Antineutrophil cytoplasmic antibodies.
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Antibodies, Antineutrophil Cytoplasmic↗
Biomedical subjects
U Specks
Publications and source records attributed to U Specks.
A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide.
ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and delta-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, delta-rPR3-S176A does not. Culture supernatants of rPR3-S176A and delta-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With delta-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.
Antibodies, Antineutrophil Cytoplasmic↗
Antineutrophil cytoplasmic antibodies.
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Antibodies, Antineutrophil Cytoplasmic↗
Cloning and functional expression of the murine homologue of proteinase 3: implications for the design of murine models of vasculitis.
Anti-neutrophil cytoplasmic autoantibodies recognizing conformational epitopes (c-ANCA) of proteinase 3 (PR3) from azurophil granules are a diagnostic hallmark in Wegener's granulomatosis (WG). Because a functional PR3 homologue has not been identified in rodents, it is difficult to assess immunopathological responses in rats or mice immunized with patients' derived c-ANCA or human PR3. Here we report the full length cDNA cloning and functional expression of murine PR3 in HMC-1 cells. Recombinant murine PR3 shows highly similar substrate specificities towards synthetic peptides and is inhibited by human alpha1-proteinase inhibitor like human PR3. However, neither human c-ANCA, rabbit sera nor mouse monoclonal antibodies to human PR3 recognize the murine homologue. Consequently, it is unlikely that disease observed in mice after immunization with c-ANCA or human PR3 is caused by pathogenic antibodies directed against mouse PR3. Recombinant human-mouse chimaeric variants will be a valuable new tool to localize the disease-specific immunodominant epitopes in human PR3.
Amino Acid Sequence↗
Human mast cells expressing recombinant proteinase 3 (PR3) as substrate for clinical testing for anti-neutrophil cytoplasmic antibodies (ANCA).
We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjogren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P = 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P = 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P < 0.01). These findings indicate that HMC-1/PR3 cells are a very sensitive antigen-specific substrate for clinical anti-PR3 ANCA testing which appears superior to standard C-ANCA testing using PMN cytospin substrates and anti-PR3 ELISA. Our results also suggest that in WG the C-ANCA fluorescence pattern is not caused by antibodies against target antigens other than PR3.
Antibodies, Antineutrophil Cytoplasmic↗
Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA.
We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by alpha 1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies.
Antibodies, Antineutrophil Cytoplasmic↗
Microtiter plate immunoassay for the evaluation of platelet adhesion to fibronectin.
Investigations of platelet adhesion to adhesive proteins have been pursued to understand the basic mechanisms of hemostasis and thrombosis. Most assays used to determine platelet adhesion under stasis conditions rely on radiolabeled platelets. We describe a new microtiter immunoassay to study platelet adhesion to adhesive proteins under stasis conditions. Direct comparison of platelet adhesion to fibronectin using a standard platelet adhesion assay based on 51Cr-labeled platelets and the new immunoassay showed that the optical density values obtained with the immunoassay are directly proportional to the number of platelets bound. The choice of platelet suspension buffer crucial for the design of such experiments, because the adhesion of resting platelets to fibronectin is increased in response to thrombin stimulation. This increase buffer rather than Tris buffer. Platelet adhesion to fibronectin is increased in response to thrombin stimulation. This increase can be inhibited by synthetic RGD peptides. The thrombin-induced increase of platelet adhesion to fibronectin could be detected with antibodies against actin and glycoprotein IIb-IIIa, but not against the alpha-granule constituent platelet factor 4 (PF4). This assay is very versatile, because it avoids the use of radioactivity, and allows the parallel processing of a large number of samples. In addition, the parallel use of antibodies against different platelet antigens allows the screening for platelet activation events associated with the measured platelet adhesion.
Amino Acid Sequence↗
Lung toxicity associated with cyclophosphamide use. Two distinct patterns.
