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PubMed1991

Determination of K88 antigens and enterotoxins of Escherichia coli strains isolated from Polish piglets with diarrhea by the use of enzyme-linked immunosorbent assays.

We studied 103 Escherichia coli strains isolated from suckling and weaned piglets with diarrhea using different ELISA tests. K88 fimbrial antigen was determined by the slide agglutination test and the ELISA inhibition method. LT and STa enterotoxins were tested directly in the microtiter plates using monoclonal antibodies. It was found that 56.3% strains possessed K88 antigen, all of which were of the K88ac type. There was 100% correlation between the slide agglutination and ELISA tests. Of the 103 strains tested 68.9% produced LT or STa or both toxins. LT-positive strains were the most common ones in both groups of piglets. All K88-positive strains were enterotoxigenic and elaborated LT (56 strains) or LT and STa (2 strains); STb production was not determined in this study. Our ELISA tests were easy to perform, specific and can be used for determination of K88 and enterotoxins in E. coli strains isolated from piglets.

Agglutination Tests↗

PubMedDate not supplied

Evaluation of the Biken test to detect heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains.

Fifty-seven strains of enterotoxigenic Escherichia coli isolated from humans and pigs and producing thermolabile (LT) enterotoxin were used to ascertain the efficiency of the Biken test compared to the passive immune haemolysis test (PIH), considered as very sensitive for detecting that enterotoxin. The two assays were carried out using anti-porcine (anti-LTp), anti-human (anti-LTh), anti-cholera toxin (anti-CT) and anti-choleragenoid (anti-Cg) antisera. Our results showed that the Biken test was very irregular, with many false-negative results. Positive results (ranging from 78.9 to 22.8) were dependent upon the antiserum used. Conversely, the PIH test was much more efficient in the detection of LT, since 100% of the LT+ strains were positive in this test whatever the antiserum used.

Animals↗

PubMed1988

Hyperproduction of heat-stable enterotoxin (STA4) of Escherichia coli and analysis of the unusual electrophoretic behavior of reduced and alkylated forms of STAs.

The methanol-soluble heat-stable enterotoxin gene (estA4) of Escherichia coli (STA4) yielded 128-fold more toxin when expressed by a T7 RNA polymerase driven system than when driven by its own promoter. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vivo [35S]cysteine radiolabeled products of the cloned gene revealed an apparent molecular mass larger than that expected for a 19 amino acid polypeptide (mol. wt. 2049). Purified [125I]radiolabeled enterotoxin, STA1 (mol. wt. 1979) showed an Mr of 3800 when reduced, 2000 when reduced and carboxylated, and 14,500 when reduced and carboxyamidated. Similar changes after carboxyamidation were obtained with two different chemically synthesized STAs. These unusual electrophoretic mobilities were shown to be common to all STAs studied. Alkylation of the reduced STA species occurred only at the six cysteine residues of the toxin. Upon gel filtration the native, reduced, and reduced and alkylated forms of STAs eluted from the column in close agreement to the molecular weight expected from the known amino acid composition of the peptides.

Alkylation↗

PubMed1990

Biological and immunological characterization of a cloned cholera toxin-like enterotoxin from Salmonella typhimurium.

A chromosomal DNA fragment, encoding an enterotoxin gene of Salmonella typhimurium Q1, was cloned into bacteriophage EMBL3 and plasmid vector pBR322. The recombinant clones lambda B8 and pC1 were identified using a synthetic oligonucleotide probe made to the B subunit region of the cholera toxin gene (ctx). Cell lysates of Escherichia coli VCS257 [lambda B8] induced fluid secretion in rabbit intestinal loops, while lysates of E. coli DH5 alpha [pC1] failed to elicit an enterotoxic response in this model. Both lysates and partially purified preparations elongated Chinese hamster ovary (CHO) cells, elevated cellular cAMP and PGE2, and bound to ganglioside GM1. The biological activity associated with the cloned enterotoxin was neutralized by monospecific antiserum to cholera toxin (CT). Immunoblots of pC1 and lambda B8 lysates probed with anti-CT, exhibited a 30 kDa protein similar to that of pJM17, which carried the ctx gene. Under non-dissociating conditions, anti-CT immunoblots of the same lysates revealed two proteins, one corresponding in size to the holotoxin and the other to CT-A. When analysed by DNA-directed protein synthesis in vitro, both pC1 and lambda B8 DNA expressed two unique proteins (30 and 11 kDa) similar to that of pJM17.

Animals↗

PubMed1996

Suppressive effect of staphylococcal enterotoxin B on murine contact hypersensitivity.

