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PubMed1984

The ability of IgA to inhibit complement consumption by complement-fixing antigens and antigen-antibody complexes.

The polymeric and monomeric (7S) forms of the nitrophenyl-specific MOPC-315 murine IgA myeloma protein were examined for their capacity to inhibit the consumption of guinea-pig complement (C) by the C-activating protein DNP46BSA and by antigen-antibody (Ag-Ab) complexes. On a molar basis, 13S IgA was slightly (2-3 fold) more efficient than 7S IgA in inhibiting C consumption by DNP-BSA. When mixed with IgG antibodies prior to incubation with a non-complement-fixing antigen (TNP-KLH), 13S IgA was considerably more effective than 7S IgA in preventing the formation of Ag-Ab complexes able to fix C. The degree of inhibition observed was related to the concentration of C used in the assay, being greater at lower C concentrations.

Animals↗

PubMed1982

Polymerization of the ninth component of complement (C9): formation of poly(C9) with a tubular ultrastructure resembling the membrane attack complex of complement.

The ninth component of complement (C9) has a marked propensity to polymerize. C9 polymers [poly(C9)] formed spontaneously in Veronal-buffered saline upon incubation of purified C9 for 64 hr at 37 degrees C or within 2 hr at 46--56 degrees C. Poly(C9) formed at 37 degrees C was visualized by electron microscopy as a tubular structure with an internal diameter of 110 A and a length of 160 A. Its ultrastructure suggested a dodecameric composition and resembled that of the membrane attack complex of complement. The wider end of the tubular structure was formed by an approximately 30-A-thick torus with inner and outer diameters of 110 A and 220 A, respectively. Because the dimensions of C9 within poly(C9) were 160 x 55 A (maximal) and 20 A (minimal) and because monomeric C9 has dimensions of approximately 80 x 55 A, it is proposed that monomeric C9 unfolds during polymerization into tubules. Polymerization also occurred upon treatment of C9 for 1 hr at 37 degrees C with 0.6 M guanidine . HCl, 0.1 M octyl glucoside, or 1.5% sodium deoxycholate. Guanidine . HCl-induced C9 polymers consisted of elongated highly curved strands 55--80 A wide, suggesting that these polymers were formed by globular C9 that had not unfolded.

Cell Membrane Permeability↗

PubMed1996

Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

Base Sequence↗

PubMed1966

Deficiency of the sixth component of complement in rabbits with an inherited complement defect.

A strain of rabbits with an inherited complement deficiency was shown to lack the sixth component of the hemolytic complement system. A method was elaborated for the partial purification of this component from normal rabbit serum. Upon injection of partially purified rabbit C'6 into C'6-deficient animals, an antibody was obtained which specifically inhibited the hemolytic activity of C'6. The data suggest that C'6-deficient serum either lacks the C'6 molecule or contains it in a chemically modified and inactive form.

Animals↗

PubMed1996

The role of complement in the pathogenesis of postpartum thyroiditis: ultrasound echogenicity and the degree of complement-induced thyroid damage.

Postpartum thyroiditis (PPT) is a transient autoimmune thyroiditis occurring during the postpartum year that is characterized by circulating antithyroid antibodies, abnormal thyroid ultrasound echotexture, and episodes of hyperthyroidism, hypothyroidism, or both. In this study we examined the relationship between lymphocytic thyroiditis, as indicated by echotexture changes, and complement-activating thyroid autoantibodies. Thyroid ultrasound echotexture, thyroid function, and bioactive (complement-activating) thyroid peroxidase (TPO) antibodies have been measured in a group of 63 TPO antibody-positive women during the postpartum year. When the maximum bioactive TPO antibody activity recorded was compared with echogenicity and thyroid status, there was a correlation between hypoechogenicity, elevated antibody activity, and abnormal thyroid status (r = 0.72, p < 0.001). However, 7 cases showed severe ultrasound changes in the absence of any thyroid dysfunction (3 of these cases showed normal bioactive antibody activity), while 4 (all hyperthyroid PPT) showed thyroid dysfunction in the absence of any ultrasound changes. Within the euthyroid ultrasound normal group, bioactive TPO antibody activity remained low throughout the postpartum year. Antibody activity in the hypoechogenic euthyroid women was significantly elevated (p < 0.001) compared with the echo normal group, but was indistinguishable from the activity curve obtained in women whose PPT included a hypothyroid phase. The determination of echotexture by thyroid ultrasonography gives a useful, noninvasive measure of the degree of thyroiditis in these women. However, as a significant number of cases with severe changes in echotexture remained euthyroid, we conclude that the development of thyroid dysfunction is not an inevitable consequence of lymphocytic thyroiditis during the postpartum and suggests that other factors must also be involved in progression to overt thyroid dysfunction.

