Atypical Antineutrophil Cytoplasmic Antibodies Research Articles

Anti-proteinase 3 antibodies in diffuse systemic sclerosis (SSc) with normotensive renal impairment: is it suggestive for an overlapping between SSc and idiopathic vasculitis?

OBJECTIVE: To test the prevalence of anti-neutrophil cytoplasmic antibodies (ANCA) in systemic sclerosis (SSc) and to verify a possible association of ANCA with normotensive renal involvement in SSc. PATIENTS AND METHODS: 51 patients affected by SSc, 35 with diffuse scleroderma (dSSc) and 16 with limited scleroderma (lSSc), were tested for ANCA by indirect immunofluorescence (IIF) on human ethanol and formalin-acetone-fixed granulocytes (before and after DNase treatment), by conventional enzyme linked immuno-sorbent assay (ELISA) and by capture-ELISA. RESULTS: Six out of 51 selected SSc patients had ANCA by IIF (11.7%) and five presented a perinuclear/nuclear atypical ANCA pattern. In all cases we only found anti-proteinase3 (aPR3) antibodies. All ANCA positive patients had diffuse form of SSc (17.1%), all were anti-Scl70 positive (aScl70), five patients had proteinuria, three had microscopic haematuria. All ANCA positive patients were normotensive with normal renin plasma levels, the mean erythrocyte sedimentation rate (ESR) was higher in this group compared to the other SSc patients. CONCLUSIONS: Our study shows that aPR3 is not rare in dSSc. According to the clinical and serological findings and to the recent literature, we can hypothesise that when ANCA are found in SSc, an overlapping of scleroderma with systemic necrotizing vasculitis should be suspected.

Antineutrophil Cytoplasmic Antibodies, Autoimmune Neutropenia, and Vasculitis

Reports of an association between antineutrophil cytoplasmic antibodies (ANCA) and autoimmune neutropenia have rarely included cases of proven vasculitis. A case of ANCA-associated vasculitis (AAV) with recurrent neutropenia is described and relevant literature on the association between ANCA, neutropenia, and vasculitis is reviewed. Longitudinal clinical assessments and laboratory findings are described in a patient with AAV and recurrent episodes of profound neutropenia from December 2008 to October 2010. A PubMed database search of the medical literature was performed for articles published from 1960 through October 2010 to identify all reported cases of ANCA and neutropenia. A 49-year-old man developed recurrent neutropenia, periodic fevers, arthritis, biopsy-proven cutaneous vasculitis, sensorineural hearing loss, epididymitis, and positive tests for ANCA with specificity for antibodies to both proteinase 3 and myeloperoxidase. Antineutrophil membrane antibodies were detected during an acute neutropenic phase and were not detectable in a postrecovery sample, whereas ANCA titers did not seem to correlate with neutropenia. An association between ANCA and neutropenia has been reported in 74 cases from 24 studies in the context of drug/toxin exposure, underlying autoimmune disease, or chronic neutropenia without underlying autoimmune disease. In these cases, the presence of atypical ANCA patterns and other antibodies were common; however, vasculitis was uncommon and when it occurred was usually limited to the skin and in cases of underlying toxin exposure. ANCA is associated with autoimmune neutropenia, but systemic vasculitis rarely occurs in association with ANCA and neutropenia. The interaction between neutrophils and ANCA may provide insight into understanding both autoimmune neutropenia and AAV.

Primary Amyloidosis Causing Diffuse Alveolar Hemorrhage

Diffuse alveolar hemorrhage is a serious complication that has been described in various disease states including several vasculitic syndromes as well as conditions that may resemble vasculitis clinically. In this article, we present a case of 79-year-old man, who was admitted with productive cough, blood-tinged sputum, and a positive atypical antineutrophil cytoplasmic antibody pattern on indirect immunofluorescence microscopy. He subsequently developed frank hemoptysis and respiratory failure. Open lung biopsy demonstrated amyloid deposition within blood vessels. He was treated with 1 g of intravenous methylprednisolone over 3 days with rapid improvement in hemoptysis and hypoxemia. This case report and a review of literature illustrate unusual clinical manifestations of vascular amyloidosis that may be confused with vasculitis. Accurate diagnosis of this condition may improve clinical outcome and spare the patient from unwarranted, potentially harmful treatments.

