Cytoplasmic Antigens Research Articles

HEMATOPOIETIC STEM CELL SEPARATION FOR EXPERIMENTAL PURPOSES - METHODIC LIMITATIONS

Protooncogene CD117 is a cytokine receptor important for hematopoietic element development. Currently, we are not able to routinely separate a sufficient quantity of bone marrow CD117+ cells for experimental purposes. The aim of this study was to establish an immunomagnetic separation method for CD117+ hematopoietic stem cell isolation and to estimate the expression of chosen BCl-2 family protein members in these elements. 120 samples of human and murine bone marrow were acquired using the magnetic separation system. The cells were stained for CD117, BCl-2, BAX, and CD33 by an indirect fluorescent immunocytochemistry. The flow cytometry analysis showed only 2.6% CD117+ cells from human as well as mouse bone marrow which is insufficient for further experiments. Cytospin was not good for morphologic characterization and immunophenotyping due to the fragility and destruction of the studied cells. Therefore, cell suspension staining was selected and by this method we found CD117 positivity in 70% of the mononuclerar (CD33 positive) elements in the case of chronic myeloid leukaemia. Labelling of the BCl-2 family in this case showed antiapoptotic BCl-2 expression in 80 %, proapoptotic BAX expression in approximately 5%. Our results show that CD117 immunomagnetic separation from bone marrow material is not acceptable for experimental purposes. They demonstrate that the only practical useful for the bone marrow cell examination (morphology and immunophenotype) is cell suspension staining which uncovers the distribution of both cytoplasmic proteins and surface antigens of immature blood elements.

Human Hendra virus infection causes acute and relapsing encephalitis

To study the pathology of two cases of human Hendra virus infection, one with no clinical encephalitis and one with relapsing encephalitis. Autopsy tissues were investigated by light microscopy, immunohistochemistry and in situ hybridization. In the patient with acute pulmonary syndrome but not clinical acute encephalitis, vasculitis was found in the brain, lung, heart and kidney. Occasionally, viral antigens were demonstrated in vascular walls but multinucleated endothelial syncytia were absent. In the lung, there was severe inflammation, necrosis and viral antigens in type II pneumocytes and macrophages. The rare kidney glomerulus showed inflammation and viral antigens in capillary walls and podocytes. Discrete necrotic/vacuolar plaques in the brain parenchyma were associated with antigens and viral RNA. Brain inflammation was mild although CD68(+) microglia/macrophages were significantly increased. Cytoplasmic viral inclusions and antigens and viral RNA in neurones and ependyma suggested viral replication. In the case of relapsing encephalitis, there was severe widespread meningoencephalitis characterized by neuronal loss, macrophages and other inflammatory cells, reactive blood vessels and perivascular cuffing. Antigens and viral RNA were mainly found in neurones. Vasculitis was absent in all the tissues examined. The case of acute Hendra virus infection demonstrated evidence of systemic infection and acute encephalitis. The case of relapsing Hendra virus encephalitis showed no signs of extraneural infection but in the brain, extensive inflammation and infected neurones were observed. Hendra virus can cause acute and relapsing encephalitis and the findings suggest that the pathology and pathogenesis are similar to Nipah virus infection.

Identification of Human Spermatozoa Antigens Using Monoclonal Antibodies and the Alkaline Phosphatase Anti-Alkaline Phosphatase-Technique/Identifizierung von Antigenen humaner Spermatozoen mit Hilfe monoklonaler Antikörper und der alkalischen Phosphatase

Both cytoplasmic and surface-membrane antigens of human spermatozoa were detected by means of monoclonal antibodies (MoAbs) and of the alkaline phosphatase anti-alkaline phosphatase- (APAAP-) technique. Several advantages of this technique for the identification of sperm could be demonstrated. The labeling of cytocentrifuge preparations from 16 ejaculates proved the presence of glycosphingolipids, nuclear and mitochondrial antigens of spermatozoa. However, there were no HLA-molecules and other leukocyte antigens on sperm cells.

