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We previously described the requirement of tumour necrosis factor-alpha (TNF- α) and the role of β2 integrins in the Fc-gamma receptor IIa (Fc γRIIa)-mediated mechanism of neutrophil activation by antiproteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies. In the present study, we assessed the involvement of Fc γRIIIb by studying the respiratory burst activation of completely Fc γRIIIb-deficient neutrophils primed by TNF- α and exposed to anti-PR3 or anti-MPO. Activation of the NADPH oxidase occurred normally in these neutrophils, which indicates that engagement of Fc γRIIIb is not essential in our model. Experiments performed with neutrophils from severe leucocyte adhesion deficiency (LAD) patients confirmed that β2 integrins play a pivotal role in this activation. We next studied whether adhesion per se, β2-integrin-mediated adhesion, or β2-integrin ligation without adhesion is necessary or sufficient for this activation. Anti-PR3 or anti-MPO induced an Fc γRIIa-dependent burst in TNF-primed neutrophils incubated in wells coated with poly- l-lysine, known to induce β2-integrin-independent adhesion, but this reaction was still inhibited by blocking CD18 antibodies. In a system with granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, which did not enhance adhesion, we measured a similar activation by anti-PR3 or anti-MPO and inhibition by CD18. We also noticed that treatment with the β2-integrin-activating CD18 MoAb KIM185 per se is insufficient for neutrophil activation by anti-PR3 or anti-MPO. We therefore conclude that ligation of β2 integrins rather than adherence per se is essential for this activation, and that TNF- α or GM-CSF is needed for priming but not for adherence. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about the remission of myasthenia gravis after proctocolectomy in a patient with ulcerative colitis.

Purpose: To assess the value of serial determinations of antineutrophil cytoplasmic autoantibodies (ANCA) for monitoring disease activity in patients with systemic vasculitis.Patients and Methods: Forty-three patients with histologically proven vasculitis (21 with Wegener's granulomatosis, 17 with microscopic polyangiitis, and 5 with renal-limited vasculitis) were studied for a median follow-up of 22 months. Disease activity was prospectively assessed and quantified by the Birmingham Vasculitis Activity Score. A total of 347 sera were analyzed for ANCA determination.Results: Relapses occurred in 23 (54%) of 43 patients. Diagnostic category (Wegener's granulomatosis vs micropolyangiitis and renal-limited vasculitis), severity of initial symptoms (mean vasculitis activity score, mean number of organs involved), and ANCA pattern [cytoplasmic-ANCA (c-ANCA) vs perinuclear-ANCA (p-ANCA)] did not significantly differ between relapsers and nonrelapsers. Lung involvement was more frequent at onset among relapsers [16 of 23 (70%) vs 6 of 20 (30%); P = 0.02]. Relapses were slightly, but not significantly, more frequent in patients with Wegener's granulomatosis or a c-ANCA pattern. The percentage of relapsers was greater in patients with persistently positive ANCA than in patients with negative or decreasing ANCA titers (86% vs 20%, P = 0.0001). However, the predictive value of an increase in ANCA titers for the occurrence of a subsequent relapse was only 28% (4 of 14) for c-ANCA, 12% (2 of 17) for anti-proteinase 3-ANCA, and 43% (6 of 14) for anti-myeloperoxidase-ANCA. An increase in ANCA occurred before or during relapse in 33% (10 of 30) of cases for c-ANCA/anti-proteinase 3 antibodies, and 73% (11 of 15) of cases for anti-myeloperoxidase antibodies.Conclusion: The persistence of ANCA positivity is strongly associated with relapses. However, an increase in ANCA titers has a poor value for the early prediction of a subsequent relapse and should not be used as a sole parameter for therapeutic intervention. In addition, our results suggest that serial anti-myeloperoxidase determination may be useful as a prognostic marker in patients who are p-ANCA positive. [ABSTRACT FROM AUTHOR]

