Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis.

BACKGROUND: Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. RESULTS: Alpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model. CONCLUSION: Although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.

Effects of Clostridium perfringens alpha and epsilon toxins in the bovine gut.

Clostridium perfringens alpha and epsilon toxins produce enterotoxaemia in sheep and goats. However, the information regarding the pathophysiology of alpha and epsilon toxins in the bovine intestine is still scanty. In this study, intestinal loops were performed in the ileum and colon of three one-week-old Holstein and two four-week-old crossbreed calves. Laparotomy was performed in all calves under anaesthesia and four loops -three cm long- were performed in the small and large intestines. For both intestines, loops were inoculated with alpha or epsilon toxins. Tissue samples from all loops were obtained and processed for routine histology and for transmission electron microscopy. Congestion was observed in toxin treated loops. Fluid accumulation in the gut lumen was prominent in all treated loops, but in epsilon treated ones the mucous was also haemorrhagic. The histology revealed large amount of exfoliated epithelial cells in the lumen of alpha toxin treated loops and severe haemorrhage was observed in the lamina propria of epsilon toxin treated colonic loops. Despite some necrotic exfoliated enterocytes, no ultraestructural changes were observed in alpha toxin treated loops, though with epsilon toxin the loops exhibited dilation of the intercellular space in the mucosa of both, small and large intestines. These observations indicate that both, alpha and epsilon toxins can alter the intestinal barrier, in calves and are pathogenic for this species.

Trigger release liposome systems: local and remote controlled delivery?

Target-specific delivery has become an integral area of research in order to increase bioavailability and reduce the toxic effects of drugs. As a drug-delivery option, trigger-release liposomes offer sophisticated targeting and greater control-release capabilities. These are broadly divided into two categories; those that utilise the local environment of the target site where there may be an upregulation in certain enzymes or a change in pH and those liposomes that are triggered by an external physical stimulus such as heat, ultrasound or light. These release mechanisms offer a greater degree of control over when and where the drug is released; furthermore, targeting of diseased tissue is enhanced by incorporation of target-specific components such as antibodies. This review aims to show the development of such trigger release liposome systems and the current research in this field.

Safety and efficacy of a maternal vaccine for the passive protection of broiler chicks against necrotic enteritis.

Necrotic enteritis is a potentially fatal multifactorial disease of chickens, which under commercial conditions is often associated with increased levels of mortality and reduced bird performance. The safety and efficacy of a Clostridium perfringens type A alpha-toxoid (Netvax™) formulated as an oil emulsion was investigated, following maternal immunization of broiler breeder hens, housed under commercial conditions, by the intramuscular route. A total of 11,234 hens were vaccinated across two integrated poultry sites. The vaccine was safe with no systemic reactions or adverse effects on bird performance detected. Vaccination resulted in a significant increase in anti-alpha toxin antibody in the hen that was maintained throughout the study, and subsequently transferred to their progeny throughout the laying period via egg yolk. Chicks hatched from eggs produced from vaccinated hens were shown to have reduced mortality specifically related to progeny flocks where gross gut lesions associated with necrotic enteritis were observed in control chicks. Further, whilst C. perfringens was isolated from control chicks with necrotic enteritis lesions, no such isolations were made at these time points from chicks from vaccinated hens. These results indicate that, under commercial conditions, maternal vaccination with Netvax™ can help to control losses related to necrotic enteritis.

Immunization with recombinant alpha toxin partially protects broiler chicks against experimental challenge with Clostridium perfringens.

