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PubMed1988

Stimulatory effect of phorbol diester on relaxin release by porcine luteal cells in culture.

The role of protein kinase C (PKC) activation in the modulation of secretion of the peptide hormone, relaxin, by porcine luteal cells was examined by use of a reverse-hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin anti-serum and complement, a zone of hemolysis--a plaque--developed around relaxin-releasing luteal cells. The rate of development of plaques in timecourse studies was then used as an index of the rate of relaxin release. The tumor-promoting agent, phorbol 12-myristate 13-acetate (PMA) activates a phospholipid- and calcium-dependent kinase, PKC. This enzyme is present in high concentrations in porcine luteal tissue, although its physiological role(s) is unknown. We report here that PMA exerted a time- and dose-dependent stimulatory effect on relaxin release by enzyme-dispersed porcine luteal cells in culture. Maximum stimulation was achieved by 50nM PMA. In contrast, the non-PKC-activating phorbol ester, 4 alpha-phorbol-12,13-didecanoate, exerted no significant effect on the rate of relaxin in doses up to 1 microM. We further observed that a synthetic 1,2-diacyl-glycerol (1-oleoyl-2-acetyl-rac-glycerol; 125 microM) mimicked the action of PMA in stimulating relaxin secretion. These results are consistent with the view that activation of PKC provides at least one intracellular mechanism that regulates relaxin secretion by porcine luteal cells.

Animals↗
PubMedDate not supplied

Immune complex-like materials in sera of patients with breast cancer: detection by complement-consumption assay.

Sera of patients with breast cancer were tested for the presence of immune complexes by a complement-consumption assay. To increase the concentration of immune complexes and thus improve the chances of their detection, sera were first ultracentrifuged and the sedimented material was assayed. Sera were considered to be positive if they consumed at least 75% of the hemolytic activity under the conditions of the test. Of the 60 patients tested, 45% were positive as compared with 13.5% positive sera among health blood bank donors. The presence of immune complexes could not be correlated with the extent of disease, the age of the patients, treatment or lack of treatment at the time of the test, or the time that had elapsed since diagnosis. In a few control tests, treatment of samples with DNase had no effect on their anticomplementary activity, whereas reduction with 2-mercaptoethanol abrogated the activity. These results strengthened the assumption that the substances binding complement were indeed immune complexes.

Adult↗
PubMed1996

Complement C4 protein expression by rat hepatic stellate cells.

Stellate cells play an important role in the production and turnover of the normal extracellular matrix of the liver and are key effector cells in the hepatic fibrogenesis that occurs in response to liver injury. In the present study, we used a rat model of long term dietary iron supplementation to identify stellate cell genes that are expressed during chronic hepatic iron overload. Using a subtraction cloning strategy, we identified a rat isoform of the complement C4 protein gene whose expression was strongly induced in stellate cells after iron overload. Highly purified, cultured stellate cells synthesized the C4 precursor protein and released its subunits into the culture medium. The C4 protein secreted in vitro was biologically active in a C4-specific hemolytic assay. C4 mRNA expression was minimal in freshly isolated stellate cells and increased between days 3 and 7 of primary culture, coincident with the expression of smooth muscle alpha-actin (alpha-SMA), a marker of cellular activation. C4 expression was absent in strongly alpha-SMA-positive, passaged cells, but was induced by IFN-gamma, which simultaneously inhibited alpha-SMA expression. Our studies establish hepatic stellate cells as a previously unrecognized source of C4 and raise the possibility that complement protein expression by the cells plays a role in the hepatic injury response and in fibrogenesis. Our in vitro data point to the presence of two distinct stimulatory pathways for C4 expression in stellate cells that differ with regard to their sensitivity to IFN-gamma and their relationship to cellular activation.

Actins↗
PubMed1980

In vitro activation of feline complement by feline leukemia virus.

Incubation of normal feline serum with purified feline leukemia virus (FeLV) at 37 degrees C for 30 min resulted in the activation of the complement system via the classical pathway as demonstrated by consumption of the C1, C4, C2, C3, and, to a lesser extent, the later C components. A similar finding was observed when normal human serum was substituted for normal cat serum. In contrast, complement-dependent lysis of FeLV with normal feline serum as assayed by the release of ribonucleic acid-dependent deoxyribonucleic acid polymerase was one-third that of complement-dependent FeLV lysis with normal human serum. The levels of total hemolytic complement and neutralizing antibody in individual feline sera were also not proportional to the degree of virolytic activity. These observations indicate that the inefficient virolysis of FeLV by normal cat serum may be one of the factors contributing to the high incidence of leukemia observed in cats.

