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U Specks

Publications and source records attributed to U Specks.

At least 19 recordsLinked to original sources
PubMed2006

Agreement of anti-neutrophil cytoplasmic antibody measurements obtained from serum and plasma.

Serum and plasma are used interchangeably to measure anti-neutrophil cytoplasmic antibodies (ANCA), even though the release of ANCA target antigens during the preparation of serum could affect ANCA assays and cause discrepancies between the results obtained from serum and plasma. To what extent ANCA test results obtained from serum agree and correlate with results from plasma remains unknown. Therefore, a comprehensive comparison was performed using serum and plasma samples which were collected in 175 patients with active Wegener's granulomatosis at enrollment of a recent randomized trial. These paired serum and plasma samples were subjected to parallel ANCA testing by standard indirect immunofluorescence on ethanol-fixed neutrophils, a direct enzyme-linked immunoassay (ELISA) for proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA, and two different capture ELISAs for PR3-ANCA. The concordance of categorical serum and plasma ANCA results was assessed using kappa-coefficients. These were > 0.8 for all assays, indicating a very good concordance between positive and negative serum and plasma results. Spearman's correlation coefficients for serum and plasma PR3-ANCA values obtained by direct ELISA and both capture ELISAs were > or = 0.95 (P < 0.0001). Our study shows that serum and plasma samples can be used interchangeably for measuring ANCA.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2005

Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments.

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3-related autoimmune processes investigated in wild-type-, mNE- and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico-chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc-AAPV-pNA and Suc-AAPV-pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by alpha(1)-antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom-designed hNE inhibitor, Val(15)-aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species-specific assessment of neutrophil protease function and inhibition.

Animals↗
PubMed2003

Interaction of proteinase 3 with CD11b/CD18 (beta2 integrin) on the cell membrane of human neutrophils.

Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoimmune vasculitis, Wegener's granulomatosis, is a serine proteinase stored in granules of human neutrophils. As previously shown, PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. The current study demonstrates that membrane-bound PR3 colocalizes with the adhesion molecule CD11b/CD18 (beta2 integrin). Immunoprecipitation experiments using plasma membranes of phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils revealed coimmunoprecipitation of PR3 with CD11b/CD18, indicating their location in the same complex. PR3 was also detected in TritonX-100-insoluble cytoskeleton of plasma membranes isolated from unactivated and activated neutrophils. Release of cytoskeletal PR3 by salt treatment implied electrostatic interaction with the enzyme. The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) augmented membrane expression of PR3 in unactivated and PMA-stimulated neutrophils. PMSF significantly reduced adhesion of neutrophils to fibrinogen-coated plates and their NADPH oxidase activity. Moreover, the addition of exogenous PR3 (1-5 microg/ml) augmented the CD11b/CD18-dependent adhesion of neutrophils. Taken together, these results implicate the beta2 integrin of neutrophils in their membrane association with PR3 and suggest a role of PR3 in the modulation of cell adhesion.

CD11b Antigen↗
PubMed2001

Antineutrophil cytoplasmic antibodies reacting with the pro form of proteinase 3 and disease activity in patients with Wegener's granulomatosis and microscopic polyangiitis.

OBJECTIVE: Antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) are diagnostic markers for the small vessel vasculitides Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Correlation of disease activity with PR3 ANCA levels, as determined by standard methods, is not apparent in every patient. PR3 ANCA react with yet to be identified conformational epitopes. We have identified PR3 ANCA subsets that react differentially with mature recombinant PR3 (rPR3; lacking the N-terminal activation dipeptide) and the pro form of this enzyme (pro-rPR3). The present study was performed to determine the association of these PR3 ANCA subsets with disease activity. METHODS: Sera from 61 PR3 ANCA-positive patients with WG or MPA were assayed by capture enzyme-linked immunosorbent assay using pro-rPR3 and rPR3 as target antigens, and were correlated with disease activity as determined by the Birmingham Vasculitis Activity Score (BVAS). RESULTS: Median levels of PR3 ANCA reacting with pro-rPR3 were higher during active (n = 32) than during inactive (n = 29) disease (P = 0.016). Reactivity with mature rPR3 was not significantly different (P = 0.71). Serial followup in individual patients also indicated better correlation of PR3 ANCA reactivity with pro-rPR3 than with mature rPR3. CONCLUSION: PR3 ANCA subsets reactive with epitopes accessible on pro-PR3 correlate better with disease activity than do subsets reactive with epitopes accessible only on mature PR3. This observation may explain why ANCA levels determined with current standard methods are suboptimal for monitoring disease activity. It raises new questions about the primary target of the PR3 ANCA immune response in patients with small vessel vasculitis.

