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Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.

Recent publications have provided confusing information on the importance of the J chain for secretion of dimeric IgA at mucosal surfaces. Using J chain-deficient (J chain-/-) mice, we addressed whether a lack of J chain had any functional consequence for the ability to resist challenge with cholera toxin (CT) in intestinal loops. J chain-/- mice had normal levels of IgA plasma cells in the gut mucosa, and the Peyer's patches exhibited normal IgA B cell differentiation and germinal center reactions. The total IgA levels in gut lavage were reduced by roughly 90% as compared with that in wild-type controls, while concomitantly serum IgA levels were significantly increased. Total serum IgM levels were depressed, whereas IgG concentrations were normal. Following oral immunizations with CT, J chain-/- mice developed 10-fold increased serum antitoxin IgA titers, but gut lavage anti-CT IgA levels were substantially reduced. However, anti-CT IgA spot-forming cell frequencies in the gut lamina propria were normal. Anti-CT IgM concentrations were low in serum and gut lavage, whereas anti-CT IgG titers were unaltered. Challenge of small intestinal ligated loops with CT caused dramatic fluid accumulation in immunized J chain-/- mice, and only 20% protection was detected compared with unimmunized controls. In contrast, wild-type mice demonstrated 80% protection against CT challenge. Mice heterozygous for the J chain deletion exhibited intermediate gut lavage anti-CT IgA and intestinal protection levels, arguing for a J chain gene-dosage effect on the transport of secretory IgA. This study unequivocally demonstrates a direct relationship between mucosal transport of secretory SIgA and intestinal immune protection.

Despite data from animal studies, seroepidemiological surveys, and controlled clinical trials, skepticism persists about immunity to pertussis conferred by serum IgG neutralizing antibodies (antitoxin). This is largely prompted by the absence of a "protective" level of antitoxin. Examination of the similarities between the pathogenesis and immunity to pertussis and diphtheria provides an explanation for this dilemma. As with pertussis, diphtheria toxoid vaccination confers only approximately 70% immunity on an individual basis, individuals with protective levels of antitoxin may contract diphtheria, and about 50% of the entire population, especially adults, have less than protective levels of antitoxin. The virtual disappearance of diphtheria followed vaccination of the entire population with diphtheria toxoid, which blocked transmission of toxigenic Corynebacterium diphtheriae and thus reduced the pathogen to almost undetectable levels. The individual and community-based immunity induced by diphtheria toxoid, we hypothesize, is similar to that of pertussis and pertussis toxoid.

Clostridium difficile is the bacterial pathogen identified as the cause of pseudomembranous colitis and is principally responsible for nosocomial antibiotic-associated diarrhea and colitis. The pathologic findings associated with this infection are believed to be caused by two large (approximately 300-kDa) exotoxins, toxins A and B. Because of the mucosal nature of this infection, vaccination strategies aimed at providing prophylactic or therapeutic immune protection have included immunization by mucosal routes. Using the hamster model of C. difficile infection, we examined the protective efficacy of inactivated toxin (toxoid) vaccine formulations prepared as either culture filtrate or partially purified toxoid. We compared combination parenteral and mucosal vaccination regimens involving intranasal, intragastric, or rectal routes of immunization and found that rectal immunization in conjunction with intramuscular (i.m.) vaccination provided full protection of hamsters from death and diarrhea while the other mucosal routes did not. Protection was associated with high levels of toxin-neutralizing antibodies in serum. The requirement for adjuvants for protection was assessed by using sequential i.m. and rectal or i.m. vaccination regimens. Unexpectedly, i.m. immunization without adjuvant conferred the highest protection from death and diarrhea; this regimen elicited the highest serum anti-toxin B titers as well as toxin B neutralizing titers. Passive transfer of mouse antitoxin antibodies protected hamsters in a dose-dependent manner, demonstrating the principal role of circulating antitoxin antibodies in immunity from this toxin-mediated mucosal disease. These results suggest that prophylactic parenteral vaccination or intravenous immunotherapy could provide protection from C. difficile disease in humans.