Animals↗
PubMed1975

Studies with human leukocyte lysosomes. Evidence for antilysosome antibodies in lupus erythematosus and for the presence of lysosomal antigen in inflammatory diseases.

Human lysosomes were isolated from normal peripheral blood leukoyctes and characterized by electron microscopy, enzyme analysis, and assays for DNA and RNA. Stored sera from 37 unselected patients with systemic lupus erythematosus (SLE), including active and inactive, treated and untreated cases, were tested in complement fixation (CF) reactions with these lysosome preparations. 23 SLE sera exhibited positive CR reactions, as did sera from two patients with "lupoid" hepatitis. The seven SLE sera with strongest CF reactivity also demonstrated gel precipitin reactions with lysosomes. Neither CF nor precipitin reactions with lysosomes were observed with normal sera or with sera of patients with drug-induced lupus syndrome, rheumatoid arthritis (RA), polymyositis, or autoimmune hemolytic anemia. By several criteria the antilysosome CF and precipitin reactions of SLE sera cound not be attributed to antibody to DNA, RNA, or other intracellular organelles. The lysosomal component reactive with SLE sera in CF assays was sedimentable at high speed and is presumably membrane associated. The CF activity of two representative SLE sera was associated with IgG globulins by Sephadex filtration. A search for lysosomal antigen in SLE and related disorders was also made. By employing rabbit antiserum to human lysosomes in immunodiffusion, a soluble lysosomal component, apparently distinct from the sedimentable (membrane-associated) antigen described above, was identified in serum, synovial fluid, or pleural fluid from patients with SLE, RA, ankylosing spondylitis, and leukemoid reaction. An antigenically identical soluble component reactive with the rabbit antiserum could be released in vitro from intact lysosomes by repeated freeze-thaw cycles..

Anemia, Hemolytic, Autoimmune↗
PubMed1980

Complement activation by Coccidioides immitis: in vitro and clinical studies.

Mycelial- or spherule-phase derivatives of Coccidioides immitis caused a decrease in vitro of total hemolytic complement in serum from a nonsensitized person. Activation involved both classic and alternative pathways as shown by deprssion of hemolytic C4 and by generation of products of activation of components C3, C4, and factor B. In addition, functional complement activity or immunoreactive levels of complement components or both were measured in 23 patients with self-limited or disseminated coccidioidomycosis. Low total hemolytic complement was found in nine, usually during the early phase of primary illness, and was transient. Hemolytic C4 was low, and the effect of inulin to decrease complement levels was blunted, suggested both classic and alternative pathways may be deficient. However, associated depression of immunoreactive levels of components assayed (C3, C4, C5, factor B, and properdin) was not consistently found. This disparity raises the possibility of enhanced in vitro inactivation analogous to activation by immune complexes.

Coccidioides↗
PubMed1996

Mechanism of aggregates generated by heating human serum.

Heating normal human serum produced aggregates that can be detected with a F(ab')2 anti-C3 enzyme-linked immunosorbent assay (FaC3 ELISA) for circulating immune complexes. Using this we studied the production mechanism of these aggregates. Heating serum with the inactivated complement system produced aggregates which included IgG and C3/C3 fragments. The aggregates also incorporated IgA, IgM, and albumin as well as IgG and C3/C3 fragments. These results indicate that the production of the aggregates was not due to activation of the complement system by aggregated IgG generated by heating, but mainly resulted from aggregation of serum proteins induced by heating. This study suggests that there are heating conditions under which the hemolytic activity of complement system is abolished, but aggregates are not detected with FaC3 ELISA.

Antigen-Antibody Complex↗
PubMed1984

Potentiation of virulence by group B streptococcal polysaccharides.

