Enterotoxic Effects Research Articles

Inulin alleviates GenX-induced intestinal injury in mice by modulating the MAPK pathway, cell cycle, and cell adhesion proteins

GenX, a substitute for perfluorooctanoic acid, has demonstrated potential enterotoxicity. The enterotoxic effects of GenX and effective interventions need further investigation. In the present study, the mice were administered GenX (2 mg/kg/day) with or without inulin supplementation (5 g/kg/day) for 12 weeks. Histopathological assessments revealed that GenX induced colonic gland atrophy, inflammatory cell infiltration, a reduction in goblet cell numbers, and decreased mucus secretion. Furthermore, a significant decrease in the protein levels of ZO-1, occludin, and claudin-5 indicated compromised barrier integrity. Transcriptomic analysis identified 2645 DEGs, which were mapped to 39 significant pathways. The TGF-β, BMP6, and β-catenin proteins were upregulated in the intestinal mucosa following GenX exposure, indicating activation of the TGF-β pathway. Conversely, the protein expression of PAK3, CyclinD2, contactin1, and Jam2 decreased, indicating disruptions in cell cycle progression and cell adhesion. Inulin cotreatment ameliorated these GenX-induced alterations, partially through modulating the MAPK pathway, as evidenced by the upregulation of the cell cycle and cell adhesion proteins. Collectively, these findings suggested that GenX exposure triggered intestinal injury in mice by activating the TGF-β pathway and disrupting proteins crucial for the cell cycle and cell adhesion, whereas inulin supplementation mitigated this injury by modulating the MAPK pathway.

Possible interactions between selected food processing and medications.

The impact of food processing on drug absorption, metabolism, and subsequent pharmacological activity is a pressing yet insufficiently explored area of research. Overlooking food-processing-drug interactions can significantly disrupt optimal clinical patient management. The challenges extend beyond merely considering the type and timing of food ingestion as to drug uptake; the specific food processing methods applied play a pivotal role. This study delves into both selected thermal and non-thermal food processing techniques, investigating their potential interference with the established pharmacokinetics of medications. Within the realm of thermal processing, conventional methods like deep fat frying, grilling, or barbecuing not only reduce the enteric absorption of drugs but also may give rise to side-products such as acrylamide, aldehydes, oxysterols, and oxyphytosterols. When produced in elevated quantities, these compounds exhibit enterotoxic and pro-inflammatory effects, potentially impacting the metabolism of various medications. Of note, a variety of thermal processing is frequently adopted during the preparation of diverse traditional herbal medicines. Conversely, circumventing high heat through innovative approaches (e.g., high-pressure processing, pulsed electric fields, plasma technology), opens new avenues to improve food quality, efficiency, bioavailability, and sustainability. However, it is crucial to exercise caution to prevent the excessive uptake of active compounds in specific patient categories. The potential interactions between food processing methods and their consequences, whether beneficial or adverse, on drug interactions can pose health hazards in certain cases. Recognizing this knowledge gap underscores the urgency for intensified and targeted scientific inquiry into the multitude of conceivable interactions among food composition, processing methods, and pharmaceutical agents. A thorough investigation into the underlying mechanisms is imperative. The complexity of this field requires substantial scrutiny and collaborative efforts across diverse domains, including medicine, pharmacology, nutrition, food science, food technology, and food engineering.

Inulin alleviates perfluorooctanoic acid-induced intestinal injury in mice by modulating the PI3K/AKT/mTOR signaling pathway