The granule enzyme myeloperoxidase (MPO) plays an important role in neutrophil antimicrobial responses. However, the severity of immunodeficiency in patients carrying mutations in MPO is variable. Serious microbial infections, especially with Candida species, have been observed in a subset of completely MPO-deficient patients. Here we show that neutrophils from donors who are completely deficient in MPO fail to form neutrophil extracellular traps (NETs), indicating that MPO is required for NET formation. In contrast, neutrophils from partially MPO-deficient donors make NETs, and pharmacological inhibition of MPO only delays and reduces NET formation. Extracellular products of MPO do not rescue NET formation, suggesting that MPO acts cell-autonomously. Finally, NET-dependent inhibition of Candida albicans growth is compromised in MPO-deficient neutrophils. The inability to form NETs may contribute in part to the host defense defects observed in completely MPO-deficient individuals.

Backgrounds: Among anti-double-strand (ds)DNA antibody assays, Farr radioimmunoassay is decreasingly used because it requires radioactive material and is labor intensive. We evaluated the performance of Farr, three commercial enzyme immunoassays (EIAs) and the Crithidia luciliae immunofluorescence test (CLIFT) in systemic lupus erythematosus (SLE)., Methods: Anti-dsDNA antibodies were determined in 99 SLE patients, 101 healthy subjects, and 53 patients with autoimmune rheumatic diseases., Results: Farr performed better than the 3 EIAs and CLIFT for the diagnosis of SLE at the manufacturer's cut off and at the cut off set to achieve a specificity of 95%. To achieve a similar level of specificity, some EIAs had a decrease in sensitivity which was dramatic for some tests. Farr was also the best at distinguishing patients with quiescent to mildly active disease from patients with more active disease at the cut off value of 93 IU/ml. Using manufacturer's cut off did not allow distinguishing between patients with quiescent and active SLE., Conclusions: Farr was the best global test to assess the level of anti-dsDNA antibodies for both diagnosis and disease activity evaluation in SLE with adequately determined cut off values. Some EIA had low performances limiting their use in decision-making regarding diagnosis and/or treatment., (Copyright 2010 Elsevier B.V. All rights reserved.)

Anti-proteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies are capable of activating human neutrophils primed by TNF-alpha in vitro. We described previously the involvement of FcgammaRIIa and beta(2) integrins in this neutrophil activation. In the literature, the requirement of TNF priming has been attributed to an effect of TNF-alpha on the expression of PR3 or MPO on the cell surface. Under our experimental conditions, TNF-alpha (2 ng/ml) increased the binding of the antibody against PR3, whereas binding of the antibody against MPO could hardly be detected, not even after TNF-alpha treatment. The aim of this study was to consider (an)other(s) role(s) for TNF-alpha in facilitating the NADPH-oxidase activation by these antibodies. We demonstrate the early mobilization of the secretory vesicles as a result of TNF-induced increase in intracellular-free calcium ions, the parallel colocalization of gp91(phox), the main component of the NADPH oxidase with beta(2) integrins and FcgammaRIIa on the neutrophil surface, and the FcgammaRIIa clustering upon TNF priming. TNF-alpha also induced redistribution of FcgammaRIIa to the cytoskeleton in a dose- and time-dependent manner. Moreover, blocking CD18 MHM23 antibody, cytochalasin B, and D609 (an inhibitor of phosphatidylcholine phospholipase C) inhibited this redistribution and the respiratory burst in TNF-treated neutrophils exposed to anti-PR3 or anti-MPO antibodies. Our results indicate direct effects of TNF-alpha in facilitating neutrophil activation by these antibodies and further support the importance of cytoskeletal rearrangements in this priming process.