Cyclophosphamide-induced lung toxicity may be difficult to recognize because of the presence of confounding variables such as concomitant use of other cytotoxic drugs, opportunistic infections, diffuse pulmonary malignancy, radiation pneumonitis, and oxygen toxicity. The purpose of this retrospective analysis was to identify the clinical spectrum of pulmonary toxicity of cyclophosphamide. In our review of case records, we sought to identify patients in whom cyclophosphamide was the only identifiable etiologic factor for lung toxicity. In a 20-yr period six patients were identified with cyclophosphamide-induced lung disease, including five men and one woman ranging in age from 42 to 78 yr. Clinical features of toxicity include dyspnea, fever, cough, new parenchymal infiltrates, gas exchange abnormalities on pulmonary function tests, and pleural thickening on chest roentgenogram. Two patterns of cyclophosphamide-induced lung toxicity were identified. A single patient presented with early-onset pneumonitis and responded to discontinuation of the drug. Five patients with late-onset pneumonitis developed progressive pulmonary fibrosis associated with bilateral pleural thickening. Patients with late-onset pneumonitis showed no response to cessation of cyclophosphamide and institution of corticosteroid therapy. Three of these patients died of respiratory failure. Careful review of the individual cases reported in the literature as cyclophosphamide lung toxicity revealed only 12 cases in whom none of the additional confounding factors could be identified. These could easily be divided in the same two categories. Early-onset pneumonitis is reversible and may respond to corticosteroid therapy. Late-onset pneumonitis, frequently associated with pleural thickening, is clinically distinct from idiopathic pulmonary fibrosis but has a chronically progressive course. It appears unresponsive to corticosteroid therapy.
Adult↗
Tracheobronchial involvement in Wegener's granulomatosis.
This study was designed to characterize the clinical spectrum and course of tracheobronchial involvement in Wegener's granulomatosis (WG). Of the 51 patients with biopsy-proven WG who underwent bronchoscopy at least once at our institution between January 1982 and November 1993, 30 (59%) had endobronchial abnormalities due to WG. Initial findings included subglottic stenosis in five (17%), ulcerating tracheobronchitis with or without inflammatory pseudotumors in 18 (60%), tracheal or bronchial stenosis without inflammation in four (13%), and hemorrhage without identifiable source in two (4%) patients. Nine patients with ulcerating tracheobronchitis on initial study had subsequent bronchoscopies for continued symptoms, which in seven cases documented the progression from ulcerating tracheobronchitis to stenosis without inflammation. Bronchoscopic interventions included dilation by rigid bronchoscope in three, YAG-laser treatment in one, and placement of silastic airway stents in three patients. Only the stents provided persistent airway patency. Endobronchial biopsies were performed on 21 occasions in 17 patients. Half of the specimens were helpful in establishing the diagnosis and in all but three in assessing disease activity. While antineutrophil cytoplasmic antibody titers reflect overall disease activity, no correlation with endobronchial inflammatory activity was apparent.
Adult↗
Increased expression of type VI collagen in lung fibrosis.
Pulmonary fibrosis is characterized by disturbances of extracellular matrix protein deposition resulting from fibroblast activation and proliferation. Collagen VI, forming microfibrillar meshworks separate from major fibrillar systems, is thought to serve as an anchoring element between collagen I/III fibrils and basement membranes and as a cell binding substrate. We report on the expression of collagen VI in normal and in fibrotic lungs. Collagen VI is present in vascular and bronchial walls and in the interstitial space of normal lungs. Its turnover is too low to generate a mRNA signal by in situ hybridization. Collagen VI expression is increased in lung fibrosis, and its degree appears independent of the etiology of fibrosis. There was no evidence for differential regulation of gene expression for the alpha 1(VI) and alpha 3(VI) constitutive peptide chains of collagen VI. Collagen VI mRNA is expressed by fibroblasts, mostly with myofibroblast characteristics. Collagen VI was coexpressed with collagen III rather than collagen I in idiopathic bronchiolitis obliterans with organizing pneumonia, acute interstitial pneumonitis of the Hamman-Rich type, and bleomycin-induced fibrosis, but collagen VI overlapped with collagen types I and III in idiopathic pulmonary fibrosis. These coexpression data suggest that collagen VI expression may be an early rather than a late phenomenon in pulmonary fibrosis.
Collagen↗
Comparison of neutrophil chemotactic factor release by human and rabbit alveolar macrophages in response to tannin exposure.
