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PubMed1991

In vivo anti-complementary activities of the cobra venom factors from Naja naja and Naja haje.

The kinetics of complement (C) depletion and recovery of C levels upon injection of BALB/c mice with cobra venom factors (CVF), from N. naja (C3- and C5-depleting) and N. haje (selectively C3-depleting) were studied. The animals received i.p. or i.v. injections of either of the two preparations. CH50 and hemolytic C3 and C5 levels were followed as parameters of residual complement activity. N. naja CVF turned out to be as efficient in depleting total complement activity as N. haje CVF. Decreased CH50 values could largely be ascribed to C3 depletion. Complement consumption after N. naja CVF, however, lasted longer than after N. haje CVF administration. Estimated functional half-lives of N. naja and N. haje CVF were 11.5 and 4.5 h, respectively. Inhibition ELISAs showed that, after in vivo administration of either of the two CVF preparations, antigenic C3 and C5 kept circulating for days.

Animals↗
PubMed1982

Mechanisms of immune haemolysis: cell-dependent destruction of autologous red blood cells in penicillin-induced haemolytic anaemia.

The mechanisms of red blood cell destruction in 2 patients with penicillin-induced immune haemolytic anaemia were investigated. Anti-penicillin antibodies of the IgG subclass were found in the patients' sera and in the eluates of their direct antiglobulin positive red blood cells. Using a rapid 51Cr in vitro assay it was shown that fresh peripheral blood mononuclear phagocytes and granulocytes but not lymphocytes from both patients lysed and phagocytosed autologous red blood cells previously treated in vivo or in vitro by penicillin and autologous anti-penicillin antibody. Antibody-dependent cell-mediated cytotoxicity (ADCC) as well as antibody-dependent phagocytosis (ADPh) were proportional to serum concentration and to the number of attacking cells. Anti-penicillin antibody from 1 patient activated the complement system in vitro but failed to induce lysis of penicillin-treated red blood cells in the presence of complement. These results suggest that ADCC as well as ADPh participate in the destruction of red blood cells in penicillin-induced haemolysis in vivo.

Adult↗
PubMedDate not supplied

Sensitive one-step haemolytic assays of the fourth (C4) and fifth (C5) components of the human complement system.

In this paper, we describe two simple "one-step" methods to assay the C4 and C5 haemolytic activities of human complement. These methods required fresh normal sera immunochemically depleted of C4 or C5 components of the complement system. These sera, called R4 and R5 respectively, were prepared by affinity chromatography on monospecific anti-C4 Sepharose-4B or monospecific anti-C5 Sepharose-4B. Resins were washed with high-ionic strength solution containing 20% saccharose, which gave this method a very high specificity. These reagents were successfully used in the detection and quantification of C4 and C5 in all preparation steps as well as in normal and pathological sera. Two different kinds of haemolytic assays were developed and optimized: a haemolytic tube assay and a haemolytic agarose plate assay.

Complement C4↗
PubMed1984

Suppressor T cells for delayed-type hypersensitivity to Japanese encephalitis virus.

The delayed-type hypersensitivity (DTH) to Japanese encephalitis virus (JEV) and the suppressor cells controlling it and the antibody-forming cells in inbred Swiss mice have been studied. JEV induces DTH, with a peak response at day 7 following infection which persists at low levels at least up to 119 days. Suppressor activity appeared on day 18. It was transferable by immune spleen cells. Treatment of spleen cells with anti-Thy-1.2 antisera and complement abrogated the suppressor activity. The homogenate of the spleen was equally effective in mediating suppression of DTH and the humoral response as measured by direct antibody plaque-forming cell (IgM-PFC) assay. The suppressor activity was antigen-specific both on DTH and T helper for antibody response as the immune responses against SRBC or Coxsackie B4 virus were not suppressed. The suppressor cells were sensitive to cyclophosphamide treatment when the drug was given 48 hr before their appearance. It is, therefore, concluded that in JEV infection of mice, antigen-specific suppressor T cells are generated, both for DTH and IgM antibody, which are cyclophosphamide-sensitive and mediate suppression through soluble product(s).