Severe autoimmune neutropenia associated with acute autoimmune hepatitis

A 49-year-old previously healthy female presented with acute hepatitis and severe neutropenia. A diagnosis of type 1 autoimmune hepatitis was made based on the histological appearance of a liver core biopsy, positive anti-smooth muscle antibodies, and positive anti-neutrophil cytoplasmic antibody (atypical ANCA). Hemogram revealed mild leukopenia with severe neutropenia (absolute neutrophil count 256/mm(3)), normal hemoglobin and mild thrombocytopenia (115000/mm(3)). A bone marrow biopsy and aspirate had a normal karyotype, increase in granulopoiesis, prominence of promyelocytes (31%) and absence of mature granulocytes. Anti-neutrophil antibodies were detected in the patient's blood. Therapy was directed at the underlying hepatitis with resolution of neutropenia without the use of colony-stimulating factors.

Autoantibodies in silicosis patients and in silica-exposed individuals

The aim of this study was to evaluate the prevalence of autoantibodies in silica-exposed patients with and without silicosis and without any known rheumatic disease. We studied 61 males exposed to silica for a mean time of 12.2 +/- 10.2 years of exposure. A total of 72.1% (44/61) of them presented with pulmonary silicosis. As control group we included 62 healthy males. In all samples we screened for rheumatoid factor (latex agglutination), antinuclear antibodies (indirect immunofluorescence), anti Scl-70 (ELISA) and ANCA (indirect immunofluorescence technique). One patient (1.6%) of the silica group had weakly positive ANA (titer 1:80, centromeric pattern); one (1.6%) had atypical ANCA and seven patients (11.4%) presented positive rheumatoid factor (values range from 8 to 32 UI/ml). One control patient had a positive RF and none of them had positive ANA or ANCA. All patients and controls were negative for anti-Scl-70. The finding of positive RF was higher in the silica-exposed patients (p = 0.032; Fisher). All patients with positive RF had pulmonary silicosis. In the silica-exposed group we could not find a relationship between the presence of RF and age (p = 0.21; Mann-Whitney), smoking habits (p = 0.25; Fisher) but a positive relationship was found with exposure time to silica dust (p = 0.005; Mann-Whitney). We conclude that there was 11.4% prevalence of low titer RF in the silica-exposed patients without known rheumatic disease. RF was more common in patients with longer exposure to silica dust and appeared only in those with silicosis. The presence of ANA, Scl-70 and ANCA was the same as in the control population.

Evaluation of the Combined Application of Ethanol-Fixed and Formaldehyde-Fixed Neutrophil Substrates for Identifying Atypical Perinuclear Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Disease

No clear guidelines for indirect immunofluorescence (IIF) detection and interpretation of antineutrophil cytoplasmic antibodies (ANCA) have been proposed for inflammatory bowel diseases (IBD). We evaluated the reliability of the combined use of ethanol- and formalin-fixed neutrophil substrates to identify atypical perinuclear ANCA (P-ANCA) by IIF under routine laboratory circumstances. A total of 204 IBD patients were assessed with four different fluorescent substrates in two distinct laboratories. Antibodies against myeloperoxidase, proteinase-3, and other specific granule proteins (elastase, lactoferrin, cathepsin G, lysozyme, and bactericidal permeability-increasing protein) were measured by an enzyme-linked immunosorbent assay. The combined application of ethanol- and formalin-fixed slides to detect atypical P-ANCA resulted in a lack of agreement between assays (kappa, < or =0.39) in the interassay study and moderate agreement in the interobserver study (kappa, 0.42). After atypical and typical P-ANCA patterns were combined, the consensus improved greatly. A total of 26.9% of patients were P-ANCA positive by at least two tests (44.3% of ulcerative colitis [UC] and 13.1% of Crohn's disease [CD] patients; P < 0.0001), while overall ANCA positivity was 22.5% to 34.8%. The combined application of ethanol-fixed and formaldehyde-fixed neutrophil substrates did not facilitate differentiation between P-ANCA and atypical P-ANCA, and the results were not consistent when substrates from different sources were used. Combining all P-ANCA ensures the highest sensitivity and specificity in differentiating UC from CD.