Perfluorooctanoic acid (PFOA) is a widely used industrial compound that has been found to induce intestinal toxicity. However, the underlying mechanisms have not been fully clarified and effective interventions are rarely developed. Inulin, a prebiotic, has been used as a supplement in human daily life as well as in gastrointestinal diseases and metabolic disorders. In this study, male mice were exposed to PFOA with or without inulin supplementation to investigate the enterotoxicity and potential intervention effects of inulin. Mice were administered PFOA at 1 mg/kg/day, PFOA with inulin at 5 g/kg/day, or Milli-Q water for 12 weeks. Histopathological analysis showed that PFOA caused colon shortening, goblet cell reduction, and inflammatory cell infiltration. The expression of the tight junction proteins ZO-1, occludin and claudin5 significantly decreased, indicating impaired barrier function. According to the RNA-sequencing analysis, PFOA exposure resulted in 917 differentially expressed genes, involving 39 significant pathways, such as TNF signaling and cell cycle pathways. In addition, the protein expression of TNF-α, IRG-47, cyclinB1, and cyclinB2 increased, while Gadd45γ, Lzip, and Jam2 decreased, suggesting the involvement of the TNF signaling pathway, cell cycle, and cell adhesion molecules in PFOA-associated intestinal injury. Inulin intervention alleviated PFOA-induced enterotoxicity by activating the PI3K/AKT/mTOR signaling pathway and increasing the protein expression of Wnt1, β-catenin, PI3K, Akt3, and p62, while suppressing MAP LC3β, TNF-α, and CyclinE expression. These findings suggested that PFOA-induced intestinal injury, including inflammation and tight junction disruption, was mitigated by inulin through modifying the PI3K/AKT/mTOR signaling pathways. Our study provides valuable insights into the enterotoxic effects of PFOA and highlights the potential therapeutic role of inulin.

Enterotoxic potential of dietary cholesterol autoxidation products

Background In both Western and Westernized diets, together with a relatively high amount of cholesterol, variable amountsof its oxidized metabolites, oxysterols, are consistently consumed. These oxysterols, mostly of non-enzymatic origin, are produced during sterol autoxidation in foodstuff manufacturing and storage. Objective This study aimed to analyze the potential enterotoxic effects of all main oxysterols of non-enzymatic origin so far identified in a variety of foods. Experimental plan Differentiated human intestinal cell monolayers (CaCo-2) were incubated up to 48 h in the presence or absence of 0.5, 1 or 5 µM with one out of seven non-enzymatic oxysterols, prior to the verification of minimal irreversible cell damage within the chosen concentration range. Results All tested oxysterols were proven to exert damaging effects on cell monolayers in vitro. The inflammatory interleukin-8 and monocyte chemotactic protein-1 were mostly upregulated by 7-ketocholesterol and 7β-hydroxycholesterol, respectively, then to a lower extent by 5α,6α-epoxycholesterol, 7α-hydroxycholesterol and 5β,6β-epoxycholesterol. 7-Ketocholesterol and 7β-hydroxycholesterol also appeared to be most effective in impairing claudin-1, occludin and E-cadherin proteins, followed by 25-hydroxycholesterol and triol. Conclusions The oxysterols consistently derived by food autoxidation were tested; they potentially impaired the integrity of the intestinal epithelial barrier and triggered an inflammatory response within 0.5–5 µM concentrations, easily reachable in a single Western meal. Significance Statement This comprehensive analysis focused on the potential impairment of the intestinal barrier by the main dietary non-enzymatic oxysterols, should guide further nutrition research aiming at defining a threshold amount of these cholesterol derivatives in order not to derange the physiological gut–brain axis.

Lacticaseibacillus rhamnosus GG Counteracts Rotavirus-Induced Ion Secretion and Enterocyte Damage by Inhibiting Oxidative Stress and Apoptosis Through Specific Effects of Living and Postbiotic Preparations.

BackgroundAdministration of Lacticaseibacillus rhamnosus GG (LGG) to children with gastroenteritis is recommended by universal guidelines. Rotavirus (RV) causes diarrhea through combined cytotoxic and enterotoxic effects. Aim of this study was to evaluate the mechanisms of efficacy of LGG in an in-vitro model of RV diarrhea in its viable form (LGG) and conditioned medium (mLGG).MethodsIon secretion corresponding to the NSP4 enterotoxic effect, was evaluated by short circuit current (Isc) and the cytotoxic effect by transepithelial electrical resistance (TEER) in Ussing chambers, upon exposure to RV in Caco-2 enterocyte monolayers treated or not with living probiotic or its culture supernatant. Mechanisms of enterotoxic and cytotoxic damage were evaluated including oxidative stress measured by reactive oxygen species, apoptosis evaluated by DAPI and nuclear staining, NFkβ immunofluorescence.ResultsRV induced Isc increase and TEER decrease, respectively indicating ion secretion and epithelial damage, the two established pathways of diarrhea. Both probiotic preparations reduced both diarrheal effects, but their potency was different. Live LGG was equally effective on both enterotoxic and cytotoxic effect whereas mLGG was highly effective on ion secretion and showed minimal protective effects on cytoskeleton, apoptosis and NFkβ.ConclusionsLGG counteracts RV-induced diarrhea by inhibiting both cytotoxic and enterotoxic pathogenic mechanisms. Namely, LGG inhibits chloride secretion by specific moieties secreted in the medium with a direct pharmacologic-like action. This is considered a postbiotic effect. Subsequently, live bacteria exert a probiotic effect protecting the enterocyte structure.