Lupus and T Cells

T-cell abnormalities and aberrant T helper cytokine profiles have been implicated in the loss of immune tolerance to nuclear and cytoplasmic antigens and linked to a variety of clinical manifestations in systemic lupus erythematosus (SLE). Here, we review the role of T cells in promoting and maintaining SLE in relation to their cellular and molecular abnormalities and provide an update on recent T cell-targeted therapeutic approaches for the restoration of T cell homeostasis in the disease.

Investigations on the nature and pathogenicity of circulating antikeratinocyte antibodies in dogs with pemphigus foliaceus

In humans with pemphigus foliaceus (PF), pathogenic autoantibodies are principally of IgG4 subclass and they cause superficial vesiculation when injected into neonatal mice. The objectives of this study were to determine the isotypes of circulating antikeratinocyte antibodies in dogs with PF, to assess whether serum antikeratinocyte antibody titres decreased during successful treatment, and to study whether such antibodies were pathogenic in passive transfers. Using indirect immunofluorescence with neonatal mouse skin substrates, circulating antikeratinocyte IgG antibodies were detected in 36 of 44 dogs with PF (82%). Serum autoantibodies belonged predominantly to IgG4 (three of 44; 80%) and IgG1 (30 of 44; 68%) subclasses. Antikeratinocyte IgG antibodies were detected in 16 of 20 normal dogs (80%), and these antibodies were IgG1 (16 of 20, 80%) but rarely IgG4 (two of 20; 10%) isotypes. In four dogs, IgG4 antikeratinocyte antibody titres decreased concomitantly to lesions nearing or reaching complete remission. In contrast, IgG or IgG1 titres remained stable or increased when lesions abated. Antikeratinocyte antibodies targeted mainly intercellular autoantigen(s) in the stratum granulosum, while in fewer dogs, such antibodies bound to cytoplasmic basal antigen(s). Intradermal injections of PF or pemphigus vulgaris (PV) IgG into neonatal mice caused subgranular or suprabasal acantholytic vesiculation without granulocyte infiltration, respectively. Similar transfers of normal dog IgG did not cause vesiculation. These observations suggest that antikeratinocyte IgG4 antibodies could be relevant to disease pathogenesis. Importantly, canine PF or PV IgG appear to be pathogenic when transferred passively into mice, causing vesiculation at epidermal levels similar to those of the natural disease.

An Antinuclear Antibody-Negative Patient With Lupus Nephritis

Systemic lupus erythematosus (SLE) is a typical autoimmune disease that's characterized by various autoantibodies to nuclear and cytoplasmic antigens. The presence of antinuclear antibodies (ANA) in serum is generally considered a decisive diagnostic sign of SLE. However, a small subset of SLE patients who had the typical clinical features of SLE was reported to show persistently negative ANA tests. Our report describes a 16-yr-old female who presented with the clinical manifestations of SLE such as malar rash, photosensitivity, arthritis, lymphopenia, pericarditis and proteinuria. The serum autoantibodies were all negative and renal biopsy showed that the histopathological changes of immune complex mediated the focal segmental necrotizing glomerulonephritis with crescent formation. She was treated with monthly pulse cyclophosphamide along with corticosteroids. During the 2-yr follow-up period, the proteinuria was markedly decreased and all of the ANA and anti-double stranded DNA antibody tests were negative. This case suggests that ANA may not be required in the pathogenesis of lupus nephritis.

Intracytoplasmic antigen study by flow cytometry in hematolymphoid neoplasm

Flow cytometric detection of intracellular antigens has become a standard method in establishing proper leukemic cell lineage affiliation. It has a non-debatable contribution to the diagnosis of hematolymphoid neoplasm as well as in minimal residual disease. Combination of analysis of fluorescence labeling and light scatter properties of cells allows rapid and better determination of target cell antigens. Regarding the detection of intracellular antigens, standardization of the procedure remains, however, a real challenge. Detection of intracellular antigens by flow cytometry (FCM) requires effective fixation and permeabilization of the cell membrane. In the available literature, some reports describe methodologies to achieve satisfactory results for detection of either cytoplasmic or nuclear antigens; however, no methodological consensus has yet been achieved among the laboratories. This article is an attempt to describe different approaches to detect intracellular molecules by FCM.