Necrotic enteritis (NE) in poultry has re-emerged as a concern for poultry producers, due in part to banning, by many countries, of the use of antimicrobial growth promoters in feeds. This re-emergence has led to a search for alternative methods for control of the disease, particularly vaccination. The objective of this work was to determine if vaccination of broiler chicks with recombinant alpha toxin protected against experimental challenge. Broiler chicks were vaccinated subcutaneously at 5 and 15 days of age, followed 10 days later by challenge with Clostridium perfringens. Birds were challenged twice daily on 4 consecutive days by mixing C. perfringens cultures with feed (three parts culture: four parts feed). Non-vaccinated birds challenged with C. perfringens developed NE at the rate of 87.8%, while only 54.9% of vaccinated birds developed lesions. In addition, non-vaccinated birds had lesion scores averaging 2.37, while average scores in vaccinated birds were 1.35. Vaccination produced an antibody response, with post-vaccination anti-alpha toxin IgG (IgY) titers in vaccinated birds more than 5-fold greater than in non-vaccinated birds. After challenge, vaccinated birds had average IgG (IgY) titers>15-fold higher than those in non-vaccinated birds. These results suggest that alpha toxin may serve as an effective immunogen, and, as such, may play a role in pathogenesis.

[Induced expression of alpha-toxin gene of Clostridium perfringens in recombinant Lactobacillus casei and their immunoprotective in mice].

OBJECTIVE: The prepared an oral vaccine by constructing recombinant Lactobacillus casei expressing alpha-toxin gene of Clostridium perfringens, for preventing poisoning by Clostridium perfringens. METHODS: The constructed cell-surface expression plasmid pPG1-alpha/L. 393 and secretion expression plasmid pPG2-alpha/L. 393, both with alpha-toxin gene, were electroporated into L. casei 393, generating recombinant bacteria pPG1-alpha/L. casei 393 and pPG2-alpha/L. casei 393. The recombinant strains were induced by 1% lactose in De Man, Rogosa and Sharp (MRS) broth, and the target protein was detected by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and indirect immunofluorescence assay. BALB/C mouse were used as animal model immunized with recombinant strains by intragastric administration, and the immune efficacy was analyzed. Specific anti-alpha-toxin protein sIgA was detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces, vaginal lavage, eye washing of mice after intragastric administration, and Specific IgG was detected by indirect ELISA in the serum of immunized mice. The resistance of immunized mice to alpha-toxin and the neutralization ability of antibodies to alpha-toxin were also tested. RESULTS: Mice immunized with pPG1-alpha/L. casei 393 and pPG2-alpha/L. casei 393 could produce remarkable anti-alpha-toxin antibodies, sIgA and circulating antibody IgG had completely neutralization ability against alpha-toxin. The test of alpha-toxin challenge in mice showed that the immunized mice could resist three times' Minimum Lethal Dose (MLD). CONCLUSION: All the results indicated that mice inmmunized by the recombinant L. casei expressing alpha-toxin gene of C. perfringens could elicit regional and systematic immunity response and neutralization ability.

HPMA copolymer-phospholipase C and dextrin-phospholipase A2 as model triggers for polymer enzyme liposome therapy (PELT).

'Polymer Enzyme Liposome Therapy' (PELT) is a two-step anticancer approach in which a liposomal drug and polymer-phospholipase conjugate are administered sequentially to target the tumour interstitium by the enhanced permeability and retention effect, and trigger rapid, local, drug release. To date, however, the concept has only been described theoretically. We synthesised two polymer conjugates of phospholipase C (PLC) and A2 (PLA2) and evaluated their ability to trigger anthracycline release from the clinically used liposomes, Caelyx® and DaunoXome®. N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-PLC and a dextrin-PLA2 were synthesised and their enzymatic activity characterised. Doxorubicin release from polyethyleneglycol-coated (PEGylated) Caelyx® was relatively slow (<20%, 60 min), whereas daunomycin was rapidly released from non-PEGylated DaunoXome® (∼87%) by both enzymes. Incubation with dextrin-PLA2 triggered significantly less daunomycin release than HPMA copolymer-PLC, but when dextrin-PLA2 was pre-incubated with α-amylase, the rate of daunomycin release increased. DaunoXome®'s diameter increased in the presence of PLA2, while Caelyx®'s diameter was unaffected by free or conjugated PLA2. Dextrin-PLA2 potentiated the cytotoxicity of DaunoXome® to MCF-7 cells to a greater extent than free PLA2, while combining dextrin-PLA2 with Caelyx® resulted in antagonism, even in the presence of α-amylase, presumably due to steric hindrance by PEG. Our findings suggest that in vivo studies to evaluate PELT combinations should be further evaluated.