Study of the role of staphylococcal infection in pyoinflammatory complications of mandibular injuries showed that immunization of this category of patients with staphylococcal antitoxin simultaneously with antibiotic therapy leads to a significant increase in the blood level of anti-alpha-toxin. The latter has a positive impact on the clinical course of disease and staphylococcal contamination of involved site and oronasal mucosa.

Objective: Constant antigenic stimulation of the large immune cell population contained within gut-associated lymphoid tissue during HIV infection may contribute to patients' total viral load. The aim of this investigation was to evaluate the effect of a mucosal antigenic challenge on HIV replication., Design: Prospective clinical study., Methods: Twelve HIV-1-infected men (mean age, 42.3 years) from the Casa de Apoio Santo Antonio, Rio de Janeiro, Brazil, were immunized with combined whole cell-toxin B subunit oral cholera vaccine. Blood was collected on days 0, 2, 4, 6, 10 and 15 after immunization and plasma was tested for cholera toxin-specific antibody response (IgG and IgA), beta2-microglobulin, and plasma viral load. CD4 lymphocyte counts were performed within 1 week before immunization. Five HIV-infected non-immunized individuals were studied as controls., Results: There were no adverse effects following immunization and no deterioration in clinical outcome during 3 months of follow-up. A transient increase in viral load that ranged from twofold to 60-fold was observed in all cases and was statistically significant on days 2, 6 and 10 (P = 0.017, P = 0.025, P = 0.021, respectively). There was no correlation with CD4 cell counts. None of the non-immunized subjects demonstrated the pattern of viraemia observed after immunization (P > 0.10 on all days)., Conclusions: Our data indicate that mucosal immunization with oral cholera vaccine induces a transient increase in HIV viraemia, regardless of clinical stage of infection and CD4 cell counts. These findings suggest that mucosal stimulation of HIV-infected patients enhances HIV replication.

Immunity conferred by vaccination with cellular pertussis vaccines and pertussis wanes so that adults are now the main reservoir of B. pertussis. Similar to diphtheria toxoid, acellular pertussis vaccines are suitable for vaccination of adults and it is probable that addition of pertussis toxoid to DT will be recommended for this age group. If this can be achieved, it is easy to predict that pertussis will be eliminated and the possibility of eradicating B. pertussis will become feasible. We propose that, despite the relative inaccuracy of correlating levels of antitoxin with individual immunity, a standardized assay and reference antiserum for pertussis toxoid will control acellular pertussis vaccines as was achieved with diphtheria toxoid.

During development of Peru-15, a new live oral vaccine for cholera, the role of buffer needed to be evaluated. Generally, oral bacterial vaccines are acid labile and need to be administered by using a formulation which protects them from gastric acid. We compared three different buffers for use with Peru-15, including a standard bicarbonate-ascorbic acid buffer, Alka-Seltzer, and a new electrolyte-rice buffer, CeraVacx. Saline served as the control. Thirty-nine healthy adult volunteers received Peru-15 (10(8) CFU) with one of the three buffers or saline in a double-masked study. The volunteers were monitored for symptoms for 7 days after the dose, serum was tested for antibody responses by vibriocidal antibody and immunoglobulin G antitoxin enzyme-linked immunosorbent assays, and stool samples were tested for excretion of the vaccine strain. Side effects were minimal in all groups. All 30 volunteers who took Peru-15 with a buffer showed significant rises in vibriocidal antibody titer. The magnitude of the rises was higher in the CeraVacx group than in the other two buffer groups. Four of nine volunteers who took the vaccine with saline also showed increased titers, but they were lower than those in any of the three buffer groups. Excretion of the vaccine strain was similar in the buffer groups, but excretion was not associated with the magnitude of the vibriocidal responses. Excretion of Peru-15 was not detected in the saline group. We conclude that buffer does amplify the serological response to Peru-15 and that CeraVacx may provide benefits not provided by other buffers.

