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PubMed1992

Functional evaluation of T helper, T suppressor, and B lymphocytes in lethally irradiated rhesus monkeys injected with autologous bone marrow.

Lethally irradiated rhesus monkeys were treated with autologous bone marrow that had either been (1) nontreated, i.e., normal; (2) depleted of T lymphocytes with a monoclonal antibody directed against rhesus T lymphocytes; (3) fractionated with the soybean agglutinin (SBA- fraction); or (4) fractionated with SBA and further depleted of T cells by E-rosetting. There was no difference in hematologic reconstitution among the animals, but all showed a marked lowering of the T helper/T suppressor ratio during the first 10 months posttransplant and reduced capability of their peripheral blood leukocytes (PBL) to produce Ig upon stimulation with pokeweed mitogen. This subnormal ability of PBL to produce Ig, as measured by plaque-forming cells in a reverse hemolytic plaque assay, was not explained entirely by the altered T-H/T-S ratio but was correlated with the functional status of the T-H, T-S, and B lymphocytes. Isolated populations of the different lymphocyte subsets from PBL of the experimental animals were cocultured with normal cells of the appropriate subset to obtain Ig synthesis when stimulated with PWM. Animals treated with normal bone marrow showed recovery of T-H cell function after 5 months, but their T-S cells showed excessive suppressor activity that persisted for 20 months posttransplant. In contrast, those animals receiving treated marrow (mAb plus complement, or SBA) showed a much-delayed (12 months or more) return to normal T-H cell function and an earlier return of T-S cells expressing a normal level of suppressor activity. Since the SBA- fraction of marrow contains very few or no T-H cells and T cell depletion of marrow with mAb also removes these cells, it is suggested that the kinetics of immune recovery of the different lymphocyte subsets of PBL is influenced by the presence or absence of T-H cells in the marrow inocula.

Animals↗
PubMed1986

A product of the C4B locus lacking hemolytic activity.

An unusual C4B allotype, C4B 4I, was identified in a study of C4 polymorphism in Japanese. This variant was defined as C4B using a murine monoclonal antibody specific for the C4d region of C4B. Hemolytic assay, however, revealed that the C4 variant, C4 BI was hemolytically inactive in contrast to other well-defined C4B locus products.

Alleles↗
PubMed1967

Deficiency of the fifth component of complement in mice with an inherited complement defect.

The inherited complement deficiency of certain inbred strains of mice was shown to be due to an isolated lack of the fifth component of complement. The protein MuB1 (or hc'), which is present in normal mouse serum but absent from the serum of complement-deficient mice, was shown to be immunochemically related to the fifth component of human complement (C'5). C'5 hemolytic activity was specifically inhibited in human serum by mouse anti-MuB1 and in normal mouse serum by mouse antiserum to human C'5. Highly purified human C'5 reconstituted the hemolytic activity of complement-deficient mouse serum. It was, therefore, concluded that he', or MuB1, constitutes the murine analogue of the fifth component of human complement. The MuB1 concentration in normal mouse serum was found to be subject to a sex-related variation. By quantitative precipitin analysis it was demonstrated that serum from male mice contains twice as much MuB1 as that of female mice. This difference in C'5 concentration was also detected by hemolytic assay. In addition, C'6 and C'7 were also found to be subject to sex-related variations.

Animals↗
PubMed1994

Monocyte chemoattractant protein production in red cell incompatibility.