Autoantibodies↗

PubMed1996

Interallelic complementation at the suppressor of forked locus of Drosophila reveals complementation between suppressor of forked proteins mutated in different regions.

The Su(f) protein of Drosophila melanogaster shares extensive homologies with proteins from yeast (RNA14) and man (77 kD subunit of cleavage stimulation factor) that are required for 3' end processing of mRNA. These homologies suggest that su(f) is involved in mRNA 3' end formation and that some aspects of this process are conserved throughout eukaryotes. We have investigated the genetic and molecular complexity of the su(f) locus. The su(f) gene is transcribed to produce three RNAs and could encode two proteins. Using constructs that contain different parts of the locus, we show that only the larger predicted gene product of 84 kD is required for the wild-type function of su(f). Some lethal alleles of su(f) complement to produce viable combinations. The structures of complementing and noncomplementing su(f) alleles indicate that 84-kD Su(f) proteins mutated in different domains can act in combination for partial su(f) function. Our results suggest protein-protein interaction between or within wild-type Su(f) molecules.

Alleles↗

PubMed1989

Immunohistochemical demonstration of age-related deposition of vitronectin (S-protein of complement) and terminal complement complex on dermal elastic fibers.

Immunoreactivity of vitronectin was investigated in 100 skin specimens from different body regions in 87 individuals of different ages using monoclonal and polyclonal anti-vitronectin antibodies in an avidin-biotin-peroxidase complex technique. Vitronectin immunoreactivity was found in conjunction with dermal elastic fibers in all subjects older than 13 years. No vitronectin immunostaining was detected in subjects younger than six years, suggesting deposition of vitronectin during late childhood or early adolescence. Using an immunogold staining procedure, vitronectin immunoreactivity was ultrastructurally localized to the periphery of elastic fibers. The blood level of vitronectin in 20 healthy newborns was 67% of the adult level, suggesting active biosynthesis already in the fetus. To investigate whether vitronectin is deposited as part of the SC5b-9 complex or as uncomplexed protein, the immunoreactivity of vitronectin was compared with that of C9, using monoclonal and polyclonal antibodies against the C9 neoantigen. Distinct C9 neoantigen immunoreactivity was demonstrated in association with dermal elastic fibers in human skin in adults but only in subjects older than 30 years. The intensity of C9 neoantigen immunoreactivity appeared to increase with age and was found to be stronger in sun-exposed skin than in sun-protected skin. These findings indicate that uncomplexed vitronectin is deposited during childhood or early adolescence and that terminal complement complexes (C5b-9 and/or SC5b-9) are deposited on elastic fibers later on in life. Hypothetically, the tissue form of vitronectin may be involved in the prevention of tissue damage in proximity to local complement activation. In addition, it may be physiologically important as substratum for cells, stimulating cell migration and anchorage.

Adolescent↗

PubMed1991

Roles of complement and complement receptor type 3 in phagocytosis of Listeria monocytogenes by inflammatory mouse peritoneal macrophages.

Listeria monocytogenes is a facultative intracellular bacterium that is phagocytosed by and can proliferate within cells of the mononuclear phagocyte system. However, the receptors used by macrophages to internalize this organism have not been identified. In the experiments described here, the contributions of serum complement component C3 and macrophage complement receptor type 3 (CR3) to opsonization and phagocytosis of L. monocytogenes by mouse inflammatory peritoneal macrophages were studied. An assay which allowed the distinction of adherent versus internalized bacteria was used to show that following mixing of L. monocytogenes with inflammatory macrophages, greater than 95% of the bacteria bound were internalized by these phagocytes. When immunofluorescent antibodies to C3 and immunoglobulin were used, C3 but not immunoglobulin was detected on L. monocytogenes following incubation in normal serum or ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetracetic acid-Mg(2+)-chelated serum. When macrophages were incubated with 5% serum and L. monocytogenes in a standard assay, approximately 80% of the phagocytosis was inhibited by heat-inactivated serum or by the addition of F(ab')2 anti-C3 antibody. The role of macrophage CR3 was demonstrated by the ability of anti-CR3 monoclonal antibody M1/70 to decrease phagocytosis to the same levels as those seen with heat-inactivated serum. These experiments indicated that in the presence of normal serum, L. monocytogenes is phagocytosed by inflammatory macrophages primarily because CR3 on these cells binds to C3 deposited on the bacterial surface.

Animals↗

PubMed1992

The coccidioidal complement fixation and immunodiffusion-complement fixation antigen is a chitinase.