Membranous Glomerulonephritis with ANCA-Associated Necrotizing and Crescentic Glomerulonephritis

Only rare cases of concurrent membranous glomerulonephritis (MGN) and antineutrophil cytoplasmic antibody (ANCA)-associated necrotizing and crescentic glomerulonephritis (NCGN) have been reported. The authors report the clinical and pathologic findings in 14 patients with MGN and ANCA-associated NCGN. The cohort consisted of eight men and six women with a mean age of 58.7 yr. ANCA positivity was documented by indirect immunofluorescence or ELISA in all patients. Indirect immunofluorescence was positive in 13 patients (seven P-ANCA, five C-ANCA, one atypical ANCA). ELISA was positive in nine of 10 patients (five MPO-ANCA, three PR3-ANCA, one MPO- and PR3-ANCA). Clinical presentation included heavy proteinuria (mean 24-hr urine protein 6.5 g/d), hematuria, and acute renal failure (mean creatinine 4.4 mg/dl). Pathologic evaluation revealed MGN and NCGN, with crescents involving a mean of 32% of glomeruli. On ultrastructural evaluation, the majority of cases showed stage I or II membranous changes. Follow-up was available for 13 patients, 12 of whom were treated with steroids and cyclophosphamide. At a mean follow-up of 24.3 mo, five patients progressed to ESRD, seven had stabilization or improvement in renal function, and one had worsening renal function. Five patients, including three with ESRD, died during the follow-up period. The only independent predictor of progression to ESRD was serum creatinine at biopsy. MGN with ANCA-associated NCGN is a rare dual glomerulopathy seen in patients with heavy proteinuria, acute renal failure, and active urine sediment. Prognosis is variable, with 50% of patients reaching endpoints of ESRD or death.

Diagnostic pitfall: Mycobacterium avium complex pulmonary infection and positive ANCA

We report the case of a 58-year-old female who presented with productive cough, weight loss, pulmonary nodular infiltrates and cavitations. She had a positive anti-neutrophil cytoplasmic antibodies (ANCA) test. A diagnosis of vasculitis was considered and a video-assisted thoracoscopic biopsy of the lung nodules was organised. However, prior to the biopsy, the sputum results revealed the presence of acid-fast bacilli, which were identified as Mycobacterium avium complex. A repeat ANCA assay was positive for atypical ANCA with negative proteinase-3 and myeloperoxidase titres. The patient was treated with rifampicin, ethambutol and clarithromycin with clinical and radiological improvement. The objective of this report is to highlight a rare association between positive ANCA titres and a non-tuberculous mycobacterial infection as a misdiagnosis and treatment of this patient with immunosuppressive therapy might have led to serious consequences.

Intravenous immunoglobulins contain naturally occurring antibodies that mimic antineutrophil cytoplasmic antibodies and activate neutrophils in a TNFα-dependent and Fc-receptor–independent way