Long-term treatment results and the immunoglobulin G subclass distribution patterns of proteinase-3-antineutrophil cytoplasm antibody (ANCA) and myeloperoxidase-ANCA in ANCA-associated vasculitis

Small vessel vasculitis associated with antibodies to neutrophil cytoplasm antigens has been denominated antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV). Ninety-eight patients with various forms of AAV with renal involvement were studied retrospectively with regard to treatment, side-effects and outcome. The immunoglobulin G (IgG) subclass distribution patterns in serum were determined in 51 patients with nephelometry and those of anti-proteinase-3 (PR3) and anti-myeloperoxidase (MPO) in 44 patients by enzyme-linked immunosorbent assay. Fifty-nine patients with a mean age of 63 years were given treatment with intermittent intravenous regimens of cyclophosphamide and continuous corticosteroids, whereas 39 patients with a mean age of 58 years were given continuous oral treatment. Malignancy, mainly due to skin tumours, was more common in AAV than in the general population. The total IgG subclass distribution pattern was asymmetric. The response to PR3 was of IgG(1), IgG(3) and IgG(4) isotypes, while IgG(1) and IgG(3) predominated in the response to MPO. The aberrant IgG subclass distribution pattern detected in the autoantibodies may be of importance in the pathogenesis of AAV.

Sortase A localizes to distinct foci on the Streptococcus pyogenes membrane

Cell wall peptidoglycan-anchored surface proteins are essential virulence factors in many gram-positive bacteria. The attachment of these proteins to the peptidoglycan is achieved through a transpeptidation reaction, whereby sortase cleaves a conserved C-terminal LPXTG motif and covalently attaches the protein to the peptidoglycan precursor lipid II. It is unclear how the sorting reaction is regulated spatially and what part sortase localization plays in determining the distribution of surface proteins. This is mainly the result of inadequate immunofluorescence techniques required to resolve these issues in certain bacterial pathogens. Here we describe the utilization of the phage lysin PlyC to permeabilize the cell wall of Streptococcus pyogenes to antibodies, thereby allowing the localization of sortase A using deconvolution immunofluorescence microscopy. We find that sortase localizes within distinct membranal foci, the majority of which are associated with the division septum and colocalize with areas of active M protein anchoring. Sortase distribution to the new septum begins at a very early stage, culminates during septation, and decays after division is completed. This implies that the sorting reaction is a dynamic, highly regulated process, intimately associated with cell division. The ability to study cytoplasmic and membrane antigens using deconvolution immunofluorescence microscopy will facilitate further study of cellular processes in S. pyogenes.