Animals↗
PubMed1971

Studies of human sera with cytotoxic activity.

42 human sera showing in vitro cytotoxic activity of restricted or broad HL-A specificities with test human lymphocytes were studied for the molecular and immunoglobulin class of cytotoxic antibody using sucrose gradient separations, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration. Sera originated from patients with previous multiple pregnancies (19), multiply transfused patients (8), subacute bacterial endocarditis (4), systemic lupus (2), and human umbilical cord sera (9). In 32 of 42 instances, predominant cytotoxic activity was found in high molecular weight gradient fractions; however, DEAE chromatographic separations revealed cytotoxic activity in initial buffer fractions containing primarily gammaG globulin. Gradient separations of cytotoxic activity within initial gammaG DEAE fractions showed localization of cytotoxicity only in high molecular weight materials. Confirmation of high molecular weight gammaG cytotoxic activity was obtained by resistance to mercaptoethanol treatment, abolition of activity after absorption only with specific anti-gammaG antisera, and by the finding that high molecular weight cytotoxic activity in gradients or gel filtration run at neutral pH 7.4 became 7S when separations were rerun at an acidic pH of 4.0. Such 7S activity again became rapidly sedimenting when the same fractions were again rerun in gradients at neutral pH.19S gammaM cytotoxic activity was documented in a panel of 15 human sera containing anti-"I" cold agglutinins. In this instance the cytotoxic activity appeared to be related to the cold agglutinin antibody since it was mercaptoethanol sensitive and could be demonstrated in monoclonal antibody eluates containing primarily gammaM. No in vitro protection again cytotoxic effect was demonstrated when isolated human 19S anti-gamma-globulins were aded to the lymphocytotoxic assay system. With the exception of cold agglutinins, human cytotoxic antibodies appear to be primarily gammaG producing in vitro lymphocyte killing either as 7S gammaG globulin or as rapidly sedimenting aggregates or complexes of gammaG molecules.

Anemia, Hemolytic↗
PubMed1986

Detection of C-reactive protein, streptolysin O, and anti-streptolysin O antibodies in immune complexes isolated from the sera of patients with acute rheumatic fever.

Circulating immune complexes (IC) of 42 patients with acute rheumatic fever from Santiago, Chile, were studied. The complexes were isolated by polyethylene glycol precipitation and were analyzed for antibodies, antigens, and C-reactive protein. We found the complexes to be enriched in antibody to streptolysin O, particularly in the group of patients with elevated levels of IC. IgM was the predominant class of Ig present in the complexes. Western blots from 12 patients to detect antigens in the complexes showed proteins of m.w. 50,000, 60,000, and 69,000, consistent with the polypeptides of streptolysin O. Such antigens were absent in the complexes from patients with post-streptococcal glomerulonephritis and pharyngitis. Eluted antibodies from these protein bands on the nitrocellulose sheets reacted with the streptolysin O in Western blots and neutralized the hemolytic activity of streptolysin O in a microhemolysin assay. In addition, isolated complexes from several sera showed the presence of C-reactive protein bound to complexes. In vitro experiments demonstrated that [125I]C-reactive protein was not precipitated by polyethylene glycol either alone or when added to monomeric IgG, whereas it precipitated significantly when added to aggregated IgG. The detectable C-reactive protein in isolated complexes and sera samples increased after treatment with sodium dodecyl sulfate. These data suggest that circulating immune complexes in acute rheumatic fever contain streptolysin O and its antibody and raise interesting questions regarding the pathogenetic significance of C-reactive protein in the complexes.

Antigen-Antibody Complex↗
PubMed1996

Do plasma anti phospholipid antibodies in patients with autoimmune haemolytic anaemia correlate with reduced levels of erythrocyte complement receptor 1?