[The correction action of Phosprenyl and Gamavit on the functional activity of mouse peritoneal macrophages in response to high doses of Staphylococcus aureus alpha-toxin].

The study of the functional activity of peritoneal macrophages of BALB/c mice at different stages of the toxic action caused by S. aureus alpha-toxin (ST) was carried out. The analysis of the dynamics of toxic reaction revealed the main critical points of triggering necrotic processes: the first hour and day 2. One hour after the injection of large doses of ST a sharp increase in the process of antigen binding with its subsequent sharp decrease. Simultaneously, a decrease in the activity of the lysosomal enzymes cathepsin D and acidic phosphatase was established, which was indicative of the destabilization of both lysosomal and cellular macrophage membranes. The increase of oxygen metabolism on day 2, together with the release of lysosomal proteases into the extracellular area, correlated with the maximum death rate of mice and served as the main index of the development of necrosis. The prophylactic and therapeutic use of the preparations Gamavit and Phosprenyl revealed their antitoxic activity and capacityfor stimulating the level of natural body resistance.

Bacterial phospholipases C as vaccine candidate antigens against cystic fibrosis respiratory pathogens: the Mycobacterium abscessus model.

BACKGROUND: Vaccine strategies represent one of the fighting answers against multiresistant bacteria in a number of clinical settings like cystic fibrosis (CF). Mycobacterium abscessus, an emerging CF pathogen, raises difficult therapeutic problems due to its intrinsic antibiotic multiresistance. METHODS: By reverse vaccinology, we identified M. abscessus phospholipase C (MA-PLC) as a potential vaccine target. We deciphered here the protective response generated by vaccination with plasmid DNA encoding the MA-PLC formulated with a tetra functional block copolymer 704, in CF (ΔF508) mice. Protection was tested against aerosolized smooth and rough (hypervirulent) variants of M. abscessus. RESULTS: MA-PLC DNA vaccination (days 0, 21, 42) elicited a strong antibody response. A significant protective effect was obtained against aerosolized M. abscessus (S variant) in ΔF508 mice, but not in wild-type FVB littermates; similar results were observed when: (i) challenging mice with the "hypervirulent" R variant, and; (ii) immunizing mice with purified MA-PLC protein. High IgG titers against MA-PLC protein were measured in CF patients with M. abscessus infection; interestingly, significant titers were also detected in CF patients positive for Pseudomonas aeruginosa versus P. aeruginosa-negative controls. CONCLUSIONS: MA-PLC DNA- and PLC protein-vaccinated mice cleared more rapidly M. abscessus than ß-galactosidase DNA- or PBS- vaccinated mice in the context of CF. PLCs could constitute interesting vaccine targets against common PLC-producing CF pathogens like P. aeruginosa.

Selective elimination of lymphocyte subpopulations by monoclonal antibody-enzyme conjugates.

A novel method for the selective depletion of lymphocyte subpopulations has been developed. Conjugates of glucose oxidase (GOx) and phospholipase-C (PL-C) coupled to a monoclonal mouse anti-rat IgG (MAR) were shown to be selectively cytotoxic for targeted lymphocyte subsets in the presence of various rat monoclonal antibodies directed toward murine cell surface antigens. The ability of both conjugates to bind specifically to rat monoclonal antibodies was demonstrated by flow cytometry. The PL-C-MAR conjugate was more stable than the GOx-MAR conjugate. The PL-C conjugate, in conjunction with primary rat anti-mouse monoclonal antibodies, produced selective killing of T or B cells as detected by a loss of proliferative capacity in response to mitogens and by specific cell depletion demonstrated by flow cytometry. Normal mouse serum protected against the cytotoxicity of free enzymes but had no protective effect on enzyme conjugates. Because the substrates of these enzymes are abundant in vivo and serum did not interfere with their cytotoxicity, these enzyme-antibody conjugates may be valuable for selective lymphocyte depletion in vivo.

