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[Systematic lupus erythematosus and pregnancy].

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects primarily women of childbearing age. 20-30 years ago it was common belief that SLE is exacerbated during pregnancy. Recent studies, however, could not support this notion. The likelihood of an increase in clinical activity of SLE is similar during pregnancy and in nonpregnant patients. It correlates directly with the activity of disease at the time of conception. The pregnancy outcome in patients with SLE has improved considerably in recent years. The frequency of fetal loss, however, still remains higher in SLE patients than in healthy women. In most of the cases fetal loss is due to habitual abortions, intrauterine death and premature labors. The main reasons are anti-phospholipid antibodies and the activity of SLE at the onset of pregnancy, respectively during the pregnancy. For monitoring disease activity and especially for distinguishing between the development of eclampsia and exacerbation of SLE, periodic determinations of complement proteins CH50, C3, C4 and C3d are helpful. A prophylactic therapy e.g. with prednisone in pregnant SLE patients is not justified. However, when SLE is exacerbating immunosuppressive therapy with prednisone and, if needed in combination with azathioprine, should be administered. In cases with high antiphospholipid antibody titers and prior fetal loss a treatment with low-dose acetylsalicylic acid can be considered.

Female↗

CD23 interacts with a new functional extracytoplasmic domain involving N-linked oligosaccharides on CD21.

Human CD21 has been described as a receptor for the C3d,g and iC3b proteins of complement, for the Epstein-Barr virus, and also for IFN-alpha. We reported recently that CD23, a low affinity receptor for IgE (Fc epsilon R2), is a new functional ligand for CD21. To determine the site of interaction of CD23 on CD21, we analyzed the ability of purified recombinant CD23 incorporated into fluorescent liposomes to bind CD21 mutants bearing various deletions of extracytoplasmic short consensus repeats (SCRs). We found that the site of interaction of CD23 on CD21 is on SCRs 5 to 8, with contribution of SCRs 1 and 2. Tunicamycin treatment of CD21-transfected K562 cells strongly inhibited the binding of CD23-liposomes, suggesting that an N-linked sugar, present on SCRs 5 to 8, is involved in the CD23/CD21 interaction. By mutating together or individually, the three asparagines present on SCRs 5 to 8, asparagines (Asn) 370 and 295, but not Asn 492, were shown to be involved critically in the binding of CD23. Furthermore, we mapped the binding sites of a panel of anti-CD21 mAbs and found that at least six epitopes can be detected on CD21. The mAbs that inhibit the most CD23 binding to CD21 map in SCRs 5 to 8. This study indicates that SCRs 5 to 8 represent a novel functional domain on the CD21 molecule, and is the first demonstration of an activity of an extracytoplasmic region of the CD21 outside of SCRs 1 to 4.

Antibodies, Monoclonal↗

Patients with systemic lupus erythematosus are deficient in complement-dependent prevention of immune precipitation.

OBJECTIVE: A functional deficiency of complement has been implicated but not conclusively demonstrated in the pathogenesis of systemic lupus erythematosus (SLE). To test this, we studied several aspects of complement in 44 patients with SLE, 46 patients with rheumatoid arthritis and 102 blood donors. METHODS: Prevention of immune precipitation (PIP) was measured by an enzyme immunoassay, levels of C1q, C4 and C3 by rocket immunoelectrophoresis, C4A, C4B and C3d by enzyme-linked immunosorbent assay (ELISA), complement haemolysis (CH50) by standard methods and C4 allotypes by high-voltage agarose electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: PIP was significantly reduced in SLE (P<0.001); the defect was revealed by a sensitive assay measuring this function of complement but not by the other tests employed. The patients were clinically well at the time of study, and levels of C3d, which have been shown to correlate with disease activity, were normal. The defect was more common in patients with early disease (P = 0.009), supporting a role in aetiology or early pathophysiology. PIP was positively correlated with levels of C4 (P = 3 x 10(-5)) and in particular the C4A isotype (P = 9 x 10(-10)) whereas C4B was redundant. CONCLUSIONS: Our results reveal a defect in prevention of immune precipitation in SLE that is apparent at an early stage in the disease and correlates with low levels of C4A. These results indicate that subtle deficiencies of complement may predispose to SLE.

Adult↗

Complement activation in chronic liver disease.

Patients with HBsAg positive chronic active liver disease (CALD) and primary biliary cirrhosis (PBC) exhibit increased C3d concentrations and changes in the serum concentrations of the complement components consistent with activation of the classical and alternative pathways. In these patients the concentrations of the regulatory proteins, C3b inactivator (C3bINA) and beta IH globulin, are normal. Patients with HBsAg negative CALD and alcohol induced liver disease (ALD) exhibit no evidence of an increased level of complement system activation. In these patients diminished serum concentrations of complement components appear to be related to diminished hepatic synthetic function. C4 synthesis may be specifically reduced in autoimmune chronic active liver disease.