Performance Comparison of Commercial Fixing and Permeabilizing Reagents

Flow cytometry is commonly used to characterize cellular antigens expressed both on the cell surface and inside the cell. As the technology advances, more researchers are using this tool to study intracellular activity to understand how cells communicate and respond to their environment. Intracellular targets of interest include cytoplasmic antigens and proteins, nuclear antigens and proteins, cytokines, etc. The quality of intracellular staining depends on the ability of the permeabilizing reagent to permeabilize the cell membrane for antibodies to get to the target of interest without destroying the cell. Five different commercial permeabilizing reagents were evaluated in a small study, Caltag Fix and Perm® Kit, BD IntraSure™ Kit**This product is produced under license from Leeds Teaching Hospitals NHS Trust., BD FACS™ Permeabilizing Solution 2 (FACS Perm 2), 1X FACS Lysing Solution, and BD Cytofix/Cytoperm™ Kit. Five categories of performance were evaluated: cell recovery, maintenance of surface staining, preservation of light scattering properties, intracellular staining, and background staining. Cell recovery was assessed using CD45 APC and BD TruCOUNT™ tube; surface staining maintenance was assessed using CD45 APC, CD45 PerCP-Cy5.5, CD19 PE, and CD20 PerCP; intracellular and background staining were assessed using anti-bcl-2 FITC, anti-Zap 70 PE, anti-TdT FITC, anti-Ki-67 FITC, and CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5 reagents. Caltag and BD IntraSure Kits performed well in the category of cell recovery. Caltag Kit, BD IntraSure Kit, and FACS Lysing Solution maintained cell surface staining after cell permeabilization. BD IntraSure Kit and FACS Lysing Solution preserved light scattering properties of cells after permeabilization. Using signal-to-noise ratio as the criterion to evaluate background and intracellular positive staining, FACS Lysing Solution and FACS Perm 2 detected anti-bcl-2 FITC; BD IntraSure Kit and FACS Lysing Solution detected anti-TdT FITC; BD IntraSure Kit, FACS Lysing Solution, FACS Perm 2, and BD Cytofix/Cytoperm Kit detected anti-Ki-67 FITC; BD IntraSure Kit, FACS Lysing Solution, and FACS Perm 2 detected anti-Zap 70 PE; all kits detected CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5. Results showed that there is no universal permeabilizing reagent for all intracellular staining assays. Each test reagent has its own strength and weakness. Choosing the best permeabilizing reagent for a particular assay depends on the target of interest and the output of the assay.

Pseudoglandular Hepatoid Differentiation in Endometrioid Carcinoma of the Ovary Simulates Oxyphilic Cell Change

A 42-year-old patient had a stage III ovarian endometrioid adenocarcinoma with areas of hepatoid carcinoma (HC) of clear cell and eosinophilic pseudoglandular type that was difficult to differentiate from endometrioid carcinoma of oxyphilic type and sex cord-stromal tumor. Immunohistochemically, endometrioid adenocarcinoma was positive for CA125, estrogen and progesterone receptors, CAM5.2, cytokeratin (CK) 7 and 19, and vimentin. HC areas were positive for hep par1, polyclonal carcinoembryonic antigens, CD10, alpha-fetoprotein, epithelial membrane antigens, and antimitochondrial antibodies and shared with endometrioid carcinoma focal CK7, and constant positive CK19, CAM5.2, and progesterone receptors. In the differential diagnosis, a hepatic immunophenotype of oxyphilic, mitochondriae-rich areas (demonstrated by antimitochondrial antibodies) was identified by HC specific (hep par1) and characteristic markers: canalicular, cytoplasmic and membranous polyclonal carcinoembryonic antigens, CD10 patterns, and alpha-fetoprotein). Trabecular and clear vacuolated areas of HC resembled luteinized cells of sex cord-stromal ovarian tumors, but the membranous positivity to CAM5.2 supported epithelial (hepatoid) identity. The partial preservation of an endometrioid immunophenotype in HC (positive CK7 and 19 and progesterone receptors) would support an origin from endometrioid carcinoma.

Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis

Pauci-immune focal necrotizing glomerulonephritis (FNGN) is a severe inflammatory disease associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Here we characterize autoantibodies to lysosomal membrane protein-2 (LAMP-2) and show that they are a new ANCA subtype present in almost all individuals with FNGN. Consequently, its prevalence is nearly twice that of the classical ANCAs that recognize myeloperoxidase or proteinase-3. Furthermore, antibodies to LAMP-2 cause pauci-immune FNGN when injected into rats, and a monoclonal antibody to human LAMP-2 (H4B4) induces apoptosis of human microvascular endothelium in vitro. The autoantibodies in individuals with pauci-immune FNGN commonly recognize a human LAMP-2 epitope (designated P(41-49)) with 100% homology to the bacterial adhesin FimH, with which they cross-react. Rats immunized with FimH develop pauci-immune FNGN and also develop antibodies to rat and human LAMP-2. Finally, we show that infections with fimbriated pathogens are common before the onset of FNGN. Thus, FimH-triggered autoimmunity to LAMP-2 provides a previously undescribed clinically relevant molecular mechanism for the development of pauci-immune FNGN.