Superantigens (SAg) possess the capacity to interact with particular V beta regions of T cell receptor (TCR) and major histocompatibility complex (MHC) class II molecules, and activate a large number of T cells and accessory cells. staphylococcal enterotoxins (SEs) are recently well known as SAg and anticipate to modulate immunological reactions. In this study, we investigated the effect of staphylococcal enterotoxin B (SEB) on contact hypersensitivity reaction (CHR) to 2,4-dinitrofluorobenzene (DNFB) in BALB/c mice. SEB-injection inhibited the induction of sensitization on CHR. Suppressor cells were not found in the spleen or lymph node cells from mice treated with SEB. Normal spleen cells cultured with SEB showed significant proliferation and tumor necrosis factor-alpha (TNF-alpha) production. CHR was suppressed by intravenous injection of the culture supernatant. In addition, anti-TNF-alpha antibodies inhibited the suppressive effect induced by the supernatant. These findings indicate that TNF-alpha produced by SEB-responding cells inhibits the induction of sensitization on CHR. Therefore, SAg may play important roles in the modulation of immune system through the stimulation of TNF-alpha production.

Animals↗

PubMed1996

Phenotypic and genotypic (pulsed-field gel electrophoresis) characteristics of enterotoxin-A-producing Staphylococcus aureus strains.

The phenotypic (antibiotype, serotype, phagetype) and genotypic (SmaI restriction patterns using pulsed-field gel electrophoresis) characters of 162 Staphylococcus aureus epidemiologically unrelated strains were studied. Eighty-two of the isolates produced enterotoxin-A (SEA+), while 80 produced none (SEA-). None of the phenotypic characters observed were characteristic of SEA+ strains. On the other hand, the electrophoretic profiles revealed a non-random distribution of the SEA+ strains (p < 0.01 in groups PI and PIII, and p < 0.03 in group PII). It can therefore reasonably be assumed that the enterotoxin-A-producing strains did not constitute a single clone, but rather, seemed to belong to strains derived from at least three clones with distinct genetic organization.

DNA, Bacterial↗

PubMed1991

Studies on the mechanism of Salmonella typhimurium enterotoxin-induced diarrhoea.

The unidirectional fluxes of Na+ and Cl- were studied in Salmonella typhimurium enterotoxin-treated rats. There was net secretion of Na+ and Cl- in toxin-treated animals, while in control animals there was net absorption of these ions. In the presence of the Ca(2+)-ionophore, there was net secretion of Na+ and Cl- in the control group, while the ionophore enhanced the secretion of these ions in experimental animals. The calcium channel blocker, verapamil, decreased the secretion induced by salmonella toxin, but could not reverse the secretion to absorption. There was no difference in the net absorption of Ca2+ in both the control and experimental animals. There was a significant increase in the intracellular free calcium concentrations in enterocytes isolated from toxin-treated rat intestines as compared to that in enterocytes isolated from control animals. In the presence of PMA (phorbol-12-myristated-13-acetate) there was net secretion of Na+ and Cl- in the control group, while in the experimental group there was no change in the fluxes of these ions. The selective, potent inhibitor of protein kinase C, H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine) reversed the secretion of Na+ and Cl- in the toxin-treated group to absorption. The addition of indomethacin also inhibited the secretion induced by salmonella toxin, but failed to reverse it to absorption. However, the addition both H-7 and indomethacin to the experimental group had a partial additive effect. These studies demonstrate that the Salmonella enterotoxin-mediated fluid secretion involves protein kinase C and the arachidonic acid metabolites and perhaps does not involve the extracellular calcium pools.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2005

The role of central corticotropin-releasing hormone in the anorexic and endocrine effects of the bacterial T cell superantigen, Staphylococcal enterotoxin A.