AbstractIntravenous immunoglobulin (IVIg) preparations are increasingly used for therapy of several neuroimmunologic diseases. IVIg therapy is considered safe, although serious side effects like aseptic meningitis, cerebral vasospasm, or ischemic encephalopathy have been reported. These side effects are frequently associated with neutrophilic pleocytosis in the cerebrospinal fluid (CSF), suggesting a neutrophil-mediated mechanism. To elucidate the potential role of neutrophil activation, we analyzed IVIg preparations from 5 different commercial sources for the presence of antineutrophil cytoplasmic antibody (ANCA)–like immunoglobulins against ethanol-fixed peripheral-blood neutrophils, purified human antigens, and a panel of human and nonhuman tissues. All IVIg batches tested (n = 13) contained atypical ANCAs (IgG titer up to 1:2048, IgA up to 1:512). Moreover, all preparations were capable of inducing hydrogen peroxide production in TNFα-primed human neutrophils, with a significant correlation (P < .005) between atypical ANCA titers in IVIg preparations and neutrophil activation. Fc-mediated binding and activation was ruled out by the use of IVIg-F(ab′)2 fragments. Our findings strongly suggest that in vivo activation of TNFα-primed neutrophils by atypical ANCAs of IVIg may contribute to the side effects of IVIg therapy and for the first time demonstrate that the activation of neutrophil granulocytes by IVIg occurs in an Fc receptor (FcR)–independent, hence antigen-dependent, way.

Low seroprevalence and poor specificity of antineutrophil cytoplasmic antibodies in tuberculosis

Recently published findings suggested that antineutrophil cytoplasmic antibodies (ANCA), particularly those with a cytoplasmic (C-ANCA) labelling pattern and targeting proteinase 3 (anti-PR3), might be markers of tuberculosis (TB). This is a critical issue, because C-ANCA/anti-PR3 were considered to be a highly specific hallmark of Wegener's granulomatosis or microscopic polyangiitis and because TB may clinically mimic Wegener's granulomatosis. We therefore undertook a study with the aim of investigating further the prevalence and specificity of ANCA in TB. We evaluated serum samples from 67 patients diagnosed with culture-proven TB and 10 previously untested control samples from patients known to be ANCA positive (four Wegener's granulomatosis and two microscopic polyangiitides) or negative. All 77 sera were screened for ANCA using commercially available indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) for anti-PR3 and antimyeloperoxidase (MPO). IIF-positive and anti-PR3- and anti-MPO-negative sera were also tested for bactericidal/permeability-increasing protein, lactoferrin, elastase and cathepsin G specificities with commercially available ELISA. IIF detected ANCA in seven (10%) of the TB sera, including three C-ANCA and four atypical perinuclear-labelling ANCA. Only one IIF-negative specimen was anti-PR3 positive in ELISA. ANCA testing of the control sera yielded IIF and ELISA results concordant with previous findings, except for one borderline ELISA. Our results indicate that TB is associated with low ANCA seroprevalence and poor specificity, with no test serum showing combined C-ANCA/anti-PR3 activity. In a clinical setting of Wegener's granulomatosis/TB mimicry, such combined reactivity would seem to be more suggestive of Wegener's granulomatosis.

Use of Immunofluorescence and Confocal Laser Scanning Microscopy in Identifying Rare Cases of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) Showing Dual Specificities to Myeloperoxidase and Proteinase3

Anti-neutrophil cytoplasmic antibodies (ANCA) are the immunodiagnostic markers for idiopathic necrotizing crescentic glomerulonephritis affecting mainly medium to small sized blood vessels. The diagnosis of ANCA associated vasculitis (AAV) is mainly based on clinical and histopathological characteristics along with the serological evidence. Immunofluorescence microscopy (IIF) is considered as the "gold standard" for ANCA detection, and ANCA showing two major patterns ie, cytoplasmic (c-ANCA) and perinuclear (p-ANCA) react with different antigenic targets of neutrophils like Proteinase3 (PR3) and Myeloperoxidase (MPO). A third unusual and rare immunofluorescence pattern called as "X- ANCA" or atypical ANCA is also sometimes seen. The difficulty in identification of ANCA immunofluorescence patterns is mainly seen due to the rare dual patterns seen in the same sera and also the additional nuclear immunofluorescence seen due to presence of anti-nuclear antibodies. ANCA testing by immunofluorescence and Confocal Laser scanning microscopy, as well as by specific ELISAs for detection of anti-PR3 and anti-MPO antibodies have helped in improving the diagnosis. Patients having dual specificities to MPO and PR3 in a patient is a rare finding. Among 425 clinically and histopathologically proven cases of AAV, eight patients (1.9%) had dual specificities, of which five patients showed mixed immunofluorescence pattern and 3 patients showed X-ANCA pattern which was confirmed by both immunofluorescence and Confocal Laser scanning microscopy and the dual specificities to MPO and PR3 were detected by individual ELISAs.

