using amphipathic VH-region peptides from four different human monoclonal anti-PR3 antibodies. Control peptides of the same length but without amphipathic characteristics along with other amphipathic peptides not derived from monoclonal anti-PR3 sequence were employed as controls. No significant lymphocyte stimulation was observed with normal controls, but positive stimulation with amphipathic VH peptides was also recorded in other connective tissue disease controls mainly patients with rheumatoid arthritis. Amphipathic peptides not derived from anti-PR3 sequence did not stimulate WG lymphocytes. Our findings indicate that lymphocyte reactivity as an element of cell-mediated immunity may be activated by amphipathic VH-region amino acid sequences of autoantibodies which are themselves associated with diseases such as WG. [ABSTRACT FROM AUTHOR]

Since proteinase 3 (PR3)-ANCA interact with PR3 on the surface of apoptotic polymorphonuclear neutrophils (PMN) and ingestion of apoptotic PMN is known to modulate macrophage inflammatory reactions, we raised the question whether PR3-ANCA-opsonized apoptotic PMN influence the uptake by macrophages and their state of activation. We therefor analysed the effects of PR3-ANCA-opsonized apoptotic PMN on the uptake process by enzymatic assay. We further investigated the production of TNF-α, IL-10, IL-12 and the secretion of lipid inflammatory mediators (TxB2, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2)) by human monocyte-derived macrophages using FACS and ELISA methods. We show that PMN-opsonization by PR3-ANCA substantially enhances phagocytosis by macrophages and thereby triggers the production of TNF-α and TxB2. These in vitro findings indicate that PR3-ANCA opsonization of apoptotic PMN might be an important mechanism in the pathogenesis of Wegener's granulomatosis (WG), prompting macrophages to produce proinflammatory mediators. These mediators, mainly TNF-α, might prime further PMN leading to perpetuation of the known priming-dependent mechanisms of ANCA action. [ABSTRACT FROM AUTHOR]

Focuses on a group of patients with a distinct disease phenotype due to a defective TAP complex, the peptide transporter complex associated with antigen presentation. HLA class I assembly and the role of the TAP complex; Clinical manifestations and immunological findings of patients suffering from TAP deficiency syndrome; Differential diagnosis and therapeutic options.

The theoretical risk of triggering vasculitis resulting from administration of G-CSF and GM-CSF to patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), such as Wegener’s granulomatosis (WG), who develop agranulocytosis due to cytotoxic therapy, is unknown. Since there is strong evidence that activation of polymorphonuclear neutrophils (PMN) induced by binding of ANCA to PR3 or myeloperoxidase (MPO) expressed on their plasma membrane is involved in the pathogenesis of systemic vasculitides (SV), we studied the surface expression of PR3 and MPO on PMN from healthy donors in response to G-CSF and GM-CSF in vitro by flow cytometric analysis. Increasing doses of G-CSF did not alter PR3 expression on either untreated or tumour necrosis factor-alpha (TNF-α)-primed donor PMN significantly. In contrast, GM-CSF significantly increased PR3 membrane expression on both intact PMN and neutrophils primed with TNF-α. MPO expression was not significantly altered by either G-CSF or GM-CSF. In summary, these data demonstrate that GM-CSF, but not G-CSF, induces plasma membrane expression of PR3 on PMN in vitro. Since in AAV accessibility of the antigen (PR3 or MPO) to the antibody (ANCA) on the plasma membrane of PMN is thought to be essential for neutrophil activation by ANCA, the results of the present study suggest that administration of GM-CSF to patients with WG with neutropenia implies a definite theoretical risk of deterioration of vasculitis via this mechanism. [ABSTRACT FROM AUTHOR]

