Role of <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> heat-stable enterotoxin (Y-STa) on differential regulation of nuclear and cytosolic calcium signaling in rat intestinal epithelial cells

The heat-stable enterotoxin (Y-STa) produced by the pathogenic strains of Yersinia enterocolitica is a causative agent of secretory diarrhea. We have reported earlier that Y-STa-induced inositol trisphosphate-mediated cytosolic calcium rise occurs in rat intestinal epithelial cells. In the present communication, the involvement of a nuclear calcium store in the action mechanism of Y-STa in rat intestinal epithelial cells has been shown. Calcium imaging with time series confocal microscopy shows that Y-STa stimulates both the nuclear and cytosolic calcium levels in rat intestinal epithelial cells where a rise in nuclear calcium precedes the cytosolic events. Moreover, Y-STa stimulates both cytosolic and nuclear inositol trisphosphate (IP₃) levels in a time-dependent manner. Western blot and immunocytochemical analysis reveal a higher density of IP₃ receptor type II in the nuclear membrane compared to the cytosol, which may be the cause of an early rise of the nuclear calcium level. Therefore, it is suggested that Y-STa regulates the nuclear and cytosolic calcium signals in a distinct temporal manner in rat intestinal epithelial cells.     

Loss of tight junction barrier function and its role in cancer metastasis

As the most apical structure between epithelial and endothelial cells, tight junctions (TJ) are well known as functioning as a control for the paracellular diffusion of ions and certain molecules. It has however, become increasingly apparent that the TJ has a vital role in maintaining cell to cell integrity and that the loss of cohesion of the structure can lead to invasion and thus metastasis of cancer cells. This article will present data showing how modulation of expression of TJ molecules results in key changes in TJ barrier function leading to the successful metastasis of a number of different cancer types.     

Staphylococcal enterotoxin A: a candidate for the amplification of physiological immunoregulatory responses in the gut

Staphylococcal enterotoxin A (SEA) is one of the bacterial products tested for modulation of unwanted immune responses. Of all the staphylococcal enterotoxins, SEA is the most potent stimulator of T cells. When administered orally, SEA acts as a superantigen (SA), producing unspecific stimulation of intra-epithelial lymphocytes (IELs) in the intestinal mucosa. This stimulation results in amplification of the normal local immunologic responses, which are mainly regulatory. This amplification is based on increased local production of IFN-γ by IELs, which acts on the nearby enterocytes. As a result, the enterocytes produce large amounts of tolerosomes, cellular corpuscles which detach themselves from the basal poles of the enterocytes and contain antigenic peptides that are conditioned to be interpreted as tolerogenic by the gut immune system. Tolerosomes are physiologically produced as a response to dietary peptides; it is already known that enterocytes posses the molecular mechanisms for processing peptides in a similar manner to lymphocytes. The fate of tolerosomes is not precisely known, but it seems that they merge with intestinal dendritic cells, conveying to them the information that orally administered peptides must be interpreted as tolerogens. SEA can stimulate this mechanism, thus favoring the development of tolerance to peptides/proteins administered subsequently via the oral route. This characteristic of SEA might be useful in therapy for regulating immune responses. The present paper reviews the current status of research regarding the impact of SEA on the enteric immune system and its potential use in the treatment of allergic and autoimmune diseases.     

Wet‐milling transgenic maize seed for fraction enrichment of recombinant subunit vaccine

The production of recombinant proteins in plants continues to be of great interest for prospective large‐scale manufacturing of industrial enzymes, nutrition products, and vaccines. This work describes fractionation by wet‐milling of transgenic maize expressing the B subunit of the heat‐labile enterotoxin of Escherichia coli (LT‐B), a potent immunogen and candidate for oral vaccine and vaccine components. The LT‐B gene was directed to express in seed by an endosperm specific promoter. Two steeping treatments, traditional steeping (TS, 0.2% SO₂ + 0.5% lactic acid) and water steeping (WS, water only), were evaluated to determine effects on recovery of functional LT‐B in wet‐milled fractions. The overall recovery of the LT‐B protein from WS treatment was 1.5‐fold greater than that from TS treatment. In both steeping types, LT‐B was distributed similarly among the fractions, resulting in enrichment of functional LT‐B in fine fiber, coarse fiber and pericarp fractions by concentration factors of 1.5 to 8 relative to the whole kernels on a per‐mass basis. Combined with endosperm‐specific expression and secretory pathway targeting, wet‐milling enables enrichment of high‐value recombinant proteins in low‐value fractions, such as the fine fiber, and co‐utilization of remaining fractions in alternative industrial applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The safety of Bacillus thuringiensis to mammals investigated by oral and subcutaneous dosage