Anti-neutrophil cytoplasmic antibody (ANCA) in malaria is directed against cathepsin G

SUMMARY Autoantibodies of diverse specificities are detected in sera of patients with acute malaria. The clinical relevance of these autoantibodies is not clear, though there are reports associating some autoantibodies with specific disease manifestations. We have investigated the occurrence of ANCA in the sera of 93 patients during episodes of acute malaria. Sera were tested by indirect immunofluorescence (IIF) and by ELISA for antibodies to neutrophil cytoplasmic components proteinase 3 (PR3), myeloperoxidase (MPO), cathepsin G (CG), human leucocyte elastase (HLE), and lactoferrin (LF). Forty-seven sera samples (50.5%) were positive by IIF, all except one with the atypical ANCA pattern (a-ANCA). When screened by ELISA, anti-CG antibodies were detected in 52 samples (56%), while anti-PR3 and anti-MPO antibodies were detected in three and one samples, respectively. Antibody binding to HLE and LF was not significant. Anti-CG antibodies were detected in 93% of the IIF-positive sera. A combination of anti-CG and anti-PR3 antibodies was noted in three samples. Our study demonstrates the presence of ANCA in sera from patients with acute malaria, almost all with the a-ANCA pattern on IIF. The antibody specificity, noted for the first time in our study, appears to be predominantly directed against CG. The significance of CG and CG-ANCA in the pathogenesis and clinical manifestations of malaria has yet to be elucidated.

Anticorps anticytoplasme des polynucléaires chez les patients atteints de polyarthrite rhumatoïde : corrélations avec les caractéristiques cliniques, biologiques et radiologiques

Objectives. – To determine the prevalence and the associations of anti-neutrophil cytoplasmic antibodies (ANCA) and subtypes with clinical, biological and radiological findings in patients with rheumatoid arthritis (RA). Materials and methods. – This is a transversal study of 85 patients with RA (followed in Ibn-i Sina Hospital of School of Medicine, Ankara University) with disease duration of 8.7 ± 6.4 years. Besides clinical, biological and radiological disease activity parameters, ANCA and ANCA against myeloperoxidase (MPO) and proteinase 3 (PR3) were examined. Results. – The prevalence of ANCA, perinuclear ANCA (p-ANCA) and atypical ANCA (a-ANCA) were 18% (15/85 patients), 6% and 12%, respectively. Anti-MPO was found in six patients while anti-PR3 was not found. No significant association could be found between clinical, biological, and radiological disease activity assessments and ANCA (including indirect immunoflorescence subtypes). Similarly, ANCA were not associated with features suggestive of underlying vasculitis (noticed in 11/85 patients), and/or other extra-articular features. Conclusions. – Our results confirm that ANCA of various specificities (mainly a-ANCA) occur in a minority of RA. However, those ANCA were not associated with more severe RA, including the 6/85 patients positive for MPO (who were all free from vasculitis). The over-representation in RA sera of a-ANCA, as compared to p-ANCA, should be further studied.

Anti-neutrophil cytoplasmic antibodies in patients with rheumatoid arthritis: clinical, biological, and radiological correlations