Bacterial superantigens, such as the staphylococcal enterotoxins, exert a strong capacity for in vivo stimulation of T cell proliferation and cytokine production. Previously, staphylococcal enterotoxin A (SEA) was shown to induce an anorexic effect under novel contextual conditions of testing, and produced an increase in plasma ACTH and corticosterone levels in C57BL/6J mice. In the present study, the role of corticotropin releasing hormone (CRH) in promoting these effects of SEA was addressed via intracerebroventricular (icv) administration of alpha-helical CRH(9-41) ((alpha)hCRH), a non-selective CRH receptor antagonist, and astressin-2B, a selective CRH receptor 2 antagonist. The efficacy of (alpha)hCRH and astressin-2B in blocking anorexic responses to CRH and urocortin under the current conditions of testing was first confirmed. Subsequently, it was found that (alpha)hCRH (20 microg icv), but not astressin-2B (10 and 25 microg icv), significantly attenuated the anorexia induced by SEA. This suggested that central CRH is involved in mediating the anorexia induced by SEA, but potentially through CRH receptor 1. Additional results revealed that plasma ACTH stimulation in response to SEA was not significantly attenuated by either antagonist administered icv. However, the plasma corticosterone elevation showed a modest, but significant, attenuation in SEA challenged mice given (alpha)hCRH. These data suggest a possible influence of central CRH on adrenocorticoid activity subsequent to SEA challenge. More importantly, it appears that central activation of CRH receptors is a consequence of SEA challenge, and this likely contributes to its anorexic effects.

Adrenocorticotropic Hormone↗

PubMed2004

A staphylococcal enterotoxin B magnetoelastic immunosensor.

A magnetoelastic immunosensor for detection of staphylococcal enterotoxin B (SEB) is described. The magnetoelastic sensor is a newly developed mass/elasticity-based transducer of high sensitivity having a material cost of approximately $0.001/sensor. Affinity-purified rabbit anti-SEB antibody was covalently immobilized on magnetoelastic sensors, of dimensions 6 mm x 2 mm x 28 microm. The affinity reaction of biotin-avidin and biocatalytic precipitation are used to amplify antigen-antibody binding events on the sensor surface. Horseradish peroxidase (HRP) and alkaline phosphatase were examined as the labeled enzymes to induce biocatalytic precipitation. The alkaline phosphatase substrate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) produces a dimer, which binds tightly to the sensor surface, inducing a change in sensor resonance frequency. The biosensor demonstrates a linear shift in resonance frequency with staphylococcal enterotoxin B concentration between 0.5 and 5 ng/ml, with a detection limit of 0.5 ng/ml.

Alkaline Phosphatase↗

PubMed2005

Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichia coli enterotoxin STa across the colon of the gerbil (Gerbillu cheesmani) in different dietary states.

The effects of mucosally added Escherichia coli heat stable enterotoxin (STa, 30 ng mL(-1)) on the basal short-circuit current (Isc in microA cm(-2)) across stripped and unstripped sheets of proximal, mid and distal colon were investigated. Samples were taken from fed, starved (4 days, water ad lib) and undernourished (50% of control food intake for 21 days) gerbils (Gerbillus cheesmani). The effect of neurotoxin tetrodotoxin (TTX 10 microM) and the effect of replacing chloride by gluconate or the effect of removing bicarbonate from bathing buffer on the maximum increase in Isc induced by E. coli heat stable enterotoxin STa were also investigated. The results showed that there is a segmental difference both in the basal and STa-induced Isc. Also, STa is more effective in the proximal than distal colon in the three feeding conditions. Undernourishment raised the STa-induced Isc in the three regions of the colon. In fed and starved gerbils part of STa-induced Isc in the proximal colon was chloride-dependent, while the other was bicarbonate-dependent; in the mid colon, the STa-induced Isc was bicarbonate-dependent only. In the three regions of the colon taken from undernourished gerbil, the STa-induced Isc was both chloride-and bicarbonate-dependent. The increase in STa-induced Isc as a results of undernourishment in proximal and mid colon was chloride-dependent, while in the distal colon, it was both chloride- and bicarbonate-dependent.

Animals↗

PubMed2006

Staphylococcal superantigens of the enterotoxin gene cluster (egc) for treatment of stage IIIb non-small cell lung cancer with pleural effusion.

There has been renewed interest in the superantigens as antitumor agents with the discovery of a group of bacterial superantigens known as the enterotoxin gene cluster (egc staphylococcal enterotoxins [SEs]). This article discusses the mechanisms by which egc SEs induce tumor killing and pleurodesis. The application of SE homolog and nucleic acid compositions as vaccines and for treatment of established tumors is reviewed. Finally, the use of native SEs ex vivo-intratumorally and intravesicularly administered superantigens against established tumors-is described and the interrelation between superantigen therapy and chemoradiotherapy.

Animals↗

PubMed2004

Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A.

This study identified a functional spo0A ORF in enterotoxigenic Clostridium perfringens type A. To evaluate the function of spo0A, an isogenic spo0A knock-out mutant was constructed. The spo0A mutant was unable to form endospores and produce enterotoxin, however, these defects could be restored by complementing the mutant with a recombinant plasmid carrying the wild-type spo0A gene. These results provide evidence that spo0A expression is essential for sporulation and enterotoxin production in C. perfringens.

