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PubMed1990

Effects of trospectomycin on serum sensitivity of Escherichia coli UC 9451.

Trospectomycin sulfate, a chemically synthesized analog of spectinomycin, exhibits a broad range of activity against both aerobes and anaerobes, including the etiological agents of sexually transmitted diseases. Its activity in vitro against Escherichia coli is considered only moderate. At subinhibitory levels, however, trospectomycin induced changes in a pathogenic strain of E. coli, UC 9451, which significantly increased its sensitivity to serum lysis. This strain of E. coli shows high-level resistance to serum in vitro, typically growing twofold within a 45-min incubation period. Following exposure to one-fifth the MIC of trospectomycin, greater than 99% of the bacteria were killed in 25% serum within 15 min. Surviving bacteria were static in this level of serum for over 3 h. Killing was due to lysis mediated by both the classical and alternative complement pathways. The bacteria exposed to trospectomycin were enlarged in both diameter and length, but they still grew at rates comparable to those of untreated bacteria. No other visible morphological changes could be directly related to the increase in serum sensitivity. The profile of outer membrane proteins obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical for trospectomycin-treated or untreated bacteria. However, the relative proportion of four major outer membrane proteins varied considerably.

Anti-Bacterial Agents↗
PubMed1979

Cryptic peptidoglycan and the antiphagocytic effect of the Staphylococcus aureus capsule: model for the antiphagocytic effect of bacterial cell surface polymers.

The antiphagocytic effect of the Staphylococcus aureus capsule is known to be related to its ability to interfere with opsonization by normal human serum. In this study, evidence is presented with isolated cell surface components which indicates that the capsule hinders opsonization by masking cell wall peptidoglycan. In contrast to intact, encapsulated S. aureus M cells, peptidoglycan particles isolated from the organism were efficiently opsonized by normal human serum and phagocytized by human polymorphonuclear leukocytes. Cell wall particles retaining capsular material were opsonized less efficiently than peptidoglycan. Studies comparing the opsonic capacities of normal, C2-deficient, and heat-inactivated sera led to the conclusion that both the classical and the alternative complement pathways contribute to the opsonization of peptidoglycan in normal human serum. It appears that the capsule interferes with opsonization via both of these complement pathways. Serum from rabbits immunized with S. aureus M had significant heat-stable opsonic activity for the intact organism and cell walls retaining capsular material, but not for peptidoglycan. A general model is proposed to explain how antiphagocytic cell surface polymers may inhibit bacterial opsonization and thereby impede natural immunity.

Cell Wall↗
PubMed1986

Pneumococcal intracellular killing is abolished by polysaccharide despite serum complement activity.

Normal human serum absorbed at 0 degrees C with pneumococcal serotype 1, 12, or 25 lost the ability to support polymorphonuclear leukocyte intracellular killing of some pneumococcal serotypes even if immunoglobulin was provided. The absorbed serum contained no organisms but had residual polysaccharide when measured by counterimmunoelectrophoresis against type-specific antisera. The influence of pneumococcal polysaccharide (PPS) on serum support of intracellular polymorphonuclear leukocyte killing was evaluated. Normal human serum was mixed with PPS serotype 1, 12, or 25 at 0 degrees C for up to 120 min and then used as the opsonic source in standard phagocytic killing assays with serotype 1, 12, or 25. Immediately after mixing, each serum combined with PPS serotype 1, 12, or 25 supported killing of all serotypes tested. With increasing time at 0 degrees C with serotype 1 PPS, serum exhibited a marked progressive decline in killing of serotype 25, a similar but less marked impairment in killing of serotype 12, and essentially no impairment for serotype 1. Serum treated with type 25 PPS did not support killing of type 25, but allowed normal killing of types 1 and 12. Incubation with type 12 PPS impaired opsonization of types 12 and 25, but not 1. Addition of PPS-specific antisera did not restore killing. Residual serum hemolytic activity of classic and alternative complement pathways was not reduced below opsonizing levels after 120 min at 0 degrees C. Furthermore, PPS treatment of normal human serum did not alter the attachment or ingestion of 14C-labeled pneumococci by polymorphonuclear leukocytes. Some serotypes of PPS reacted slowly with serum at 0 degrees C, diminishing its ability to support intracellular killing of pneumococci despite serum hemolytic complement activity. Phagocytosis was not inhibited. Specific antibody did not overcome inactivation. Different requirements for the inactivated factor among serotypes may be a characteristic related to organism virulence.

Blood Bactericidal Activity↗
PubMed2000

Bactericidal activity of a monoclonal antibody against a recombinant 40-kDa outer membrane protein of Porphyromonas gingivalis.

