Synthesis of peptide based epsilon toxin vaccine by covalent anchoring to tetanus toxoid.

The epsilon toxin (Etx) produced by Clostridium perfringens type B and D causes severe enterotoxaemia associated with a general edema and neurological alterations, leading to subsequent death and is listed as one of the most lethal toxins. Currently employed vaccines against C. perfringens epsilon toxin include toxoid based vaccines. Use of peptide vaccines has become an interesting approach for vaccination after the successful licensing of peptide vaccines against Haemophilus influenza, Neisseria meningitides and Streptococcus pneumonia that have demonstrated the potential and effectiveness of these vaccines. Therefore, the present study was undertaken to develop a peptide based vaccine against epsilon toxin. Peptides were selected on the basis of epitope mapping by making 35 overlapping peptides of 15 amino acid residues in length specific to the primary amino acid sequence of the toxin, with a 7 amino acid residues overlaps between sequential peptides. Chemically synthesized peptides that were recognised by the antibody against the full length epsilon toxin were further assessed for vaccine potential. The selected peptides were chemically conjugated to partially reduced tetanus toxoid (TT) using of N-succinimidyl-3(2-pyridyldithio) propionate. Immunization of BALB/c mice with TT-peptide conjugates by sub-cutaneous route induced sustained high level mixed immune response as analyzed by antibody isotyping. Immunoblot analysis and ELISA clearly indicated generation of Etx-specific antibodies. Further, neutralization studies with the antisera generated against the TT-conjugated peptide(s) demonstrated that the antisera were able to neutralize the lethal dose of epsilon toxin in vitro demonstrating its potential as a promising vaccine candidate against enterotoxaemia.

The crystal structure of shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor.

Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.

Simvastatin inhibits inflammatory properties of Staphylococcus aureus alpha-toxin.

BACKGROUND: Simvastatin, a 3-hydroxy-methylglutaryl coenzyme A reductase inhibitor, has been shown to lower serum cholesterol levels in clinical use. Moreover, statins exert beneficial effects in vascular diseases by inhibition of leukocyte rolling, adherence, and transmigration. The aim of this study was to determine if pretreatment with simvastatin attenuates Staphylococcus aureus alpha-toxin-induced increase in leukocyte-endothelial interactions during exotoxemia. METHODS AND RESULTS: The effects of simvastatin on leukocyte-endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation. Simvastatin (50 or 100 microg/kg) was administered 18 hours before the study. Activation of microcirculation was induced by bolus administration of 40 microg/kg S aureus alpha-toxin. Exotoxemia resulted in a significant and time-dependent increase in leukocyte rolling, adherence, and transmigration of leukocytes as well as P-selectin expression on the intestinal vascular endothelium. Pretreatment with simvastatin significantly inhibited exotoxin-induced leukocyte rolling from 71+/-10 to 14+/-4.7 cells/min (P<0.01) and adherence from 14+/-3.5 to 0.4+/-0.2 cells (P<0.01). In addition, simvastatin pretreatment significantly inhibited transmigration of leukocytes from 10.5+/-1.2 to 4.2+/-0.9 (P<0.05) cells. Immunohistochemical detection of endothelial cell adhesion molecule P-selectin showed a 50% decrease in endothelial cell surface expression after simvastatin treatment. Furthermore, simvastatin treatment resulted in enhanced expression of endothelial cell NO synthase III in the intestinal microcirculation. CONCLUSIONS: These results demonstrate that simvastatin interferes with exotoxin-induced leukocyte-endothelial cell interactions, which may be relevant in various infectious diseases. Statin treatment may offer a new therapeutic strategy for these clinical conditions.

Inhibition of endogenous TNF formation by pentoxifylline.

During the last decade cytokines were recognized as focal components in acute and chronic inflammatory processes. The growing knowledge about these agents stimulated efforts to pharmacologically control their synthesis and action in clinical situations. Various rational approaches to these issues including selective antibodies or receptor antagonists are at present under clinical investigation. Recently, in our institute evidence was raised that pentoxifylline is able to suppress the synthesis of tumor necrosis factor-alpha in cell cultures, and in vivo, and to protect experimental animals against endotoxin shock. Extended studies in human experimental endotoxemia showed that pentoxifylline decreased circulating TNF without affecting endogenous formation of interleukins. The potency of this drug to interfere with TNF synthesis could also be demonstrated in cases of acute and chronic cytokine release-syndromes such as OKT3 first-dose reaction and severe pulmonary tuberculosis, respectively. In conclusion, we suggest that pentoxifylline may improve therapeutic strategies in septic syndrome and other diseases in which TNF represents a causative pathophysiological factor.

Prevention of endotoxaemia by non-absorbable antibiotics in heat stress.