Anti phospholipid antibody (APA), of both IgG and IgM class, was assayed in plasma obtained from patients with autoimmune haemolytic anaemia (AHA) and active haemolysis. Although plasma APA was elevated in some AHA patients, when compared to a normal control population, there was no apparent correlation with levels of erythrocyte complement receptor 1 (RBC-membrane CR1) in this patient group. However, there appeared to be a correlation between RBC-membrane CR1 and plasma IgG APA in a subset of the AHA patients with low RBC-membrane CR1. The potential significance of this observation was complicated by the majority of these patients having a plasma APA within the normal range. These results suggest that the reported inverse correlation between APA and RBC-membrane CR1 may not be definitive.

Adult↗
PubMed1991

Autoantibodies against CD4 cells are associated with CD4 helper defects in human immunodeficiency virus-infected patients.

To investigate whether autoantibodies against CD4-positive lymphocytes might induce helper dysfunction, autoantibody formation and T-cell function was examined simultaneously in 61 hemophilia patients. Twenty patients were human immunodeficiency virus (HIV)-negative, 26 HIV-positive stage CDC II or III, and 15 were HIV-positive stage CDC IV. T lymphocytes, CD4-positive, or CD8-positive T subsets were cocultured with B lymphocytes and pokeweed mitogen (PWM) for 6 days and Ig-secreting cells were assessed in a reverse hemolytic plaque assay. The presence of IgM, IgG, C3d, or gp120 on the surface of T cells or T subsets was analyzed by flow cytometry. Autoantibodies against CD4-positive T cells were not detected in controls or HIV-negative patients, but were common in HIV-positive patients (20 of 41 patients). In patients with autoantibodies we found an increased incidence of CD4 helper defects (P less than .0001 in CDC II or III patients; P less than .02 in CDC IV patients). 12 of 13 patients with IgM autoantibodies and 4 of 4 with IgG autoantibodies showed CD4 helper defects. Complement fixation had no relevance. Autoantibody formation against CD4 cells was not due to increased in vivo B-cell stimulation (spontaneous plaque formation: 611 +/- 204 PFC/10(6) B cells in autoantibody-negative patients v 650 +/- 202 PFC/10(6) B cells in autoantibody-positive patients; not significant). Thus, our results suggest that autoantibody formation is not caused by a general state of in vivo B-cell activation. Rather, the production of autoantibodies appears to coincide with defects in B-cell proliferation or differentiation, as shown by reduced mitogen-stimulated B-cell responses in CDC II and III patients (P less than .05). Autoantibodies against CD4 cells appear to be involved in the pathogenesis of CD4 helper defects of HIV-infected patients.

Antigens, CD↗
PubMed1977

Isolation and characterization of Guinea pig properidin.

Guinea pig properdin was purified to homogeneity by employing as an assay during isolation its capacity to augment the hemolytic activity of a heterologous human C3b-dependent C3 convertase, C3B. The purified protein elicited a monospecific antibody response in a rabbit. The antiserum, by immunodiffusion, gave a reaction of identity between a protein in whole guinea pig serum and the immunogen. A solid phase immunoadsorbent prepared with the antiserum removed properdin function from the purified protein. The purified guinea pig protein exhibited the classical properdin function of reconstituting a human RP for zymosan-induced C3 inactivation. The guinea pig properdin also agglutinated red cell intermediates bearing either guinea pig or human C3b and retarded the decay of homologous C3 convertase, C3B. These activities are the same as those observed for purified human properdin and validate the amplification function of properdin on terminal component activation in a second species.

Animals↗
PubMed1975

A plaque technique for assay and characterization of antibody-dependent cytotoxic effector (K) cells.