Cutaneous vasculitis in breast cancer treated with chemotherapy

A patient from the University of California Los Angeles Medical Center who developed cutaneous vasculitis during the course of treatment for metastatic breast cancer is presented (status: post-lumpectomy and radiation therapy). Since the onset of vasculitis occurred during the course of therapy for the neoplasm, it was difficult to differentiate between drug-induced vasculitis and paraneoplastic vasculitis. The patient had been exposed to medications including gabapentin, methimazole, trastuzumab, fulvestrant, and letrozole — which could cause endothelial cell toxicity. Drug-induced small vessel vasculitis usually attacks the skin or subcutaneous parts of the skin. In cancer therapy, there have been case reports that hormonal drugs such as estrogen receptor antagonists, aromatase inhibitors, and epidermal growth factor receptor (EGFR) inhibitors can induce cutaneous vasculitis. On the other hand, paraneoplastic syndromes manifested as cutaneous vasculitis have been documented, possibly mediated by unknown immunological mechanisms associated with the tumor such as formation of immune complexes, direct antibody-mediated effects on endothelial cells, or direct effects of tumor cells on the vascular wall. Some patients with drug-induced cutaneous vasculitis have antineutrophil cytoplasm antibodies (ANCA) directed to one or more neutrophil cytoplasm antigens — the most common being granule protein myeloperoxidase (MPO), human leukocyte elastase (HLE), cathepsin G and lactoferrin. Some patients also have antibodies against histones and antiphospholipid. Serologic testing and measurements suggest an influence of therapy on vasculitis, yet the lack of sensitivity and specificity for a biomarker in endothelial injury indicate the need to search and evaluate new markers for improved predictive value of the tests, and to provide guidance in therapy.

CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis.

BackgroundWegener's Granulomatosis and Microscopic Polyangiitis are life-threatening systemic necrotizing vasculitides of unknown aetiology. The appearance of circulating antibodies to neutrophil cytoplasmic antigens (ANCA) is strongly associated with the development of the disease. A link between infection and disease has long been suspected, and the appearance of ANCA antibodies has been reported following bacterial and viral infections. The depletion of circulating B cells with monoclonal antibody therapy can induce remission, and this observation suggests a pathogenic role for B cells in this disease. As bacterial DNA is known to induce B cell proliferation and antibody production via TLR-9 stimulation, we have explored the possibility that unmethylated CpG oligodeoxynucleotide, as found in bacterial and viral DNA, may play a role in stimulating circulating autoreactive B cells to produce ANCA in patients with vasculitis.ResultsWe have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC in vitro. The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls. In spite of this, PR3 ANCA+ patients synthesised significantly higher amount of IgG ANCA than normal controls. In MPO ANCA+ patients, there was a tendency for patients to produce higher amount of ANCA than controls, however, the difference did not reach significance. Furthermore, we were able to detect circulating MPO-reactive B cells by ELISpot assay from the peripheral blood of 2 MPO+ ANCA vasculitis patients. Together, this indicates that circulating anti-neutrophil autoreactive B cells are present in ANCA+ vasculitis patients, and they are capable of producing antibodies in response to CpG stimulation. Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases.ConclusionCirculating ANCA autoreactive B cells are present in patients with ANCA+ vasculitis. The production of ANCA from these cells in response to unmethylated CpG stimulation lead us to propose that stimulation of these cells by immunostimulatory DNA sequences such as CpG oligodeoxynucleotide during infection may provide a link between infection and ANCA associated vasculitis. This phenomenon may also apply to other antibody mediated autoimmune diseases.

Flow cytometric immunophenotyping of cerebrospinal fluid.