BACKGROUND: Mononuclear phagocytes play a central role in hemolytic transfusion reactions by erythrophagocytosis and production of inflammatory mediators. Factors that affect the number or function of monocyters would be expected to alter the clinical course of hemolytic transfusion reactions, and thus the production of monocyte chemoattractant protein-1 (MCP-1), a recently described chemotactic and activating cytokine specific for monocytes, was investigated in two distinct settings of red cell (RBC) incompatibility. STUDY DESIGN AND METHODS: Fresh heparinized whole blood was incubated with ABO-compatible or -incompatible RBCs. Isolated peripheral blood mononuclear cells were incubated with anti-D-coated or uncoated RBCs. MCP-1 was measured in the plasma or culture medium by enzyme-linked immunosorbent assay. MCP-1 gene expression was detected by Northern blot analysis of buffy coat or mononuclear cell total RNA. RESULTS: Significant levels of MCP-1 protein in plasma or medium were detected 24 hours after the addition of incompatible RBCs, but not in the first 6 hours. Nonimmune hemolysis of added RBCs did not stimulate MCP-1 production. The inactivation of complement by heat treatment of plasma prior to the addition of RBCs to whole blood did not prevent MCP-1 production. Nor did neutralizing antibodies to tumor necrosis factor prevent MCP-1 production in ABO incompatibility. MCP-1 production was associated with increased steady-state levels of white cell MCP-1 mRNA, which occurred more rapidly in ABO than Rh incompatibility. CONCLUSION: The monocyte-specific chemotactic cytokine MCP-1 is produced by peripheral blood leukocytes in response to RBC incompatibility. MCP-1 may act in a positive feedback loop to recruit and activate monocytes during hemolytic transfusion reactions, thus contributing to the maintenance of these reactions.

ABO Blood-Group System↗
PubMed1988

Pathogenesis of Campylobacter fetus infections. Failure of encapsulated Campylobacter fetus to bind C3b explains serum and phagocytosis resistance.

Campylobacter fetus ssp. fetus strains causing systemic infections in humans are highly resistant to normal and immune serum, which is due to the presence of high molecular weight (100,000, 127,000, or 149,000) surface (S-layer) proteins. Using serum-resistant parental strains (82-40 LP and 23D) containing the 100,000-mol wt protein and serum-sensitive mutants (82-40 HP and 23B) differing only in that they lack the 100,000-mol wt protein capsule, we examined complement binding and activation, and opsono-phagocytosis by polymorphonuclear leukocytes. C3 consumption was similar for all four strains but C3 was not efficiently bound to 82-40 LP or 23D even in the presence of immune serum, and the small amount of C3 bound was predominently the hemolytically inactive iC3b fragment. Consumption and binding of C5 and C9 was significantly greater for the unencapsulated than the encapsulated strains. Opsonization of 82-40 HP with heat-inactivated normal human serum caused greater than 99% killing by human PMN. Similar opsonization of 82-40 LP showed no kill, but use of immune serum restored killing. Findings in a PMN chemiluminescence assay showed parallel results. Association of 32P-labeled 82-40 HP with PMN in the presence of HINHS was 19-fold that for the 82-40 LP, and electron microscopy illustrated that the difference was in uptake rather than in binding. These results indicate that presence of the 100,000-mol wt protein capsule on the surface of C. fetus leads to impaired C3b binding, thus explaining serum resistance and defective opsonization in NHS, mechanisms that explain the capacity of this enteric organism to cause systemic infections.

Blood Bactericidal Activity↗
PubMed1985

Specific anti-erythrocyte focus formation as a measure of autoantibody secreting cells in NZB mice.

Using a modification of the antibody forming cell (AFC) focus assay, it is possible to quantitate the spontaneous anti-erythrocyte autoantibody response of New Zealand Black (NZB) mice at the level of the autoantibody secreting cell. This complement independent assay is specific for an antigen(s) present on unmodified mouse erythrocytes. A comparison with the direct Coombs' test showed that the focus assay detected autoantibody responses earlier and demonstrated a wide range of AFC levels in responding mice. The focus assay provides a method for evaluating autoimmunity at the cellular level and for investigating the activities of cells responding to autoantigens.

Anemia, Hemolytic, Autoimmune↗
PubMed2006

De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement factor H associated with atypical hemolytic uremic syndrome.