Culture filtrates and autolysates of Coccidioides immitis have provided suitable crude antigens for the serodiagnosis and prognosis of coccidioidomycosis. One of these, a heat-labile antigen which participates in the immunodiffusion reaction corresponding to the complement fixation reaction (IDCF), has been characterized as a 110-kDa native protein that, when subjected to reducing conditions and heat, yields a 48-kDa component. The present report provides serologic and biochemical evidence that this antigen is a chitinase. This chitinase, isolated from 48-h culture filtrate of the spherule-endospore-phase C. immitis by affinity adsorption to chitin, formed a line of identity with the IDCF reference antigen and participated in the complement fixation reaction with human serum. It lost its enzymatic as well as antigenic activity when heated, but when not heated it retained its enzymatic activity even when precipitated with coccidiodal antibody present in human serum. This chitinase represents a significant serodiagnostic substance and may be important in the morphogenesis of C. immitis.

Antigens, Fungal↗

PubMed2000

DNA-Based immunization with Trypanosoma cruzi complement regulatory protein elicits complement lytic antibodies and confers protection against Trypanosoma cruzi infection.

A complement regulatory protein (CRP) of Trypanosoma cruzi was evaluated as a vaccine candidate in a murine model of experimental T. cruzi infection. Recombinant CRP derived from an Escherichia coli expression system and a plasmid encoding the full-length crp structural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice. Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles. Only mice immunized with the crp DNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge with T. cruzi trypomastigotes. These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP. The work also supports the further investigation of CRP as a component of a multigene, anti-T. cruzi DNA vaccine.

Animals↗

PubMed1968

Nature of the complementation products formed by a complementing mutant of neurospora crassa.

The mutant am-14 produces no active nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase (GDH) and no protein showing immunological cross-reaction with the enzyme. Nevertheless, it shows complementation with several other am mutants in heterokaryons. Active GDH can be extracted from heterokaryons formed from am-14 and other mutants which, by themselves, produce more or less inactive varieties of the enzyme. The enzyme from am-14 + am-3 heterokaryons can be partially separated from am-3 mutant GDH on a diethylaminoethyl cellulose column. It is characterized by abnormally high thermolability and by a capacity for activation by glutamate. By the same procedure as brings about hybridization between mutant GDH proteins, it has been possible to recover enzyme with the properties of pure am-3 GDH from a partially purified am-14 + am-3 GDH preparation which was initially substantially free of unhybridized am-3 enzyme. This is interpreted as evidence that the active complementation product is a hybrid oligomer containing am-3 monomers and also am-14 monomers, the latter being unable to aggregate by themselves. Heterokaryons formed from am-14 and wild type produce GDH of abnormally high thermolability, presumably due to the formation of am-14 + am(+) hybrids.

Alleles↗

PubMed1994

Characterization of strains of Neisseria meningitidis recovered from complement-sufficient and complement-deficient patients in the Western Cape Province, South Africa.

Complement deficiency has been associated with increased susceptibility to meningococcal disease. In order to determine whether special meningococcal strains caused disease in complement-deficient (CD) patients, 17 Neisseria meningitidis strains recovered from patients in the Western Cape Province, South Africa, known to be CD were compared with 124 routine isolates obtained from patients living in the same area. Serogrouping of the strains from the CD subjects revealed that the common serogroups, particularly serogroup B, predominated. However, the prevalence of rare serogroups among isolates from CD subjects was significantly higher than that found among isolates from the control group. Sero- and subtyping of the class 1 and class 2 or 3 outer membrane proteins showed no significant difference between isolates from CD subjects and the routine clinical isolates. Multilocus enzyme electrophoresis of the 141 isolates revealed six clusters of electrophoretic types (ETs) and two unrelated ETs. The same degree of genetic diversity existed in ETs of isolates from CD subjects and the control group. However, the ET-5 complex, which is known to be associated with epidemic disease, was found in 22 (18%) of the routine clinical isolates but in none of the isolates from the CD subjects. This difference was marginally significant. What was highly significant was the finding that 8 of the 17 isolates from CD subjects were in one ET cluster, cluster F, which comprised a total of 20 isolates. Thus, our results show a difference in the clonal compositions of the strains that infect CD subjects in comparison with the clonal compositions of those that cause clinical infections in the population at large.

Adolescent↗

PubMed1986

Complementation of a bovine papilloma virus low-copy-number mutant: evidence for a temporal requirement of the complementing gene.

We identified a bovine papilloma virus function encoded by the E6/E7 gene, which is required for both BPV high-copy-number replication and maintenance of transformation of cultured cells. A cDNA copy of this gene was isolated and expressed from a retrovirus vector. We found that complete complementation of a BPV low-copy-number mutant (dl576) by the cDNA encoding the E6/E7 gene was temporally dependent. When both the E6/E7 cDNA and dl576 were introduced together into cells, wild-type replication and stable transformation resulted. In contrast, introduction of the complementing cDNA into cells already carrying dl576 led to only partial amplification of the resident mutant DNA accompanied by a restoration of the transformed phenotype. These results, along with other findings, suggest that the establishment of BPV plasmid replication occurs in two stages: an initial amplification of the incoming DNA followed by stable homeostatic replication which maintains the existing copy number.

