Biomedical subjects
Publications and source records attributed to J Lüdemann.
Elafin and antileukoprotease are potent peptide-like inhibitors of human leukocyte elastase and have been isolated from human skin and bronchial mucus. Elafin proved to be a potent inhibitor of proteinase 3, whereas other inhibitors of human leukocyte elastase such as antileukoprotease and eglin C proved to be much less effective.
Sera of 108 patients with chronic inflammatory bowel disease (IBD) and 13 control sera were screened for antibodies against neutrophil cytoplasmic antigens (ANCA) by an indirect immunofluorescence test. 37 out of 64 sera (58%) from patients with ulcerative colitis (UC) produced a fine granular and perinuclear ANCA staining pattern (p-ANCA) clearly different from the typically diffuse and granular cytoplasmic ANCA fluorescence (c-ANCA) seen in active Wegener's granulomatosis (WG). Only 1 of the 44 sera from patients with Crohn's disease and none of the control sera showed positive p-ANCA reactions. Only 1 of the 64 CU sera was positive for antinuclear antibodies, and another one showed a positive reaction in the anti-proteinase-3 ELISA which is specific for WG. Antibodies against myeloperoxidase were negative in the CU sera. 31 out of the 37 p-ANCA-positive sera (84%) were obtained from patients with high disease activity. p-ANCA titers became negative after long-term steroid therapy and after complete colectomy. These preliminary data suggest that ANCA screening may be of value in differentiating IBD and in monitoring disease activity and drug effects in UC patients.
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The target antigen of anti-neutrophil cytoplasm antibodies (ACPA; also known as ANCA) was isolated by affinity chromatography from supernatants of human neutrophils, stimulated with phorbol ester to induce degranulation. Sequence analysis of the antigen revealed 17 NH2-terminal amino acids (IVGGHEAQPHIRPIYMA), which have considerable sequence homology with known serine proteinases. Investigation of the enzymatic activity showed that the antigen is a neutral serine proteinase that is able to cleave elastin. Since the molecular weight of the antigen, its substrate specificity, and its inhibitor profile reported in this study are identical with those reported recently for proteinase 3, we conclude that ACPA are most probably directed against proteinase 3.
Sera of 64 patients with chronic inflammatory bowel disease (IBD) were screened for antibodies against neutrophil cytoplasmic antigens (ANCA) using an indirect immunofluorescence technique on ethanol-fixed human neutrophil granulocytes. 20 of 34 sera (59%) from patients with ulcerative colitis (UC) produced a fine-granular and perinuclear ANCA staining pattern (p-ANCA) clearly different from the typical diffuse and granular cytoplasmic ANCA fluorescence (c-ANCA, synonym ACPA) seen in active Wegener's granulomatosis (WG). The majority of the 20 p-ANCA positive UC patients had a high inflammatory disease activity. Among the 14 p-ANCA negative UC patients nine were without steroids; five of them had active disease, two were inactive and two had previously undergone colectomy. The remaining five patients still had active disease but received steroids for more than 4 weeks. Only 3 of the 30 sera from patients with Crohn's disease (CD) showed positive p-ANCA reactions. To narrow the specificity of the p-ANCA reaction all 64 sera were tested by ELISA for antibodies against anti-proteinase-3 (WG specific) and on HEp-2 cells for antinuclear (ANA) and anticytoplasmic antibodies. Ten p-ANCA positive UC sera were also tested in a myeloperoxidase ELISA. Only one UC serum reacted positively in the proteinase-3-ELISA and another one produced a weakly positive anti-nucleolar ANA fluorescence on HEp-2 cells. None of the tested sera reacted with myeloperoxidase suggesting that the p-ANCA staining pattern of granulocytes is not restricted to anti-myeloperoxidase antibodies as reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)
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To investigate the distribution of proteinase 3, the target antigen of anti-cytoplasmic antibodies (ACPA or C-ANCA), within the organelles of resting normal human polymorphonuclear leukocytes and monocytes, the authors used immunocytochemical techniques on thin frozen sections. To obtain valuable tools for immunolabeling, two murine monoclonal antibodies (MAbs) directed against the ACPA antigen were produced. In neutrophils, the authors observed immunogold label for the ACPA antigen, predominantly in myeloperoxidase-positive azurophil granules, and in smaller amounts on the plasma membrane. In monocytes, the ACPA antigen could be detected in small granules, which occasionally also contained myeloperoxidase, and rare labeling was found on the monocyte membrane. The finding that the ACPA antigen is expressed on the plasma membrane of neutrophils and monocytes, thereby becoming accessible to circulating autoantibodies, supports the supposition that ACPA are not only markers of disease activity, but also are involved in the pathogenesis of Wegener's granulomatosis.