Four anaesthetised monkeys were given oral kanamycin (15 mg 1 kg 12 hourly) over five consecutive days before being heat stressed. Four other anaesthetised monkeys served as controls. The plasma lipopolysaccharide concentration in control primates increased initially from 0.044 (SEM 0.004) ng/ml to 0.062 (0.006) ng/ml as the rectal temperature increased from 37.5 to 39.5 degrees C. A second increase in lipopolysaccharides started at 42 degrees C and reached 0.308 (0.038) ng/ml (p less than 0.01) at 44.5 degrees C. Before heat stress the plasma lipopolysaccharide concentration in the primates who had been pretreated with kanamycin was 0.007 (0.006) ng/ml, and despite heating these animals to 44.5 degrees C no increase in plasma lipopolysaccharide concentrations were seen in this group. The cardiovascular variable during heat stress were more unstable in the control group and began to deteriorate at a lower temperature than in the group receiving antibiotic. These data suggest that the increased plasma lipopolysaccharide concentration during heat stress originates mainly from the gut.

Endotoxemia, complement, and white blood cell activation in cardiac surgery: a randomized trial of laxatives and pulsatile perfusion.

Endotoxin activates complement and white blood cells and all are implicated in the pathologic effects of cardiopulmonary bypass (CPB). We investigated if reduction in intestinal bacterial load with a laxative and/or pulsatile perfusion to improve bowel circulation during CPB reduced endotoxemia and complement and white blood cell activation. Sixty patients were randomized to four groups in a 2 x 2 factorial structure: group 1 (no laxative, nonpulsatile perfusion); group 2 (laxative, nonpulsatile perfusion); group 3 (no laxative, pulsatile perfusion); and group 4 (laxative, pulsatile perfusion). Plasma concentrations of endotoxin, C3a and C5a, and granulocyte elastase (GE) were measured before anesthesia, skin incision, and heparin administration; during CPB (1, 30, 60, 90, and 120 minutes and after protamine administration); and after CPB at 3, 6, 12, 24, and 48 hours and 7 days. In all groups there was a small increase in the concentration of endotoxin (overall from 6 ng/L before CPB to 11 ng/L at 90 to 120 minutes; p < 0.001) and significant increases in C3a, C5a, and GE levels but no significant differences among the groups. Endotoxin levels did not correlate with activation of complement or white blood cells. There was a weak correlation between duration of CPB and levels of C3a (r = 0.14; p < 0.03) and GE (r = 0.25; p = 0.001) but not endotoxin or C5a. There was a general correlation between levels of C3a and GE but not in individual patients. In conclusion, CPB results in statistically significant increases in endotoxin, C3a, C5a, and GE during CPB.(ABSTRACT TRUNCATED AT 250 WORDS)

Prophylactic use of human endotoxin-core hyperimmune gammaglobulin to prevent endotoxaemia in colostrum-deprived, gnotobiotic lambs challenged orally with Escherichia coli.

The efficacy of human IgG polyclonal antibody to endotoxin-core in preventing endotoxaemia and subsequent disease was studied in colostrum-deprived gnotobiotic lambs challenged orally at about 5 h old with 10(9) cfu Escherichia coli. Human endotoxin-core hyperimmune gammaglobulin was given intravenously to 5 lambs at 1.9 g IgG/kg bodyweight prior to challenge. Human albumin was given intravenously to 3 control lambs. Bacteraemia was observed in all lambs, but the incidence was lower (P < 0.01) and the onset later (P < 0.05) in gammaglobulin pre-treated lambs. These lambs showed no signs of disease, whereas clinical endotoxaemia, manifesting as watery mouth disease, was diagnosed in 2 of the 3 control lambs which were killed between 18 and 22 h after challenge. Thus, prophylactic treatment of colostrum-deprived lambs with human IgG enriched in endotoxin-core antibodies was effective in reducing the degree of bacteraemia and preventing endotoxaemia, leukopenia and clinical disease following oral challenge with E. coli.

Lactulose--a drug with antiendotoxin effect.

Lactulose (beta-galactosido-fructose) was found to have anti-endotoxin properties: 670 mg lactulose abolished the gelating activity of mg E. coli endotoxin on Limulus lysate in vitro. When lactulose was fed to rats (6.3 +/- 1.1 g/kg/day) over a period of 4 or 8 days before i.v. administration of 0.5 g/kg galactosamine, the liver damage that normally develops was prevented. Since galactosamine-induced necrosis of hepatocytes and inflammatory reaction of the liver are mediated by systemic endotoxemia of intestinal origin, an anti-endotoxin effect of lactulose was demonstrated in vivo. We suggest that lactulose might offer a therapeutic basis in clinical situations in which endotoxemia is of pathogenetic significance, such as certain gastrointestinal and liver diseases, shock states and gram-negative sepsis.

