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PubMed1991

Kinetics of the biosynthesis of complement subcomponent C1q by murine macrophages: effects of stimulation by interferon-gamma.

The effects of interferon-gamma (IFN-gamma) on the kinetics of biosynthesis of complement subcomponent C1q by mouse inflammatory peritoneal macrophages were determined. Stimulation of macrophages with various concentrations of IFN-gamma produced a dose-dependent increase in C1q mRNA accumulation which was detected as early as 3 h and sustained through 24 h, as determined by Northern blot analysis. A corresponding early increase in the extracellular accumulation of functional C1q was detected in culture supernatants after 3-9 h stimulation of macrophages with IFN-gamma that was sustained for 24-48 h as determined by a complement hemolytic assay. Autoradiographic analysis of [35S]methionine-labeled secretory C1q confirmed the protracted dose-dependent secretion of C1q by IFN-gamma stimulated macrophages during 24-48 h of culture. Western blot analysis of macrophage lysates indicated no significant changes in endogenous C1q levels following stimulation with IFN-gamma either after 3-9 h or 24-48 h when both C1q mRNA and extracellular accumulation were at their peak. Our results indicate that IFN-gamma promotes early and protracted mRNA accumulation and secretion of C1q by macrophages without intracellular accumulation, presumably due to the rapid rate of secretion of newly synthesized C1q. It is apparent that priming of macrophages with IFN-gamma provides a rapid and abundant source of secretory C1q for potential interaction with various macrophage triggering agents which also bind C1q.

Animals↗
PubMed1992

Discordant secretion of relaxin by individual porcine large luteal cells: quantitative analysis by a reverse haemolytic plaque assay.

Individual large luteal cells (LLC) derived from pregnant swine differ conspicuously in their ability to secrete the peptide hormone relaxin under basal and stimulated conditions--the phenomenon of functional heterogeneity. The purpose of this study was to quantitate knowledge of this phenomenon through use of a reverse haemolytic plaque assay, a technique that utilizes antibody-directed, complement-mediated erythrocyte lysis to detect hormone secretion by single LLCs in culture. Measurement of individual plaque areas (an index of the amount of relaxin secreted) demonstrated an approximate 100-fold range in the amount of relaxin secreted by a single cell under basal conditions. This range was doubled by exposure to the phorbol ester, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA; 50 nmol/l). Under basal conditions, 50 and 80% of the total amount of relaxin was secreted by approximately 10 and 30% of all LLCs respectively. The size of these fractions was not influenced by the time of incubation (1-8 h), or by the presence of either of two non-specific stimulatory relaxin secretagogues, PMA (50 nmol/l) or arachidonic acid (1 mumol/l). The unimodal frequency distribution of plaque areas (under basal or stimulated conditions) suggests that relaxin-secreting LLCs comprise a discrete functional population of secretory cells, at least under these experimental conditions. We conclude that a remarkably small fraction of LLCs secretes the majority of relaxin, and that the size of this fraction was not influenced by time or secretagogues.

Animals↗
PubMed1990

Cytotoxic antibody against trophoblast and lymphocytes present in pregnancy with intrauterine fetal growth retardation and its relation to anti-phospholipid antibody.

To elucidate immunological mechanisms involved in the genesis of intra-uterine fetal growth retardation (IUGR), an in vitro cytotoxicity assay against normal trophoblast and lymphocytes was performed. The data demonstrated the existence of cytotoxic antibody directed against trophoblast exclusively in the IgG fraction of the sera of 9 out of 15 mothers with IUGR, but in none of the sera from normal pregnant women. This antibody showed differential reactivity patterns that may be indicative women. This antibody showed differential reactivity patterns that may be trophoblast in common. Out of 9 IUGR mothers with this cytotoxic antibody, in 6 cases (66.7%) chronic villitis was evident upon microscopic examination of the placenta, the frequency being significantly higher than that in IUGR mothers without cytotoxicity or in control mothers (P less than 0.02). It is suggested that in situ inflammatory change triggered by this antibody might lead to IUGR through chronic villitis. This antibody showed cross-reactivity with anti-negatively charged phospholipid antibody, as confirmed by an absorption experiment, indicating that the trophoblast antigenic stimuli in pregnancy can induce the production of various autoantibodies including anti-phospholipid antibodies. These results are of interest in relation to the pathogenesis of autoimmune diseases.