The plaque forming cell (PFC) and proliferative responses of human peripheral blood lymphocytes and highly purified blood B cells induced by pokeweed mitogen (PWM) and group A streptoccocal cell membranes (A-ScM) were compared with the responses triggered by various cell preparations of Klebsiella pneumoniae K 43 (Klebs). The number of PIT was determined by a protein A plaque assay, and lymphoproliferation was measured by ³H-thymidine incorporation. In cell cultures stimulated with PWM and A-ScM. lymphocyte proliferation appeared to be associated with the generation of PEC. Klebs caused development of PFC without measurable prior proliferation. Whereas the response to PWM and A-ScM was absolutely T cell-dependent. highly purified H cells generated PFC when incubated with Klebs. Moreover, restitution of T cells to the B cell fraction did not augment (or diminish) the number of plaques. These studies establish that Klebs cell envelope structures contain a T cell-independent polyclonal B cell activator for human B lymphocytes in a high stage of differentiation. Use of this probe should provide further insight into the cellular interactions involved in the differentiation of antibody forming cells in humans. [ABSTRACT FROM AUTHOR]

The HLA phenotype of fifty-four patients with myasthenia gravis (MG) was compared to that of 600 controls. When male and female patients were compared separately with the control group, HLA-B12 was increased in MG males (P<0023) and HLA-A1, B8 in MG females (PcO023). In addition, HLA-A1, B8 was correlated with the early onset type of MG and with a more severe clinical course (Osserman scale IIb-III) in female patients. Cell-mediated immune responses were studied in a twenty-one hospitalized MG patients. Specific cell-mediated immunity to highly purified muscle proteins was investigated using the two-step-leucocyte migration agarose test and general cell-mediated immunity was studied by determining the cutaneous response to four extrinsic antigens. Cellular immune reactivity to muscle antigens occurred in fourteen out of twenty-one patients (67%) with MG and with one exception, in none of the controls. There was no significant difference in the LIF inducing ability of the different muscle antigens. A statistically significant correlation between HLA-A1 and B8 and either cell-mediated immune reactivity to muscle antigens or humoral autoimmune phenomena or clinical parameters was not found. Only a trend toward an association between HLA-A1 and B8 and a cell-mediated immune response to muscle antigens could be demonstrated. Positive delayed skin reactions to extrinsic antigens were observed with the same frequency as in healthy blood donors. [ABSTRACT FROM AUTHOR]

The past decade has seen an explosion of data on the new group of autoantibodies known collectively as ANCA (anti-neutrophil cytoplasmic antibodies). ANCA are specific for granule proteins of granulocytes and monocytes and induce distinct fluorescence patterns, e.g. the cytoplasmic (classic) cANCA and the perinuclear pANCA. cANCA is induced by antibodies directed against Proteinase 3 (PR3; PR3-ANCA) in about 90% of all ANCA-positive sera, and pANCA is induced by antibodies against myeloperoxidase (MPO; MPO-ANCA) in about 40%. A further staining pattern, which does not have a clear cut association with a distinct granule protein, is sometimes seen in chronic inflammatory bowel diseases. PR3-ANCA are serological markers for Wegener's granulomatosis (WG) and MPO-ANCA are associated with certain subtypes of primary vasculitides. Evidence exists that both the autoantigen and ANCA participate in the pathogenesis of at least the group of'ANCAassociated vasculitides'. [ABSTRACT FROM AUTHOR]

Autoantibodies against cytoplasmic components of neutrophil granulocytes (ACPA) were detected in 18 of 32 patients with Wegener's granulomatosis (WG), but in none of the controls (n = 900), including patients with glomerulonephritis, sarcoidosis. tuberculosis, polyarteritis nodosa, and connective tissue diseases, and healthy blood donors. The presence and. to a lesser extent, the titre of ACPA correlated with the severity and activity of the disease. ACPA could be detected in only three of 11 patients with the limited form of the disease and in none in complete remission. In contrast, in all patients with active extensive disease. ACPA were present in a higher titre, and in most of the patients in partial remission (eight of 12) antibodies were demonstrable, especially in those with frequent relapses. Furthermore, the antibody titre correlated significantly with the Creative protein concentration (P<0.05), but with none of the other laboratory parameters. In conclusion. ACPA have proven lo be a highly specific disease marker of great clinical significance that provides us with a useful tool to confirm, or even establish, the diagnosis of WG, [ABSTRACT FROM AUTHOR]