To define the alterations provoked by malnutrition during suckling (20 pups/dam) in the gut-associated lymphoid tissues of rats, Peyer patch (PP) and mesenteric lymph node (MLN) cells were studied by flow cytometry. After weaning (21 days of age), rats malnourished during suckling (MNR) showed an increase in the CD4+ CD45RC+ subset together with a decrease in the CD4+ CD45RC- subset (P < 0.01). These alterations remained even after 3 weeks of refeeding with stock diet. The CD4+CD8+ subset was not increased in the MNR, indicating that a release of cortical thymocytes did not occur. At weaning the percentage of CD4+Thy1+ cells was decreased in the MNR, indicating a low number of cells released from the thymus. When the B cell lineage was studied, we found a decreased percentage of precursors in the bone marrow and a decreased percentage of mature B cells in the periphery. When the MNR were immunized intra-PP with cholera toxin (CT) after 1 week of refeeding, the specific IgG and IgA and IgM antibody-forming cells (measured by ELISPOT) were diminished in the PP, MLN, and spleen when compared to the age-matched controls (P < 0.001). These results were coincident with the ELISA titers obtained in the sera and in the intestinal fluids. When CT was administered after 2 weeks of refeeding, the number of IgM anti-toxin AFC approached control values, but the number of IgA and IgG AFC continued to be low. When 3 weeks of refeeding was allowed before the CT delivery, the immune response in the MNR approached control values. These results indicate that malnutrition during suckling provokes alterations in B and T lymphocytes and produces a lack in the induction of the primary and secondary immune responses in the GALT which reversed after 3 weeks of refeeding.

The effectiveness, both immunological (by an increase in the titers of antitoxins) and protective (by resistance to the inoculation of the absolute lethal dose of infective agents), of the regional (wound) revaccination with tetratoxoid (Clostridium perfringens, C. oedematiens, C. septicum, C. histolyticum) was demonstrated on the experimental model of wound infection (gas gangrene) of guinea pigs. The schedule of rapid immunization with tetratoxoid was developed, which made it possible to create good immunological preparedness (basic immunity) for subsequent revaccination in case of traumas within 6 days. The effectiveness of rapid immunization by the application of tetratoxoid on the wound was shown. This immunization ensured a considered increase in the titers of antitoxins within the first 6 days, which increased the protection of the animals from infection with each of the four causative agents of gas gangrene.

Objective: To assess the value of measuring circulating concentrations of mediators (endotoxin, tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], and interleukin-6[IL-6]) and their endogenous antagonists (antiendotoxin core antibody [EndoCAb], interleukin-1 receptor antagonist [IL-1ra], and soluble TNF receptors [sTNF-R]) in predicting mortality and organ failure in sepsis syndrome., Design: Cohort study with a follow-up period of 30 days., Setting: Intensive therapy units of five tertiary referral centers in Scotland., Subjects: A total of 146 intensive therapy unit patients with sepsis syndrome underwent repeated sampling during a 10-day period following admission to an intensive therapy unit., Main Outcome Measures: Circulating concentrations of mediators and antagonists were compared in survivors and nonsurvivors., Results: Median Acute Physiology and Chronic Health Evaluation II score was 23 (range, 8 to 40). Mortality at 30 days was 49%. On entry to the study, circulating endotoxin was detected in 66% of patients, TNF-alpha in 14%, and IL-1 beta in 29%. Levels did not predict mortality or organ failure. Patients with IL-6 concentrations in excess of 3000 pg/mL had an increased mortality rate (64% vs 40%, P = .02). The incidence of IgG EndoCAb depletion on entry to the study was 26% in nonsurvivors and 10% in survivors (P = .02). Initial concentrations of both type I and type II sTNF-R were significantly higher in nonsurvivors (P < .01). Initial circulating IL-1ra concentrations were not of value in predicting mortality. Cytokine antagonists were present in concentrations 30- to 100,000-fold greater than their corresponding cytokine., Conclusion: The observed high circulating levels of the cytokine antagonists IL-1ra and sTNF-R and the relatively small proportion of patients developing EndoCAb depletion may contribute to the limitations of therapies that aim to augment natural defenses against endotoxin or the proinflammatory cytokines.