Adult↗
PubMed1993

Interactions in the complement-mediated lysis of blood group AB erythrocytes sensitized simultaneously with anti-A and anti-B monoclonal antibodies.

The lysis of group AB erythrocytes by human complement was studied by different anti-A and anti-B IgM monoclonal antibodies (mabs) in a 51Cr-release assay. The concentration of membrane-bound immunoglobulin was detected by ELISA, and the amount of C1q and C3 bound to sensitized red cells was measured by using purified, 125I-labelled molecules. We have demonstrated that there is an exponential relationship between the concentration of the sensitizing IgM mabs and C1q binding to the sensitized AB cell. The efficiency of binding was related to the number of antibodies bound; thus, anti-A sensitized cells bound 3-6 times more C1q than anti-B sensitized cells did. AB cells, on the other hand, bound similar amounts of C3 whether anti-A or anti-B was present. The lytic efficiencies of the various IgM mabs during short incubation times were different, suggesting that the complement activation rates vary widely with different antibodies on the AB cell membrane. The binding of C1q to an antibody-sensitized target activates a cascade, whose components may migrate away from the sensitizing antibody; interactions between the activation processes generated by the anti-A and anti-B antibodies may thus occur. Choosing appropriate pairs of anti-A and anti-B mabs for the simultaneous sensitization of AB cells has indeed resulted in stimulation in some and inhibition in other combinations of mabs. It is suggested that stimulation is observed when the activated intermediates are produced in excess, whereas inhibition occurs when a shortage of activated intermediates prevents mutual utilization.

ABO Blood-Group System↗
PubMed1991

Characterization of C1 inhibitor binding to neutrophils.

In a previous study we have isolated neutrophil membrane proteins that non-covalently bind to native C1-INH (105,000 MW) and a non-functional, degraded C1-INH (88,000 MW; C1-INH-88). To further characterize the binding nature, we have designed a novel kinetic C1 titration assay which enables not only a quantification of the removal of fluid-phase C1-INH by neutrophils, but also a concomitant measure of residual C1-INH function. Native C1-INH, when adsorbed to EDTA-pretreated neutrophils, lost its function in the inhibition of fluid-phase C1. The non-functional C1-INH-88, which is probably devoid of a reactive centre, was found to block the binding of native C1-INH to neutrophils. Pretreatment of neutrophils with serine esterase inhibitors did not abrogate binding capacity of the cells for C1-INH, whereas the binding affinity for C1-INH was lost when the cells were pretreated with trypsin. An array of human peripheral blood leucocytes and several lymphoid cell lines has surface binding sites for C1-INH, but not on human erythrocytes and U937 cells. Binding was further confirmed using (i) C1-INH-microsphere beads to neutrophils, in which the binding was blocked when pretreating neutrophils with excess C1-INH or with trypsin, and (ii) radiolabelled C1-INH to neutrophils, which was competitively blocked by unlabelled non-functional C1-INH-88. Desialylation of C1-INH significantly reduced its binding affinity for neutrophils, indicating that the membrane receptor sites on neutrophils could be specific for the binding of sialic acid residues on C1-INH. Overall, our studies indicate that neutrophils or other leucocytes possess specific surface binding sites for the sialic acid-containing portion of C1-INH.

Binding, Competitive↗
PubMed1990

Antibody- and complement-mediated lysis of HIV-infected cells and inhibition of viral replication.

HIV-1-positive antisera were tested for their ability to lyse HIV-1-infected cells in the presence of active complement. Cytolytic effects caused by sera derived from infected humans were slower than those observed with sera from immunised chimpanzees. Lytic but also negative sera were found among HIV-1-infected asymptomatic men as well as among clinical AIDS cases. Human antisera that lysed infected cells reacted similarly irrespective of whether the complement was heterologous or autologous. Analysis of complement-mediated lysis using defined antisera against recombinant HIV-1 env or core antigens suggested that gp160/gp120 and p24 can act as target antigens for an antibody- and complement-mediated cytolysis of infected cells. Complement alone reduced the spread of HIV-1 infection in CD4+ cells and the ability of HIV-1 and HIV-2 to form plaques in CD4-transfected HeLa cells. Co-operative effects of specific antibodies and complement were the most effective in inhibiting HIV infections.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Enhanced immunoglobulin levels correlate with infectious complications after surgery in esophageal cancer.