Animals↗

PubMed1978

A comparison of complement fixation, indirect immunofluorescence for viral late antigens, and anti-complement immunofluorescence tests for the detection of cytomegalovirus specific serum antibodies.

It was shown that, on average, the titres of the sera obtained by both the anti-complement immunofluorescence (ACIF) and indirect immunofluorescence for viral late antigens (IFA-LA) procedures were approximately eight times higher than those obtained by complement fixation. There was no significant difference between the mean ACIF and IFA-LA titres, but the former method was preferred because it was not affected by non-specific staining. When 406 sera were screened for the presence or absence of antibodies, only five sera (1.2%) gave discordant results with the three tests. None of the tests could detect all of the sera that contained specific antibodies while each test detected over 98% of these sera. Thus, the increased sensitivity of the immunofluorescence procedures did not result in an appreciably increased rate of detection of cytomegalovirus seropositive sera.

Antibodies, Viral↗

PubMed1996

Complements of the season: the hazards of testing for complement activation.

As some of the subtleties of the new era of biological testing become appreciated, a few of the more recently introduced tests are starting to cause concern. The evaluation of the ability of a material to activate complement comes into this category. This article explains complement and its activation and attempts to rationalize the need for testing in this complex area.

Complement Activation↗

PubMed1976

Complement receptor lymphocytes in the rabbit. II. The effect of immunization on the complement receptor lymphocyte population in the craining lymph node.

In an attempt to determine the effect of immunization on the size and nature of the complement receptor lymphocyte (CRL) and surface immunoglobulin (SIg)-bearing lymphocyte (B cell) populations, rabbits were injected with 1 mg alum-precipitated keyhole limpet hemocyanin and the cells of the draining popliteal lymph nodes were examined at various intervals for these populations. Comparison to unimmunized control lymph nodes revealed that immunization resulted in an approximate doubling of the percentages of both CRL and B cell populations by the peak of the response, while the cell number increased about 7-to 8-fold. Kinetic analysis of the response indicated that the percentages of CRL and B cells increased essentially in parallel beginning within 40 h after immunization. Simultaneous assay of complement receptor (CR) and SIg disclosed that CRL remained a B cell subpopulation throughout the response. To determine whether cell division could account for the increases in total cell number and per cent CRL and B cells, immunized rabbits were given 3H-thymidine in vivo, and the fraction of labeled (dividing) cells was determined by autoradiography. Results of such experiments were consistent with the possibility that the early increase in cell number and the increase in CRL and B cell percentages were a result of trapping and/or homing of lymphocytes migrating from the peripheral blood. A significant amount of lymphocyte division occurred during later stages of the response.

Animals↗

PubMed1985

The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens.

Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.

Animals↗

PubMed1975

Complement sensitivity of somatic hybrids of a complement-resistant murine leukemia cell line.

RADA-1 cells (H-2a), a murine leukemia cell line maintained by serial transfer in histocompatible recipients expressing thymus-leukemia (TL) 1, 2, 3 antigenic determinants, resisted the cytotoxic effects of guinea pig complement (GPC) and TL antiserum. The cells expressed a lower density of TL antigens than did ASL-1 cells, another TL(+) leukemia cell line expressing the same determinants and susceptible to complement (C)-mediated lysis. Stable somatic cell hybrids of RADA-1 cells and LM(TK)- cells (H2k), a TL(minus) thymidine kinase-deficient mutant of mouse L cells, were selected in hypoxanthine-aminopterin-thymidine medium. The hybrid cells expressed the H-2 antigens of both parents and shared a hybrid karyotype. They formed TL 1, 2, 3 antigens as determined by immunofluorescence, mixed hemagglutination methods, the direct isolation of TL antigens from Nonidet P40 extracts of the cells, and the capacity of the cells to reduce by absorption the known titers of TL antiserum. These hybrid cells lost the capacity to resist lysis by TL antiserum and GPC. They were susceptible to the cytotoxic effects of TL 1, 2, 3; TL 2; OR TL 1, 3 antiserum and GPC, even though the density of TL antigens associated with the cells was approximately 25% of their resistant RADA-1 parental cells. These results indicated that the mechanism of resistance to C-mediated lysis was genetically separable from the expression of TL antigens by the cells and that the susceptibility of the cells to the cytotoxic effects of antiserum was related only in part to the density of TL antigens expressed by the cells.

Animals↗