The gelatin-particle-agglutination (PA) test for titrating antibodies against diphtheria, pertussis and tetanus toxins was developed and used for assaying 65 sera from healthy children to assess the antitoxin acquisition in relation to the administration of adsorbed diphtheria-purified pertussis-tetanus (DPT) combined vaccine. The antitoxin titers obtained by the PA test and the conventional methods were correlated well; the correlation coefficient of the diphtheria antitoxin titers between the PA test and the cell culture method was 0.908, that of the tetanus antitoxin titers between the PA test and the passive hemagglutination test 0.968, and that of anti-pertussis toxin titers between the PA test and polystyrene-ball ELISA 0.885. The PA test was shown to be useful in both developed and developing countries, since it is simple to perform, sensitive and specific, and the three antitoxins can be titrated by the same procedure.

Conditions for the desorption of immunoglobulins from the surface of blood cells of animals used for the production of antitoxic therapeutic and diagnostic sera have been established. The possibility of obtaining, in principle, additional amounts of immunoglobulins and specific antibodies (in comparison with their initial content in sera) without increasing the number of producer animals has been demonstrated. The possibility of using desorbed immunoglobulins and specific antibodies in the production of therapeutic and diagnostic immunobiological preparations is discussed.

This study investigated whether differences in fecal and serum antitoxin A antibody levels may account for the duration of Clostridium difficile-associated diarrhea (CDAD) and the occurrence of relapses. By an enzyme linked-immunosorbent assay, we tested 40 patients with CDAD including 25 patients without immunodeficiency and 15 patients receiving antineoplastic drugs. Two hundred eighty serum samples and 80 normal stool samples were investigated as controls. In nonimmunocompromised patients, serum immunoglobulin (IgG) and fecal IgA antitoxin A antibody titers were significantly higher in patients who suffered a single episode (n = 21) than in those with relapsing CDAD (n = 4) whose titers were at control levels. Of these 25 patients, eight suffered from diarrhea which lasted for more than 2 weeks. These patients had significantly lower serum- and feces-specific antibody levels than the others who presented symptoms of shorter duration. In cytostatic-treated patients, antitoxin A antibody levels were similar to controls, but relapses occurred in a single case. These data suggest an association between a defective humoral response to toxin A and a more severe form of C. difficile infection. They also indicate that other host-related factors control the severity of CDAD and remain to be elucidated.

In tetanus immunization, the need for a booster vaccination can be easily determined by a serological analysis of tetanus antitoxin. Vaccination without knowledge of the immune status carries a high risk of postvaccinal complications. We have used the Pro. M.D. expert system shell to produce the knowledge base TETANUS, to improve the diagnostic interpretation of tetanus antitoxin findings. This knowledge base is comprehensive, runnable, useful, and modifiable by anyone who works with Pro.M.D. and is able to provide medical knowledge in this field.

CVD 103-HgR is an attenuated, AB+, live, recombinant vaccine strain, developed by deletion of the toxA gen in a virulent Vibrio cholerae 01, Inada classical strain (569B). In phase II studies conducted to date, CVD 103-HgR has been well tolerated and immunogenic in volunteers from both industrialized countries and cholera-endemic areas. In this study of safety, immunogenicity and excretion, 81 Chilean adults were randomly allocated to receive, in a double blind fashion, a single oral dose of 5 x 10(9) FU of CVD 103-HgR or placebo, (5 x 10(9) heat-killed E. Coli K12 organisms), in 100 ml of buffered water. Side effects were assessed by daily visits to the participants. Immunogenicity, (vibriocidal seroconversion), was investigated in blood drawn before and on days 8 and 28 after immunization, while stool cultures to assess excretion of the vaccine strain were performed on specimens obtained on days 1 and 7. None of the participants, (40 vaccinees and 41 placebo recipients), experienced untoward effects during 30 minutes of close surveillance after ingestion of the preparation; upon follow up, neither adverse events were more frequently reported by the vaccinees. 34/40 vaccinees, and 2/41 participants receiving placebo had a significant raise, (> = fourfold), in their vibriocidal titers; (85 vs 2%, p < 0.001). The peak postimmunization geometric mean titer, (222), was ten fold higher than the baseline vibriocidal titer. The vaccine strain was recovered in stool cultures from 8 participants, one of them excreted the strain in both specimens. We conclude that CVD-103-HgR is safe and immunogenic in Chilean adults.