Severe septic complications account for the high mortality of patients with esophageal cancer. We examined the levels of immunoglobulins and complements together with infection-related complications in a large number of patients. Enhancements of IgG, IgA, C3, C4, and CH50 were evident in patients with esophageal cancer and were more predominant compared to findings in cases of gastric cancer. Average levels of IgG and IgA immediately before surgery were significantly higher in esophageal cancer patients with postoperative septic complications than in those without such problems. Preoperative radiation therapy and total parenteral nutrition did not significantly alter the levels of immunoglobulins and complements. It would thus appear that the enhancement of IgG and IgA is associated with the occurrence of infectious complications following surgery for patients with esophageal cancer.

Adenocarcinoma↗
PubMed1997

Cysteine-specific radioiodination of proteins with fluorescein maleimide.

A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label is then removed by gel-permeation chromatography. The procedure avoids exposition of the protein to oxidative conditions and does not require purification of the labeled carrier reagent. Suitability of the method for a given protein can be evaluated spectrophotometrically without employing radioactivity. It can be applied under denaturing conditions and may be particularly useful with mutant proteins carrying engineered single cysteine residues at sites that are not functionally critical.

Bacterial Proteins↗
PubMed1995

Immunological function in post-traumatic splenosis.

A 28-year-old male medical student underwent splenectomy at 8 years of age due to traumatic rupture of the spleen sustained in a motor vehicle accident. Eighteen years later the patient had major abdominal surgery performed for an unrelated condition and, at the time of surgery, over 100 splenic nodules were found embedded throughout the patient's omentum, small bowel, and mesentery. An extensive study of immunological functions was carried out during the following 2 years. Through the course of this investigation, it was determined that the patient's peripheral blood smear lacked Howell-Jolly bodies and deformed or damaged erythrocytes, indicating that the splenotic tissue had the capacity to remove intranuclear inclusions from circulating red cells and to phagocytose old erythrocytes. The patient's levels of complement, serum immunoglobulins and the numbers of circulating T and B lymphocytes, helper T cells, and cytotoxic/suppressor T cells all were within the normal range. The response to Streptococcus pneumoniae polysaccharides was also normal, with increased levels of specific antibodies to all serotypes included in the vaccine 4 months after immunization. Finally, histological examination of his biopsied splenotic nodules revealed tissue that was indistinguishable from normal spleen.

Adult↗
PubMed1989

Further characterization of the impaired protective function in mice fed with lipid diet.

Female mice were maintained on lipid diet for 20 days. The nonspecific and immunological defense capability was determined by in vitro and in vivo methods. It was found that mice held mostly on a lipid diet demonstrate an all-round lowered response. Following 20 days of lipid diet the splenocytes exhibit: (1) an inversed lipid-protein ratio; (2) an inability to respond to sheep erythrocytes; (3) a reduction in [3H] thymidine incorporation in splenocytes stimulated with lipopolysaccharide (LPS) or with concanavalin A; (4) a reduction in the number of cells bearing surface immunoglobulins in splenocytes stimulated with LPS; (5) an inhibition of phagocytosis and intracellular killing in macrophages; (6) a lowering in granulocyte chemotaxis and adherence capacity; (7) a higher mortality to LPS after loading with galactosamine; and (8) a lowered complement activity even following LPS activation.

Animals↗
PubMed1996

Antineutrophil cytoplasmic antibodies (ANCA) in rheumatoid arthritis: a prospective study.

