Method of determinating the protective capacity of C1. perfringens toxoid]

The authors describe a model of experimental gas gangrene in guinea pigs; it was produced by the administration of the vegetative form of C. perfringens; the cells were completely washed of the lethal toxin and no toxic or necrotizing agents were added. A possibility of development of gangrenous process without any preliminary depression of the resistance of body tissues in the area of injection of the causative agent was revealed. Apart from the local process and general intoxication gas gangrene, caused by intramuscular injection of C1. perfringens to guinea pigs, was accompanied by bacteriemia and microbial contamination of the internal organs. A method of the animal infection was ascertained and the causes of their death was assessed. The method is recommended for determination of the immunological efficacy of C1. perfringens toxoids.     

Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis

Clostridium perfringens (C. perfringens) has been developed as a potential oral delivery vehicle to deliver antigens or therapeutic compounds to Gut-Associated Lymphoid Tissue (GALT). However, this recombinant C. perfringens carries a plasmid-encoded expression system, which raises several safety concerns regarding possible horizontal plasmid transfer and spread of plasmid-associated antibiotic resistant genes. Furthermore, this bacterium produces the extracellular theta toxin, which poses a potential safety issue for general administration. Using a Clostridium-specific-targetron donor plasmid, we inserted the Simian Immunodefiency Virus (SIV) p27 gene into the theta toxin gene (pfoA) on the C. perfringens chromosome, which simultaneously inactivated the theta gene and introduced SIV p27 gene onto the bacterial chromosome. Such mutant C. perfringens without an input plasmid or antibiotic resistant gene stably produced a large amount of SIV p27 protein during sporulation and did not produce theta toxin. Upon oral feeding of the mutant bacteria to mice, intact p27 protein was detected in the lower GI tract. The re-engineered C. perfringens provides a biosafe efficient oral vehicle to deliver antigen to the gastrointestinal tract.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Detection of Clostridial Hemolysin Formed In Vivo

Experimental clostridial myonecrosis closely resembling gas gangrene was produced in goats by the intramuscular injection of washed clostridia suspended in adrenalin. The preparation provides a model for the study of clostridial myonecrosis that is relatively isolated from preceding trauma. A new test for the demonstration of clostridial exotoxins, the hemolysin indicator plate test, was described. This test gave reliable results within 6 hr, and only minute quantities of test material were required. Application of this test to would exudates of goats with experimental clostridial myonecrosis demonstrated the presence of the gamma toxin of Clostridium novyi, alpha toxin of C. septicum, and the alpha toxin of C. perfringens in exudates from animals injected with washed Clostridium bacilli suspended in adrenalin. Exudates from goats injected with C. perfringens were lethal for mice and produced necrosis of guinea pig skin typical of C. perfringens. The convincing demonstration of clostridial exotoxins formed in vivo in this experimental model could be the basis for definition of clostridial exotoxin actions which have evaded study in the past.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin.

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Protective properties of theta-hemolysin obtained by affinity chromatography

The data on the study of the protective activity of theta hemolysin and Cl. perfringens lecithinase preparations and the corresponding antitoxic sera obtained by indirect immune affinity chromatography are presented. Experiments in mice and guinea pigs indicate that the injection of antihemolytic serum and immunization with anatheta hemolysin ensures the protection of the animals from theta toxin. The enrichment of analecithinase preparation with anatheta hemolysin has been found to increase its protective properties against Cl. perfringens culture and toxin.     

CD34 mediates intestinal inflammation in Salmonella‐infected mice

CD34 is a highly glycosylated sialomucin expressed on a variety of cells, ranging from vascular endothelial cells to haematopoietic stem cells. Depending on its glycosylation state, CD34 has been shown to promote or inhibit cell adhesion and migration; however, a functional role for CD34 in the gut has not been determined. Using a model of Salmonella‐induced gastroenteritis, we investigated the role of CD34 in the context of infection. Upon oral infection, the number of CD34+ cells detected in the submucosa, vascular endothelium and lamina propria significantly increased in S. Typhimurium‐infected C57Bl/6 mice. The pathology of S. Typhimurium‐infected C57Bl/6 mice was characterized by recruitment of neutrophils to the site of inflammation, submucosal oedema and crypt destruction. In contrast, Cd34^?/? mice showed a delayed pathology, a defect in inflammatory cell migration into the intestinal tissue and enhanced survival. Importantly, this was not due to a lack of chemotactic signals in Cd34^?/? mice as these mice had either similar or significantly higher levels of pro‐inflammatory cytokines and chemokines post infection when compared with infected C57/Bl6 control mice. In summary, we demonstrate a novel role for CD34 in enhancing migration of inflammatory cells and thereby exacerbating host‐mediated immunopathology in the intestine of S. Typhimurium‐infected mice.     