Effects of polymyxin B and Salmonella typhimurium antiserum on horses given endotoxin intravenously.

Polymyxin B and an antiserum against an Re mutant Salmonella typhimurium were evaluated for protective effect in an equine model endotoxemia. Six 3- to 5-month-old foals were given endotoxin (0.25 micrograms/kg of body weight) IV after no pretreatment, or pretreatment with polymyxin B (6,000 U/kg, IV) or S typhimurium antiserum (1.5 ml/kg, IV). When given without pretreatment, endotoxin caused transient recumbency and increases in rectal temperature, and heart and respiratory rates. In addition, leukopenia and increases in circulating tumor necrosis factor (TNF) and interleukin 6 (IL-6) activities were detected. Compared with results obtained when endotoxin was given alone, pretreatment with polymyxin B resulted in significantly (P < 0.05) lower maximal plasma TNF and IL-6 activities, and significantly lower rectal temperature and respiratory rate. In contrast, compared with effects of endotoxin given without pretreatment, use of antiserum was associated with significantly (P < 0.05) higher respiratory rate, maximal plasma IL-6 activity, and total TNF response (as determined by areas under curves of plasma TNF vs time). These results indicate that polymyxin B may have potential as a treatment for equine endotoxemia. Salmonella typhimurium antiserum had no positive effect in this model, and, under certain conditions, may exacerbate the actions of endotoxin.

Hypoxia-induced endotoxemia in primates: role of reticuloendothelial system function and anti-lipopolysaccharide plasma.

Acute hypoxia is known to cause a marked reduction in intestinal and peripheral blood flow, in favor of blood flow to the brain and heart. Complete occlusion of the intestinal circulation is known to damage the gut wall, allowing potentially lethal endotoxins present within the intestines to escape into the circulation. We examined here whether the breathing of a hypoxic gas mixture could lead to sufficient damage of the intestinal wall to cause endotoxemia. Six anesthetized monkeys breathed air for 1 hr, then an hypoxic mixture (FIO2 = 0.13) containing N2O for 1 h and, finally, 100% O2. Plasma endotoxin concentrations were determined by two methods. After approximately 40 min of hypoxia, the plasma endotoxin level rose significantly from 0.39 to 1.60 ng X ml-1 (p less than 0.001) and then subsided to near control levels. Control monkeys breathing air only or 70% N2O in oxygen (FIO2 = 0.3) for 3 h showed no such elevation in plasma endotoxin concentration. We conclude that hypoxia leads to a temporary endotoxemia in primates. Reticuloendothelial system depression by whole body X-irradiation (200 rads) increased both the magnitude and duration of the hypoxia-induced endotoxemia. Prior administration of equine anti-lipopolysaccharide (anti-LPS) hyperimmune plasma greatly reduced the magnitude of the induced endotoxemia. Since endotoxemia may be lethal, the use of anti-LPS as possible prophylaxis should be considered in persons breathing artificial atmospheres or where acute hypoxia may be a danger.

[Intestinal endotoxin translocation in endotoxemic rats].

It has been known that enterogenic endotoxemia is an important pathogenetic factor in multiple organ failure. However, pathogenetic mechanism of enterogenic endotoxemia is yet unclear. In this work, experiments were performed on intact animal or isolated rat ilea-vascular perfusion apparatus and membrane vesicles produced from the extraction of brush border of rat intestinal mucosa. It is found that circulating endotoxin prompted intestinal endotoxin translocation in a positive feedback manner. Various regulatory peptides modulated the endotoxin translocation: endothelin stimulated and atrial natriuretic peptide inhibited the endotoxin translocation, and the pathway of enterogenic endotoxin translocation is mainly not transcellular, probably paracellular. It is also found that taurine significantly inhibited intestinal endotoxin translocation and protected the animals from endotoxemic injury.

[Ofloxacin in comprehensive treatment of infections in burn patients]. / Ofloksatsin v kompleksnom lechenii infektsii u obozhzhennykh.

The experience with ofloxacin in the prophylaxis and treatment of infected burn wounds in 40 patients was investigated. High clinical and microbiological efficacy of the drug was stated (82.5 and 83 per cent respectively). The highest efficacy of ofloxacin was observed when the burned area did not exceed 25 per cent of the body surface. It was concluded that the prophylactic use of the drug during acute burn toxemia was not expedient.

[Photomodification of autologous blood in surgery]. / Fotomodifikatsiia autokrovi v khirurgii.