To investigate a possible relationship between the presence of antineutrophil cytoplasmic antibodies (ANCA), rheumatoid factor (RF), antinuclear antibodies (ANA), complement, disease activity and disease severity, 111 clinically well-documented RA patients were studied prospectively for ANCA, RF, ANA, C-reactive protein (CRP), total haemolytic complement (CH50) and complement split product C3d. Disease activity and severity were also assessed clinically, as well as anamnestically, using the Hannover Activity of Daily Living Questionnaire, the functional Steinbrocker grades, and numeric and verbal rating scales. At a serum dilution of 1:50, 20% of the 111 sera showed predominantly an atypical perinuclear staining pattern. There was no correlation between ANCA positivity and serological markers, disease activity and disease severity. Regarding previous therapies with disease-modifying antirheumatic drugs, ANCA+ patients took sulphasalazine significantly more often than ANCA- patients.

Activities of Daily Living↗
PubMed1992

Hyperprolactinemia inhibits natural killer (NK) cell function in vivo and its bromocriptine treatment not only corrects it but makes it more efficient.

We studied NK cell function in eight patients with pathological hyperprolactinemia by measuring 51Cr release by K562 cells exposed to their mononuclear cells and found it decreased compared to normal controls (P less than 0.01). Bromocriptine (BrC) treatment corrected NK function but also made it more efficient at 12:1 than at 25:1 or 50:1 effector:target ratios (ANOVA; P = 0.01). The study of NK cell function in agarose revealed that its decrease in hyperprolactinemia is due to their low active binding to target cells, active killing, and recycling capacity. BrC tended to correct them but also increased recycling capacity to levels higher than those of controls (P less than 0.05). Sequential studies in three hyperprolactinemic patients before and after BrC showed correction of NK function within 1 week but its increased efficiency at the 12:1 effector:target ratio required 8 weeks. We conclude that hyperprolactinemia decreases NK cell function. BrC corrects this by decreasing prolactin levels but also makes NK function more efficient by increasing the capacity of NK cells to recycle after killing.

Adult↗
PubMed1992

Cold-dependent activation of complement: recognition, assessment, and mechanism.

Cold-dependent activation of complement (CDAC) is a phenomenon characterized by low hemolytic complement activity in chilled serum. Complement component levels are normal when measured immunologically, and there is normal hemolytic activity in EDTA plasma or serum maintained at 37 degrees C. Little attention has been paid to CDAC except in Japan, and current unfamiliarity with it, even by clinical immunologists, can lead to confusion and unnecessary laboratory tests. A 66-year-old patient with a complex medical history is described whose complement tests showed abnormalities characteristic of CDAC. Evidence for classical complement pathway activation in the cold was obtained by CH50 measurements, by hemolytic C4 determinations, by C4a, C3a, and C4d generation, and by quantitating C1s-C1r-(C1 inhibitor)2 complexes. A good correlation was observed among these parameters. Cryoprecipitates were absent. CDAC activity has persisted for over 5 years and is greater at 13 than at 4 degrees C. Activation is ablated by heating at 56 degrees C and restored by the addition of C1 to the heated serum. Adsorption by streptococcal protein G-Sepharose and precipitation by 2.5% polyethylene glycol support the hypothesis that CDAC is caused by aggregated IgG. The CDAC factor(s) also induces complement activation in normal serum but has not interfered with Raji cell or C1q binding tests or with FACS analysis. More limited studies of a second individual experiencing CDAC yielded similar results.

Adult↗
PubMed1993

Activity of C1 esterase inhibitor in patients with vascular leak syndrome after bone marrow transplantation.

Vascular-leak syndrome (VLS) is a common complication in the first 3 weeks after bone marrow transplantation (BMT). The patients present with weight gain, generalized edema, ascites, pericardial or pleural effusions, tachycardia, arterial hypotonia, and/or pre-renal failure. The aim of our study was to investigate the role of the complement system in VLS. The protein concentrations of C3 and C4 were studied by immunodiffusion, and total hemolytic complement activity was studied by assessment of CH50. C1 esterase inhibitor (C1 Inh), the major inhibitor of the classical pathway of complement, was assessed by a functional test. Activation of complement was assessed by C4d (a C4 activation product). Twelve patients were followed prospectively from start of conditioning therapy to day +21 after bone marrow transplantation. Eight of 12 patients did not develop VLS. These patients had an increase of C3 between day +9 and day +13 (range: 1.3- to 1.5-fold, median: 1.4-fold), C4 (range: 1.3- to 1.9-fold, median: 1.4-fold), CH50 (range: 1.3- to 1.6-fold, median: 1.4-fold), and C1 Inh (range: 1.2- to 1.5-fold, median: 1.3-fold). Four of 12 patients developed VLS. C1 Inh activity was decreased to 0.60- to 0.80-fold. This decrease began 2-6 days prior to clinical diagnosis of VLS (n = 3), or at onset of VLS (n = 1). Patients with VLS showed elevated C4d concentrations (up to 2.4 mg/dl, upper normal threshold value: 0.9 mg/dl). Patients with VLS reveal an activated state of the complement system which is accompanied by a reduced activity of C1 Inh. Insufficient control of complement activation may contribute to VLS in patients after BMT.