Background: Crohn's disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel diseases (IBD), have been associated with disturbances in vascular physiology, including permeability and angiogenesis, that are in part regulated by the endothelial intercellular junctions. These junctions are composed of several adhesion molecules including the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) and the more recently described CD146 (S-Endo1 Ag, MUC18)., Aim: To study the expression of tissue and soluble form of CD146 in patients with CD or UC in relation to disease activity and location. This study was made in comparison with the soluble form of CD31 (sCD31)., Results: In active disease, a high expression of CD146 was observed on endothelial cells in intestinal biopsies from both CD and UC. In addition, we observed a decrease of sCD146 in relation to active disease and extensive location of CD and UC. Lower levels of sCD31 were also detected in active and extensive location of UC, but no difference could be observed in CD., Conclusion: sCD146 is a novel marker of the endothelial intercellular junction that reflects endothelial remodeling more effectively than soluble CD31. Further studies are warranted to determine whether sCD146 will provide a serological assay reflecting alterations in vascular permeability and vessel proliferation in the inflamed IBD intestine.

Little is known about the etiologies of diseases associated with circulating antineutrophil cytoplasm autoantibodies (ANCA), such as primary vasculitides and inflammatory bowel diseases. However, the understanding of immune mechanisms supposedly involved in the pathogenesis of these diseases is still growing. In the present review, we first focus on the mechanisms triggering the development of ANCA, including the potential role of microbial superantigens and the possible defect(s) in the progression of apoptosis or in the removal of apoptotic cells. We next concentrate on the contribution of ANCA to the clinical symptoms and on the pathogenic role of ANCA, including the accessibility of ANCA antigens as targets for circulating antibodies and the mode of action of ANCA. Mechanisms of neutrophil activation by ANCA include the engagement of Fcgamma receptors, the possible mechanisms of neutrophil-mediated tissue damage, and the neutrophil-endothelial interaction.

Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase-3 and myeloperoxidase (MPO) activate tumor necrosis factor-alpha-primed neutrophils in vitro. We used neutrophils from one completely and one partially MPO-deficient donor to assess the requirement of MPO expression for neutrophil activation by anti-MPO antibodies. The MPO deficiencies were defined enzymatically, by immunocytochemistry and by immunoblotting. The mutations in the MPO genes of these donors were identified as a combination of a novel splice-site mutation at the 3' end of intron 11 (A-2-->C), a deletion of 14 nucleotides in exon 9 (A1555-C1568), and a novel C1907 --> T (636Thr-->Met) substitution in exon 11 in the completely MPO-deficient donor and as the same splice-site mutation and a novel C995 --> T (332Ala-->Val) substitution in exon 7 in the partially MPO-deficient donor. Monoclonal antibody 4.15 against MPO and MPO-ANCA-immunoglobulin G induced no superoxide anion production in these MPO-deficient neutrophils despite a normal production induced by other stimuli. Thus, the presence of MPO is a conditio sine qua non for neutrophil activation by anti-MPO antibodies. Moreover, we demonstrated that by means of these MPO-deficient cells, hydrogen peroxide may diffuse from neutrophils to surrounding cells, which may contribute to the damage induced by oxygen radicals in the pathology of systemic vasculitides.

This chapter is an overview of the literature on serological markers of inflammatory bowel diseases (IBD), focusing on anti-neutrophil cytoplasm autoantibodies (ANCA) and anti- Saccharomyces cerevisiae mannan antibodies (ASCA). The methodology for ANCA and ASCA testing is first introduced. The value of these markers as diagnostic tools is then discussed. Other chapters are devoted to the potential role of ANCA and ASCA in disease monitoring, disease stratification and as subclinical markers in families. Finally reviewed are other antibodies recently tested in clinical trials such as pancreatic antibodies and antibodies directed against bacterial antigens. The role of these antibodies in the pathophysiology of IBD still needs to be assessed. We also need to identify the ASCA immunogen(s) eliciting the antibody response.

The relationship between anti-neutrophil cytoplasmic auto-antibodies (ANCA) and disease activity in inflammatory bowel diseases remains controversial. The aim of this study was to highlight the relationship between ANCA presence or titers and disease activity in ulcerative colitis (UC). Three groups of patients with UC were studied: 1) group A included 39 patients who had not undergone colectomy, 2) group B, 43 patients with subtotal colectomy and ileo-rectal anastomosis, 3) group C, 98 patients with proctocolectomy and ileo-anal anastomosis, including 88% with pouchitis and 12% without pouchitis at the time of the study. Determination of ANCA was performed using the standardized indirect immunofluorescence assay. ANCA were positive in 59%, 65%, and 54% of patients from groups A, B, and C, respectively (NS). No relationship between ANCA presence or titers and UC activity could be detected within groups A and B. In group C, 45 of 86 patients (52%) without pouchitis and 8 of 12 patients (67%) with pouchitis, were ANCA positive (NS). These results do not support a relationship between ANCA and UC activity in this cohort of 180 patients.

