Interactions of the Complement System with Native and Chemically Modified Endotoxins
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Endotoxic lipopolysaccharides (LPS) isolated from Serratia marcescens, Veillonella alcalescens , and Salmonella typhosa were potent in their ability to induce fixation of complement (C′) in normal guinea pig, rabbit, mouse, and human serum. The C′-fixing ability of LPS was pronounced even when assays were performed in undiluted serum, and was lost after each of four chemical modifications which resulted in loss of biological toxicities. The detoxification procedures had in common the cleavage of ester-bound, long-chain carboxylic acids. The ability of biologically active LPS to fix C′ in normal guinea pig serum was reflected chiefly in dramatic uptake of classical C′3 (C′3t); fixation of C′1, C′4, and C′2 was virtually undetectable. Hence, it was the capacity for fixation of C′3t which was lost most overtly during detoxification. Addition of immune serum to the assay mixtures resulted in detectable fixation of C′1 and C′4. Biologically active LPS also fixed more of these components than did detoxified LPS. Immune serum restored the ability of detoxified LPS to fix C′3t, but whether this is by the original pathway is not yet clear. We concluded that the loss of certain biological activities and the loss of ability to fix C′3t in normal serum after LPS detoxification involved loss or rearrangement of substrates on LPS which either initiated or supported, or both, its interaction with the complement system. It was apparent that the ability to fix C′ can serve as a valuable in vitro indicator of the integrity of the toxic conformation of biologically active LPS membrane fragments. These experiments supported the hypothesis that certain of the biological activities induced by endotoxins are mediated via the complement system.Keywords:
Veillonella
Complement fixation test
Detoxification
In normal human serum chelated with magnesium (10 mM) and ethylene glycotetraacetic acid (10 mM) (MgEGTA), the classical pathway of complement activation is blocked; however, the alternate pathway of complement activation is intact. Diplococcus pneumoniae, Staphylococcus albus, Streptococcus viridans, Streptococcus faecalis , and Serratia marcescens were opsonized in normal human serum containing 10 mM MgEGTA. In contrast, Staphylococcus aureus and Pseudomonas aeruginosa were poorly opsonized in human serum chelated with 10 mM MgEDTA. Therefore certain bacterial species appear to require the classical pathway of complement activation to be opsonized and other bacterial species utilize the alternate pathway of complement activation.
Antibody opsonization
Lectin pathway
Complement factor B
Complement C1q
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Endotoxic lipopolysaccharides (LPS) isolated from Serratia marcescens, Veillonella alcalescens , and Salmonella typhosa were potent in their ability to induce fixation of complement (C′) in normal guinea pig, rabbit, mouse, and human serum. The C′-fixing ability of LPS was pronounced even when assays were performed in undiluted serum, and was lost after each of four chemical modifications which resulted in loss of biological toxicities. The detoxification procedures had in common the cleavage of ester-bound, long-chain carboxylic acids. The ability of biologically active LPS to fix C′ in normal guinea pig serum was reflected chiefly in dramatic uptake of classical C′3 (C′3t); fixation of C′1, C′4, and C′2 was virtually undetectable. Hence, it was the capacity for fixation of C′3t which was lost most overtly during detoxification. Addition of immune serum to the assay mixtures resulted in detectable fixation of C′1 and C′4. Biologically active LPS also fixed more of these components than did detoxified LPS. Immune serum restored the ability of detoxified LPS to fix C′3t, but whether this is by the original pathway is not yet clear. We concluded that the loss of certain biological activities and the loss of ability to fix C′3t in normal serum after LPS detoxification involved loss or rearrangement of substrates on LPS which either initiated or supported, or both, its interaction with the complement system. It was apparent that the ability to fix C′ can serve as a valuable in vitro indicator of the integrity of the toxic conformation of biologically active LPS membrane fragments. These experiments supported the hypothesis that certain of the biological activities induced by endotoxins are mediated via the complement system.