The use of the method of autologous blood photomodification in surgery is discussed on basis of generalized experience of the Leningrad Research-Practical Center of Blood Photomodification (3,000 procedures conducted in over 2,000 patients). The current state of the problem, the main mechanisms of the therapeutic action of the procedure, and the clinical efficacy of the method in various surgical diseases are dealt with. The possible complications and the means of their prevention are deal with. The methodological aspects of research are discussed. The authors define the main problems which must be solved in order to raise the efficacy of using photomodification of autologous blood in surgery.

[Bile sorption in hepatic insufficiency in patients with mechanical jaundice]. / Zhelchesorbtsiia pri pechenochnoi nedostatochnosti u bo'lnykh s mekhanicheskoi zheltukhoi.

The author describes and substantiates the performance of bile sorption in the prevention and treatment of hepatic insufficiency in patients with obstructive jaundice who were subjected to external drainage of the bile tract. A total of 357 sessions of bile sorption were conducted in the pre- and postoperative periods in 51 elderly and old-aged patients with obstructive jaundice of benign (23 patients) and malignant (18 patients) character. Four patients died.

[Ultraviolet irradiation of blood in surgery]. / Ultrafioletovoe obluchenie krovi v khirurgii.

The results of complex treatment of 81 patients with pyoinflammatory diseases with the use of blood ultraviolet irradiation are discussed. A marked clinical effect was noted, the terms of treatment reduced by 5-10 days, the outcomes improved, and the number of complications decreased. Irradiation of autologous blood by ultraviolet rays led to modulation of the indices of antimicrobial protection, increase of the intensity of the histochemical reaction to peroxidase up to 40-50%, and diminution of pH in the neutrophil phagosomes to 5.0. The ultrastructure and ability of thrombocytes to store serotonin were restored, and intensity of their metabolic processes increased, the membrane phospholipid composition changed, and juvenile platelet forms appeared.

[Plasmapheresis in severe forms of suppurative-septic diseases in children]. / Plazmaferez pri tiazhelykh formakh gnoino-septicheskikh zabolevanii u detei.

Under analysis is the first experience with the application of discrete plasmapheresis in 44 children aged from 6 months to 14 years for detoxication of severe forms of pyo-septic diseases. The method of discrete plasmapheresis is described. The method was shown to have a favourable influence upon hemodynamics and some parameters of the cell and humoral immunity. The application of discrete plasmapheresis resulted in rapid liquidation of endotoxicosis, less lethality and shorter time of treatment at the hospital. Wider introduction of discrete plasmapheresis in children with pyo-septic diseases is recommended.

[Experience with extracorporeal use of xenospleen in patients with traumatic shock]. / Opyt ékstrakorporal'nogo podkliucheniia ksenoselezenki postradavshim s shokogennoi travmoi.

The article presents results of using the extracorporal connection with the donor (porcine) spleen (ECCDS) for the treatment of 14 patients with critical mechanical traumas and their complications. An experience with 30 ECCDS-s has shown them to possess a pronounced detoxicating and immunomodulatory effect followed by a positive clinical result. In 6 cases of ECCDS a temporary chill was noted. There were no other complications.

The management of central venous catheters and infection control: is it time to change our approach?

Catheter related bloodstream infections (CR-BSIs) can lead to a number of serious conditions for the patient, including death. There is much recent evidence both in the UK and abroad which identifies the sources of CR-BSIs, yet they continue to occur. This article seeks to review some of the current evidence in relation to the prevention of CR-BSIs at insertion point.

A low-toxic site-directed mutant of Clostridium perfringens ε-toxin as a potential candidate vaccine against enterotoxemia.

Clostridium perfringens epsilon toxin (ETX), one of the most potent toxins known, is a potential biological weapon; therefore, the development of an effective vaccine is important for preventing intoxication or disease by ETX. In this study, genetically detoxified epsilon toxin mutants were developed as candidate vaccines. We used site-directed mutagenesis to mutate the essential amino acid residues (His106, Ser111 and Phe199). Six site-directed mutants of ETX (mETX (H106P) , mETX (S111H) , mETX (S111Y) , mETX (F199H) , mETX (F199E) , mETX (S111YF199E) ) were generated and then expressed in Escherichia coli. Both mETX (F199E) and mETX (H106P) with low or non-cytotoxicity that retained their immunogenicity were selected to immunize mice 3 times, and the mouse survival data were recorded after challenging with recombinant wild-type ETX. mETX (F199E) induces the same protection as mETX (H106P) , which was reported previously as a promising toxin mutant for vaccine, and both of them could protect immunized mice against a 100× LD50 dose of active wild-type recombinant ETX. This work showed that mETX (F199E) is another promising candidate vaccine against enterotoxemia and other diseases caused by ETX.