A Case of Fatal Intestinal Infarct Preceded by Recurrent Ischaemic Colitis due to the Enterotoxic Effect of Sodium Polystyrene Sulfonate.

A 64-year-old patient with chronic renal failure and persistent hyperkalaemia not corrected by dialysis, was prescribed sodium polystyrene sulfonate (SPS) at a low dose (30 g/day for 2 days a week during the long interdialytic interval). After 3 months of therapy, the patient developed intense abdominal pain with non-specific colitis identified with a colonoscopy. In addition, the biopsy specimens showed rhomboid SPS crystals in the intestinal mucosa. Fourteen months after discontinuing therapy, the patient again presented with colitis and persistent biopsy finding of SPS crystals. The patient died a few months later due to intestinal infarction. SPS is a cation exchange resin used to treat hyperkalaemia resistant to dialysis, but may cause inflammation and ischaemia of the colon. In our patient, a short 3-month course of low-dose SPS therapy (without sorbitol, which is used to counter iatrogenic constipation caused by SPS) induced relapsing colitis, which was followed by massive intestinal infarction a few months later. In light of frequent reports of its enterotoxic effects, SPS should be replaced with the new potassium chelators (patiromer and sodium zirconium cyclosilicate). Sodium polystyrene sulfonate can cause life-threatening colitis.Alternatives medications should be used for the long-term reduction of potassium levels.

Curcumin Blocks Cytotoxicity of Enteroaggregative and Enteropathogenic Escherichia coli by Blocking Pet and EspC Proteolytic Release From Bacterial Outer Membrane.

Pet and EspC are toxins secreted by enteroaggregative (EAEC) and enteropathogenic (EPEC) diarrheagenic Escherichia coli pathotypes, respectively. Both toxins are members of the Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) family. Pet and EspC are important virulence factors that produce cytotoxic and enterotoxic effects on enterocytes. Here, we evaluated the effect of curcumin, a polyphenolic compound obtained from the rhizomes of Curcuma longa L. (Zingiberaceae) on the secretion and cytotoxic effects of Pet and EspC proteins. We found that curcumin prevents Pet and EspC secretion without affecting bacterial growth or the expression of pet and espC. Our results show that curcumin affects the release of these SPATEs from the translocation domain, thereby affecting the pathogenesis of EAEC and EPEC. Curcumin-treated EAEC and EPEC did not induce significant cell damage like the ability to disrupt the actin cytoskeleton, without affecting their characteristic adherence patterns on epithelial cells. A molecular model of docking predicted that curcumin interacts with the determinant residues Asp1018-Asp1019 and Asp1029-Asp1030 of the translocation domain required for the release of Pet and EspC, respectively. Consequently, curcumin blocks Pet and EspC cytotoxicity on epithelial cells by preventing their release from the outer membrane.

Differential effects of Clostridium difficile toxins on ion secretion and cell integrity in human intestinal cells.

Toxin A (TcdA), toxin B (TcdB), and binary toxin (CDT) produced by Clostridium difficile (CD) are thought to play a key role in inducing diarrhea. The aim of this study was to investigate the individual and combined roles of CD toxins in inducing enterotoxic and cytotoxic effect. Ion secretion and epithelial damage were evaluated in the Ussing chambers as measure of enterotoxic or cytotoxic effect, respectively, in human-derived intestinal cells. When added to the mucosal side of Caco-2 cells, TcdB, but not TcdA, induced ion secretion and its effects increased in the presence of TcdA. CDT also induced ion secretion when added to either the mucosal or serosal compartment. Serosal addition of TcdB induced epithelial damage consistent with its cytotoxic effect. However, mucosal addition of TcdB had similar effects, but only in the presence of TcdA. CDT induced epithelial damage when added to the serosal side of cell monolayers, and this was associated with a late onset but prolonged effect. All data were replicated using human colon biopsies. These data indicate that CD, through the combined and direct activity of its three toxins, induces integrated and synergic enterotoxic and cytotoxic effects on the intestinal epithelium.