Using monolayers of erythrocytes as target cells, a plaque assay was developed by which individual antibody-dependent cytolytic lymphoid cells (K cells) could be identified at the cellular level. The extent of plaque formation depended on the concentration of sensitizing antibody, time of incubation, and number of lymphocytes added. Most of the plaque-forming cells (50% to 70%) were shown to possess receptors for activated complements, a small but variable fraction (5% to 25%) had surface Ig detectable by indirect immunofluorescence, and 5% to 10% bound sheep erythrocytes at 5 degrees C. The plaque-forming cells appeared as villous, small to medium-sized lymphocytes when studied by transmission or scanning electron microscopy. Under standardized experimental conditions the minimal number of active, plaque-forming K cells in purified lymphocyte preparations could be estimated.

Antibody Formation↗
PubMed1983

Complement factor D concentrations in normal sera: comparison of immunochemical and functional determinations.

The concentration of factor D (D) was determined in the sera of 144 healthy adults and 29 healthy children by enzyme amplified electroimmunoassay (EAE), and by a hemolysis in gel (HIG) assay for D. The 95% geometric confidence areas for D in adults were 75-143% as determined by EAE, and 65-171% by the functional assay. The concentrations were given in per cent of a reference serum pool containing 1.6 mg/l of D as measured by EAE. The correlation coefficient between D values obtained by the two methods was r = 0.52 (p less than 0.001). D levels were lower in children than in adults. In the EAE purified D, and pathological sera with very high D Concentrations, gave lower precipitation peaks with dilutions performed in buffer than with dilutions in D depleted serum or human serum albumin.

Adult↗
PubMed1991

Association of activated properdin with complexes of properdin with C3.

An ELISA using antibody to properdin (P), followed by antibody to C3 to detect complexes of P with C3 (P-C3), detected low levels of P-C3 complexes in human serum and plasma samples. Incubating serum for 1 h at 37 degrees C increased the amount of P-C3 and diminished factor B hemolytic activity without altering total alternative pathway activity or C3 activity in serum. When P and C3 in incubated serum were analyzed by size exclusion HPLC, complexes of P-C3 were detected at retention times corresponding to molecular mass measuring in excess of 2 x 10(6) Da. Activation of serum with zymosan or cobra venom factor greatly increased the level of P-C3 and decreased alternative pathway hemolytic activity. Chromatography of proteins eluted from serum-treated zymosan detected a peak of P at 9.7 x 10(5) Da and a peak of P-C3 at 1.5 x 10(6) Da. Functional assays for activated properdin also revealed a peak of activity at 1.5 x 10(6) Da, congruent with the peak of P-C3. Native properdin was detected at 3.9 x 10(5) Da. When native properdin was added to properdin-depleted serum and incubated for 1 h at 37 degrees C, activated properdin was detected at the same position in the chromatograph as were P-C3 complexes. We conclude that incubation of serum at 37 degrees C produces complexes of P with C3, that exposure of serum to alternative pathway activators increases the amount of P-C3, and that generation of P-C3 complexes is associated with the presence of activated P.

Chromatography, Gel↗
PubMed1988

The second component of human complement: detection of two hemolytic forms in plasma by pH variation.

The second component of human complement (C2) in pseudoglobulin prepared from normal plasma eluted as a single peak at high conductivity (30 mS) and pH 4.5 from the cationic exchangers S-Sepharose or Mono S in the Fast Protein Liquid Chromatography (FPLC) System. The C2 was stable at pH 4.5 and 0 degrees C if enzyme inhibitors were used and the pH was raised to 6.0 after elution from the columns. After rechromatography on Mono S in the FPLC System at the median isoelectric point of 5.5 or pH 6.0, the C2 eluted as two distinct hemolytic forms: the first peaked at 16 mS, the second at 30 mS. The two forms of C2 did not correlate with the allotypic variant of C2 in individual, normal human plasmas. After elution at pH 4.5 from S-Sepharose and rechromatography at pH 5.5 or 6.0 on Mono S, the hemolytic activities of the two forms in individual plasmas eluted in 3 patterns: 1) high activity at 16 mS, low activity at 30 mS; 2) low activity at 16 mS, high activity at 30 mS; 3) high activity at 16 mS, high activity at 30 mS. The specific activities of both forms were approximately the same; both eluted the same after gel filtration at pH 5.5, and both had the same pattern on SDS-PAGE and immunoblots. The pattern of elution was characteristic for each individual plasma, and the first hemolytic form appeared to elute independent of the second form. At pH 4.5, C2 was completely separated from Factor B, a functionally and structurally similar protein of the alternative complement pathway, whereas at pH 5.5 or 6.0, the two proteins eluted together. From these results, the two forms of hemolytic C2 can be purified for structural and functional analyses.