Amino Acid Sequence↗

PubMed2004

Use of novel PCR primers specific to the genes of staphylococcal enterotoxin G, H, I for the survey of Staphylococcus aureus strains isolated from food-poisoning cases and food samples in Taiwan.

Data regarding the incidence of the newly found enterotoxigenic Staphylococcus aureus strains in food poisoning cases and in food samples were to date not available in Taiwan. In this study, PCR primers specific for the detection of SEG, H and I genes, i.e., seg, seh and sei, were used for the assay of 55 human isolates of S. aureus negative to the classical enterotoxins (SEA-->SEE) detection. These isolates were from the fecal specimens of the patients suffering from food poisoning outbreaks. Only eight strains were found to have the seg, seh and sei. The presence of other bacterial pathogens, such as Vibrio parahaemolyticus, Bacillus cereus and Salmonella spp. and perhaps, strains producing other new staphylococcal enterotoxins, in the fecal specimens of these patients, may account for these food poisoning cases. For 139 strains from food samples, such as frozen Chinese foods, Chinese sausages and lunch meals, sea strains accounted for the major portion and it seemed to be the most common SE type to coexist with seg, seh and sei. Only two strains had sec and none of them had seg, seh or sei. For strains without the classical SE genes, only 13 strains had seg, seh and/or sei. The above results imply that seg, seh and sei S. aureus strains play only a minor role in food-borne outbreaks in Taiwan.

DNA Primers↗

PubMed2006

Staphylococcal enterotoxin B inhibits regulatory T cells by inducing glucocorticoid-induced TNF receptor-related protein ligand on monocytes.

BACKGROUND: Superantigens inhibit naturally occurring CD4+CD25+ regulatory T-cell (nTreg) activity, yet the mechanism for this is unknown. OBJECTIVE: We tested the hypothesis that staphylococcal enterotoxin B (SEB), a prototypic superantigen, inhibits the ability of nTregs to suppress T-effector cell (Teff) proliferation by an antigen-presenting cell-dependent cell contact mechanism and the induction of glucocorticoid-induced TNF receptor-related protein ligand (GITR-L) on monocytes. METHODS: Cell proliferation assays were performed on immunomagnetic-bead separated CD4+CD25+, CD4+CD25- T cells and CD14+ monocytes using anti-CD3, SEB, or a neutralizing anti-GITR-L antibody for stimulation. A cytokine permeable membrane insert separating cells in culture was used to evaluate the role of cell contact. Proliferation was measured by [3H]-thymidine incorporation or a carboxyfluorescein diacetate succinimidyl ester assay. GITR-L expression was determined by RT-PCR and immunostaining. RESULTS: Antigen-presenting cell contact was required for proliferation of nTregs and loss of their ability to suppress Teff proliferation as confirmed by carboxyfluorescein diacetate succinimidyl ester. At baseline, monocytes expressed no GITR-L, but after SEB stimulation, GITR-L gene and protein expression increased significantly over a period of 24 hours (P<.05). When GITR-L activity was neutralized with blocking antibody, as opposed to isotype control, nTregs suppressed Teff proliferation despite the presence of SEB (P<.05). Likewise, loss of GITR-L at 48 hours was associated with restoration of nTreg activity (P<or=.05). CONCLUSION: Staphylococcal enterotoxin B upregulates GITR-L on monocytes and inhibits nTreg ability to suppress Teff proliferation via a cell contact interaction. CLINICAL IMPLICATIONS: Prevention of GITR-L induction on monocytes by SEB may be a novel target for control of inflammation in superantigen-driven diseases such as atopic dermatitis.

Adult↗

PubMed2006

Human dendritic cells conditioned with Staphylococcus aureus enterotoxin B promote TH2 cell polarization.