Many of the complement regulatory genes within the RCA cluster (1q32) have arisen through genomic duplication and the resulting high degree of sequence identity is likely to predispose to gene conversion events. The highest degree of identity is between the genes for factor H (CFH) and five factor H-related proteins--CFHL1, CFHL2, CFHL3, CFHL4, and CFHL5. CFH mutations are associated with atypical hemolytic uremic syndrome (aHUS). In the Newcastle cohort of 157 aHUS patients we have identified CFH mutations in 25 families or individuals. Eleven of these 25 independent mutations are either c.3226C>G,Q1076E; c.3572C>T,S1191L; c.3590T>C,V1197A or combined c.3572C>T,S1191L/c.3590T>C,V1197A. Sequence analysis shows that all four of these changes could have arisen as a result of gene conversion between CFH and CFHL1. Analysis of parental samples in two patients with S1191L/V1197A has shown that the changes are de novo thus providing conclusive evidence that gene conversion is the mutational mechanism in these two cases. To confirm that S1191L and V1197A are disease predisposing we examined their functional significance in three ways - analysis of the C3b/C3d binding characteristics of recombinant mutant S1191L/V1197A protein, heparin affinity chromatography and haemolytic assays of serum samples from aHUS patients carrying these changes. The results showed that these changes resulted in impaired C3b binding and a defective capacity to control complement activation on cellular surfaces. We, therefore, provide conclusive evidence that gene conversion is responsible for functionally significant CFH mutations in aHUS.

Animals↗
PubMed1993

Differential cytokine regulation of complement C3, C4, and factor B synthesis in human intestinal epithelial cell line, Caco-2.

In the intestinal tract, the local synthesis of C3 and components of both the classical (C4) and alternative (factor B) C activation pathway has previously been demonstrated in vivo. However, the cellular source of this local C synthesis has not been identified. In this study, we demonstrated the syntheses of C3, C4, and factor B in the human colonic adenocarcinoma cell line Caco-2, which is regarded as a good experimental model of normal human intestinal epithelial cells. The results of metabolic labeling experiments indicated that the intra- and extracellular molecular sizes and subunit structures of Caco-2-derived C3, C4, and factor B were compatible with previously reported values for these components in other cells. The functional activities of C3 and C4 in the supernatants were also demonstrated by hemolytic titration assay. Furthermore, C syntheses in this line were independently upregulated by several human cytokines: C3 synthesis was dose-dependently enhanced by the addition of IL-1 beta or TNF-alpha; C4 synthesis was enhanced by the addition of IL-6 or IFN-gamma in the same manner; and the addition of IL-1 beta or IL-6 also induced a dose-dependent increase in factor B synthesis. These enhancing effects were confirmed to be specific for individual cytokines by experiments using anti-human cytokine antibodies. It is likely that intestinal epithelial cells are local production sites of C3, C4, and factor B, and that local C syntheses in the intestine are independently regulated by several cytokines, derived from monocytes/macrophages and T cells resident in the mucosal microenvironment.

Adenocarcinoma↗
PubMed1976

Diagnostic procedures in immunodermatology.

Most immunologic diseases are caused by the derailment of the humoral or cellular pathways of the immunologic defense system. This derailment results from numerous factors such as the inability of the patient to remove the pathogen; the consumption, defect, or deficiency in any component of these pathways, and the overproduction of any of the components. To diagnose these immunologic disorders one has to detect the pathogen and the reactions caused by it and to determine the cause of its nonclearance. The immunofluorescence techniques has been invaluable in detecting both the antigen that causes the disease and the reactions initiated by the antigen, such as the production of antibodies and the activation of the complement system. The immunoperoxidase technique has also been used for these purposes in certain instances. For detecting the circulating immune complexes which occur as intermediates in the chain of reactions initiated by the antigen, various physiochemical and biologic techniques have been used. However, none of these tests seems to be totally reliable for determining whether circulating immune complexes are present. The consumption of complement was detected by hemolytic estimations and radial immunodiffusion or rocket electrphoresis. These techniques were also useful in detecting the hereditary deficiencies in immunoglobulins and components of classical and alternative pathways of complement activation. Since these techniques cannot be used to estimate IgE, the radioallergosorbent test was used to measure such levels in the atopic patients. Cellular hypersensitivity was detected with skin tests together with methods which assess the ability of lymphocytes to produce mediators in response to antigen. Many of these mediator assays, however, are not suitable for this purpose. A satisfactory substitute appears to be to determine the factor in antigen-stimulated, lymphocyte culture supernatants which activates macrophages to take up radiolabeled colloidal gold or radiolabeled glucosamine. In contact allergic dermatitis, an increase in the IgD-bearing lymphocytes and granulocytes has also been correlated with cellular hypersensitivity. Lymphocytes and polymorphonuclear leukocytes coated with antibodies mainly directed against nuclear antigens of the basal layer cells of the noninvolved epidermis have invariably been encountered in psoriasis. The use of these findings for diagnostic purposes and for understanding the mechanisms of certain diseases is being explored.