Inhibition of implantation by the intrauterine administration of phospholipases in the rat.

Phospholipases A2 and C (93 and 500 mU) were administered to uterine horns of Long-Evans adult rats during the first five days of pregnancy. As control material, saline (0.15 M) was administered to contralateral uterine horns. The animals were sacrificed on the ninth day of pregnancy, the uterine horns were removed and the number of implanted embryos were counted. Both horns were examined with light and electron microscopes. For electron microscopy, Ruthenium red was used to visualize possible changes of the outer coat (glycocalix) of the plasma membrane of endometrial epithelial cells. Implantation was inhibited when phospholipases A2 and C were administered during the first three days of pregnancy. Ultrastructural modifications included decrease of glycoproteins as demonstrated by diminution of the Ruthenium red staining that may indicate a decrease in the negative surface charges of endometrial surface epithelium.

Host defense against bacterial keratitis.

PURPOSE: To define factors that protect the eye from Staphylococcus aureus keratitis and limit tissue damage once keratitis occurs. METHODS: Rabbit tears were analyzed for bactericidal and phospholipase A(2) (PLA(2)) activities on S. aureus. Inhibition by spermidine of PLA(2) anti-staphylococcal activity in tears was tested in vitro and in vivo. Rabbits immunized with heat-inactivated alpha-toxin were challenged with intrastromal injection of S. aureus. RESULTS: Arachidonic acid was cleaved from S. aureus by purified PLA( 2) or rabbit tears. Spermidine inhibited these reactions in vitro and facilitated keratitis in vivo. PLA(2) activity decreased with advanced age and shortly following sleep, but increased with keratitis. Antibody to alpha-toxin significantly reduced corneal damage and epithelial cell sloughing during keratitis. CONCLUSIONS: PLA(2) is a major host-defense component of rabbit tears. Alpha-toxin is a major mediator of corneal damage, and antibody to alpha-toxin reduces pathologic changes during keratitis.

Evaluation in broilers of the probiotic properties of Pichia pastoris and a recombinant P. pastoris containing the Clostridium perfringens alpha toxin gene.

The probiotic properties of Pichia pastoris and of a recombinant P. pastoris containing the Clostridium perfringens alpha toxin gene were evaluated in broilers. One-day-old chicks randomly divided in four groups were fed with commercial feed devoid of antibacterials. The control group (1) received plain food, while the other groups were supplemented with either P. pastoris (2), the recombinant P. pastoris (3) or Bacillus cereus var. Toyoi (4). At day 49, live weights, feed efficiency and seroconversions were higher (P<0.05) in the supplemented groups than in the control groups. Group 3 showed the best results, while group 2 had lower weight gain than groups 3 and 4 although food conversion was better than in group 4. Seroconversions were not different (P>0.05) among the supplemented groups. Adverse reactions were not observed in histopathologic evaluation. We concluded that P. pastoris and the recombinant P. pastoris could be used as probiotics in broilers.

Phospholipase C gamma in distinct regions of the ventral tegmental area differentially regulates morphine-induced locomotor activity.

Neurotrophic factors and the signaling pathways they activate play a role in mediating long-term molecular, cellular, and behavioral adaptations associated with drug addiction. Here we mimicked the biological response of phospholipase C-gamma (PLC gamma) induction in the ventral tegmental area (VTA) observed after chronic morphine using viral-mediated gene transfer. Using a behavioral sensitization paradigm, we demonstrate that microinjections of PLC gamma 1 into distinct (rostral vs. caudal) regions of the VTA result in differential locomotor responses to morphine.

A cellular deficiency of gangliosides causes hypersensitivity to Clostridium perfringens phospholipase C.

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

Immunization with alpha-toxin toxoid protects the cornea against tissue damage during experimental Staphylococcus aureus keratitis.