The pathogeny of ulcerative colitis (UC) is not yet elucidated, but some arguments suggest the implication of genetic factors. Among the candidate genes, those encoding for HLA class II genotypes have been extensively studied in UC; however, discordant data may be imputable to heterogeneity, characterized by immunological markers such as atypical ANCA (p-ANCA), or to inclusion of more or less intractable UC. The aim of our study is to evaluate the interest of HLA class II and TAP genetic markers to identify different clinical forms of UC, according to p-ANCA status. Unrelated patients with a history of UC (n = 91) and healthy control subjects with no personal or family history of inflammatory bowel diseases (IBD) (n = 200) were included. HLA-DRB1*03 was less frequent in UC patients than in healthy controls (8% vs 28%, PC < 0.03). No association was found with any TAP genotypes. Moreover, there was no association with the HLA-DR2 specificity, either in the entire group of UC patients (38% vs 28%) or in the p-ANCA-positive subgroup of patients (30%). The most consistent finding in the present study is that some genetic markers may characterize intractability in UC patients. HLA-DR2 was associated with poor prognosis, regardless of p-ANCA status. In HLA-DR2 and non-HLA-DR2 groups, colectomy was done in 55% and 27% of patients, respectively, (PC < 0.05). Furthermore, in non-HLA-DR2 patients, p-ANCA could be of interest to characterize those with more severe prognosis. Our results confirm the interest of genetic studies to define UC genetic susceptibility, taking into account intractability of the disease. They do not support the hypothesis that p-ANCA is a subclinical marker of genetic susceptibility to UC.

Despite extensive research, the pathogenesis of inflammatory bowel disease (IBD) is still unclear. Immunological disorders have been described in patients with both Crohn's disease (CD) and ulcerative colitis (UC). In this work serum samples collected from 58 patients with CD and 55 patients with UC were tested in ELISA against a panel of nuclear and cytoplasmic proteins and peptides in order to determine whether specific autoantibodies are produced in these patients. Low levels of IgG antibodies to histones H1, H2A, H2B, H3, and H4, to Hsp-70 and ubiquitin stress proteins, Ro/SSA and La/SSB proteins and myosin were detected in some of these sera. In contrast, the following antibodies of IgG isotype could be much more frequently demonstrated: antibodies to ubiquitinated H2A (U-H2A) peptide T4 (51.7% in CD; 18.2% in UC), antibodies to the zinc-finger peptide F2 of poly-(ADP-ribose polymer)ase (PARP) involved in DNA repair (58.6% in CD; 25.5% in UC) and actin antibodies (43.1% in CD; 7.3% in UC). In a follow-up study of 12 patients with CD and UC (75 additional samples), we found IgG antibodies to several histone peptides occurring essentially in the serum of patients with CD. Although we found no obvious correlation between the presence or level of these various antibodies and C-reactive protein, or the location of the disease, in a number (but not all) of patients, we observed a strikingly good relationship between antibodies to histone peptides, U-H2A peptide T4, and PARP peptide F2 and the Crohn's disease activity index. The mechanism of induction of these antibodies still remains obscure.(ABSTRACT TRUNCATED AT 250 WORDS)

IgG anti-endothelial cell antibodies (AECA) have been described in sera from patients with vasculitis and other immune disorders such as systemic lupus erythematosus. Presence of AECA may be relevant to the hypothesis that Crohn's disease (CD) is a form of intestinal vasculitis. The aim of this study was to search for IgG AECA among 141 patients with CD, 94 patients with ulcerative colitis (UC) and 71 healthy blood donors and to assess the relationship between AECA and demographic or disease data. The cut-off point was defined from the mean OD values + 2 SD obtained from healthy blood donors. Seventeen percent of sera from patients with CD were positive for IgG AECA, whereas 24.5% of sera from patients with UC were positive. Among disease data, only a significant relationship between presence of IgG AECA and CD activity was noticed. These results might reinforce the hypothesis that intestinal vascular injury may be an important event in CD. However, detection of AECA in an almost similar percentage of patients with UC is more suggestive of an immune response to hidden endothelial self-antigen exposed after endothelial cell damage or a further marker of disturbed immunoregulation in inflammatory bowel disease.