Acetylsalicylic acid in the cardiological practice: the focus on enterotoxicity

Acetylsalicylic acid (ASA) is one of the most commonly prescribed drugs all over the world. The constant ASA administration results in the considerable side effects, especially associated with the gastrointestinal tract. The review evaluates the enterotoxic ASA effects as well as the current understanding of the frequency, pathogenesis and approaches to the treatment of ASA­associated enteropathy.

Enterotoxic effects of Aeromonas hydrophila infection in the catfish, Clarias gariepinus: Biochemical, histological and proteome analyses

Aeromonas hydrophila is considered as a potential risk to fish populations in the aquaculture industry and could also pose a serious threat to humans. In this study, the impact of A. hydrophila infection in the air-breathing catfish, Clarias gariepinus was analyzed using a multidimensional approach. Aeromonas hydrophila (1 × 107 cells) was injected into C. gariepinus intraperitoneally and maintained at an ambient temperature and photoperiod with periodical monitoring for morphological changes. After 7 days post-infection, tissue samples of the gills, liver, intestine, and kidney were subjected to biochemical, histological, transmission electron microscope (TEM) and proteomic analyses. Observed results indicated distinct morphological changes with the significant increase of ROS and oxidative stress enzymes (CAT and SOD) in tissues of the infected group when compared to the control. Histological analysis in infected fish revealed the presence of pyknotic nuclei, early stages of necrosis in the liver, degradation of renal tubules and widened sinusoidal space in kidneys along with enlargement of the epithelial region in the intestine. TEM analysis of the infected intestine showed degeneration of villi and the presence of multinucleated erythrocytes. Two-dimensional proteomic and mass spectrometry analysis of intestine and liver displayed up-regulation of several immune regulatory proteins such as proteasome subunit 3 protein, prolactin and intermediated filament protein; and down-regulation of proteins including actin, serine/arginine-rich splicing factor and carbonic anhydrase. Taken together, these results suggest that the identified proteins may have a role in immune regulation against A. hydrophila infection in C. gariepinus and support further investigations of host-pathogen interactions.

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia

BackgroundDiarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity.MethodsDiarrhea was induced by intracolonic administration of HIV-1 Tat protein in rats at day 1. PEA alone or in the presence of peroxisome proliferator-activated receptor (PPAR) antagonists was given intraperitoneally from day 2 to day 7. S100B, iNOS, NF-kappaB, TLR4 and GFAP expression were evaluated in submucosal plexi, while S100B and NO levels were measured in EGC submucosal plexi lysates, respectively. To verify whether PEA effects were PPARα-mediated, PPARα−/− mice were also used. After 7 days from diarrhea induction, endogenous PEA levels were measured in submucosal plexi homogenates deriving from rats and PPARα−/− mice.ResultsHIV-1 Tat protein induced rapid onset diarrhea alongside with a significant activation of EGCs. Tat administration significantly increased all hallmarks of neuroinflammation by triggering TLR4 and NF-kappaB activation and S100B and iNOS expression. Endogenous PEA levels were increased following HIV-1 Tat exposure in both wildtype and knockout animals. In PPARα−/− mice, PEA displayed no effects. In wildtype rats, PEA, via PPARα-dependent mechanism, resulted in a significant antidiarrheal activity in parallel with marked reduction of EGC-sustained neuroinflammation.ConclusionsEGCs mediate HIV-1 Tat-induced diarrhea by sustaining the intestinal neuroinflammatory response. These effects are regulated by PEA through a selective PPARα-dependent mechanism. PEA might be considered as an adjuvant therapy in HIV-1-induced diarrhea.