BACKGROUND: We have cloned the gene for a 40-kDa outer membrane protein (40-kDa OMP) from Porphyromonas gingivalis 381. The recombinant (r)40-kDa OMP has become the subject of considerable interest because of its potential role in the development of a vaccine useful for passive immunization. To develop such a vaccine, it is essential to fully understand the functions of anti-r40-kDa OMP antibody in the host defense against P. gingivalis. To that end, we developed a panel of monoclonal antibodies by immunizing mice with purified r40-kDa OMP. The objective of this study was to determine the bactericidal activity on P. gingivalis by the IgG1 monoclonal antibody Pg-ompA2. METHODS: Bacterial growth measurement, a complement-mediated anti-P. gingivalis assay based on [3H]thymidine uptake, and a 14C-release assay were performed to test the bactericidal activity of Pg-ompA2 to P. gingivalis. RESULTS: In the presence of complement, Pg-ompA2 was lethal to P. gingivalis 381 as well as to the more virulent P. gingivalis strains, including ATCC 53977 and W83. Using component-deficient complement, we determined that Pg-ompA2 killed P. gingivalis by activating both the classical and alternative complement pathways. CONCLUSIONS: Pg-ompA2 has an in vitro complement-mediated bactericidal activity to P. gingivalis. Pg-ompA2 may contribute to the development of a local immunotherapy that can be applied in the gingival crevice of a patient with P. gingivalis-related periodontitis, or be a vaccine candidate.

Animals↗
PubMed1992

[Complement system in status asthmatics--analysis of anti-complementary effects induced by methylprendisolone].

Complement system was investigated in 7 patients with status asthmatics treated with large doses of methylprednisolone (MPS). Complement hemolytic activities, complement protein profile, complement fragments and circulating immune complexes were measured before, 3 and 8 hours after and 14 days after MPS administration. MPS normalized C4 and C1INH activities 6 hours after administration. MPS also decreased ACH50 6 hours after administration and D activity 3 and 6 hours after, but these activities recovered to their previous normal range within 14 days. P and H were decreased at each measurement time, and C1s was transiently decreased 6 hours after MPS administration. Complement fragment iC3b was increased at each measurement time, but fragment Bb tended to be decreased 14 days after MPS administration. The increment of anaphylatoxin C3a recovered to normal 14 days after MPS administration. In vitro experiments, MPS inhibited D and C1s activation directly, and decreased the decay of B and C4. Inhibition of C1s might also increase C1INH activity clinically. These results clarified that the alternative complement pathway was activated, and suggested that the C1 bypass pathway might be also activated in status asthmatics. It was further considered that these anti-complementary effects induced by MPS, brought about an improvement in asthmatic symptoms. Studies to identify the complement activators continued, and circulating immune complexes may possibly be one of those agents activating complement cascade.

Adult↗
PubMed1985

Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system.

The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol-reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.

Blood Platelets↗
PubMed1989

Antibody complement-dependent bacteriolysis in experimentally induced pasteurellosis in mice.

Affinity-purified bovine immunoglobulin isotypes were bacteriolytic for Pasteurella haemolytica biotype A, serotype 1 (PHA-1). This bacteriolysis was specific and complement-dependent. The IgM and IgG1 were the most active isotypes in the classic complement cascade. These isotypes also induced bacteriolysis through the alternative complement cascade. The comparative bacteriolytic activities of IgG1 and IgM were equal within each cascade; however, the bacteriolytic activities of IgG1 and IgM were lower in the alternative cascade than in the classical cascade. The IgG2 was more bacteriolytic than IgA in the classic and alternative complement pathways. Bovine immunoglobulins passively protected C57BL/6 mice from experimentally induced pasteurellosis. There were no major differences in the protection among hyperimmune sera, purified IgM, or purified IgG. Mice were protected from PHA-1 by approximately 1.9 micrograms of IgG and 1.2 or 0.1 micrograms of IgM. Elimination of murine complement with cobra venom factor 3 reduced PHA-1 clearance in passively immunized C57BL/6 mice. The protective effect of IgM mediated resistance was highly dependent on an intact complement system. The intact complement cascade was associated with enhanced clearance of PHA-1 from the liver. Although PHA-1 was susceptible to antibody complement-mediated bacteriolysis in vitro, the dependence on an intact complement cascade was not absolute in experimentally induced murine septicemic pasteurellosis.

Animals↗
PubMed1983

Complement activity in the cord blood of term neonates with the amniotic fluid infection syndrome.

Cord blood samples from 11 term neonates whose placentas showed histological changes typical of the amniotic fluid infection syndrome were analysed in order to determine haemolytic activity of the classic and alternative complement pathways and serum levels of complement proteins and immunoglobulins. Although the mean values of all these parameters were higher in this group than in an age-related control group, only classic haemolytic pathway activity was significantly elevated (P less than 0,025).