Complement Activation↗

The effect of anti-reactional drugs on complement components in the type II, erythema nodosum leprosum, reaction.

Seventeen patients with the type II (erythema nodosum leprosum) (ENL) reaction were studied. They received multidrug therapy and also the anti-reactional drugs prednisolone clofazimine or chloroquin, and we measured serum levels of complement components before treatment and after the reaction had subsided. Factor B was significantly elevated after treatment with each of the three drugs. C3 levels were significantly increased after treatment, the largest change being in patients treated with clofazimine. In these patients there was also a concomitant decrease in C3d levels. This suggests that clofazimine has complement modulating activity and we would recommend it as the drug of choice in treatment of the ENL reaction.

Chloroquine↗

Binding of soluble immune complexes to human lymphoblastoid cells. I. Characterization of receptors for IgG Fc and complement and description of the binding mechanism.

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423b(hu) were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells.

Anemia↗

CD21 augments antigen presentation in immune individuals.

CD21 (complement receptor 2, CR2) binds the C3 degradation products, iC3b and C3d, which are covalently linked to antigen or immune complexes in the process of complement activation. The ability of antigen-nonspecific B cells to present immune complexes containing high titers of acquired antibodies was tested. Influenza virus was incubated with serum from immune donors to create complement-containing complexes. These bound specifically to CD21 on transfected fibroblasts and B cell lines, as measured by microcytofluorimetry. Binding of immune complexes was ablated by inactivation of serum complement. In addition, the immunoglobulin in immune human serum blocked influenza binding to cells in the absence of complement, implying a minimal role for immunoglobulin-Fc receptor interactions in this system. Significant immune complex binding required a threshold level of CD21 expression, suggesting that only those cells with the highest levels of CD21 are likely to participate in the processing of macromolecular antigens. B cells pulsed with complement-influenza complexes elicited an augmented response from a panel of influenza-specific, class II-restricted T cell clones, as compared with those which had bound immunoglobulin-influenza complexes lacking complement. This enhanced response did not require CD35. In addition, B cell lines expressing higher levels of CD21 were more efficient in processing antigen than those with lower levels. These findings suggest that presentation of antigen by B cells in immune individuals is dependent on the binding of complement-antigen immune complexes to CD21.

Antigen-Antibody Complex↗

The complement system in hemolytic-uremic syndrome in childhood.

A dynamic estimation of the complement system was obtained by immunochemical estimation of C3, C4, C5, C1q, C3b + C3c, C3d, Ba in children with hemolytic-uremic syndrome. The presence of increased breakdown products of C3 (C3b + C3c, C3d) and of factor B (Ba) suggests an activation of the complement system possibly by the alternative pathway. No definite explanation for these complement abnormalities can be given.

Child↗

Diving decompression fails to activate complement.

The present study evaluated complement activation during decompression after air dives in a hyperbaric chamber. Intravascular bubbles were quantified by Doppler ultrasound scoring. Eighteen subjects completed 92 dives, of which 74 produced bubbles. Complement activation was assessed by plasma C3a des Arg and red-cell-bound C3d before and after each dive. These parameters of in vivo complement activation failed to show significant activation. In vitro complement activation susceptibility tests on pre-dive sera were performed to explore their association with in vivo complement activation and intravascular bubbles. Such tests failed to identify a distinct complement-sensitive group and did not correlate with in vivo complement activation during the dives and/or intravascular bubble appearance. Two subjects developed decompression sickness but were not different from the rest of the group regarding in vitro complement sensitivity or complement activation during dives.

Adult↗

Molecular cloning and chromosomal localization of human membrane cofactor protein (MCP). Evidence for inclusion in the multigene family of complement-regulatory proteins.

Membrane cofactor protein (MCP), a regulatory molecular of the complement system with cofactor activity for the factor I-mediated inactivation of C3b and C4b, is widely distributed, being present on leukocytes, platelets, endothelial cells, epithelial cells, and fibroblasts. MCP was purified from a human T cell line (HSB2) and the NH2-terminal 24-amino acid sequence obtained by Edman degradation. An oligonucleotide probe based on this sequence was used to identify a clone from a human monocytic (U937) cDNA library. Nucleotide sequencing showed a 43-bp 5'-untranslated region, an open reading frame of 1,152 bp, and a 335-bp 3'-untranslated region followed by a 16-bp poly(A) track. The deduced full-length MCP protein consists of a 34-amino acid signal peptide and a 350-amino acid mature protein. The protein has, beginning at the NH2 terminus, four approximately 60-amino acid repeat units that match the consensus sequence found in a multigene family of complement regulatory proteins (C3b-receptor or CR1, C3d-receptor or CR2, decay-accelerating factor, C4-binding protein, and factor H), as well as several other complement and non-complement proteins. The remainder of the MCP protein consists of 25 amino acids that are rich in serine and threonine (probable site of heavy O-linked glycosylation of MCP), 17 amino acids of unknown significance, and a 23-amino acid transmembrane hydrophobic region followed by a 33-amino acid cytoplasmic tail. The MCP gene was localized to human chromosome 1, bands 1q31-41, by analysis of human x rodent somatic cell hybrid clones and by in situ hybridization. This same genetic region contains the multigene family of complement-regulatory proteins, which is thereby enlarged to include the functionally and structurally related MCP.