Mechanisms of antidiarrhoeal effects by diosmectite in human intestinal cells

BackgroundRotavirus (RV) induces diarrhoea through a sequence of enterotoxic and cytotoxic effects. The former are NSP4-dependent, induce calcium-dependent chloride secretion and involve oxidative stress. Diosmectite (DS) is a natural clay that has been recommended as an active therapy for diarrhoea, but the mechanism of its effect is not clear. Electrical parameters may be used to measure the direct enterotoxic and cytotoxic effects in polar epithelial intestinal cells. To investigate the effects of DS on RV-induced enterotoxic and cytotoxic damage. Caco-2 cells were used as a model of RV infection to evaluate chloride secretion, epithelial integrity, oxidative stress and viral infectivity in Ussing chambers.ResultsDiosmectite reduced the expression of NSP4 and oxidative stress, resulting in a strong inhibition of chloride secretion. Preincubating RV with DS reduced the cytotoxic effect. Finally, the viral load was reduced by DS but not by control clay. This result suggests that DS specifically affects the early events of RV infection protecting the enterocyte, whereas it does not restore already-established cell damage.ConclusionThese findings indicate that DS exerts an anti-diarrhoeal effect by inhibiting viral replication and the expression of NSP4. Both ion secretion and cell damage induced by RV are strongly inhibited consequent to the antiviral effect, which explains its clinical efficacy.

How Do the Virulence Factors of Shigella Work Together to Cause Disease?

Shigella is the major cause of bacillary dysentery world-wide. It is divided into four species, named S. flexneri, S. sonnei, S. dysenteriae, and S. boydii, which are distinct genomically and in their ability to cause disease. Shigellosis, the clinical presentation of Shigella infection, is characterized by watery diarrhea, abdominal cramps, and fever. Shigella's ability to cause disease has been attributed to virulence factors, which are encoded on chromosomal pathogenicity islands and the virulence plasmid. However, information on these virulence factors is not often brought together to create a detailed picture of infection, and how this translates into shigellosis symptoms. Firstly, Shigella secretes virulence factors that induce severe inflammation and mediate enterotoxic effects on the colon, producing the classic watery diarrhea seen early in infection. Secondly, Shigella injects virulence effectors into epithelial cells via its Type III Secretion System to subvert the host cell structure and function. This allows invasion of epithelial cells, establishing a replicative niche, and causes erratic destruction of the colonic epithelium. Thirdly, Shigella produces effectors to down-regulate inflammation and the innate immune response. This promotes infection and limits the adaptive immune response, causing the host to remain partially susceptible to re-infection. Combinations of these virulence factors may contribute to the different symptoms and infection capabilities of the diverse Shigella species, in addition to distinct transmission patterns. Further investigation of the dominant species causing disease, using whole-genome sequencing and genotyping, will allow comparison and identification of crucial virulence factors and may contribute to the production of a pan-Shigella vaccine.

The Delta Peptide of Ebola Virus has Potent Viroporin Activity

The Delta peptide, cleaved from the soluble glycoprotein (sGP) of Ebola virus (EBOV), is a partially conserved, 40-residue nonstructural polypeptide that has no known function. This peptide shows high similarity in amino acid composition to membrane permeabilizing peptides, including a peptide in the diarrhea-inducing viroporin, NSP4, of Rotavirus. In addition, EBOV disease (EVD) survivors from West Africa have antibodies against the Delta peptide. Therefore, we hypothesize that the Delta peptide has an important role as a viroporin in EVD pathology. Here we show that a peptide corresponding to the serum-stable, conserved C-terminal sequence of Delta peptide permeabilizes and kills nucleated mammalian cells, and potently increasing ion permeability across synthetic lipid bilayers. Importantly, this permeabilizing activity is eliminated by the reduction of the disulfide bond between conserved cysteines. We conclude that EBOV Delta peptide does not form large unregulated pores through membranes, but rather increases ionic permeation, which is similar to other viroporins. By this mechanism, it could contribute to EVD pathology in human through vascular leakage, enterotoxic effects, or other cytotoxic pathways.

Vaccinomics Approach for Designing Potential Peptide Vaccine by Targeting Shigella spp. Serine Protease Autotransporter Subfamily Protein SigA.