Adolescent↗
PubMed2001

A disease-specific activity index for Wegener's granulomatosis: modification of the Birmingham Vasculitis Activity Score. International Network for the Study of the Systemic Vasculitides (INSSYS).

OBJECTIVE: To refine and validate the Birmingham Vasculitis Activity Score (BVAS) as a disease-specific activity index for Wegener's granulomatosis (WG). METHODS: Sixteen members of the International Network for the Study of the Systemic Vasculitides (INSSYS) revised the BVAS, with 3 goals: to reduce the redundancy of some component items, to enhance its ability to capture important disease manifestations specific to WG, and to streamline the instrument for use in clinical research. We defined the items and weighted them empirically as either minor (e.g., nasal crusting = 1 point) or major (e.g., alveolar hemorrhage = 3 points). We then validated the new, disease-specific BVAS/WG in 2 simulation exercises and a clinical case series that involved 117 patients with WG. RESULTS: We removed 38 items from the original BVAS, revised 9 items, and added 7 new items. Correlations between the scores on the BVAS/WG and the physician's global assessment (PGA) of disease activity were high, even when patients in remission were excluded. In the clinical case series, Spearman's rank correlation coefficient between the BVAS/WG and the PGA was r = 0.81 (95% confidence interval 0.73-0.87). The interobserver reliability using intraclass (within-case) correlation coefficients in the 2 simulation exercises was r = 0.93 for the BVAS/WG and r = 0.88 for the PGA in the first and r = 0.91 for the BVAS/WG and r = 0.88 for the PGA in the second. There was no significant observer effect in the scoring of the BVAS/WG or the PGA. The discriminant validity of the BVAS/WG was good: r = 0.73 (95% confidence interval 0.43-0.83). CONCLUSION: The BVAS/WG is a valid, disease-specific activity index for WG. Tested in simulation exercises and in actual patients, the BVAS/WG correlates well with the PGA, is sensitive to change, and has good inter- and intraobserver reliability. The INSSYS will use the BVAS/WG to assess the primary outcome in a phase II/III trial of etanercept in WG.

Granulomatosis with Polyangiitis↗
PubMed2001

Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy.

We report on the successful, compassionate use of the anti-CD20 chimeric monoclonal antibody rituximab in a patient with chronic, relapsing cytoplasmic antineutrophil cytoplasmic antibody (cANCA)-associated Wegener's granulomatosis (WG). The patient initially responded to treatment with glucocorticoids and cyclophosphamide. However, bone marrow toxicity during cyclophosphamide treatment of a relapse precluded its further use. Azathioprine and mycophenolate mofetil treatment had failed to maintain remission of the WG, and methotrexate was contraindicated. Because the patient's 5-year course was characterized by close correlation of cANCA levels with disease activity, selective elimination of cANCA was deemed a treatment option for his latest relapse. He was given 4 infusions of 375 mg/M2 of rituximab and high-dose glucocorticoids. Complete remission was associated with the disappearance of B lymphocytes and cANCA. Glucocorticoid treatment was then discontinued. After 11 months, the cANCA recurred, and rituximab therapy was repeated, without glucocorticoids. At 8 months after the second course of rituximab (18 months after the first course), the patient's WG has remained in complete remission. Elimination of B cells by rituximab therapy may prove to be an effective and safe new treatment modality for ANCA-associated vasculitis and possibly other autoimmune diseases. This modality warrants closer examination in a carefully conducted clinical trial.

Aged↗
PubMed2001

Diabetes insipidus and anterior pituitary insufficiency as presenting features of Wegener's granulomatosis.

Central diabetes insipidus (DI) is a rare complication of Wegener's granulomatosis (WG), which usually presents after pulmonary or kidney involvement. Anterior pituitary dysfunction secondary to WG has been extremely rare, documented in only three cases. We report a case of a 47-year-old postmenopausal woman who was diagnosed with hypopituitarism in November 1999 and started on vasopressin, thyroxine, and hydrocortisone. She sought treatment at the Mayo Clinic in February 2000 with a purpuric rash, fever, cough, shortness of breath, and blood in the sputum. Computed tomography of the chest showed a 6-cm irregular mass in the right lower lobe, and a biopsy of the mass showed marked reactive atypia and necrosis. Positive C-antineutrophil cytoplasmic antibodies (ANCA) and skin biopsy of a purpuric lesion showing leukocytoclastic vasculitis confirmed the diagnosis of WG. Hormonal studies showed low gonadotropins, thyroid-stimulating hormone (TSH), and prolactin. Magnetic resonance imaging (MRI) of the head showed cystic enlargement of the pituitary gland that did not enhance with gadolinium. Two months into the treatment with cyclophosphamide and prednisone, she had persistent pituitary dysfunction, despite the normal appearance of the pituitary gland on repeat MRI. We conclude that WG should be included in the differential diagnosis of DI and anterior pituitary dysfunction in the proper clinical setting. Early diagnosis and treatment may be crucial in preventing pituitary gland destruction and long-term endocrine sequelae. We suggest screening for anterior pituitary failure in the presence of the WG-associated DI.