Amino Acid Sequence↗

Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures.

BACKGROUND: Detection of immunoglobulin or complement bound to RBCs by using the DAT is valuable in the diagnosis of immune-mediated hemolytic anemia. Traditionally, the DAT has been performed by tube agglutination using anti-IgG or anti-C3d. The purpose of this study was to compare the tube agglutination DAT to gel microcolumn, affinity microcolumn, and flow cytometric DATs on RBCs coated in vitro and on patient RBC samples. STUDY DESIGN AND METHODS: RBCs from 84 patients were assessed by tube agglutination DAT, one gel microcolumn DAT, and two affinity microcolumn DATs. One affinity microcolumn assay was unmodified and one was modified by the addition of polyspecific antiglobulin or anti-IgG as a secondary antibody. RBCs from 15 of the 84 patients underwent analysis by flow cytometry with fluorescence-labeled anti-IgG. The assays were also compared by using D+ RBCs sensitized with serially adjusted concentrations of anti-D. RESULTS: Both tube agglutination and gel microcolumn DATs were positive in 49 patient samples; both assays were negative in 20 samples, and the results were discordant in 15. Gel microcolumn DATs were more likely than were tube agglutination DATs to detect IgG on RBCs. Affinity microcolumn DATs were less likely than gel microcolumn or tube agglutination DATs to detect IgG on RBCs. Flow cytometry results were the same as gel microcolumn results in 12 of 15 patient samples and the same as tube agglutination results in 13 of 15. Tube agglutination and both affinity microcolumn assays reacted with RBCs coated with anti-D that was diluted 1-in-100. The gel microcolumn and flow cytometry assays reacted with RBCs coated with anti-D diluted 1-in-400. There was no correlation between tube agglutination and gel microcolumn DATs in detecting bound C3d. CONCLUSION: Detection of IgG bound to RBCs was not consistent with the methods described. Gel microcolumn DATs were more sensitive than tube agglutination and affinity microcolumn DATs. Given the varied results of these assays, reference laboratories should not rely on a single method for DATs. More comprehensive testing should be performed when the tube agglutination DAT is negative in a patient with suspected immune-mediated hemolytic anemia. Further comparisons are necessary to determine the proficiency of flow cytometric assays.

Agglutination Tests↗

Immune complexes in erythema multiforme and the Stevens-Johnson syndrome.

Early cutaneous lesions of erythema multiforme or mucosal lesions of the Stevens-Johnson syndrome contain delicate granular deposits of immune reactants and/or complement components lodged in the walls of vessels of the papillary dermis. Such deposits are not present in normal, unaffected skin although they can be caused to occur there by injection of substances which increase vascular permeability. Factors which cause accumulation of mononuclear cells in the papillary dermis have not been elucidated but we suggest that receptors for the C3d fragment of the third component of complement could be responsible. Factors which cause intercellular edema in the epidermis, epidermal-dermal separation, or necrotic keratinocytes are unknown. We, and others, may now measured immune complexes in the serum of patients experiencing these reaction patterns by means of Raji cell radioimmunoassay, C1q precipitation, 125I-C1q binding, cryoprecipitation and with monoclonal rheumatoid factor. Cryoimmunoglobulins have also been described and antigens of Herpesvirus hominus have been measured in the cryoprecipitate of 2 patients who experienced erythema multiforme following recurrent herpesvirus infection. Erythema multiforme commonly accompanied serum sickness reactions which occurred following injection of immune horse serum in the 1980s (von Pirquet and Schick). Such reactions are now well established as due to circulating immune complexes in antigen excess.

Antigen-Antibody Complex↗

The presence of complements in amyloid plaques of Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker disease.

The presence of complements Clq, C4, C3, C3b, C3c and C3d, as well as amyloid P component, in the amyloid plaques of Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSS) brains was demonstrated by means of immunofluorescent and immunoperoxidase techniques. Positive reaction was not observed in other tissue elements, including the blood vessels. These findings may not be due to an adsorption, but to the immunological binding of complements to the amyloid. Proteins such as scrapie associated fibrils or prion in the brain of patients with "unconventional' slow virus diseases are related to the amyloid plaques. It is conceivable that the complements in amyloid are related to these proteins.