BACKGROUND: Immune surveillance against microbes at sites of interface with environment involves immediate recognition of pathogen-associated molecular patterns by dendritic cells (DCs). According to their first-line position, DCs are key parameters for the establishment of an appropriate innate and adaptive response against pathogens to avoid disease development. Even though their role in pathogenesis is well known, bacterial toxins have been less examined for their ability to drive DC activation and T-cell polarization. OBJECTIVE: We made the assumption that early conditioning of DCs with Staphylococcus aureus enterotoxins could take part in T-cell polarization. METHODS: Human monocyte-derived DCs were stimulated with S aureus enterotoxin B (SEB) and characterized with respect to secretion of inflammatory cytokines and their ability to drive polarization of naive allogenic T cells. RESULTS: We demonstrated that SEB induced maturation of DCs and that SEB-activated DCs secreted high levels of IL-2 but no IL-12p70, contrary to LPS-activated ones. Accordingly, we further showed that SEB-activated DCs were able to drive polarization of naive T cells into the T(H)2 subset. By using highly purified SEB and Toll-like receptor (TLR)-2 stably transfected Human Embryonic Kidney (HEK) 293 cells, we demonstrated for the first time the ability of SEB to induce TLR2 signaling. Furthermore, the involvement of SEB-TLR2 interaction in activation of dendritic cells was supported by neutralizing activity of anti-TLR2 antibodies. CONCLUSION: Altogether, our findings reinforce the notion that bacterial toxins may appear as new pathogen-associated molecular patterns, which could play a major role in inflammation and bacterial pathologies.

Antigens, Bacterial↗

PubMedDate not supplied

Evaluation of staphylococcal enterotoxin-specific IgE antibody in tears in allergic keratoconjunctival disorders.

PURPOSE: To investigate the presence of staphylococcal enterotoxin A (SEA) and B (SEB)-specific IgE antibodies in tears from patients with allergic conjunctival disorders. METHODS: The study included 8 eyes of 4 patients with perennial allergic conjunctivitis (PAC), 14 eyes of 7 patients with vernal keratoconjunctivitis (VKC), 12 eyes of 6 patients with atopic keratoconjunctivitis (AKC), and 10 eyes of 10 healthy volunteers as controls. Tears were sampled by the method of the Schirmer test I. Sampled tears were eluted and SEA- and SEB-specific IgE antibodies were analyzed by the AlaSTAT-IMMULIZE method. RESULTS: SEA-specific IgE antibodies in tears were positive in 9 of 14 eyes in VKC patients and in 1 of 12 eyes in AKC patients. SEB-specific IgE antibodies in tears were positive in 7 of 14 eyes in VKC patients and in 2 of 12 eyes in AKC patients. Values for antibodies were higher in patients with severe clinical findings. However, all the cases in the normal control and the PAC groups were negative for both antibodies. CONCLUSION: Our data strongly suggested that staphylococcal enterotoxin may cause type I allergy, and may be an exacerbating factor for vernal keratoconjunctivitis and atopic keratoconjunctivitis.

Adult↗

PubMed2006

Acute systemic immune activation following vaginal exposure to staphylococcal enterotoxin B--implications for menstrual shock.

Menstrual toxic shock syndrome (mTSS) is an acute systemic inflammatory disease associated with the superantigenic exotoxin, toxic shock syndrome toxin (TSST)-1, produced by Staphylococcus aureus and the use of high absorbency tampons. Even though S. aureus is capable of elaborating several other superantigenic exotoxins, only TSST-1 has been implicated in the pathogenesis of mTSS possibly because most other superantigenic exotoxins are known enterotoxins. Nonetheless, we have shown recently that one of the enterotoxigenic staphylococcal superantigens, staphylococcal enterotoxin B (SEB), can cause robust systemic immune activation following exposure through non-enteric mucosa, including nasal or conjunctival routes. In a similar manner, we show here that vaginal administration of SEB in HLA class II transgenic mice can cause robust systemic immune activation characterized by profound elevation of proinflammatory cytokines in the serum, activation and expansion of SEB-reactive CD4(+) and CD8(+) T cells in peripheral lymphoid organs and SEB-induced deletion of immature thymocytes. Vaginal administration of SEB also caused leukocytic infiltration in major organs, such as liver and lung, reminiscent of human toxic shock syndrome. Systemic immune activation following vaginal superantigen delivery was independent of the stage of the estrus cycle in the mouse. Using HLA class II transgenic mice, we have shown that exposure to SEB through the vaginal canal can cause robust systemic immune activation. SEB could thus play a role in the pathogenesis of mTSS.

Animals↗

PubMed2004

The NheA component of the non-hemolytic enterotoxin of Bacillus cereus is produced by Bacillus anthracis but is not required for virulence.

A non-hemolytic enterotoxin (NHE) is one of the two enterotoxins thought to cause diarrhea produced by Bacillus cereus. We identified genes in Bacillus anthracis homologous to the B. cereus nheAB genes encoding proteins of the NHE complex. The NheA component was detected immunologically in culture supernatants from B. anthracis but not from a NheA(-) mutant, suggesting that B. anthracis produces and secretes the NheA subunit of NHE. A NheA deletion mutant was not attenuated in the guinea pig suggesting that NheA is not absolutely required for virulence.

Amino Acid Sequence↗