Alpha-toxin is a major virulence factor in Staphylococcus aureus keratitis. Active or passive immunization with alpha-toxin toxoid could protect against corneal damage. Results show that either form of immunization did not kill bacteria but did significantly protect against corneal pathology, especially epithelial erosion.

S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C.

Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected (100-700 micrograms/ml) into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by [3H]thymidine incorporation into nuclear DNA. In addition, approximately to 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration (1 mg/ml) was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC (approximately 10,000 molecules per cell) to override the cellular G0 block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.

Combination chemotherapy with Clostridium perfringens phospholipase C and cytosine antimetabolites: complementary inhibition directed at membrane lipids.

Tumor cell membranes were susceptible to the action of Clostridium perfringens phospholipase C, and this was reflected by inhibition of cellular replication in culture. The differential susceptibility of two neoplastic cell lines to this enzyme was studied in detail. The growth of sarcoma 180 cells cultured in Fischer's medium was markedly inhibited by phospholipase C; whereas, in contrast, cultured L1210 leukemia cells were relatively resistant to the cytotoxic effects of this enzyme. The differential sensitivity of these two neoplastic cell lines to phospholipase C was corroborated by dye-exclusion tests. Thus, leukemia L1210 cells exposed to a concentration of 0.2 mg of phospholipase C per ml of Fischer's medium for 30 min at 37 degrees C were able to exclude Trypan Blue; whereas, only about 21% of sarcoma 180 cells treated under identical conditions were able to exclude the dye. That the cytotoxicity of phospholipase C to sarcoma 180 was the result of hydrolysis of phospholipids of the plasma membrane was supported by measurements of the rate of hydrolysis of radioactivity from the phospholipid of neoplastic cells prelabeled with [3H]choline. Eighty-two percent of incorporated radioactive choline was released from sarcoma 180 cells treated with phospholipase C in Fischer's medium, whereas, only 20% of the label from [3H]choline was solubilized from L1210 leukemia cells treated with the enzyme under similar conditions. Scanning electron microscopy revealed significant damage to sarcoma 180 cells exposed to phospholipase C in Fischer's medium, which was characterized by alterations in size and shape of cells, disappearance of microvilli, and appearance of fistulas in cell membranes; relatively resistant L1210 leukemic cells did not appear to be markedly damaged by comparable enzyme treatment. Exposure of leukemia L1210 cells to phospholipase C in Puck's saline A increased the sensitivity of these cells to enzymatic action. Under these conditions, a comparable amount of phospholipid was hydrolyzed from surface membranes of sarcoma 180 and leukemia L1210 cells, and the degree of membrane damage appeared to be similar, as measured by the capacity of the tumor cell lines to exclude Trypan Blue and by scanning electron microscopy. The extensive damage to membranes by hydrolysis of phospholipids was not accompanied by a change in the degree of specific binding of [3H]concanavalin A(ConA).(ABSTRACT TRUNCATED AT 400 WORDS)

Immunization with the C-Domain of alpha -Toxin prevents lethal infection, localizes tissue injury, and promotes host response to challenge with Clostridium perfringens.

Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, impaired host response, and, often, death. Phospholipase C (alpha -toxin) is the virulence factor most responsible for these pathologies. The present study investigated the efficacy of active immunization with the C-terminal domain of alpha -toxin (Cpa247-370) in a murine model of gas gangrene. Primary end points of the study were survival, progression of infection, and tissue perfusion. Secondary end points, which were based on findings of histologic evaluation of tissues, included the extent of tissue destruction and microvascular thrombosis, as well as the magnitude of the tissue inflammatory response. Survival among C-domain-immunized animals was significantly greater than that among sham-immunized control animals. Furthermore, immunization with the C-domain localized the infection and prevented ischemia of the feet. Histopathologic findings demonstrated limited muscle necrosis, reduced microvascular thrombosis, and enhanced granulocytic influx in C-domain-immunized mice. We conclude that immunization with the C-domain of phospholipase C is a viable strategy for the prevention of morbidity and mortality associated with C. perfringens gas gangrene.