The IgG-subclass distribution of IgG-anti-cytoplasmic autoantibodies (ACPA) was investigated. ACPA appeared in all IgG subclasses of 25 patients with Wegener's granulomatosis (WG), but at different quantities and frequencies. No IgG subclass with ACPA specificity is closer connected to activity in WG than IgG-ACPA. The data suppose a polyclonal induction of ACPA which are not directed against epitopes of polysaccharide origin most probably. Serum-IgG4 concentration was significantly elevated in WG patients (n = 25) compared to serum of healthy controls (n = 23).
Immuno-augmentation with substances of bacterial origin was studied in vitro for its ability to induce polyclonal B-cell activation. Biostim, Broncho-Vaxom, Omnadin, Paspat and OK 432 were compared for their B-cell mitogenicity with classical polyclonal B-cell activators (Staph. aureus Cowan I, KlebsM, Pokeweed mitogen). B-cell mitogenicity, as measured by 3H-thymidine incorporation into proliferating blood B-cells, was not induced by any of the studied preparations. On the other hand, OK 432 produced a T-cell dependent and Biostim a T-cell independent blood B-cell differentiation in immunoglobulin producing cells. However, the extent of immunoglobulin production was clearly less than with the polyclonal B-cell activator KlebsM. These results demonstrate that, in some of the preparations, in vivo polyclonal B-cell activation can be expected to occur.
STUDY OBJECTIVE: To determine disease specificity and sensitivity of anticytoplasmic autoantibodies (ACPA) for Wegener granulomatosis, as well as their value as a marker of disease activity. DESIGN: Blind analysis of serum samples, retrospective analysis of clinical data on patients, and prospective follow-up of a subgroup of patients with Wegener granulomatosis. PATIENTS: The study included 277 patients with Wegener granulomatosis (222 with biopsy-proven disease) and 1657 control patients. SETTING: University hospital and academic medical center. LABORATORY INVESTIGATIONS: Analysis of 2653 serum samples from 1934 patients for ACPA. Antibody detection was by indirect immunofluorescence and a new type of enzyme-linked immunoadsorbent assay (ELISA). Prospective follow-up was on 172 patients with Wegener granulomatosis. MEASUREMENTS AND MAIN RESULTS: Specificity of ACPA for Wegener granulomatosis measured by indirect immunofluorescence was 99% (CI, 98.9% to 99.7%) and 98% (CI, 97.4% to 99.2%) by ELISA. Sensitivity of ACPA depended on disease activity and extent: It was 67% (CI, 38% to 89%) by immunofluorescence and 60% (CI, 32% to 84%) by ELISA for patients with active locoregional symptomatology (n = 15); and 32% (CI, 14% to 54%) by immunofluorescence and 40% (CI, 21% to 61%) by ELISA for patients in full remission after initial locoregional symptoms (n = 25). The sensitivity was 96% (CI, 89% to 99%) by immunofluorescence and 93% (CI, 86% to 98%) by ELISA for patients with active generalized disease (n = 92). Serial testing was done; every patient with active generalized disease eventually had at least one positive serum sample. Sensitivity decreased to 41% (CI, 22% to 62%) by both immunofluorescence and ELISA for patients in full remission after active generalized disease (n = 27). Levels of ACPA expressed both as immunofluorescence titers and ELISA values (U/mL) correlated well with disease activity. CONCLUSIONS: Testing for ACPA in serum of patients with Wegener granulomatosis is valuable for differential diagnosis; furthermore, APCA can be used as a marker to follow disease activity. A new type of ELISA yielded the same results as indirect immunofluorescence for the specificity, sensitivity, and correlation with disease activity of ACPA.