Objectives. – To determine the prevalence and the associations of anti-neutrophil cytoplasmic antibodies (ANCA) and subtypes with clinical, biological, and radiological findings in patients with rheumatoid arthritis (RA). Materials and methods. – This is a transversal study of 85 patients with RA (followed in Ibn-i Sina Hospital, Ankara University School of Medicine) with disease duration of 8.7 ± 6.4 years. Besides clinical, biological, and radiological disease activity parameters, ANCA and ANCA against myeloperoxidase (MPO) and proteinase 3 (PR3) were examined. Results. – The prevalence of ANCA, perinuclear ANCA (p-ANCA) and atypical ANCA (a-ANCA) were 18% (15/85 patients), 6% and 12%, respectively. Anti-MPO was found in six patients while anti-PR3 was not found. No significant association could be found between clinical, biological, and radiological disease activity assessments and ANCA (including indirect immunoflorescence subtypes). Similarly, ANCA were not associated with features suggestive of underlying vasculitis (noticed in 11/85 patients), and/or other extra-articular features. Conclusions. – Our results confirm that ANCA of various specificities (mainly a-ANCA) occur in a minority of RA. However, those ANCA were not associated with more severe RA, including the 6/85 patients positive for MPO (who were all free from vasculitis). The over-representation in RA sera of a-ANCA, as compared to p-ANCA, should be further studied.

Antineutrophil cytoplasmic antibodies in patients with early rheumatoid arthritis: an early marker of progressive erosive disease.

To evaluate the clinical associations of antineutrophil cytoplasmic antibodies (ANCA) in patients with early rheumatoid arthritis (RA), as well as the possible predictive role of ANCA. We also assessed the overlap of ANCA with other specific serologic markers of RA. Eighty-two RA patients with symptoms for < or = 12 months were studied for the presence of ANCA by immunofluorescence and specific enzyme immunoassays. ANCA were determined and clinical, radiographic, and laboratory data were collected at study entry and later at 12, 36, 60, and 84 months. In 2 patients, the first serum samples (obtained at study entry) were no longer available for the determination of ANCA. Perinuclear ANCA (pANCA) were found in 40 patients (50%), and atypical cytoplasmic ANCA were found in 3 patients (4%) at study entry. Perinuclear ANCA-positive patients were significantly more frequently positive for rheumatoid factor (78%) than were ANCA-negative patients (54%) (P = 0.0297). Fifty-five percent of pANCA-positive patients and 22% of ANCA-negative patients were positive for antiperinuclear factor (P = 0.0044). Similarly, pANCA-positive patients had antikeratin antibodies more frequently than did ANCA-negative patients (35% versus 20%). During a 7-year followup, the progress of radiographic joint destruction, assessed with Larsen scores, was significantly more rapid in patients who were pANCA positive at study entry than in those who were ANCA negative (P = 0.0015). Also, the mean titer of pANCA at study entry was significantly higher in those patients who subsequently had advanced radiographic joint destruction at 60 and 84 months. The association of pANCA with rapid radiographic destruction in patients with early RA was further corroborated by a logistic regression analysis that selected pANCA positivity as an independent and statistically significant predictor of rapid radiographic joint destruction. In patients with early RA, pANCA are associated with specific serologic markers of RA and predict rapid radiographic joint destruction.

Anti-neutrophil cytoplasmic antibodies directed against the bactericidal/permeability-increasing protein (BPI) in pediatric cystic fibrosis patients do not recognize N-terminal regions important for the anti-microbial and lipopolysaccharide-binding activity of BPI.