Amniotic Fluid↗
PubMedDate not supplied

Immune complexes in polyarthritis after Salmonella gastroenteritis.

In a case of polyarthritis after salmonella gastroenteritis the levels of circulating immune complexes (CIC) (C1q-binding assay) correlated closely with disease activity. This observation suggests that systematically absorbed salmonella antigens may lead or contribute to the formation of CIC. CIC activate both the classical and alternate complement pathway and may be important in the pathogenesis of the arthritis associated with salmonella infection.

Adult↗
PubMed1993

The role of the complement system in the neutrophil functions stimulated in vitro by an alkali-insoluble cell wall fraction of Paracoccidioides brasiliensis.

We investigated the capacity of an alkali-insoluble cell wall polysaccharide fraction (F1) of Paracoccidioides brasiliensis to induce rat polymorphonuclear neutrophil (PMN) migratory and chemiluminescence (CL) responses. Normal rat serum pre-incubated with F1 induced a chemotactic neutrophil response which was fully abolished by heat-inactivation. The participation of the alternative complement pathway was more effective than that of the classical pathway since depletion of factor B by heating at 50 degrees C reduced PMN migration, whereas blockade of the classical pathway with EGTA left the migratory response practically unchanged. Opsonized serum F1 induced a significant release of oxygen radicals from PMN as measured by CL. The complement system was also found to be involved in this activity since serum inactivation at 56 degrees C altered the CL response. In addition to complement-derived fragments, other serum opsonins, probably cross-reacting antibodies, were required for optimal interaction between PMN and opsonized particles. These results contribute to the understanding of the role of fungal components and of the complement system in the inflammatory response observed in paracoccidioidomycosis.

Animals↗
PubMed1984

Factor D of the alternative pathway of bovine complement: isolation and characterization.

Factor D of the bovine alternative complement pathway has been purified by chromatography on CM-Sephadex C-50, Sephacryl S-200 and hydroxylapatite. The isolated factor D (0.25 mg from 1 litre of bovine serum) had an apparent molecular weight of 27,500 and a pI of 7.2. In whole bovine serum the pI of factor D was also 7.2. The isolated protein caused the Mg++-dependent cleavage of bovine factor B in the presence of cobra venom factor (CVF) to generate a haemolytically active C3-convertase as shown by SDS-polyacrylamide gel electrophoresis and haemolytic diffusion plate assays. Bovine factor D and human factor D were interchangeable in restoring the alternative pathway haemolytic activities of both bovine RD and human RD (factor D deficient sera). The haemolytic activity of bovine serum factor D was completely inhibited by 20 mM diisopropylfluorophosphate (DFP) but only 25% inhibited by 1 mM DFP. Serum heated at 56 degrees C for 10 min completely lost factor D activity but purified factor D was relatively more heat stable.

Animals↗
PubMed1984

Susceptibility of Giardia lamblia trophozoites to the lethal effect of human serum.

To define the potential role of complement and antibody in host defense against Giardia lamblia, the effect of human serum on axenically cultured G. lamblia trophozoites was studied. Sera from patients without a history of giardiasis and with no detectable antibody by an indirect immunofluorescence antibody (IFA) assay (IFA titers less than 1:2) killed from 8 to 76% of trophozoites (n = 23 sera). Whereas less than 10% of parasites incubated in phosphate-buffered saline alone were killed, 16 sera killed from 10 to 25% and six sera killed from 25 to 75%. One serum with an anti-G lamblia antibody titer of 1:128 killed greater than 98% of the parasites. The complement dependency of killing was demonstrated by abrogation of the lethal effect when serum was chelated with EDTA (7 mM) or was heat-inactivated (56 degrees C, 30 min), and, in an immunofluorescence assay, by detection of C3 on parasites killed by normal serum. When the classical complement pathway was selectively blocked by using serum chelated with Mg+2 (2 mM)-EGTA (8 mM), or serum congenitally deficient for the second component of complement, there was no killing; thus, killing was dependent on the presence of an intact classical pathway. In each of three sera from donors with negative IFA titers, an absorbable factor specific for G. lamblia, possibly antibody not detected by IFA, was required for classical pathway activation. To determine if alteration of the surface of G. lamblia would render it an activator of the alternative pathway of complement, trophozoites were studied after cell death or after treatment with neuraminidase or trypsin. In MgEGTA-chelated serum, dead trophozoites activated the alternative pathway as determined by consumption of Factor B and deposition of C3 on their surface. In contrast, untreated or enzyme-treated living parasites did not activate the alternative pathway.

Adult↗
PubMed1986

Eimeria tenella: quantitative in vitro and in vivo studies on the effects of mouse polyclonal and monoclonal antibodies on sporozoites.