The diagnostic value of c-ANCA as a specific marker of systemic vasculitis (particularly Wegener's granulomatosis) is well established. The prognostic value of c-ANCA for determining disease activity is controversial. We have prospectively studied in ten patients with systemic vasculitis over a mean period of 34 months (extreme 2-61 months). All patients had c-ANCA at the moment of the diagnosis: four patients had high titer of c-ANCA all over the period study; three clinical and biological exacerbations of the disease was observed without variation of the c-ANCA titer. In four patients c-ANCA disappeared within 6 months after the beginning of the treatment correlated with disease activity. Sometimes a rise of c-ANCA titer was observed with or without disease activity. In one case c-ANCA titer had a serrated evolution. The sensitivity and the specificity of the c-ANCA for disease activity in the ten studied patients were respectively 1 and 0.28. In patients with systemic vasculitis and c-ANCA at the time of the diagnosis, variation in c-ANCA titer alone is of limited prognostic value for predicting disease course.

Presence of antineutrophil cytoplasmic auto-antibodies (ANCA) in ulcerative colitis could be an epiphenomenon related to colonic inflammation and/or may reflect a primitive disturbance of immune regulation. In this regard, study of ANCA status after the whole colorectal mucosa has been removed could favor one of these two hypothesis. We compared the prevalence of ANCA in a first group of 70 patients with non operated UC and in a second group of 32 patients with UC having had a proctocolectomy with ileoanal anastomosis. Perinuclear ANCA (p-ANCA) were found in 34/70 (49%) of the first group as compared to 11/32 (34%) in the second group (NS). Our results further support that the presence of ANCA in UC reflects an immune disturbance not linked to the presence of the target organ.

Antineutrophil cytoplasmic autoantibodies have been recently reported in sera from patients with inflammatory bowel disease. We report our experience based on 90 patients with ulcerative colitis, 148 patients with Crohn's disease, and 60 controls. Determination of antineutrophil cytoplasmic autoantibodies was performed by the indirect immunofluorescence technique on ethanol fixed leucocytes. The specificities for proteinase 3, myeloperoxidase, and lactoferrin were tested by enzyme-linked immunosorbent assay. Forty-three out of 90 (48%) patients with ulcerative colitis, 10 out of 148 (7%) patients with Crohn's disease, and none of controls were positive by indirect immunofluorescence technique. All patients but two with positive immunofluorescence exhibited a perinuclear staining pattern. Among patients with ulcerative colitis, there was no relationship between the presence of perinuclear antineutrophil cytoplasmic autoantibodies and disease location or activity. Seven out of 20 (35%) patients with ulcerative colitis who had a previous colectomy (including 1 with ileoanal anastomosis) had perinuclear antineutrophil cytoplasmic autoantibodies. Antineutrophil cytoplasmic autoantibody specificity was not directed against myeloperoxidase, proteinase 3 or lactoferrin in sera from patients with inflammatory bowel diseases. Inflammatory bowel diseases are associated with a new subset of antineutrophil cytoplasmic autoantibodies. Among patients with Crohn's disease and ulcerative colitis, the sensitivity and specificity of the presence of perinuclear antineutrophil cytoplasmic autoantibodies for ulcerative colitis was 46% and 93%, respectively. Their presence reinforces the likelihood of underlying immunologic dysregulation in ulcerative colitis. Identification of the autoantigen(s) to which these antibodies are directed might facilitate the understanding of inflammatory bowel disease pathophysiology.