TGF-β is a multifunctional cytokine modulating the onset and course of autoimmune diseases as shown in experimental models. The aim of this study was to investigate TGF-β expression in ANCA-associated vasculitis (AAV), and the possible interactions of this cytokine with lysosomal enzymes identified as ANCA autoantigens (e.g. PR3). This included TGF-β effects on the translocation of the lysosomal enzymes to the cell surface of polymorphonuclear neutrophils (PMN), and the presumed activation of non-bioactive, latent TGF-β by these enzymes. Patients with various types of systemic vasculitis (SV) were studied, including three different types of AAV (Wegener's granulomatosis (WG), Churg-Straß syndrome (CSS) and microscopic polyangiitis (MPA)). Regardless of the type of assay applied, the TGF-β1 isoform was found to be overexpressed in SV, including AAV, and to correlate with disease activity as shown for WG. Mean TGF-β1 plasma levels in AAV patients ranged from 8.9 ng/ml (WG) to 13.3 ng/ml (CSS) (control 4.2 ng/ml; P < 0.01), while TGF-β2 levels were not elevated. Flow cytometry analysis showed TGF-β1 to be a potent translocation factor for PR3 comparable to other neutrophil-activating factors such as IL-8. PR3 membrane expression on primed PMN increased by up to 51% after incubation with TGF-β1. PR3 itself was revealed as a potent activator of latent TGF-β, thus mediating bioeffects of this cytokine. These findings, together with other features of TGF-β such as induction of angiogenesis and its strong chemotactic capacity, indicate that TGF-β might serve as a proinflammatory factor in SV, especially in AAV. [ABSTRACT FROM AUTHOR]

It was our purpose to determine the immunodiagnostic value of ANCA directed against BPI in diseases known to he associated with ANCA. such as ANCA-associated vasculitides, inflammatory bowel disease (IBD) and the associated condition primary selerosing cholangitis. The immunoreactivity of recombinant BP1 (rBP1) was established in order to an ELISA specific for rBPI. By means of this assay. BPI-ANCA were assessed in sent of 178 patients with IBD or the associated disorder primary selerosing cholangitis. 112 patients with ANCA-associated vasculitides, arid in sera ol 182 disease and 140 healthy controls. BPI-ANCA were found to he closely associated with IBD and primary selerosing cholangitis (34% and 44% of ANCA-positive sera respectively). By contrast, BPI-ANCA positivity was low t<10%) in the double-negative sera of patients with ANCA-associated vasculitides and in disease and healthy controls. BP1-ANCA appear to constitute an important marker for IBD and primary selerosing cholangitis, but not for the ANCA-associated vasculitides. [ABSTRACT FROM AUTHOR]

The article presents information on the European therapeutic trials in antineutrophil cytoplasmic antibodies-associated systemic vasculitis. The systemic vasculitides are a significant health issue in the European Union (EU) with an annual incidence of approximately 40 per million and estimated direct health costs to the EU of 500 million ECU per year. The relative rarity and heterogeneous nature of systemic vasculitis have impeded controlled clinical trials and dictate a mulidisciplinary and multicentre approach to their clinical investigation, which has been assisted by recent advances in classification and diagnosis.

Cell-mediated immune responses in 28 hospitalized patients with psoriasis and in 36 healthy controls were studied using the two-step leukocyte migration agarose test. Specific cell-mediated immunity to A-streptococcal cell wall and cell membrane antigens occurred significantly more often in patients with psoriasis than in the control group. A statistically significant correlation between psoriasis- associated antigens of the HLA-B locus and cellular immune reactivity to A-streptococcal antigens or clinical course was not found. When patients with guttate psoriasis were compared separately with the control group, leukocyte migration inhibition induced by cell-free supernatants of A-streptococcal antigen-exposed mono-nuclear cell cultures was found to be more frequent than in other forms of psoriasis. [ABSTRACT FROM AUTHOR]