Six strains of Bacillus thuringiensis were tested with two commercially available kits for their ability to produce Bacillus cereus-type enterotoxin and by dipteran bioassay for the production of -exotoxin. All of the strains were positive for enterotoxin production including three which have been used world-wide for many years to control pest insects. Rats given oral doses totalling 1 × 10¹² spores ( +crystals), over three weeks, or a single subcutaneous dose of 1 × 10⁶ spores ( +crystals) showed no ill-effects in terms of their condition or in the pathology of their internal organs: this was in spite of the strain of B. thuringiensis used (13B) being an active producer of both -exotoxin and enterotoxin. A commercial insecticide containing B. thuringiensis was sprayed onto spinach leaves. After normal food preparation regimes some leaves retained residual spore loads sufficient for a strongly enterotoxic strain to cause food poisoning in humans. These findings suggest that the agricultural use of some, previously unvalidated, strains of B. thuringiensis could give rise to cases of food poisoning and that rodents are unsuitable for testing the safety to humans of oral exposure to this organism.     

Molecular characterization and distribution of virulence-associated genes amongst Aeromonas isolates from Libya

AIMS: The aim of the study was to type 52 Aeromonas spp. isolates from chicken carcasses, children with diarrhoea and a hospital environment in Libya, and to determine the distribution of putative virulence genes amongst them. METHODS AND RESULTS: Macrorestriction analysis using pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S rRNA and aroA genes were used to type the isolates. Whereas 30 of 32 chicken isolates were identified as Aeromonas veronii, eight of 12 environmental isolates were Aer. caviae. Three species were identified amongst the eight isolates from children. Aeromonas veronii and Aer. caviae isolates could be divided into eight and five PFGE types, respectively. All species could be further subtyped into one of 21 aroA PCR-RFLP groups. Aerolysin-like haemolysin or enterotoxin gene sequences were detected in all the isolates. Overall carriage rates for hlyA and alt were 77 and 75%, respectively. CONCLUSIONS: Seven of eight isolates from children were of different subtypes, indicating a lack of any common source of acquisition. Isolates of common molecular type did not share identical distributions of putative virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the effectiveness of using molecular typing to identify and study genetic variation amongst Aeromonas isolates.     

Superantigen-induced apoptotic death of tumor cells is mediated by cytotoxic lymphocytes, cytokines, and nitric oxide.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of CTL activity and cytokine production in vivo. Protein A (PA) of Staphylococcal aureus has been found to have diverse biological response modifying properties and to possess antitumor, antitoxic and antiparasitic effects. In this study we examined the anti-tumor effect of these two superantigens used separately as well as in combination in mice carrying the Ehrlich ascites tumor. With combined treatment, DNA cell cycle analysis of tumor cells showed a significant (P < 0.05) percentage of tumor cell death. Levels of the soluble mediators TNF-alpha, IFN-gamma and IL-1 as well as NO were elevated. Additionally, CD4(+) and CD8(+) specific T cells in spleen, thymus and PBMC in tumor carrying mice were increased (P < 0.01). Our data altogether suggests that enhanced tumor cell death is caused by the increased CTL activity, cytokine and nitric oxide levels, in response to the combined effect of SEA + PA.     

Pig Intestinal Membrane-Bound Receptor (Guanylyl Cyclase) for Heat-Stable Enterotoxin: cDNA Cloning,Functional Expression,and Characterization

A cDNA encoding the receptor protein for a heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli was cloned from intestinal epithelial cells of a 10-week-old pig. The cDNA had an open reading frame of 3,219 base pairs and coded for a protein with 1,073 amino acid residues. The mature protein consisted of 1,050 amino acid residues with a molecular mass of ca. 121 kDa and was 87% and 82% identical with the human and rat protein, respectively. The CHO cell line overexpressing the pig recombinant STa receptor specifically bound to a photoaffinity-labeled analog of STa and showed marked elevation of the cellular content of cGMP in response to STa.     

Identification and Characterization of Heat-Stable Enterotoxin II-Producing Escherichia coli from Patients with Diarrhea

Stock strains of Eschericia coli isolated from patients with traveller's diarrhea were examined for production of heat-stable enterotoxin II (STII). Of 400 strains examined, 3 were found to produce STII. The nucleotide sequence of the STII gene of these human strains was shown to be identical to that of porcine strains. Cultured cells of these strains induced fluid accumulation in ligated mouse intestinal loops and the activity was neutralized by anti-STII antiserum. These results suggest that STII-produciing enterotoxigenic E. coli can cause human diarrhea.