Medium for Toxin Production by Clostridium perfringens in Continuous Culture

A tryptone-salts medium for continuous growth and alpha toxin production of Clostridium perfringens type A in an adapted chemostat is described. In such steady-state cultures, fermentative and biochemical activity of C. perfringens remained unchanged. Toxigenic ability to produce alpha, theta, and nu toxins was preserved.     

Clostridium perfringens alpha-toxin: characterization and mode of action

Clostridium perfringens type A strains that produce alpha-toxin cause gas gangrene, which is a life-threatening infection with fever, pain, edema, myonecrosis and gas production. Intramuscular injection of the toxin or Bacillus subtilis carrying the alpha-toxin gene causes myonecrosis and produces histopathological features of the disease. Immunization of mice with alpha-toxin or fragments of the toxin prevents gas gangrene caused by C. perfringens. The toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis, lethality and dermonecrosis. These biological activities are closely related to PLC and/or SMase activities. However, there is yet some uncertainty about the biological activities induced by the PLC and SMase activities of alpha-toxin. Based on the isolation and characterization of the gene for alpha-toxin and a comparison of the toxin with enzymes of the PLC family, significant progress has been made in determining the function-structure of alpha-toxin and the mode of action of the toxin. To provide a better understanding of the role of alpha-toxin in tissue damage in gas gangrene, this article summarizes current knowledge of the characteristics and mode of action of alpha-toxin.     

Neutrophil influx in response to a peritoneal infection with Salmonella is delayed in lipopolysaccharide-binding protein or CD14-deficient mice

The induction of an adaptive immune response to a previously unencountered pathogen is a time-consuming process and initially the infection must be held in check by the innate immune system. In the case of an i.p. infection with Salmonella typhimurium, survival requires both CD14 and LPS-binding protein (LBP) which, together with Toll-like receptor 4 and myeloid differentiation protein 2, provide a sensitive means to detect bacterial LPS. In this study, we show that in the first hours after i.p. infection with Salmonella a local inflammatory response is evident and that concomitantly neutrophils flood into the peritoneum. This rapid neutrophil influx is dependent on TNF since it is 1) abolished in TNF KO mice and 2) can be induced by i.p. injection of TNF in uninfected animals. Neutrophil influx is not strictly dependent on the presence of either LBP or CD14. However, in their absence, no local inflammatory response is evident, neutrophil migration is delayed, and the mice succumb to the infection. Using confocal microscopy, we show that the neutrophils which accumulate in CD14 and LBP null mice, albeit with delayed kinetics, are nevertheless fully capable of ingesting the bacteria. We suggest that the short delay in neutrophil influx gives the pathogen a decisive advantage in this infection model.     

Essential involvement of IFN-gamma in Clostridium difficile toxin A-induced enteritis

Clostridium difficile has emerged as the important causative agent of antibiotics-associated pseudomembranous colitis; especially its toxin A is presumed to be responsible for the colitis. We examined the pathophysiological roles of IFN-gamma in toxin A-induced enteritis using IFN-gamma knockout (KO) mice. When toxin A of C. difficile was injected into the ileal loops of BALB/c wild-type (WT) mice, massive fluid secretion, disruption of intestinal epithelial structure, and massive neutrophil infiltration developed within 4 h after the injection. IFN-gamma protein was faintly detected in some CD3-positive lymphocytes in the lamina propria and submucosa of the ileum of untreated WT mice. On the contrary, at 2 and 4 h after toxin A injection, IFN-gamma protein was detected in infiltrating neutrophils and to a lesser degree in CD3-positive lymphocytes. In the ileum of WT mice, toxin A treatment markedly enhanced the gene expression of TNF-alpha, macrophage inflammatory protein-1alpha and -2, KC, and ICAM-1 >2 h after treatment. In contrast, the histopathological changes were marginal, without enhanced fluid secretion in the ileum of toxin A-treated IFN-gamma KO mice. Moreover, toxin A-induced gene expression of TNF-alpha, neutrophil chemotactic chemokines, and ICMA-1 was remarkably attenuated in IFN-gamma KO mice. Furthermore, pretreatment of WT mice with a neutralizing anti-IFN-gamma Ab prevented toxin A-induced enteritis. These observations indicate that IFN-gamma is the crucial mediator of toxin A-induced acute enteritis and suggest that IFN-gamma is an important molecular target for the control of C. difficile-associated pseudomembranous colitis.     

Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes

Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of alpha-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in alpha-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with L-glucose) or to increased alpha-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of alpha-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of alpha-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na+ (estimated using 22Na+) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled alpha-aminoisobutyric acid (assay for Na+-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of L-glucose, 3-O-methylglucose and alpha-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5--20 min the decay of alpha-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of alpha-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular 22Na+, and on the exodus (from preloaded cells) of alpha-aminoisobutyric acid, L-glucose, and 3-O-methylglucose.     

Cessation of neutrophil influx in C5a-induced acute experimental arthritis is associated with loss of chemoattractant activity from the joint space

Neutrophil emigration is a critical component of the inflammatory process and is generally thought to play a role in host defense as well as in the tissue injury that often accompanies inflammation. Most inflammatory reactions exhibit a sequence of emigrating cell types, thus clearly demonstrating that the neutrophil influx eventually ceases and that the neutrophils are then removed from the lesion. It has been our premise that in order to understand the processes that lead to the progressive inflammatory reactions that underly so many disease processes, it is important to determine the mechanism by which the "normal" inflammatory response resolves. The purpose of this study was to identify the time of cessation of neutrophil influx in experimental arthritis induced by the injection of C5 fragments (C5f) and to investigate mechanisms underlying the cessation process. The migration of i.v. delivery pulses to inflamed joints was assessed by lavage of the joint space and by external scintigraphy. We found no evidence for the development of inhibitory systems against chemotactic factors or "desensitization" of the inflamed site, because a second injection of C5f into joints which had been injected previously with C5f resulted in enhancement rather than inhibition of migration. Neither was evidence found for altered tissue barriers to migration or for desensitization of neutrophils as possible explanations for cessation of influx. The major mechanism appeared to be a loss of chemoattractant activity in the joint space between 2 h and 6 h after C5f injection which was detected by transfer into a fresh joint. Radiolabeled C5a des-Arg had a t1/2 of disappearance from the joint of less than 1 h, which suggested that the transferred chemoattractant must, in part, have been due to the generation of new chemotaxins by C5f injection. These observations suggest that continued generation of chemoattractants or failure of their subsequent removal may be mechanisms leading to persistent neutrophil influx in chronic inflammation.     

Cholera Toxin (Choleragen) - Polymorphonuclear Leukocyte Interactions: Effect on Migration in Vitro and FcγR - Dependent Phagocytic and Bactericidal Activity

PMNL leukocytosis is a feature common to many types of infectious and inflammatory diseases. How PMNL are recruited to tissues is not yet clear although it is a question that has considerable clinical importance. We investigated the function of PMNL which migrated through an artificial barrier (Chinese hamster ovary (CHO) cells, collagen and nylon cloth membrane) subjected to CT or choleragenoid treatment toward plain medium (the same RPMI in the upper and lower chamber) or medium containing chemotactic factor (fMLP or LPS or ZAS). CT treatment significantly (P<0.01) reduced the FcγR expression on the surface of PMNL. The PMNL functions, namely, migration, phagocytic activity and intracellular killing of staphylococci, also have been reduced significantly (P<0.01). FcγR expression and some functions of PMNL that migrate to chemoattractants were reduced, irrespective of the presence or absence of CT; however, the inhibitory effect of CT on PMNL function was observed only when PMNL migrate to the lower chamber without chemotactic factor. On the other hand choleragenoid treatment of CHO cells did not have any significant influence on PMNL function and FcγR expression. In conclusion, our experiments demonstrate that CT reduces EA_Fc rosetting and the FcγR-dependent phagocytic and bactericidal activity of bovine blood PMNL.     

Hyperthermia,antipyretics and function of polymorphonuclear leukocytes.

Whether hyperthermia (temperature, 40 degrees C), salicylates, acetaminophen or phenacetin has an adverse effect on polymorphonuclear leukocyte (PMNL) function was examined. Migration experiemnts were carried out in Boyden chambers with bacterial chemotactic factor as the attract, and bactericidal assays were done with Staphylococcus aureus and serum from an AB blood group donor as a source of opsonins. PMNL viability was determined by the trypan blue exclusion method. Neither hyperthermia nor any of the drugs tested affected PMNL viability adversely, but sodium salicylate and phenacetin suppressed PMNL migration. Early staphylococcal killing was greater at 40 degrees C; however, after 2 hours the converse was true. Bactericidal activity was suppressed by acetylsalicylic acid, sodium salicylate and phenacetin. Hence it appears PMNL function is similar at 37 degrees and 40 degrees C but that some commonly used antipyretics have an adverse effect on PMNL activity.     