Animals↗
PubMed1995

Chimeric horse/human recombinant C9 proteins identify the amino acid sequence in horse C9 responsible for restriction of hemolysis.

Equine C9, in contrast to human C9, has extremely low hemolytic activity against most mammalian erythrocytes, although the amino acid sequences of both proteins show 77% identity. In an attempt to define the region of human C9 responsible for conferring its lytic activity, or conversely, the region of equine C9 responsible for its restriction, recombinant human and equine C9 and four chimeric human/equine C9 proteins were constructed and expressed in COS-7 cells. Recombinant human and equine C9 displayed hemolytic profiles similar to those of the purified native proteins. Exchange of a fragment extending from residues 145 to 290 in horse C9 with the corresponding one from human C9 created a fully hemolytic protein. This region contains the putative hinge region but not the membrane-interacting domain. Nonlytic chimeric C9 proteins inhibited hemolysis and binding of human C9 to EAC1-8 cells, indicating that they bind to their receptor, but subsequent unfolding or insertion into the membrane is impaired. These results suggest that restriction factors, such as glycophorin, CD59, or homologous restriction factor, on erythrocytes may limit the activity of horse C9 by interacting with its hinge region. In support of this conclusion direct binding of CD59 to immobilized horse C9 was detected by ligand blotting, and it was observed that a polyclonal anti-CD59 Ab enhanced human and horse C9-mediated hemolysis of human EAC1-7, but the increase in hemolytic activity of horse C9 by inhibition of CD59 was less than what could be achieved by insertion of the human C9 hinge region into horse C9.

Amino Acid Sequence↗
PubMed1994

Role of sialic acid in the resistance of Trypanosoma cruzi trypomastigotes to complement.

Trypomastigotes of Trypanosoma cruzi, mammalian infective forms of the parasite, express an unusual cell surface trans-sialidase. This enzyme enables the parasite to rapidly sialylate its surface when supplied with alpha(2,3)-linked sialic acid from glycoconjugates in serum or on cell surfaces. Here we used a novel fluorescence-based, trypomastigote lysis assay to evaluate the role of sialic acid on the parasite's plasma membrane in providing protection against the complement cascade. Trypomastigotes were desialylated, and sialic acid removal was confirmed by a chemical assay and also by flow cytometry with the use of a mAb that recognizes a T. cruzi-sialylated epitope. Compared with sialylated trypomastigotes, which were completely refractory to lysis by human serum, only about 5% of the desialylated trypomastigotes were lysed by complement. However, further analysis revealed that the desialylated parasites had been resialylated during exposure to serum complement. Next we incubated desialylated trypomastigotes with samples of desialylated human serum. Although the sialidase-treated serum retained its full hemolytic activity, lysis of trypomastigotes increased only from 5 to 24%. This increase correlated with an enhanced deposition of complement protein C3 on the parasite surface. The ratio of C3b to lytically inactive iC3b was increased for desialylated, compared with sialylated, parasites. We conclude that although parasite sialic acid promotes C3b cleavage into iC3b, this mechanism alone does not account for the robust resistance of these parasites to complement lysis.

Animals↗
PubMed1993

[C2 deficiency discovered in pneumococcal meningitis].