The ability of group B streptococcal (GBS) capsular polysaccharides to potentiate virulence was examined. Incubation of soluble type Ia or III polysaccharide in hypogammaglobulinemic human serum inhibited classical complement-dependent opsonophagocytic killing of type Ia strain 515. When functional complement components were measured, C1 activity increased in sera with added polysaccharide whereas C4 activity decreased 80%-90%. Incubation of purified C1 with type Ia polysaccharide inhibited lysis of EAC4 cells in a C1 transfer assay. In a mouse lethality model, tail-vein injection of 50 micrograms of type Ia or III polysaccharide decreased the 50% lethal dose (LD50) from 3.2 X 10(6) to 2.2 X 10(5). Total hemolytic complement levels in mice immediately after polysaccharide injection were increased over levels in control mice, but this difference was not seen 30 min later. The LD50 in mice depleted of C3 (with cobra venom factor) was 6 X 10(5) and simultaneous injection of polysaccharide did not further lower the LD50.

Animals↗
PubMed1982

C4 does not bind to human and rabbit IgM during activation of the classical complement pathway on the red cell.

It has been known for many years that IgG-C4 complexes are generated during the activation of the classical complement pathway. Information regarding binding of C4 by IgM is not available. We studied the binding of human C4 by natural human and rabbit anti-hapten IgM under conditions in which the IgM was bound to hapten coupled to sheep red cells. We eluted the IgM by excess fluid phase hapten, reattached the eluted IgM to red cells coupled with hapten, and transferred the IgM from red cell-IgM-C4 complexes to red cells coupled with hapten. Analysis of the cells by radioimmunoassay and/or by hemolytic assays showed that no C4 was eluted from the cells with the IgM, that no C4 transferred from cell to cell with the IgM, and that the C4 remaining on the cells was hemolytically active. We concluded that in contrast to IgG, natural human and rabbit IgM at a cell surface failed to bind C4.

Animals↗
PubMed1986

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis.

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.

Animals↗
PubMed1986

Increased hemolytic activity of the trypsin-cleaved ninth component of complement.

Human C9 treated with trypsin is initially cleaved into two fragments with relative mol. wts of 53,000 and 20,000. This limited cleavage of C9 induces a 2.4-times increase in the hemolytic activity of C9 when compared to untreated C9. This difference diminishes when C9 activity is tested in an assay using a prolonged incubation time of C9 with C5b-8-bearing red blood cells. Trypsinization of C9 also promotes spontaneous C9 polymerization. SDS-resistant tubular C9 complexes are formed at a C9 concn of 1 mg/ml within 8 hr at 37 degrees C. Our data indicate that specific limited proteolysis of C9 not only induces spontaneous C9 polymerization but also increases the hemolytic activity of C9, suggesting that a similar molecular mechanism is involved in both processes.

Biopolymers↗
PubMed1986

A case of hereditary angioneurotic edema associated with systemic lupus erythematosus.

A pedigree of C1 inhibitor (C1 INH) deficiency associated with positive LE cell and an elevated titer of DNA antibodies and antinuclear factor (ANF) and nephropathy was presented. The proband of this family was diagnosed as having definite systemic lupus erythematosus (SLE) after a clinical course of several year since her first visit to our hospital and because of the lack of hemolytic activity of complement (CH50), in spite of the absence of idiopathic edema, C1 INH levels were 1.2 mg/dl (3.8% NHS) determined as antigen and 65 site forming unit (SFU) (5.8% NHS) determined by hemolytic assay in her blood. Her mother and brother had characteristic idiopathic edema of her face, larynx, hand and bowel and they had low levels of C1 INH of 1.8 mg/dl (5.8% NHS) and 4.8 mg/dl (15.5% NHS) respectively in their blood. On the basis of these findings, this family was diagnosed as having a pedigree of hereditary angioneurotic edema (HANE) which is supposedly an inherited autosomal positive trait. Actually, however, the proband's serological and hematological indices became positive and progressed year by year, which implies that SLE was absent for the first several years. It might be said that this interesting clinical course indicates that SLE appeared chronologically as a hereditary deficiency in one of the complement components in this case. In concurrence with the general observation that recurrent viral infections due to the deficiency of complement components are presumed to be responsible for SLE-like disease. Her levels of several kinds of anti-virus antibodies were high. Methylprednisolone helped normalize her level of C1 INH and ameliorate the clinical course.