Amyloidosis↗

Clearance of complement by human vascular endothelial cells: effects of hypoxia/reoxygenation and IL-1beta activation.

Antibody-mediated rejection is characterized by deposits of complement (C) C4 and C3 split products on endothelial cells (ECs). C3 split products are critical mechanistically and diagnostically because they are deposited in amplified quantities, bind covalently to ECs and act as ligands for leukocytes. This study was designed to determine whether cultured vascular human ECs could clear covalently bound C3 split products from their surface. An immunoglobulin M (IgM) antibody against beta(2)-microglobulin of major histocompatibility complex class I antigens was used to activate C in human serum. Some cells were exposed to hypoxia/reoxygenation and/or interleukin 1beta (IL-1beta) prior to incubation with antibody. C3b/iC3b and C3d deposition on the cell surface was measured by flow cytometry. Incubation with antibody followed by human serum caused a dose-dependent deposition of C3b/iC3b and C3d. Over half of deposited C3b/iC3b and one-third of C3d were cleared from the cell surface during a 3-7-h incubation period with human serum. Neither hypoxia/reoxygenation nor IL-1beta further increased the deposition of C3b/iC3b and C3d, and only slightly modulated their rates of clearance. In summary, human ECs rapidly clear iC3b and C3d from their surface. This finding may have important diagnostic and mechanistic implications to transplantation because C3d is used as a marker of antibody-mediated rejection.

Antibodies, Monoclonal↗

Evidence for activation of the complement cascade in the hypoglossal nucleus following peripheral nerve injury.

Following hypoglossal nerve transection in adult rats, immunoreactivity for complement factor C3 and one of its degradation products C3d as well as C4d and immunoglobulin G (IgG) was observed in the ipsilateral hypoglossal nucleus. Double-labelling experiments indicated that these antigens were present in perineuronally located reactive microglial cells. In addition, increased levels of complement factor C3-mRNA was found in perineuronally located cells ipsilateral to nerve lesion. These results suggest that the complement cascade is locally activated in the vicinity of axotomized neuronal perikarya and that microglial cells have a key role in this process, alternatively that C3, C3d, C4d and IgG are involved in other so far unknown processes.

Animals↗

Complement (C3)-receptor-mediated phagocytosis of agarose beads by mouse macrophages. I. Intracellular degradation of agarose-bound C3bi and C3b by lysosomal enzymes.

The phagocytosis by macrophages of C3bi-coated agarose beads reached a plateau after 15 min, compared with 30 min for C3b-coated beads. By using 125I-labelled C3bi or C3b coupled to the agarose beads, we found that 70% and 95% of total radioactivity were removed from the beads after 12 h and 36 h of intracellular digestion, respectively. Intracellular degradation of C3bi linked to agarose beads was also demonstrated by testing binding of monoclonal antibodies against human C3c, C3g and C3d to beads extracted from the cells after phagocytosis. Such extracted beads also showed reduced attachment to new macrophages compared with non-ingested beads. Treatment of the cells with leupeptin, an inhibitor of the lysosomal enzyme cathepsin B, or with dextran sulphate to inhibit phagosome-lysosome fusion greatly reduced the release of labelled protein from the agarose during the first 12 h. These findings show that C3bi and C3b on agarose is destroyed intracellularly by lysosomal enzymes.

Animals↗

Complement consumption in a patient with necrobiotic xanthogranuloma and paraproteinemia.

Necrobiotic xanthogranuloma and paraproteinemia have been reported to be associated with acquired deficiency of the early components of the classic pathway of complement. We describe a patient in whom activation of complement was suggested by a high level of C3d, an accelerated catabolism of C1q, and a rapid consumption of C4 after an intravenous infusion of fresh-frozen plasma. This infusion was followed by an episode of pulmonary edema. Patients with complement deficiency or depletion should be monitored closely while receiving plasma, to avoid this potential complication.

Complement C1q↗

Circulating immune complexes and C3d in human parasitosis.

Using the Raji cell radioimmune assay, we found low levels of circulating immune complexes (IC) in a small percentage of patients with schistosomiasis and filariasis. C3d levels, measured by immunoprecipitation, were elevated in a large number of these patients, whereas complement levels were within normal limits. Proteinuria was not found in any of the 55 patients studied. Circulating IC or elevated C3d levels were not found in any of the 19 patients with hydatidosis. The increased C3d levels, apparently not related to circulating IC, may be due to direct complement activation by parasite antigens or to sequestered IC. The latter hypothesis appears more attractive because the highest levels of C3d were found in schistosomiasis whereas schistosome antigens were unable to activate complement in vitro.

Antigen-Antibody Complex↗