It is essential to determine whether the results obtained from animal models actually reflect human disease processes. Tannin is a component of cotton dust that acts as a polyclonal cell activator in vitro. Most of the effects of tannin on alveolar macrophages (AM phi) have been studied in rabbit AM phi. Therefore, we compared tannin-mediated in vitro neutrophil chemotactic factor (NCF) secretion from normal human and rabbit AM phi. For both species the NCF secretion from AM phi was dependent on tannin dose and time of exposure. The NCF released was a lipid with a molecular weight of less than 800 daltons, suggesting that it may be a metabolite of arachidonic acid. Tannin stimulation of both human and rabbit AM phi resulted in the release of 90% unmetabolized arachidonic acid derived from both phosphatidyl choline and phosphatidyl inositol membrane lipids. The NCF secreted was not leukotriene B4 or platelet-activating factor. In conclusion, tannin mediates the release of a so far undescribed NCF from resident AM phi in rabbits and human subjects that may contribute to the pathogenesis of the acute neutrophilic alveolitis associated with cotton dust inhalation. The similarity of results obtained from human and rabbit cells supports the pertinence of using rabbit cells to study tannin-mediated effects.
Animals↗
Recombinant expression and properties of the Kunitz-type protease-inhibitor module from human type VI collagen alpha 3(VI) chain.
The Kunitz-type inhibitor motif (domain C5) present at the C-terminus of the human collagen alpha 3(VI) chain was prepared in a recombinant form from the culture medium of stably transfected kidney cell clones. The 76-residue protein was disulfide bonded and showed a high stability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikrein and several other proteases, which could be due to a few unusual substitutions in the region crucial for inhibitor binding. A sensitive radioimmunoassay detected low concentrations of C5 epitopes in normal human serum and fibroblast culture medium and showed a lack of cross-reaction with aprotinin. Antibodies against C5 immunoprecipitated collagen VI obtained from fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several alpha 3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N-terminal recombinant fragment. Immunofluorescence with anti-C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicate the rapid loss of the C5 domain after biosynthesis of collagen VI.
Amino Acid Sequence↗
Lesions of the respiratory tract associated with the finding of anti-neutrophil cytoplasmic autoantibodies with a perinuclear staining pattern.
OBJECTIVE: To characterize the clinicopathologic spectrum of respiratory tract involvement in patients with positive results of immunofluorescence microscopy for anti-neutrophil cytoplasmic autoantibodies with a perinuclear staining pattern (p-ANCA) and to assess the clinical value of p-ANCA testing. DESIGN: We retrospectively reviewed the medical records of all patients at Mayo Clinic Rochester in whom p-ANCA were detected by indirect immunofluorescence microscopy during 1992. MATERIAL AND METHODS: Additional target antigen identification was performed with use of enzyme-linked immunosorbent assays for antibodies against myeloperoxidase (MPO) and proteinase 3. We summarized the clinical findings in MPO-positive and MPO-negative patients. RESULTS: Sera were positive for p-ANCA in 42 of 2,381 patients (1.8%). In 13 of these 42 patients (31%), the respiratory tract was involved. Twelve patients had chest roentgenographic abnormalities, including a diffuse alveolar filling pattern (N = 8), a diffuse interstitial pattern (N = 2), and a combined interstitial and alveolar pattern (N = 2); three others had nasal inflammation. Ten of 16 sera tested were positive for MPO, and proteinase 3 antibodies were detected in 1 specimen. All patients with alveolar hemorrhage were positive for MPO antibodies. CONCLUSION: Testing for p-ANCA by immunofluorescence microscopy discloses a wide range of clinical disorders distinct from the main cytoplasmic-staining ANCA-associated disease--namely, Wegener's granulomatosis. In particular, the respiratory tract is affected much less frequently. Further evaluation of positive p-ANCA immunofluorescence test results by enzyme-linked immunosorbent assay to determine whether MPO is the target antigen is necessary to obtain clinically useful information from this test.
Adolescent↗
Anti-neutrophil cytoplasmic antibodies.
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Antibodies, Antineutrophil Cytoplasmic↗
Cutaneous Wegener's granulomatosis: clinical, histopathologic, and immunopathologic features of thirty patients.