Veillonella
Complement fixation test
Detoxification
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The ability of MIC to induce complement activation in vitro and in vivo was investigated. For the in vitro studies, both human and guinea pig serum or EDTA-plasma samples were exposed to 1167 to 1260 ppm MIC vapor for 15 min at room temperature. The human serum samples exposed to MIC showed significant reductions in Factor B, C2, C4, C3, C5, and total hemolytic complement CH50 activity levels. C6 functional activity was unaffected. The C3, C5, and CH50 functional activities in guinea pig serum (the only functional tests conducted on these samples) were more sensitive to MIC-mediated reduction than the corresponding activity reductions observed in the human serum samples. The human and single guinea pig EDTA-plasma samples exposed to MIC vapor showed no evidence of C3 consumption but did show significant reductions in CH50 levels. Thus, MIC vapor was able to activate, and thereby reduce serum complement C3 activity in vitro by a complement-dependent process. However, the data suggest at least one complement component other than C3 was inactivated in EDTA-plasma by a complement-independent mechanism. For the in vivo studies, five pairs of guinea pigs were exposed to 644 to 702 ppm MIC vapor until one of the pair died (11-15 min). MIC exposure was then discontinued, the surviving guinea pig was sacrificed, and EDTA-plasma was obtained from both animals and analyzed for complement consumption.(ABSTRACT TRUNCATED AT 250 WORDS)
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Immunofluorescence
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Purpose: The purpose of this study was to examine the ability of Gram‐negative bacteria isolated from the non‐infectious condition contact‐lens‐induced acute red eye (CLARE) and infectious microbial keratitis (MK) to infect the eyes of mice. Methods: One cornea of BALB/c mice was scratched with a needle and 5 × 10 6 bacteria applied. Bacterial types used were Pseudomonas aeruginosa, Haemophilus influenzae, Serratia marcescens, Stenotrophomonas maltophilia and Aeromonas hydrophilia . The mice were killed after 1, 4 and 24 h and examined by slit lamp, histology and microbiology. Results: CLARE strains were unable to infect the mouse corneas. The eyes inoculated with the CLARE strains had low levels of polymorphonuclear leucocytes (PMN) in the stroma. All eyes that had MK contained bacteria and large numbers of PMN after 24 h. Conclusions: The development of CLARE or MK is dependent on the type of bacteria with specific bacterial types being responsible for CLARE or MK.
Stenotrophomonas maltophilia
Stenotrophomonas
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Complement
Immune complex
Immune complex disease
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It was previously demonstrated that predatory bacteria are able to efficiently eliminate Gram-negative pathogens including antibiotic-resistant and biofilm-associated bacteria. In this proof-of-concept study we evaluated whether two species of predatory bacteria, Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus, were able to alter the survival of Gram-negative pathogens on the ocular surface. Clinical keratitis isolates of Pseudomonas aeruginosa (strain PAC) and Serratia marcescens (strain K904) were applied to the ocular surface of NZW rabbits followed by application of predatory bacteria. At time intervals, surviving pathogenic bacteria were enumerated. In addition, B. bacteriovorus and S. marcescens were applied to porcine organ culture corneas under contact lenses, and the ocular surface was examined by scanning electron microscopy. The ocular surface epithelial layer of porcine corneas exposed to S. marcescens, but not B. bacteriovorus was damaged. Using this model, neither pathogen could survive on the rabbit ocular surface for longer than 24 h. M. aeruginosavorus correlated with a more rapid clearance of P. aeruginosa but not S. marcescens from rabbit eyes. This study supports previous evidence that predatory bacteria are well tolerated by the cornea, but suggest that predatory bacteria do not considerably change the ability of the ocular surface to clear the tested Gram-negative bacterial pathogens from the ocular surface.
Bdellovibrio
Pathogenic bacteria
Gram-Negative Bacteria
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The ability of MIC to induce complement activation in vitro and in vivo was investigated. For the in vitro studies, both human and guinea pig serum or EDTA-plasma samples were exposed to 1167 to 1260 ppm MIC vapor for 15 min at room temperature. The human serum samples exposed to MIC showed significant reductions in Factor B, C2, C4, C3, C5, and total hemolytic complement CH50 activity levels. C6 functional activity was unaffected. The C3, C5, and CH50 functional activities in guinea pig serum (the only functional tests conducted on these samples) were more sensitive to MIC-mediated reduction than the corresponding activity reductions observed in the human serum samples. The human and single guinea pig EDTA-plasma samples exposed to MIC vapor showed no evidence of C3 consumption but did show significant reductions in CH50 levels. Thus, MIC vapor was able to activate, and thereby reduce serum complement C3 activity in vitro by a complement-dependent process. However, the data suggest at least one complement component other than C3 was inactivated in EDTA-plasma by a complement-independent mechanism. For the in vivo studies, five pairs of guinea pigs were exposed to 644 to 702 ppm MIC vapor until one of the pair died (11-15 min). MIC exposure was then discontinued, the surviving guinea pig was sacrificed, and EDTA-plasma was obtained from both animals and analyzed for complement consumption.(ABSTRACT TRUNCATED AT 250 WORDS)
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Abstract The precise mechanisms and tissue sites involved in the in vivo detoxification of endotoxin remain unresolved. Although a number of studies have indicated that circulating plasma is a principal site of endotoxin inactivation (1–3), it was believed that the complement system was not required for detoxification of endotoxin (1). However, a recent report which demonstrated the increased susceptibility of C6 deficient rabbits to the injection of bacterial endotoxin suggests that complement may be important (4). Endotoxin can activate the complement system either by the classical mechanism proceeding through the activation of C1, C4 and C2 to activate C3, or via the alternate or bypass pathway proceeding through another series of proteins, as yet incompletely defined, to enter the complement sequence directly at the C3 step (5–7). In this study a further attempt is made to evaluate the role of complement in the detoxification of endotoxin, with particular reference to the contribution of the early (C1, C4 and C2) and late acting (C3–C9) complement components.
Detoxification
Complement
Defence mechanisms
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