Diabetes Insipidus↗
PubMed2001

Are antineutrophil cytoplasmic antibodies pathogenic? Experimental approaches to understand the antineutrophil cytoplasmic antibody phenomenon.

Antineutrophil cytoplasmic antibodies (ANCA) directed against the neutrophil enzymes PR3 and MPO are tightly associated with the development of small vessel vasculitis. This article reviews the large body of data derived from in vitro experiments documenting many different proinflammatory effects of these ANCA on neutrophils, monocytes, and endothelial cells. Taken in conjunction with clinical observations and data from animal models, a concept of the pathogenicity of ANCA emerges.

Animals↗
PubMed2001

Diffuse alveolar hemorrhage syndromes.

Diffuse alveolar hemorrhage (DAH) is a rare yet serious and frequently life-threatening complication of a variety of conditions. DAH may result from coagulation disorders, inhaled toxins, or infections. Most cases of DAH are caused by capillaritis associated with systemic autoimmune diseases such as antineutrophil cytoplasmic antibodies-associated vasculitis, anti-glomerular basement membrane disease, and systemic lupus erythematosus. Early recognition is crucial, because the prompt institution of supportive measures and immunosuppressive therapy is required for survival. Our understanding of DAH and its management is largely empiric and based on small case series and individual reports, many dating back more than one decade. To provide the practicing specialist with a rational diagnostic and management approach to the patient with DAH, this review summarizes the most recent publications and salient information derived from older publications.

Anti-Glomerular Basement Membrane Disease↗
PubMed2000

Recombinant proteinase 3 produced in different expression systems: recognition by anti-PR3 antibodies.

Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase 3 (PR3) are highly sensitive and specific markers for Wegener's granulomatosis (WG). Consequently, antigen-specific assays for detection of PR3-ANCA are helpful for the diagnosis and follow-up of patients with WG. Purification of PR3 is laborious and requires large amounts of granulocytes. Therefore, several attempts have been made to produce recombinant PR3 that is recognized by PR3-ANCA. The purpose of this study was to compare the recognition of different recombinant forms of PR3 (rPR3) by anti-PR3 antibodies. Recombinant PR3 produced in E. coli (rcPR3), P. pastoris (rpPR3), insect cells using the baculovirus system (rbPR3), the human mast cell line, HMC-1 (HMC-1/PR3-S176A), or the human epithelial cell line, 293 (Delta-rPR3-S176A) as well as purified neutrophil PR3 (nPR3) were used. Recognition of these rPR3s by anti-PR3 antibodies was determined by direct and capture ELISA with 19 PR3-ANCA sera, 13 anti-PR3 mAbs and a rabbit serum raised against human PR3. In the capture ELISA rabbit anti-PR3 strongly bound nPR3 and all rPR3 products. By capture ELISA rcPR3 and rpPR3 were recognized by 11 (57%) and 13 (68%) of the 19 PR3-ANCA sera, respectively, whereas rbPR3, HMC-1/PR3-S176A, Delta-rPR3-S176A and nPR3 were recognized by all PR3-ANCA sera. By direct ELISA rabbit anti-PR3 strongly bound nPR3 and all tested rPR3 products. Using the direct ELISA none of the PR3-ANCA sera recognized rcPR3, whereas rpPR3 and rbPR3 were recognized by two (11%) and 17 (89%) of the 19 PR3-ANCA sera, respectively. All 13 anti-PR3 mAbs recognized nPR3 in the direct as well as in the capture ELISA. The rcPR3 was recognized by two mAbs in the capture ELISA but by none of the mAbs in the direct ELISA. The rpPR3 was recognized by seven mAbs in the capture ELISA and only by two mAbs in the direct ELISA. All but one of the anti-PR3 mAbs recognized rbPR3, whereas HMC-1/PR3-S176A and Delta-rPR3-S176A were recognized by all anti-PR3 mAbs. In conclusion, rPR3 expressed in insect cells, HMC-1 and 293 cells is recognized by anti-PR3 antibodies, whereas conformational epitopes recognized by anti-PR3 mAbs and PR3-ANCA are not well preserved on rPR3 expressed in E. coli or P. pastoris.