Leptomeningeal disease is an important adverse complication occurring in patients with B and T cell lymphomas and acute leukemias of lymphoid and myeloid origin. Recent reports suggest that multiparameter flow cytometry immunophenotypic assessment of spinal fluid samples could improve the efficiency of detection of CNS involvement, due to its high specificity and greater sensitivity. However, spinal fluid samples are frequently paucicellular with a rapidly decreasing cell viability. Staining of spinal fluid therefore requires dedicated sample storage/transport, staining, and preparation protocols. The Basic Protocol in this unit outlines a consensus multiparameter (3- to 8-color) flow cytometry immunophenotypic protocol for the evaluation of CNS involvement of cerebrospinal fluid (CSF) samples by neoplastic cells. A Support Protocol describing the simultaneous assessment of surface and cytoplasmic antigens is also provided. Finally, in the Alternate Protocol, we describe a method to calculate absolute numbers of both normal and pathological cell subpopulations by adding counting beads to the assay.

Monoclonal autoantibodies from insulin-dependent diabetic patients: autoantibodies against β-cell surface or cytoplasmic antigens

To investigate the target antigen(s) recognized during the autoimmune process in insulin-dependent diabetes mellitus (IDDM), we produced human monoclonal antibodies by Epstein-Barr virus transformation of peripheral blood lymphocytes from a large number (n = 50) of newly diagnosed IDDM patients. Screening by indirect immunofluorescence assay, using the RINm5F rat insulinoma cell line and eight other human or rat tumour cell lines, was performed to identify monoclonal antibodies that reacted with either membrane or cytoplasmic antigens. Eighteen IgM monoclonal antibodies reacting with cytoplasmic antigens of RIN cells were obtained; 14 of them also showed a staining of the cytoplasm of various non-beta-cell lines, while four displayed a binding restricted to beta-cells among the panel tested. However, among three monoclonal antibodies reacting with the membrane of RIN cells, one (HMD-1) produced an IgG antibody with a binding restricted to the membrane of beta-cells (RIN, HIT, and normal rat islet cells). The membrane antigens of HMD-1 were identified in Western blotting as proteins with molecular weights of 64 and 70 kD. This antibody had no apparent cytotoxic effect on RIN cells. These data suggest that, apart from 'natural autoantibodies,' it is feasible to obtain human monoclonal antibodies from IDDM patients that bind specifically to the beta-cell cytoplasm or to the beta-cell membrane.

Anti-idiotypes against anti-neutrophil cytoplasmic antigen autoantibodies in normal human polyspecific IgG for therapeutic use and in the remission sera of patients with systemic vasculitis

Anti-neutrophil cytoplasmic antigen (ANCA) activity was inhibited in 15 out of 21 sera from patients with acute systemic vasculitis following incubation with normal polyspecific IgG for therapeutic use (IVIg). ANCA antibodies reacted with IVIg through idiotypic-anti-idiotypic interactions, as shown in competitive binding assays using F(ab')2 fragments from IVIg and affinity chromatography of ANCA IgG on Sepharose-bound F(ab')2 fragments from IVIg. Co-incubation of sera from patients with acute systemic vasculitis with paired autologous remission stage sera also resulted in inhibition of ANCA activity in acute sera. Remission sera contain IgM and IgG capable of interacting with beta and or alpha idiotypes of ANCA IgG from acute sera. Anti-idiotypic IgM may account for the lack of expression of ANCA activity in whole serum from patients in remission from systemic vasculitis, which were found to contain high titres of ANCA IgG. These observations suggest that remission of systemic vasculitis is associated with the generation of anti-idiotypes against autoantibodies rather than the suppression of production of ANCA autoantibodies. IVIg may modulate the activity of systemic vasculitis in vivo.

Anti-neutrophil cytoplasmic antibodies (ANCA) in inflammatory bowel disease: characterization and clinical correlates.