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Autoantibodies directed against cytoplasmic components of neutrophil granulocytes and monocytes (ACPA) have previously been described as a disease-specific marker for Wegener's granulomatosis (WG). We have developed an ELISA for determining and quantifying ACPA using an affinity-purified antigen preparation. The antigen was purified from supernatants of human neutrophils stimulated with phorbol ester to induce degranulation, by means of affinity chromatography with naturally occurring human autoantibodies. The established ELISA was sufficiently specific and sensitive, and the ACPA concentrations obtained with it correlated significantly with the ACPA titres determined by an indirect immunofluorescence technique (Spearman's rank correlation coefficient = 0.85, P less than 0.001, n = 105 WG patients). This ELISA provides precise ACPA quantitation and should prove valuable for monitoring disease activity in WG.
Anticytoplasmic antibodies (ACPA) against myelomonocytic cells were demonstrable in 54 of 72 patients with Wegener's granulomatosis (WG). Other autoantibodies (including ANA, AMAb, RF and anti-ENA) were only found in a low frequence and titre. The presence of ACPA correlated with the severity and activity of the disease. These results demonstrated that ACPA is a valuable tool to establish the diagnosis and to monitor disease activity in WG.
Like "true" polyclonal B-cell activators (PBA) for murine B cells, crude membrane preparations of Klebsiella pneumoniae (Klebs M) and some other enterobacteriaceae stimulate human B cells to mature into immunoglobulin (Ig) secreting cells without significant prior proliferation and in the absence of T cells. To investigate the biochemically defined membrane component with this unique PBA property, we studied lipoprotein and murein isolated from E. coli, since other components (e.g., a variety of lipopolysaccharide (LPS) fragments) failed to imitate Klebs M as a PBA. Mononuclear cells (MNC) and B cell-enriched cell populations from healthy blood donors were stimulated with various doses of lipoprotein and murein and, in comparison, Klebs M and pokeweed mitogen (PWM). Cell cultures exposed to either lipoprotein, murein, or Klebs M failed to incorporate [3H]thymidine significantly after 5 days in culture. In contrast, there was significant DNA synthesis (stimulation index greater than 3) when PWM was given to the same MNC population. All stimulants, with the exception of lipoprotein, induced B-cell differentiation in MNC cultures, as measured by an ELISA quantitating secreted Ig in the culture supernatants. In cultures with B cell-enriched cell populations, however, only Klebs M and murein were able to induce the production of significant amounts of IgM. Thus, the actual PBA moiety contained in the crude membrane fraction (Klebs M) appears to be associated with murein. It is important to note that murein induced considerably weaker Ig secretion than Klebs M did.
The in vitro analysis of B-cell function has become more important recently. For this purpose, polyclonal B-cell activators (PBAs) and adequate test systems are now available. We compared the protein A plaque assay which is applied to determine the number of immunoglobulin(Ig)-secreting cells and the ELISA (enzyme linked immunosorbent assay) which is employed for the measurement of the amounts of secreted Ig. The number of Ig-secreting cells and the amounts of Ig in the culture supernatants after stimulation of mononuclear cells with PBAs correlated significantly, i.e. a B-cell activation can be detected by both test systems. The variation coefficients of the protein A plaque assay were higher than those of the ELISA. Furthermore, the protein A plaque assay is a very time-consuming method; therefore, an ELISA should be applied for the routine diagnostic of immunodeficiencies. Nevertheless, the protein A plaque assay remains important for the research of immunopathological diseases and for the determination of in vivo activated B-cells (spontaneous plaques) which can increasingly be observed in immunological disorders like AIDS (acquired immunodeficiency syndrome) or systemic lupus erythematosus.