Chromatography, Ion Exchange↗
PubMed1991

The presence of an anti-erythrocyte autoantibody in C3HeB/FeJ mice after lymphocytic choriomeningitis virus infection.

Lymphocytic Choriomeningitis (LCM) virus, substrain Docile, causes a chronic infection in adult C3HeB/FeJ mice. The virus also induces a severe anemia which, unlike the viremia, eventually resolves. Initially, there is frank bone marrow deficit, but the anemia persists well beyond a strong erythroid compensatory response. An immune-mediated basis for the hemolytic anemia was suggested by its abrogation in cyclophosphamide-treated mice, as well as an abnormal number of spherocytes in the circulation. We now show by ELISA assay, using either anti-mouse Ig or RBC membrane ghosts as catching antigen, that unusually high quantities of antibodies can be eluted from the RBCs of virus-infected mice. Furthermore, the high transient antibody concentration correlates with the severity of the anemia. With no evidence for complement playing a role in the anemia, these data indicate that erythrophagocytosis (via macrophage FcRs) may be the mechanism for RBC elimination. The possibility of molecular mimicry (antibody cross-reactivity between LCM and RBC membrane epitopes) was considered but appeared unlikely since the RBC antibody eluates gave no signal in an LCM-specific ELISA (which showed an ever increasing serum titer of virus-specific antibody). Isotype determination of the RBC eluates revealed the following: IgG2a much greater than IgG1 greater than IgG2b greater than IgG3 greater than IgM. The precise role, if any, of LCM-virus induced polyclonal activation (most strikingly in the IgG2a subclass) has yet to be determined.

Anemia↗
PubMed1999

[Detection of bactericidal antibody in the breast milk of a mother infected with enterohemorrhagic Escherichia coli O157:H7].

A 21 years-old pregnant woman developed diarrhea, fresh bloody stools and abdominal pain on April 6th 1997 at 32 weeks of gestation, and was admitted to the hospital on April 11th. The stool culture on admission was positive for enterohemorrhagic Escherichia coli (EHEC) O157:H7 (Stx1 and 2). Clinical laboratory data during admission showed only slight elevation of beta-microglobulin and N-acetyl glucosaminidase in the urine, and no neurological or hemolytic symptoms were seen. After the antibiotic and lactobacillus administration, all her symptoms were relieved and no abnormal findings in pregnancy were observed. She delivered a baby girl normally on May 30th. Serum (between 41 and 120 days from the onset) and milk (between 4 and 64 days post partum) samples from the mother, and serum (64 days of age) from a baby and cord blood were obtained to monitor the immune status against EHEC O157:H7 and against Shiga toxins (Stx). Anti-E. coli O157 LPS antibodies (IgA, G and M) were assayed by the ELISA method. Neutralizing anti-Stx antibodies were measured by using ACHN cell cytotoxicity assay. In the colostrum and mature milk, high levels of IgA and IgM, and no IgG antibodies against EHEC O157 LPS were detected. In one of the control colostrum samples obtained from 4 healthy mothers IgA antibody against EHEC O157 LPS was detected. To assess the potency of protection against EHEC O157:H7 by the breast milk, we monitored it by the bactericidal activity for the organism under complement-coincubation experiment, and by the neutralization test for the Stx cytotoxicity. As a result, breast milk samples (both colostrum and mature milk) from a patient were demonstrated to kill the organisms. One of 4 healthy milk samples, showed bactericidal activity though it was negative in O157-LPS antibody. This bactericidal activity seen in one healthy colostrum is possibly due to a nonspecific reaction caused by non-O157 E. coli infection. From these observations, it was suggested that the bactericidal activity was due to the IgM class antibody against EHEC O157:H7. However, the neutralizing antibody against Stx1 and 2 could not be detected in any sample. EHEC infection at late gestation did not cause adverse effects to a fetus, and breastfeeding may have advantage for the protection of a baby against EHEC infection.