BACKGROUND: Congenital deficiencies of complement system proteins are rare. Patients with C2 deficiency have a high incidence of vascularitis syndromes. Most patients with this deficiency have no problems with increased susceptibility to infection, most commonly due to pneumococci, presumably because of the protective function of the alternative pathway. CASE REPORT: A 22 month-old girl was admitted because of acute meningitis and otitis. She had had 2 episodes of otitis media at the age of 1 year. Analysis of the CSF showed that this meningitis was due to pneumococcal infection. Recovery was complete after 15 days of antibiotic therapy. Total hemolytic complement activity (CH50) was low during the infection; one month later, the CH50 value was about zero as was C2, while C3 and C4 were normal. The patient was given polyvalent pneumococcal and anti-Haemophilus vaccines plus prophylactic penicillin G. Laboratory tests for systemic lupus erythematosus were negative. CONCLUSION: A defect of complement function should be suspected in any patient with severe of recurring pyogenic infections. Complement disorders can be detected one month later by means of the relatively simple hemolytic complement assay.

Complement C2↗
PubMed1975

Initiation of C3 cleavage in the alternative complement pathway.

The capacity of C3 and 125I-C3 to interact with B and D to generate a C3 convertase was recognized by identification of C3b and Bb on immunoelectrophoresis and 125I-C3b on disc gel electrophoresis. Since the C3 was devoid of C3b as assessed by immunoelectrophoresis, alkaline disc gel electrophoresis, and isoelectric focusing, the initial C3 cleavage was not by the C3b-dependent C3 convertase. In addition, on isoelectric focusing C3 hemolytic activity and the capacity to permit B cleavage by D were isoelectric at pH 6.4, WHEREAS THE CAPACITY OF C3b to permit cleavage by D was isoelectric at pH 5.65. Comparison of the dose-response effects of C3b and C3 for D revealed linear and sigmoidal relationships, respectively, consistent with formation of an initial C3 convertase independent of C3b followed by generation of the C3b-dependent convertase in the reaction initiated with native C3. Further, preincubation of C3 with C3bINA did not diminish its subsequent capacity to permit B inactivation by D as compared to the introduction of C3bINA during the assay, thus supporting the view that native C3, B, and D can form a convertase capable of generating initial C3b.

Chromatography, DEAE-Cellulose↗
PubMed1977

Studies of C1 subcomponents in chronic urticaria and angioedema.

C1q, C1r, C1s, C3, C4 and C-1 IA were determined by electroimmunoassay in sera from 150 patients with chronic urticaria or angioedema. Abnormal C1q and C1s levels were found in about 30% of the patients. In seven sera C1r was not measurable due to the appearance of diffuse precipitates. The levels of C3 and/or C4 were decreased in five sera with aberrations of C1 subcomponents in the electroimmunoassay. None of the patients showed reduced C-1 IA levels in the electroimmunoassay. The presence in sera of abnormal C1 subcomponent complexes was studied by crossed immunoelectrophoresis. Sera from 11% of the patients contained C1r-C1s complexes. Increased amounts of alpha2 complexes (C-1r-C-1-S-C-1 IA) were found in 33% of the patients. A major part of the C1q in sera yielding abnormal C1r precipitates had the same electrophoretic mobility as isolated C1q and was not associated with the C1qrs complex. C1 activity in hemolytic tests was low in these sera as well as in sera with decreased C1q levels. In the esterolytic assay for C-1 IA low values were found in 14 patients. Repeated sampling and family studies in appropriate cases gave no evidence for genetically determined deficiencies of C1q, C1r or C-1 IA.

Angioedema↗
PubMed1986

C1 activation, with C1q in excess of functional C1 in synovial fluid from patients with rheumatoid arthritis.

Free Clq, in functionally active form was present in increased amounts in the synovial fluid of patients with rheumatoid arthritis. The presence of free Clq was associated with low concentrations of hemolytic C1, low C4 and raised amounts of C3dg/d fragments in the synovial fluid. The findings suggested intra-articular C1 activation with dissociation of C1 into free C1q and complexes containing C1r, C1s, and C1 inactivator. However, the immunochemical properties of synovial fluid C1r-C1s-C1 inactivator complexes appeared to differ from those of the complexes formed in serum, which hampered quantification with the assay used. Control patients with osteoarthritis or spondylarthritic syndromes did not show evidence of intra-articular complement activation, even though 1 patient with Reiter's disease had unexplained low concentrations of synovial fluid C4 and C3. The concentrations of circulating complement components were largely normal in the patients. Slightly increased concentrations of free C1q and C1r-C1s-C1 inactivator complexes in serum and C3dg/d fragments in EDTA plasma were observed, particularly in the patients with rheumatoid arthritis.