Murine, polyclonal and monoclonal antibodies, raised against sporozoites of Eimeria tenella, were tested for their ability to neutralize sporozoite infectivity in vitro and in vivo. Neutralization was effected via three mechanisms. Firstly, sporozoites fixed complement, at low titres, and lysis occurred by the alternative pathway of complement activation. Secondly, in the absence of complement activity, the murine heat-inactivated, hyperimmune antiserum neutralized sporozoites at relatively low titres. At high titres, even though sporozoites were agglutinated, neither the heat-inactivated hyperimmune antiserum nor the monoclonal antibody neutralized sporozoites. Finally, in the presence of complement and specific antibodies, at titres which by themselves would not neutralize sporozoites, neutralization was effected due to lysis via the classical pathway of complement activation.

Agglutination↗
PubMed1993

The specificity of alternative complement pathway-mediated lysis of erythrocytes: a survey of complement and target cells from 25 species.

Sera from 20 species of mammals were tested for their ability to lyse erythrocytes from 18 species of mammals and birds by the alternative complement pathway. Erythrocytes were not lysed by homologous complement, with one minor exception, but all erythrocytes tested were lysed by at least one complement source, and all sera tested except that of the horse lysed at least one type of erythrocyte. Control experiments indicated that lysis was via the alternative complement pathway and that antibodies were not involved. Complement from the various species could be ranked from most active to least active, and erythrocytes could be ranked from most susceptible to least susceptible. There was an inverse correlation between complement activity and erythrocyte susceptibility. The ranking of the orders of placental mammals, from strongest to weakest complement, was carnivore > artiodactyl (ruminants and swine) > primate = armadillo > rodent > rabbit > horse. Opossum serum had activity that placed it in the centre of this range. Ferret complement, the most potent tested, lysed all erythrocytes tested except for homologous erythrocytes, with APCH50 titres as high as 4000. Although the overall reactivity pattern was clear, there were several striking exceptions. For example, the only complement source which lysed ferret erythrocytes was sera of the mouse. The amount of sialic acid present on erythrocytes of 14 mammals was determined, and was, in general, directly correlated with resistance to alternative complement pathway lysis, although there were prominent exceptions to this correlation, involving erythrocytes of the horse, burro and human. All 20 types of complement were also tested for their ability to lyse antibody-coated human tumour cells, under conditions in which both the classical and alternative complement pathways were functional. The data obtained suggest that alternative pathway activation is, in some cases, a major factor determining the effectiveness of a particular complement source in the lysis of xenogeneic tumour cells.

Animals↗
PubMed1986

Brugia malayi: rat cell interactions with infective larvae mediated by complement.

Albino rat macrophages and neutrophils, in the presence of fresh normal rat serum as a source of complement, adhered to and promoted killing of Brugia malayi infective larvae in vitro. Eosinophils, by themselves, were marginally cytotoxic at a high cell-target ratio but promoted cytotoxicity when mixed with macrophages. Eosinophil culture supernatants enhanced the macrophage mediated killing of infective larvae. The complement of fresh normal rat serum was found to act by the alternate pathway. Fresh normal rat serum depleted of alternate pathway complement activity by treatment with zymosan A, or of Factor B by heating at 50 C for 20 min, or of Factor D by passing through Sephadex G75 column, failed to promote cell adherence to the parasite. C3 molecules were detected on the surface of infective larvae by immunofluorescence. There was a significant consumption of complement when Brugia malayi infective larvae were incubated in fresh normal rat serum. Albino rat cells were more potent in inducing cytotoxicity to infective larvae in vitro than those from jird or Mastomys natalensis, which may reflect the greater resistance offered by the rat to B. malayi infection. There was much less cellular infiltration on introduction of Brugia malayi infective larvae into the peritoneal cavity of jirds compared to rats and Mastomys natalensis indicating the greater susceptibility of jirds to intraperitoneally induced infections.

Animals↗
PubMed1997

A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis.

The demand for convenient and sensitive means of measuring cytotoxicity and complement-mediated killing is likely to be increased by the recent identification of Complement Factor H, an important regulatory protein of both the classical and alternate pathways of complement, as a tumor-associated antigen. Here we describe a simple luminometric assay capable of detecting the death of approximately 0.03 nucleated human-cell equivalent or approximately 1 rabbit-erythrocyte equivalent. The assay measures the release of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from dead or damaged cells by coupling its enzymatic activity to production of ATP, which in turn is measured by well-known methods involving firefly luciferase. This is accomplished by means of a reaction series in which the activity of G3PDH is coupled with that of phosphoglycerate kinase, the next enzyme in the glycolytic pathway. As described, the assay uses inexpensive, commercially available reagents. This coupled assay was used to demonstrate that an anti-factor-H antibody is capable of enhancing complement-mediated killing of the Raji cancer cell line by > 1000%.

Adenosine Triphosphate↗