Adult↗
PubMed1971

In vitro studies of complement function in sera of C4-deficient guinea pigs.

In vitro studies were performed utilizing sera from a strain of guinea pigs with a total absence of hemolytically active C4. Previous studies in these animals have demonstrated normal complement-dependent inflammatory reactions, suggesting that they are able to bypass their deficiency of C4. In vitro studies with C4-deficient serum also indicate normal activation of late-acting C components. Thus, endotoxin was capable of fixing normal amounts of the late components of complement (C3-9) in these sera, but did not fix C1 and C2. Antigen-antibody complexes fixed both early and late components of complement, although components beyond C4 were fixed less efficiently than in normal sera. Therefore, both in vivo and in vitro evidence indicates that the C4-deficient guinea pigs possess an alternate pathway for activation of late-acting complement components. Antigenic analysis of C4-deficient serum utilizing both guinea pig anti-C4 antibody and rabbit anti-C4 antibody suggests an absolute deficiency of C4-like molecules. Sera from animals with C4-deficiency were found to have one-half the normal level of C2. Sera from five of eight animals tested had 10-20% normal C1 activity. C3-9 assayed as a complex was normal.

Animals↗
PubMed1988

Expression of functional cell surface C1-inactivator by U937 cells.

We have previously shown that the human monocyte-like cell line U937 synthesizes C1-INA and expresses cell surface C1-INA. In this report we provide evidence that this surface-expressed C1-INA is functionally active. Intact U937 cells demonstrated functional C1-INA activity in a hemolytic assay. This activity was blocked when the cells were incubated with monospecific antibody to C1-INA, and was not detectable in cell-free supernatants of U937 cells. SDS-PAGE analysis of radiolabeled U937 cell surface proteins purified by anti-C1-INA affinity chromatography revealed two distinct bands. One protein had a Mr of 105 kDa identical to plasma C1-INA, and the second had a Mr of 200 kDa. We were unable to determine the identity of the 200 kDa protein by Western blotting with anti-C1-INA. However, the possibility exists that this 200 kDa molecule may represent a C1-INA receptor, a dimeric form of C1-INA, or an unrelated cell surface protein with affinity for C1-INA. Furthermore, we show that treatment of U937 cells with phorbol ester resulted in an increase in the percentage of cells expressing surface C1-INA. These results suggest that U937 cells express functional cell surface C1-INA, which could function in vivo to protect these human tumor cells from lysis by host complement.

Blotting, Western↗
PubMedDate not supplied

Inhibition of human factor B activity of the properdin system by human and guinea-pig sera.

The activity of factor B of the alternative pathway of complement activation was assayed in human and guinea-pig sera using the radial haemolytic assay with various MgCl2 and RB concentrations. RB were prepared by heating at 50 degrees C human or guinea-pig sera to render them deficient in factor B. Whatever concentrated RB and MgCl2 were, guinea-pig sera seemed unable to react while human sera gave an haemolytic reaction with both human and guinea-pig RB. We described an inhibition of human factor B activity by human and guinea-pig RB under certain conditions. This inhibition is more pronounced in the case of guinea-pig RB.

Animals↗