This study was performed to examine the prevalence and clinical correlates of bactericidal/permeability-increasing protein anti-neutrophil cytoplasmic antibodies (BPI-ANCA) in pediatric cystic fibrosis (CF) patients and to elucidate their possible role in CF pulmonary pathology. Sera of 27 CF patients were tested for ANCA by indirect immunofluorescence (IFT) and by enzyme-linked immunosorbent assay (ELISA) for ANCA sub-specificities. BPI-ANCA were examined by using standard ELISA for BPI, lipopolysaccharide-binding protein (LBP), and BPI/LBP fusion proteins to epitope map the main binding sites and look for cross-reactivity with LBP. Pulmonary function and serum concentrations of total immunoglobulin G (IgG) were measured and infections were diagnosed. In addition, release of reactive oxygen species (ROS) by neutrophil granulocytes was measured after stimulation with monoclonal BPI-ANCA. Using IFT, two patients showed atypical ANCA staining, six patients exhibited perinuclear ANCA staining, and no cytoplasmic ANCA staining was detected. Of 27 patients, 13 (48%) were BPI-ANCA (IgG) positive, and three were also immunoglobulin A (IgA) BPI-ANCA positive; one patient had ANCA against lactoferrin; and no proteinase 3 ANCA was detected in any of the patients. All BPI-ANCA bound to the C-terminal region of the molecule; none bound to the N-terminus or to LBP. There was no significant correlation between clinical data and the occurrence of BPI-ANCA in this cross-sectional study. Release of ROS from granulocytes was induced by monoclonal BPI-ANCA. Activation of neutrophils and possible modulation of BPI-mediated opsonophagocytosis and disposal of Gram-negative bacteria and lipopolysaccharides by BPI-ANCA raise the possibility that they contribute to pulmonary pathology in pediatric CF patients but intervention longitudinal studies in large groups of patients are needed to establish a causative association.

Diagnostic value of classical and atypical antineutrophil cytoplasmic antibody (ANCA) immunofluorescence patterns.

BACKGROUND: The "classical" antineutrophil cytoplasmic antibody (C-ANCA) pattern seen on indirect immunofluorescence (IIF) is characterised by granular cytoplasmic staining showing central or interlobular accentuation, and is strongly associated with antiproteinase-3...

Antineutrophil cytoplasmic antibodies in primary Sjögren's syndrome: prevalence and clinical significance.

To evaluate the prevalence of cytoplasmic (c) and perinuclear (p) antineutrophil cytoplasmic antibodies (ANCA) in patients with primary Sjogren's syndrome (SS), and to correlate the presence of ANCA with extraglandular and immunological manifestations related to SS. In a cross-sectional study, we included 82 consecutive patients (75 female and seven male; mean age 61 yr; range 33-87 yr) attending our unit. All patients fulfilled four or more of the diagnostic criteria for SS proposed by the European Community Study Group in 1993. Extraglandular manifestations such as arthralgia and/or arthritis, Raynaud's phenomenon, autoimmune thyroiditis, peripheral neuropathy, renal involvement and cutaneous vasculitis were also recorded. Serum samples were examined by indirect immunofluorescence (IIF) and by ELISA using as substrates myeloperoxidase (MPO) and proteinase 3 (PR3). ANCA were detected in nine (11%) patients: seven had pANCA and two an atypical pattern. These two atypical ANCA became cANCA when paraformaldehyde fixation was applied. ELISA findings showed that two patients had antibodies against MPO, and no patient had antibodies to PR3. The most common extraglandular manifestations in the ANCA-positive patients were articular involvement in six (66%) patients, peripheral neuropathy in five (55%), Raynaud's phenomenon in four (44%) and cutaneous vasculitis in four (44%). Of the four patients with cutaneous vasculitis and ANCA, two had a mononuclear inflammatory vascular disease (MIVD) in the biopsy specimen. When compared with patients without ANCA, those with these antibodies had a higher prevalence of cutaneous vasculitis (44% vs 8%, P = 0.01), Raynaud's phenomenon (44% vs 8%, P = 0.01) and peripheral neuropathy (55% vs 7%, P < 0.001). ANCA positivity can be found in patients with primary SS and its detection is associated with the presence of clinical manifestations attributable to vascular involvement (cutaneous vasculitis, peripheral neuropathy and Raynaud's phenomenon).

Atypical antineutrophil cytoplasmic antibodies with perinuclear fluorescence in chronic inflammatory bowel diseases and hepatobiliary disorders colocalize with nuclear lamina proteins.