A modified theta-toxin produced by limited proteolysis and methylation: a probe for the functional study of membrane cholesterol.

A derivative of cytolytic theta-toxin from Clostridium perfringens was prepared by limited proteolytic digestion of the native toxin followed by methylation. Among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes, the proteinase-nicked and methylated derivative (MC theta) specifically binds to cholesterol. While MC theta retains binding affinity comparable to that of intact toxin, it causes no obvious membrane damage, resulting in no hemolysis at temperatures of 37 degrees C or lower. Using MC theta, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes at both 37 degrees C and 10 degrees C. The number of high-affinity sites on sheep erythrocytes was estimated to be approximately 3-times larger at 37 degrees C than that at 10 degrees C. In addition, high- and low-affinity sites were demonstrated in human erythrocytes and a lymphoma B cell line, BALL-1 cells. Both binding sites disappear upon simultaneous treatment of cells with sublytic doses of digitonin, suggesting that cholesterol is an essential component of both the high- and low-affinity sites and that the mode of cholesterol existence in plasma membranes is heterogeneous in these cells. Because of its high affinity for membrane cholesterol without causing any obvious membrane changes at physiological temperatures, MC theta may provide a probe for use in the functional study of membrane cholesterol.     

Group B streptococci inhibit the chemotactic activity of the fifth component of complement

Infection with group B streptococci (GBS) is associated with a poor acute inflammatory response in which neutrophils fail to localize at the site of invasion. In the present studies, we have examined the effects of group B streptococci on C-derived chemotactic activity in human serum. Fresh human serum was activated to form C5a and C5adesarg by incubation with zymosan. The activated serum was then incubated with group B organisms, centrifuged, and the supernatants tested for chemotactic activity for human polymorphonuclear leukocytes. Group B organisms caused a dose-dependent decrease in C-dependent chemotactic activity. The degree of inhibition was profound with 1 X 10(9) bacteria/ml (10% of control). Experiments indicated that significant chemotactic factor inactivation occurred within 2 min of exposure to GBS organisms, while maximal inhibition occurred after 30 min incubation. A number of different strains of GBS of types I, II, and III possessed inhibitory activity. In contrast, group D streptococci, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae failed to inhibit the C-derived chemotactic activity in human serum. Group A streptococci that were M protein positive also inactivated C-dependent chemotactic activity in serum, as previously reported. The inhibitory activity of the GBS strains could be abolished by heat or trypsin treatment but not by neuraminidase, pronase, or pepsin. C5a levels in zymosan-activated serum as measured by RIA were not decreased after incubation with an inhibitory strain suggesting that absorption was not involved. SDS-PAGE analysis revealed that group B streptococci degrade the C5a molecule, increasing its electrophoretic mobility by removing a fragment with a m.w. of approximately 650 Da. Thus, one of the reasons for the poor inflammatory response at the site of GBS infection may reside in the ability of these pathogens to inactivate C-derived inflammatory mediators. The GBS C5a-ase activity probably serves as an additional virulence factor for these organisms contributing to the poor inflammatory response characteristic of group B streptococcal infection.     

Cyclooxygenase regulates cell surface expression of CXCR3/1-storing granules in human CD4+ T cells

Efficient migration of CD4+ T cells into sites of infection/inflammation is a prerequisite to protective immunity. Inappropriate recruitment, on the other hand, contributes to inflammatory pathologies. The chemokine/chemokine receptor system is thought to orchestrate T cell homing. In this study, we show that most circulating human CD4+ T cells store the inflammatory chemokine receptors CXCR3 and CXCR1 within a distinct intracellular compartment. Equipped with such storage granules, CD4+ T cells coexpressing both receptors increased from only 1% ex vivo to approximately 30% within minutes of activation with PHA or exposure to the cyclooxygenase (COX) substrate arachidonic acid. Up-regulation was TCR independent and reduced by COX inhibitors at concentrations readily reached in vivo. The inducible inflammatory CXCR3(high)CXCR1+ phenotype identified nonpolarized cells, was preferentially triggered on CCR7+CD4+ T cells, and conferred increased chemotactic responsiveness. Thus, inducible CXCR3/1 expression occurs in a large fraction of CD4+ T cells. Its dependency on COX may explain a number of established, and point toward novel, effects of COX inhibitors.     

ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.