Adult↗
PubMed1990

Two distinct abnormalities in patients with C8 alpha-gamma deficiency. Low level of C8 beta chain and presence of dysfunctional C8 alpha-gamma subunit.

The sera from three C8 alpha-gamma deficient patients previously reported to have a selective C8 alpha-gamma defect were analyzed by SDS-PAGE and Western blot using two polyclonal antisera to C8 alpha-gamma and a monoclonal antibody to C8 alpha. All three sera exhibited C8 alpha-gamma bands that dissociated into alpha and gamma chains under reducing conditions. Quantitation of the alpha-gamma subunit in these sera by a sensitive ELISA revealed an amount approximately 1% of that found in normal human serum. A similar assay performed with a specific antiserum to C8 beta showed unexpectedly low levels of C8 beta in these sera, which were confirmed by hemolytic titration of C8 beta. The remarkable differences between C8 alpha-gamma and C8 beta in the C8 alpha-gamma deficient sera was that in spite of their comparable immunochemical levels, C8 beta still exhibited functional activity whereas C8 alpha-gamma was totally inactive. That the residual C8 alpha-gamma was inactive was also proved by its inability to show lytic bands in an overlay system after SDS-PAGE and subsequent removal of SDS. The implications of these findings for a novel concept of C8 deficiency are discussed.

Blotting, Western↗
PubMed1994

[Conformational analysis and hemolytic activity of immunoglobulin G fragment 285-292 and its analogs].

Theoretical conformational analysis was carried out for the 285-292 fragment of human immunoglobulin G (His-Asn-Ala-Lys-Thr-Lys-Pro-Arg) and its analogues containing Arg, Glu, Gly, Lys, or Trp residue instead of the His residue in position 1. Spectropolarimetic investigation of these peptides showed the analogues to have different activities in the C1q-mediated erythrocytes hemolysis assay. Comparison of the low-energy structures sets of the compounds tested allowed to suggest a model of the "biological active" conformation for the peptide molecule in the course of the C1q complement component binding.

Complement C1q↗
PubMed1985

Pregnancy-associated growth factor. II. A T-dependent polyclonal activator of human adult peripheral blood lymphocytes (PBL).

This study examined the ability of pregnancy-associated growth factor (PAGF), a substance found in crude human chorionic gonadotropin (hCG), to induce plaque-forming cells (PFC) in cultured human peripheral blood lymphocytes (PBL). PAGF, 0.25 to 1 mg/ml, induced maximal PFC at 6 to 7 days as measured by the staphylococcal protein A-coupled SRBC reverse hemolytic plaque assay with a rabbit anti-human Ig antiserum. PAGF-induced PFC/culture ranged from 1800 to 39,000 with a mean of 11,524 in unfractionated PBL (N = 24), as compared to 540 to 77,840 with a mean of 17,303 for pokeweed (PWM) (N = 22). Comparison of PAGF- and PWM-induced PFC showed that both induced specific IgG, IgA, and IgM PFC. In most individuals, PAGF induced more IgM and PWM more IgG PFC. The kappa: lambda ratio was 1.5 for unstimulated PBL, and approximately 3.5 for PAGF and PWM. To see if PAGF was a T-dependent polyclonal activator of B cells, T and non-T populations were obtained by SRBC rosettes and negatively selected T4 and T8 cells by complement-mediated lysis of SRBC+(T) cells. Only the recombined subsets which included T4 cells and non-T cells supported PAGF- and PWM-induced PFC. These data indicate that PAGF, a substance derived from commercial extracts of pregnancy urine, is a T4-dependent polyclonal activator of normal human B cells.

Adult↗