Adult↗
PubMed1980

Depletion of C2 and C4 in sera from hamsters bearing herpes simplex type 2-induced tumors.

Total hemolytic complement activity (CH50) was compared in sera from normal and tumor-bearing hamsters. CH50 ranged from 160-216 in normal animals and from 82 to 152 in tumor-bearing animals. Preliminary screening assays indicated that of 9 complement components assayed, only C2 and C4 showed differences in titer in both normal and tumor-bearer sera. In an expanded study, examination of sera from 12 normal and 14 tumor-bearing hamsters showed that the mean C4 and C2 levels were approximately 88 and 70% below those seen in normal sera, respectively.

Animals↗
PubMed1995

[Rheumatoid factor and cryoglobulin are not efficiently related to cold activation of complement].

Cold activation of complement (CA) is a phenomenon characterized by a low hemolytic complement activity in chilled serum. Cryoglobulin and RF have been reported as main causes of CA, and we previously reported the close association between CA and HCV infection in 21 CA patients. Therefore, we assayed CA, RF, cryoglobulin and anti-HCV antibody titers in 304 randomly selected patients with unknown diseases. CA was positive in 26 of the 304 patients, and among them, RF and/or cryoglobulin were found positive in 20 patients, and anti-HCV antibody were positive in all of them. RF was positive in 41 of the 304 patients. 37% of them were anti-HCV antibody positive and 63% were negative. Cryoglobulin was positive in 61 out of 304 patients. Anti-HCV antibody positivity was observed in 59 out of 304 patients, and all CA-positive cases were included in the anti-HCV antibody-titer-positive group. However, either RF or cryoglobulin positive patients were observed in the same degree in both anti-HCV antibody titer positive and negative groups. Therefore, RF or cryoglobulin is not considered the main cause of CA.

Cold Temperature↗
PubMed1984

Defective binding of the third component of complement (C3) to Streptococcus pneumoniae in multiple myeloma.

Patients with multiple myeloma (MM) are at an increased risk for infections with bacteria that require opsonization with complement. Because Streptococcus pneumoniae is the most frequently encountered pathogen in these patients, we investigated the ability of serum from patients with MM to mediate the binding of C3b, the major opsonin of the complement system, to S. pneumoniae. S. pneumoniae types 3, 14, and 25 were chosen for study, since S. pneumoniae type 3 activates primarily the classical complement pathway (CCP), type 25 primarily the alternative complement pathway (ACP), and type 14 both pathways. S. pneumoniae were treated with normal serum or serum from 17 patients with MM, and the bound C3b was quantified with fluorescein-conjugated anti-C3 in a spectrophotofluorometric assay. Despite normal or elevated serum concentrations of C3, total hemolytic complement, and C-reactive protein in all of the MM sera, factor B in 16/17 such sera, and C4 in 14/17 MM sera studied, all 17 sera demonstrated a defect in C3b binding to type 3 (32.7% +/- 6% of normal). In addition, serum from 15/17 patients bound decreased amounts of C3b to types 14 (39.6% +/- 8%) and 25 (52.2% +/- 8%). Mixing normal serum with MM serum restored MM C3b binding activity to all three S. pneumoniae types, suggesting that the defect was related to a deficiency rather than an inhibitor of C3 activation. Although MM patients are unable to produce specific antibodies to bacterial antigens, the addition of anti-S. pneumoniae antibodies to MM serum did not enhance C3b binding to any of the S. pneumoniae types. However, when S. pneumoniae were opsonized in a mixture of MM serum and C3-depleted normal serum, C3b binding was restored to all three S. pneumoniae types, demonstrating that MM C3 functions normally in the presence of other normal serum factors. In the present studies, the MM C3b binding defect appeared to correlate with the incidence of S. pneumoniae infections. Serum from patients with a history of an S. pneumoniae infection bound significantly less C3 (20.5% +/- 4%) than those study patients without a history of an S. pneumoniae infection (55.8% +/- 8%) (p less than 0.0025). Thus, MM serum has a defect in the activation of C3, and this may contribute to the increased susceptibility of MM patients to S. pneumoniae infections.

Complement C3↗
PubMed1975

Activation by some T-independent antigens and B cell mitogens of the alternative pathway of the complement system.