Adult↗
PubMed2000

What you should know about PR3-ANCA. Conformational requirements of proteinase 3 (PR3) for enzymatic activity and recognition by PR3-ANCA.

The neutrophil azurophil granule constituent proteinase 3 (PR3) is the principal antigen for anti-neutrophil cytoplasmic antibodies (ANCA) in Wegener's granulomatosis. The conformation of the mature PR3 enzyme results from intracellular post-translational processing. The nascent molecule undergoes proteolytic cleavage of the amino-terminal signal peptide and activation dipeptide and of a carboxy-terminal peptide extension. The conformation of PR3 is stabilized by four disulfide bonds and, to a lesser extent, by asparagine-linked glycosylation. Most anti-neutrophil cytoplasmic antibodies directed against proteinase 3 (PR3-ANCA) recognize conformational epitopes. The expression of recombinant PR3 has provided a better understanding of the significance of the various intracellular processing steps for enzymatic activity and recognition by PR3-ANCA.

Animals↗
PubMed2000

Are animal models of vasculitis suitable tools?

Several rodent models have been proposed for various forms of systemic vasculitis. The MRL-lpr mouse has been studied extensively as a model for systemic lupus erythematosus. Backcross experiments in combination with genetic linkage studies have firmly established that the phenotype of autoimmune disease is dependent on the combination of various background genes. It has also become apparent that environmental factors, particularly infections, modulate the disease phenotype. Specific interventions, such as the treatment of Brown Norway rats with agents resulting in polyclonal B cell stimulation or immunization with human myeloperoxidase and subsequent localized perfusion with neutrophil lysosomal extract and H2O2, have provided substantial insights into the cellular and molecular mechanisms leading to the development of vasculitis and glomerulonephritis. Even though the existing models may not exactly mirror any specific human disease, they offer reproducible, highly controlled conditions to answer specific questions about pathogenesis and novel therapeutic approaches.

Animals↗
PubMed2000

Meningeal involvement in Wegener granulomatosis.

We describe 2 cases and review the literature on the spectrum of clinical manifestations associated with meningeal involvement in patients with Wegener granulomatosis (WG). A 31-year-old man and a 60-year-old woman with WG in complete clinical remission developed severe chronic headaches. No inflammatory activity was detectable at sites of previous disease activity, and nonspecific markers of inflammation were within normal limits. However, both patients had persistently elevated titers of antineutrophil cytoplasmic antibodies (cytoplasmic staining pattern). Cerebrospinal fluid examinations showed no abnormalities that suggested inflammation, infection, or malignancy. Head magnetic resonance imaging with gadolinium contrast revealed enhancement of the dura in both patients, and a meningeal biopsy in 1 patient confirmed active WG. Both patients' symptoms resolved after reinstitution of aggressive immunosuppressive therapy.

Adult↗
PubMed1998

Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes.

Proteinase 3 (PR3), a constituent of azurophil granules of neutrophils (polymorphonuclear cells, PMNs), is the target antigen for most anti-neutrophil cytoplasmic antibodies (c-ANCA) in Wegener's granulomatosis (WG). We have recently developed an expression system for recombinant PR3 (rPR3) that is recognized by c-ANCA. Here, we report on the development and characterization of two monoclonal antibodies (moABs) and a rabbit polyclonal antiserum generated against this rPR3. Epitope competition analysis indicates that the moABs MCPR3-1 and MCPR3-2 recognize overlapping epitopes on the PR3 molecule that are distinct from the ones recognized by moABs 4A5 and 6A6 developed by others. Since MCPR3-2 does not appear to compete for epitopes recognized by a sizable proportion of PR3-ANCA, we used it to develop a sensitive capture enzyme linked immunosorbent assay (ELISA) for clinical PR3-ANCA testing. Both purified PMN PR3 and crude human mast cell line (HMC-1)/PR3-S176A cell lysates were used as sources of PR3 target antigen in this assay with equal analytical sensitivity and specificity. Of 109 patients with ANCA-associated disease, 91 (83.5%) and 90 (82.6%) were PR3-ANCA positive by capture ELISA when PMN-PR3 and HMC-1/PR3-S176A cell lysates were used as antigen, respectively. When HMC-1/PR3 and HMC-1/PR3-S176A cells were used as indirect immunofluorescence (IIF) substrate, 88 (80.7%) and 92 (84.4%) were PR3-ANCA positive, respectively. These differences were not statistically significant. Only 1 of 151 controls without defined ANCA-associated disease tested positive by capture ELISA with either target antigen (both negative by PR3-ANCA specific IIF). The capture ELISA can also be used to detect of PR3-ANCA immunecomplexes and, in combination with the rabbit antiserum, for the quantitative measurement of PR3 in biological fluids.

Animals↗