Pasteurella multocida isolates from 271 nasal swabs of pigs were tested in EBL cell culture for toxin production. Mixed bacteria cultures of the same swabs were examined in the P. multocida toxin ELISA K462 (Dakopatts). In the ELISA 114 swabs reacted positive, whereas toxigenic P. multocida were detected by the EBL cell test in 86 swabs. In a neutralization test (SNT) combined with EBL cell culture and with the ELISA 111 sera were examined for P. multocida antitoxin. The toxin had to be more concentrated for the ELISA than for the cell culture; therefore the SNT with EBL cells was more sensitive. Whereas 101 sera had titres of 1:4 or higher in the cell culture, 68 of these sera were positive in the ELISA.

Different immunization schedules for oral B subunit-whole cell (B-WC) cholera vaccine were evaluated in Swedish volunteers to obtain information for recommendations of vaccine use in non-endemic areas. Two peroral doses of B-WC vaccine were as effective as three doses in inducing IgA and IgG antitoxin as well as vibriocidal antibody responses in serum. Administration of two vaccine doses either at 7, 14 or 28-42 day intervals resulted in comparable antitoxin responses in serum, whereas a 3-day immunization interval resulted in significantly lower titre increases. Vibriocidal antibody responses were comparable after the different time intervals tested (3-42 days). The B-WC vaccine can be effectively administered together with a cheap, commercially available sodium bicarbonate powder dissolved in water to protect the vaccine from gastric acid.

Attenuated Vibrio cholerae oral vaccine CVD 103-HgR was well tolerated by 324 Thai soldiers and civilians. Most received a single 5 x 10(8) cfu dose, while 40 each received one or two 5 x 10(9) cfu doses. Vibriocidal antibody (the best correlate of immunity) seroconversion was lower in soldiers than civilians (P less than .001). Increasing the vaccine dose to 5 x 10(9) cfu raised the geometric mean titer (P less than .001). A second 5 x 10(9) cfu dose one week later did not notably increase seroconversions. Likelihood of seroconversion was inversely correlated with baseline vibriocidal titer (P less than .001). CVD 103-HgR caused seroconversion in most subjects with baseline titers less than or equal to 1:40, including 100% of civilians after one 5 x 10(8) cfu dose, 79% of soldiers after one 5 x 10(9) cfu dose, and 45% of soldiers after one 5 x 10(8) cfu dose. In persons with elevated baseline titers, vibriocidal antibody seroconversion is not a sensitive measure of whether vaccine has boosted intestinal immunity; for such subjects, other measurements must be used. Study regimens in endemic areas should use a single 5 x 10(9) cfu dose.

The possibility of raising a humoral immune response able to induce protection from the lethal effects of scorpion toxins was evaluated in the mouse model. A toxic fraction from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes which yielded a conveniently detoxified immunogen. After three injections of this immunogen, all but three of a group of 18 mice developed an IgG response which was shown to be both specific and of good affinity for the toxic antigen. In vitro neutralization assays indicated that pre-incubation of a lethal dose of the toxic fraction with immune sera strongly diminished its toxicity. In vivo protection assays showed that mice with the highest levels of circulating anti-toxin antibodies could resist the challenge by double the normal LD50 of the toxic fraction, which killed all control non-immune mice. The protection was, however, found to be limited both in its duration and its effectiveness against higher amounts of toxin.

The intravenous injection of terrilytin-treated lymphocytes into rats infected with staphylococci enhances the formation of staphylococcal alpha antitoxin in the animals and the development of immune response to T-dependent antigen, such as sheep red blood cells (SRBC), but produces no effect on the development of immune response induced by T-independent antigen (lipopolysaccharide). Terrilytin-treated lymphocytes induce the release of the factor promoting the development of immune response to staphylococcal antigens and SRBC by spleen cells, incapable of adherence to plastic, but have no influence on the development of immune response to lipopolysaccharide in rats infected with staphylococci. At the same time in such rats spleen cells adhering to plastic take part in the transfer of signals from terrilytin-treated lymphocytes to nonadhering spleen cells of recipients.