A number of T-independent antigens and B cell mitogens were examined for their ability to activate C3 via the alternative pathway of the complement system. Loss of hemolytically active C3, generation of anaphylatoxin activity, and immunoelectrophoretic conversion of C3 and factor B, were checked in normal and C4-deficient guinea pig serum, and, in some cases, in normal human serum. As judged by their activity in these assays, 10 lipopolysaccharides of different origin and constitution, pneumococcus type III polysaccharide, levan, dinitrophenylated aminoethyl-dextran, dinitrophenylated (D-glutamic acid, D-lysin) copolymer, polymerized flagellin, and pokeweed mitogen were all capable of initiating the alternative pathway, but differed with respect to their potency, their relative activity in the presence or absence of C4, and their ability to inhibit C3-turnover at high concentrations. Polyvinylpyrrolidone of intermediate molecular weight (4 x 10(4) daltons) was only active if the most sensitive assay was used (anaphylatoxin generation). Other species of polyvinylpyrrolidone, depolymerized pneumococcal polysaccharide, aminoethyl-dextran, [D-glutamic acid, D-lysin] copolymer, phytohemagglutinin and concanavalin A failed to activate C3. C3-consumption by concanavalin A was due to nonspecific binding.

Anaphylaxis↗
PubMed1983

Enzyme immunoassay for antibodies to native DNA. Specificity and quality of antibodies.

An enzyme immunoassay was developed to detect antibodies to native DNA; DNA coating conditions that maximized sensitivity, specificity, and reproducibility were selected. Sera of patients with systemic lupus erythematosus (SLE) were positive more frequently by this immunoassay than by the Crithidia luciliae assay or by counterimmunoelectrophoresis. By enzyme immunoassay, 94% of sera with active SLE and 70% of sera from patients with inactive SLE were positive, as were 16% from those suspected of having SLE, and 2.5% of normal persons. Specificity for native DNA was shown for both SLE and normal sera by inhibition studies and by S1 nuclease treatment of polystyrene-bound native DNA. The enzyme immunoassay correlated more with serum hemolytic complement levels that did the other 2 assays, suggesting that it detects biologically more relevant anti-DNA antibodies than do the other 2 tests.

Antibodies, Antinuclear↗
PubMed2005

High dietary vitamin C affects growth, non-specific immune responses and disease resistance in Asian catfish, Clarias batrachus.

Studies were conducted to determine the immunomodulatory effects of high dietary ascorbic acid (vitamin C) on growth, serum concentration, non-specific immune response and disease resistance of a commercially important Asian catfish, Clarias batrachus. Four practical diets were formulated to contain 0, 500, 1000 and 2000 mg ascorbic acid (AA) equivalent/kg diet, supplied as L-ascorbyl-2-polyphosphate (LAPP) and were fed for 2, 4, 6 and 8 weeks. Each diet was fed to triplicate groups of catfish with initial body weight of 15.47+/- 0.59 g. After 2, 4, 6 and 8 weeks, growth, serum concentration of AA, oxidative respiratory burst, lysozyme and natural hemolytic complement activities, myeloperoxidase (MPO) content and natural haemagglutination titre were measured. Ten numbers of fish in duplicate were challenged with Aeromonas hydrophila to measure the level of protection against aeromoniasis at each one of the assayed times. The results showed that AA concentration in serum correlated positively with those in the diets and reached its saturation level after the time period directly proportional to the increase in dose level. Fish fed AA-supplemented diets showed significantly (p < 0.05) higher specific growth rate after 2 weeks of feeding. The superoxide production was enhanced after 8 weeks of feeding fish at a supplemented dose level of 2000 mg/kg. Similarly, MPO content, haemagglutination titre and alternative complement activity in serum enhanced with the increase of dietary AA levels at different duration of feeding. The lysozyme activity was not affected by the dietary AA treatment. On the other hand, feeding of AA at all concentrations significantly increased percent survival against A. hydrophila challenge after 4 weeks compared to control. The non-specific immune parameters as well as percent survival were enhanced as a result of high AA supply particularly at 500 mg/kg diet, although the increase was not maintained but returned to the initial levels after 4 weeks. These results support the possible use of AA as an immunostimulant at a dose of 500 mg/kg diet for a period of 4 weeks in catfish farming.

Animals↗