Adolescent↗
PubMed1994

Oxfendazole treatment of non-parasitized lambs and its effect on the immune system.

Ten parasite-free lambs were drenched with oxfendazole on days 0 and 28 and, one day after each drench, were injected with human erythrocytes and ovalbumin. Ten other antigen-injected lambs were not drenched (controls). Lymphocytes collected 3 days after each antigen injection and cultured in RPMI 1640 plus 5% fetal calf serum (FCS) and lymphocytes collected 3 days after the first and 3 and 7 days after the second antigen injection and cultured in 50% autologous serum had decreased blastogenic activity compared with control lymphocytes. After the second drench, decreased blastogenesis was seen with lymphocytes collected on days 3 and 7 and cultured in 5% FCS and concanavalin A (Con A) and on day 3 when cultured in 5% FCS and phytohaemagglutinin (PHA). Decreased blastogenesis was also seen with lymphocytes collected 7 and 29 days after the second injection of antigen and cultured in 50% autologous serum plus Con A and on days 3, 7 and 29 when cultured in 50% autologous serum and PHA. Significantly depressed antibody responses to both antigens were seen after the second drench. The serum complement level was depressed 3 days after the second injection of antigen. Serum nitric oxide levels were significantly depressed 3 and 21 days after the first and 7 and 21 days after the second injection of antigen. There were no differences in levels of growth-promoting hormones but the drenched lambs gained significantly more weight than the controls.

Animals↗
PubMed1992

Allotypes of the fourth component of complement in Koreans.

The analysis of genetic polymorphism in C4 was performed on EDTA-plasma from 169 healthy unrelated Koreans. Plasma samples were subjected to high-voltage agarose gel electrophoresis followed by immunofixation. C4B allotypes were further detected by a hemolytic overlay method. The allele frequencies of C4A and C4B were as follows; for C4A, C4A*3 = 0.6099, C4A*4 = 0.1702, C4A*Q0 = 0.1525, C4A*2 = 0.0461, and C4A*R = 0.0213; for C4B, C4B*1 = 0.6406, C4B*2 = 0.2740, C4B*5 = 0.0569, C4B*Q0 = 0.0178, and C4B*R = 0.0107. C4A*3 and C4B*1 were among the most common alleles at each locus. C4A*6 was not detected in this study, but this allele is relatively common in both Caucasoid and Negroid populations. C4B*5 is a common allele in Asian, which is rare in Caucasoids and Negroids. C4B*5 appeared to be a characteristic allele of Oriental. In the C4A locus, five individuals with duplicated allotypes (three C4A 3,3 + 2, one C4A 4,3 + 2, and one C4A 3,3 + 3) were observed, and in the C4B locus, one individual with duplicated allotype (C4B 1,1 + 1) was detected.

Alleles↗
PubMed1992

Complement component deficiencies and infection: C5, C8 and C3 deficiencies in three families.

Three families are described with complement component deficiencies. In one family, five children had C5 deficiency; in a second family, two children had C8 deficiency and one child in a third family had C3 deficiency. The index cases were identified during screening of patients with recurrent pyogenic infections, recurrent meningitis and meningococcaemia. Two of the five C5 deficient patients had recurrent meningitis and meningococcaemia, two had recurrent respiratory tract infections and otitis and one was healthy. One of the C8 deficient patients had meningitis, meningococcaemia and pneumonia, whereas his sibling with the same deficiency was healthy. The patient with C3 deficiency had four episodes of meningitis and recurrent otitis.

Child↗