ANCA were detected by indirect immunofluorescence in 34 out of 67 patients with ulcerative colitis (UC, 51%) and in 14 out of 35 patients with Crohn's disease (CD, 40%). All but one ANCA-positive sera produced a perinuclear pattern of fluorescence (P-ANCA) on ethanol-fixed neutrophils. On paraformaldehyde-fixed neutrophils 76% of P-ANCA-positive sera in UC and 50% of P-ANCA-positive sera in CD produced cytoplasmic fluorescence, indicating that, indeed, cytoplasmic antigens are recognized by a considerable number of these sera. By Western blot analysis using whole neutrophil extract as a substrate 46% of sera from patients with UC and 32% of sera from patients with CD showed reactivity with either lactoferrin, polypeptides occurring as a doublet of 66/67 kD mol. wt, or polypeptides occurring as a doublet of 63/54 kD mol. wt, respectively. Identical patterns of reactivity have been observed among P-ANCA-positive sera from patients with autoimmune liver disease and rheumatoid arthritis. These data suggest that ANCA of restricted specificities are not specific for inflammatory bowel disease (IBD), but are present in diverse conditions characterized by chronic idiopathic inflammation.

Autoantibodies to endothelial cells and neutrophil cytoplasmic antigens in systemic vasculitis

The interaction of circulating autoantibodies with the endothelium may be an important mechanism in the pathogenesis of systemic vasculitis. In a prospective study, we looked for circulating antiendothelial cell autoantibodies (AECA) and anti-neutrophil cytoplasm autoantibodies (ANCA) in 80 patients with suspected systemic vasculitis. AECA were measured using an isotype-specific cellular ELISA incorporating human umbilical vein endothelial cells. ANCA activity was determined by indirect immunofluorescence and radioimmunoassay. Sequential studies were performed on sera from four cases with dual positivity, where autoantibody binding was compared with von Willebrand factor (vWF) concentration and disease activity. IgG AECA were significantly higher in the 27 ANCA-positive sera as compared with normal controls (P = 0.027) with IgG (P = 0.009) and IgA (P = 0.046) AECA isotypes correlating with ANCA positivity; in contrast, no differences were found between AECA levels in the ANCA-negative sera and the normal controls. Cross-inhibition studies pointed to the co-existence of two autoantibody populations. An association between autoantibody binding, disease activity and vWF concentration was found for both ANCA and AECA. Some patients with systemic vasculitis have detectable AECA that recognize different epitopes to ANCA and like ANCA, their titre correlates with disease activity and thus they may have a pathogenetic role in these conditions.

The specificity of anti-neutrophil cytoplasm autoantibodies in systemic vasculitides

Autoantibodies to neutrophil cytoplasmic antigens are found in patients with small or medium-size vessel systemic vasculitis such as Wegener's granulomatosis and microscopic polyarteritis. We studied the specificity of anti-neutrophil cytoplasm autoantibodies (ANCA) by examining the binding of sera from patients with different forms of systemic vasculitis, to neutrophil extract size-fractionated by gel filtration for high performance liquid chromatography. Sequential fractions were collected for solid-phase radioimmunoassay. Patients with Wegener's granulomatosis showed two peaks of activity over fractions of 12 and 2 kD. Patients with microscopic polyarteritis had two different peaks of activity over fractions of 100 and 25 kD. Patients with Takayasu's arteritis and dermal lymphocytic vasculitis were studied as controls (all were ANCA negative by indirect immunofluorescence studies and solid-phase radioimmunoassay). However, the patient with lymphocytic vasculitis showed a distinct peak of activity over 22 kD and patients with Takayasu's arteritis had a single peak of activity over 200 kD. These results suggest the binding pattern of sera to fractionated neutrophil extract could be useful in differentiating between different diseases within the spectrum of systemic vasculitis. Cryptic antigen recognition in certain vasculitides may only be revealed by this procedure. We conclude that the association of ANCA and systemic vasculitis may not be limited to diseases of small or medium-size vessels, but could also include large vessel disease such as Takayasu's arteritis.