In this uncontrolled study 15 patients with ANCA-associated systemic vasculitis, who were poor responders to conventional therapy, were treated with single or multiple courses of intravenous immunoglobulin (IVIG). 30g/day over 5 days. Clinical and serological evaluation was performed before and 4 weeks after IVIG. Six of the 15 patients experienced clinically significant benefit from IVIG. Improvement was confined to single organ manifestations (skin. ENT findings), no improvement was seen with conjunctivitis and scleritis, pericarditis or nephritis. No patient experienced complete remission after IVIG. Repeated courses of IVIG at 4-week intervals were no more effective than single courses. In six anti-proteinase 3 (PR3)-positive patients pretreatment sera were incubated with F(ab')2 fragments of the IVIG preparation in vitro to measure the inhibitory effect of IVIG on tinti-PR3 activity. An inhibition of anti-PR3 activity by 25-70% was observed; this did not correlate with clinical effects. Approximately 40% of patients benefited from IVIG treatment, though complete remission of disease activity did not occur. Neither clinical characteristics nor the inhibitory effect of the IVIG preparation on serum anti-PR3 activity in vitro predicted clinical response to this treatment modality. [ABSTRACT FROM AUTHOR]

Apart from the diagnostic value of anti-neutrophil cytoplasmic antibodies (ANCA), their detailed characterization and that of their corresponding antigens have opened new ways for the exploration of the pathogenesis of primary systemic vasculitis. ANCA arc now thought to play an important functional role via activation of phagocytic cells (e.g, polymorphonuclear neutrophils (PMN)). In this study we examined the mechanisms by which ANCA could gain access to proteinase 3 (PR3) in intact PMN, at two levels: ex vivo by analysing the presence of PR3 on the plasma membrane of PMN from patients with ANCA-associated vasculitis, and in vitro by stimulation of PMN using different cytokines, including recombinant tumour necrosis factor-alpha (rhTNF-α) and two forms of IL-8 (produced by monocytic and endothelial cells). Using immunocytochemical staining techniques (FACS and immunoelectronmicroscopy) PR3 has been detected on the plasma membrane of PMN from patients with active ANCA-associated vasculitis. However, this phenomenon is also seen in patients with sepsis who do not have ANCA. In addition, TNF-α and both forms of IL-8 act synergistically and induce a translocation of PR3 from the intragranular loci to the cell surface of PMN. These results provide strong evidence for the hypothesis that ANCA are directly pathogenic by binding to PR3 which is expressed on the cell surface of primed/activated PMN. [ABSTRACT FROM AUTHOR]

In addition to T cell abnormalities, patients with haemophilia A show a separate B cell dysfunction. Characteristic are elevated spontaneous IgG (not IgM) levels in the patient's sera and in the culture supernatants of peripheral blood mononuclar cells. It is also observed that these cells fail to show a differentiation response to T cell-independent B cell activators. The B cell dysfunction correlates with the amount of factor VIII concentrates given prophylactically to patients with severe haemophilia. In contrast to the acquired immune deficiency syndrome (AIDS), the proliferation response to B cell mitogens is not affected. [ABSTRACT FROM AUTHOR]

The effect of the tumour-promoting agent TPA (12-0-tetra-dodecanoyl-phorbol-13- acetate) on the proliferation and Ig secretion response of blood and tonsil lymphocytes was investigated and compared to that of the T-cell-dependent polyclonal activators pokeweed mitogen (PWM) and group A streptococcal cell membranes (A-ScM) or the T cell- independent B cell mitogen Staphylococcus aureus Cowan I (SAC) and a T-cell independent B cell activator Klebsiella pneumoniae (Klebs M), In blood mononuclear cells (MNC), a rather weak, monocyte-dependent DNA synthetic response was observed after exposure to TPA, in comparison to PWM, A-ScM or SAC, Whereas highly purified B cells did not respond to TPA, purified T cells proliferated to a similar degree as unseparated MNC; moreover, the addition of T to B lymphocytes enhanced proliferation rates proportionally to the number of T cells added. This suggests that TPA acts as a polyclonal T cell activator (PTA) for human blood and tonsil cells. Similarly, TPA induced only small amounts of Ig secretion in blood and in tonsil MNC, as determined by an ELISA assay, and no significant Ig secretion in highly purified B cells. The rather weak B cell differentiation response was not due to a monocyte suppressor effect, since partially monocyte-depleted MNC or B cells responded similarly to the non-depleted cells. Thus, TPA cannot be considered as an alternative to other B cells stimulators, both with regard to DNA synthesis and Ig secretion. [ABSTRACT FROM AUTHOR]