BACKGROUND: Wegener's granulomatosis (WG) is a systemic disease characterized by necrotizing granulomatous inflammation and vasculitis. Its cutaneous manifestations vary. OBJECTIVE: We reviewed and characterized the clinical, pathologic, and immunopathologic features of the specific cutaneous manifestations of WG and investigated the sensitivity and the specificity of anti-neutrophilic cytoplasmic antibody (c-ANCA) in the cutaneous manifestations of this disease. METHODS: A retrospective analysis was conducted of 244 cases of WG observed between 1988 and 1992. RESULTS: Skin involvement occurred in 14% of the patients and was more frequent in generalized WG. Skin lesions may be an early premonitory sign of renal disease. Necrotizing ulcerations resembling pyoderma gangrenosum were not uncommon. Leukocytoclastic vasculitis was the most common cutaneous pathologic pattern. Findings of c-ANCA were positive in 81% of patients with cutaneous WG. CONCLUSION: Skin involvement usually occurred at presentation with generalized disease. c-ANCA is a valuable adjunct to diagnosis and follow-up of WG.
Adolescent↗
Immunohistochemical localization of collagen VI in diabetic glomeruli.
Late stage diabetic nephropathy is histologically characterized by either diffuse or nodular expansion of the glomerular matrix. This is presumed to represent the morphological correlate for the functional impairment of the kidney. The exact matrix composition of the nodular glomerulosclerosis lesion of end-stage diabetic nephropathy is not known. Biochemical studies have provided evidence that the microfibrillar collagen type VI is increased in diabetic nephropathy. Consequently, this immunohistochemical study was designed to evaluate the extent and exact morphologic location of increased collagen VI deposition at various stages of diabetic glomerulosclerosis (GS). An irregular, sometimes spot-like staining of collagen VI was observed in diffuse GS in the mesangial portion. The uninterrupted staining which was evident along the glomerular basement membrane in normal glomeruli was discontinuous in diffusely sclerotic glomeruli. In nodular GS, the markedly increased deposition of collagen VI appeared to be evenly distributed throughout the entire nodular lesion. At the same time, mesangial staining for collagen IV was reduced in nodular GS, suggesting that in the expanded mesangial matrix collagen IV is progressively substituted by collagen VI during the transition from diffuse to nodular GS. The colocalization of PAS staining with collagen VI deposition in nodular GS suggests that the typical Kimmelstiel-Wilson lesions at least in part consist of collagen VI. Biochemical analysis confirmed the increased collagen VI deposition in glomeruli extracted from diabetic patients with nodular GS. Application of two antisera, recognizing primarily the alpha 1(VI)- and alpha 2(VI)-chains and the N-terminal part of alpha 3(VI)-chain, respectively, revealed no difference in staining pattern.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Integrin and Arg-Gly-Asp dependence of cell adhesion to the native and unfolded triple helix of collagen type VI.
Pepsin-solubilized collagen VI in triple-helical and heat-denatured, unfolded form was shown to promote Mg(2+)- and Mn(2+)-dependent attachment and spreading of various cell lines. On the triple-helical substrate no inhibition of cell adhesion was observed with several synthetic RGD peptides except in the case of A375 melanoma cells. In contrast, adhesion to the unfolded substrate was highly sensitive to RGD inhibition. Nine synthetic peptides were designed according to 10 RGD sequences present in the triple-helical sequence of human collagen alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. Only one peptide, corresponding to the C-terminal end of alpha 3(VI) chain, showed substantial inhibitory activity, whereas several peptides were active in direct adhesion assays when used as albumin conjugates. Inhibition tests with antibodies to integrin subunits, affinity chromatography, and ligand binding with purified integrins (alpha 1 beta 1, alpha 2 beta 1, alpha V beta 3, and alpha IIb beta 3) were used to identify collagen VI receptors. Binding to the triple-helical substrate is mediated by alpha 1 beta 1 and alpha 2 beta 1 integrins. Binding of both integrins to collagen VI was weaker than that to collagens I and/or IV. Recognition of the denatured substrate is mediated by beta 1 and beta 3 integrins. Activity was shown for alpha 5 beta 1 and alpha V beta 3 and weakly for alpha IIb beta 3 but not all alpha subunits possibly involved were identified. Distinct sets of receptors were also involved in A375 cell binding to triple-helical (beta 1-mediated) and denatured (beta 3-mediated) collagen VI, even though in this case both interactions could be efficiently inhibited by RGD peptides.
Amino Acid Sequence↗
Implications of c-ANCA testing for the classification of Wegener's granulomatosis: performance of different detection systems.
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Aged↗