The reactogenic properties and immunological potency of modified cholera chemical vaccine (choleragen-toxoid + O-antigens Inaba and Ogawa) were tested in 278 volunteers aged 18 years and over in comparison with those of a commercial batch of monovalent cholera vaccine (choleragen-toxoid + O-antigen Inaba). The cholera vaccine, enriched with O-antigen Ogawa, was found to be safe; vaccination with this vaccine was not accompanied by the development of systemic and local reactions whose frequency and intensity met the requirements for the reactogenic properties of commercial cholera vaccine. The immunological potency of the bivalent vaccine with respect to strain Inaba was not inferior to that of the commercial vaccine; at the same time in persons immunized with the new preparation the titers of vibriocidal antibodies to strain of serovar Inaba were five-fold higher. The conclusion on the expediency of using cholera chemical vaccine enriched with O-antigen Ogawa was made.

Specific antibody to Pseudomonas pseudomallei exotoxin was detected in sheep sera exposed to natural infection. An enzyme-linked immunosorbent assay (ELISA) was used. Serum antitoxin was present in 49.3% of sera obtained from a flock of sheep naturally exposed to P. pseudomallei infection. Among these sera, 17.0% gave titers of 10,000. In contrast, serum antitoxin was present in only 6.0% of sera collected from sheep kept on a melioidosis-free farm. The ELISA reactivity of all positive sera could be completely absorbed with purified P. pseudomallei exotoxin. Similarly, preincubation of the exotoxin-coated wells with specific antiserum inhibited the ELISA reactivity of sheep sera. The results indicate that exotoxin is produced in vivo during infection by P. pseudomallei.

The preparation of cholera toxin obtained from Vibrio cholerae strain 1310 has been used for producing solid-phase immunosorbent intended for the enzyme immunoassay (EIA). The use of EIA and the vibriocidal antibody test (VAT) in the serological study of former cholera patients and persons having contacts with them has made it possible to show the excess of the antitoxic activity of sera over their vibriocidal activity in all subjects covered by the dynamic study (from 5-14 days to 8-10 months). EIA and VAT can be used as auxiliary methods in epidemiological survey and analysis.

Preparation of Clostridium difficile antibacterial and antitoxic sera is presented. Fifty one strains (72%) were typeable within Delmee scheme. Twenty strains (28%) belonged to new Polish serogroups designated 18, 27, 70, 71, 72, 88, 89 and NICH. Supernatants of all toxigenic Clostridium difficile strains were neutralized by gamma-globulin fraction of goat Clostridium difficile antitoxin in neutralization assay when it was performed on McCoy cell line. Only 8 toxigenic strains (21%) were positive in counterimmunoelectrophoresis.

Quantitative determinations were made of the capacity of isogenic bone marrow, spleen, and thymic cells from primed and/or nonprimed mice to repopulate the hemopoietic tissues and to mount an inflammatory and antibody response to specific antigen (tetanus toxoid) in heavily irradiated and reconstituted recipients. Spleen cells from primed mice but not from normal mice had the capacity to adoptively transfer an anamnestic antitoxin titer in irradiated animals in the absence of transplanted bone marrow cells, and during retarded myeloid regeneration. Spleen cells alone or bone marrow cells alone produced an insignificant and a moderate peritoneal eosinophil response, respectively, to antigen. In the presence of bone marrow cells, normal spleen cells augment the capacity of recipient animals to mount an eosinophil response to antigen. A much greater augmentation occurs in animals reconstituted with splenic or thymic cells from primed animals. The increase in antitoxin titers appears to be independent of the response of eosinophils since: (a) marked accumulation of eosinophils can occur in animals with no measurable humoral antitoxin, and (b) high antitoxin titers can occur in animals which do not have marked eosinophil responses. It is suggested that a thymic-derived or thymic-dependent mononuclear cell population is necessary for optimal eosinophil response to antigen. The neutrophil and mononuclear cell responses to antigen are determined by different mechanisms from those which determine the eosinophil response. These studies together with earlier findings strongly indicate that the eosinophil granulocytes play a role in the immune response to antigen.