Antineutrophil cytoplasmic antibodies (ANCA) are frequently associated with chronic inflammatory bowel diseases (IBD) and hepatobiliary disorders. However, their target antigens have not been identified yet. Recently, we observed an atypical perinuclear ANCA fluorescence (p-ANCA) together with an intranuclear staining using ANCA-positive sera from patients with IBD and hepatobiliary disorders. This observation suggests that the target antigens are localized within the nucleus of neutrophilic granulocytes. To further investigate this hypothesis, we examined sera from patients with ulcerative colitis, primary sclerosing cholangitis, autoimmune hepatitis or systemic vasculitis on ethanol or formaldehyde-fixed neutrophils using confocal laser scanning microscopy and immunoelectron microscopy. Counterstaining with propidium iodide, a DNA-specific dye, showed that ANCA-positive sera in IBD and heptobiliary disorders react with intranuclear antigens at the nuclear periphery of the neutrophils. Double immunolabeling techniques revealed that nuclear lamina proteins, lamins A, C and B1, and lamin B receptor were colocalized with the antigen(s) recognized by atypical p-ANCA. No colocalization was observed with classical p-ANCA and antibodies against histones (H1-H4). Our study showed that atypical p-ANCA are antinuclear antibodies reactive with granulocyte-specific antigens present in the nuclear lamina.

Three-diaengional radiation therapy treatment planning and delivery

Anti-neutrophil cytoplasmic antibodies (ANCA) are antibodies directed against enzymes that are found mainly within the azurophil or primary granules of neutrophils. There are 3 types of ANCA that can be distinguished by the patterns they produce by indirect immunofluorescence when tested on normal ethanol-fixed neutrophils. Diffuse fine granular cytoplasmic fluorescence (cANCA) is typically found in Wegener’s granufomatosis, in some cases of microscopic polyarteritis and Churg Strauss syndrome, and in some cases of crescentic and segmental necrotising glomerulonephritis, but it is rare in other conditions. The target antigen is usually proteinase 3. Perinuclear fluorescence (pANCA) is found in many cases of microscopic polyarteritis and in other cases of crescentic and segmental necrotising glomerulonephritis. These antibodies are often directed against myeloperoxidase but other targets include elastase, cathepsin G, lactoferrin, lysozyme and beta-glucuronidase. The third group designated “atypical” ANCA includes neutrophil nuclear fluorescence and some unusual cytoplasmic patterns, and while a few of the target antigens are shared with pANCA, the others have not been identified. Sera that produce a pANCA or atypical ANCA pattern on alcohol-fixed neutrophils result in cytoplasmic fluorescence when formalin acetone fixation is used. pANCA or atypical ANCA occur in about 2/3 of all individuals with ulcerative colitis or primary sclerosing cholangitis, and they are found in a third of patients with Crohn’s disease. The reported incidence of ANCA in rheumatoid arthritis and SLE varies considerably but the patterns are predominantly pANCA and atypical ANCA. Where ANCA are associated with a small vessel vasculitis, such as Wegener’s granulomatosis and microscopic polyarteritis, antibody titres may parallel disease activity and have been used to monitor the response to treatment. In other non-vasculitic conditions ANCA levels do not necessarily reflect disease activity or the presence of a vasculitis. ANCA are also common in HIV infections where all fluorescence patterns are found, but the presence of these antibodies has no clinical significance. There have been many reports of ANCA occurring occasionally in other diseases.ANCA have not yet been demonstrated to be pathogenetic in the small vessel vasculitides. However there is in vitro evidence for 2 mechanisms, one of which focuses on neutrophil activation as the primary event and the other on antibody binding to *An update from the recent International and Australian Workshops. This paper is based on contemporary published data and observations presented at the 4th International Workshop on ANCA (Lubeck, May 1992) and 1st Australian Workshop on ANCA (Melbourne, October, 1992). vascular endothelium. Both mechanisms assume increased levels of circulating tumor necrosis factor or another cytokine that might result from the infections that often precede the onset of Wegener’s granulomatosis and microscopic polyarteritis. Successful new regimens in the treatment of Wegener’s granulomatosis and microscopic polyarteritis include the use of pooled immunoglobulin, and cyclosporin in combination with low dose prednisolone, but one report of 3-weekly pulse cyclophosphamide as a single agent indicated that it was effective in only 42% of patients.