Shigellosis, a bacillary dysentery, is closely associated with diarrhoea in human and causes infection of 165 million people worldwide per year. Casein-degrading serine protease autotransporter of enterobacteriaceae (SPATE) subfamily protein SigA, an outer membrane protein, exerts both cytopathic and enterotoxic effects especially cytopathic to human epithelial cell type-2 (HEp-2) and is shown to be highly immunogenic. In the present study, we have tried to impose the vaccinomics approach for designing a common peptide vaccine candidate against the immunogenic SigA of Shigella spp. At first, 44 SigA proteins from different variants of S. flexneri, S. dysenteriae, S. boydii, and S. sonnei were assessed to find the most antigenic protein. We retrieved 12 peptides based on the highest score for human leukocyte antigen (HLA) supertypes analysed by NetCTL. Initially, these peptides were assessed for the affinity with MHC class I and class II alleles, and four potential core epitopes VTARAGLGY, FHTVTVNTL, HTTWTLTGY, and IELAGTLTL were selected. From these, FHTVTVNTL and IELAGTLTL peptides were shown to have 100% conservancy. Finally, IELAGTLTL was shown to have the highest population coverage (83.86%) among the whole world population. In vivo study of the proposed epitope might contribute to the development of functional and unique widespread vaccine, which might be an operative alleyway to thwart dysentery from the world.

Histoenzymological Observations on Acid Phosphatase Activity in the Posterior Intestine of HGCL2–Treated Fish, Channa striatus

Present studies incorporate enterotoxic effects of HgCl2 on the relative distribution of acid phosphatase (ACP) activity in the posterior intestine of fish, Channa striatus. It was observed that there appears to be slightly increased ACP activity accompanied by histolytic changes in the columnar epithelial cells lining of villi and intestinal glands of the posterior intestine of treated carnivorous fish. However, in control, these changes were invisible.

Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC).

Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit’s potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.

Effect of host defenses on Clostridium difficile toxin–induced intestinal barrier injury

The severity of Clostridium difficile-associated infection depends on the virulence factors of the organism and host factors, including intestinal barrier function. The intestinal mucus layer has recently been recognized as the first line of defense against enteric pathogens. Its interaction with mucosal humoral immunity provided by secretory immunoglobulin A (SIgA) is unknown as it relates to C. difficile disease severity. This was studied in vitro. Confluent HT29 (non-mucus-producing) and HT29-MTX (mucus-producing) intestinal epithelial cells (IECs) with and without transcytosed SIgA were exposed to C. difficile toxin A (6 hours), and IEC toxin internalization, permeability (fluorescein isothiocyanate dextran), and necrosis (propidium iodide staining) were determined. In other experiments, colostral SIgA was added to the apical surface of IEC, and cleavage was determined by measurement of intact SIgA and secretory component fractions by enzyme-linked immunosorbent assay. Tumor necrosis factor α and interleukin 6 were measured from basal chamber culture supernatants by enzyme-linked immunosorbent assay. Toxin A uptake and subsequent enterotoxic effects on IEC were decreased by both the mucus layer and SIgA. Similar findings were noted with the effects of toxin A on IEC monolayer permeability, cytoskeleton changes, and proinflammatory cytokine release. The combination of the mucus layer and SIgA afforded the best protection against the adverse effects on IEC by toxin A. It seems that the mucus layer was also protective against SIgA cleavage, resulting in reduced protease activity by HT29 cells exposed to toxin A. Both intestinal mucus and SIgA were important in limiting C. difficile-associated disease severity in this model. A synergistic effect of mucus and IgA was also noted and may be due to protection of SIgA from proteolytic cleavage.

The Immunogenic SigA Enterotoxin of Shigella flexneri 2a Binds to HEp-2 Cells and Induces Fodrin Redistribution in Intoxicated Epithelial Cells

BackgroundWe have previously shown that the enterotoxin SigA which resides on the she pathogenicity island (PAI) of S. flexneri 2a is an autonomously secreted serine protease capable of degrading casein. We have also demonstrated that SigA is cytopathic for HEp-2 cells and plays a role in the intestinal fluid accumulation associated with S. flexneri infections.Methods/Principal FindingsIn this work we show that SigA binds specifically to HEp-2 cells and degrades recombinant human αII spectrin (α-fodrin) in vitro, suggesting that the cytotoxic and enterotoxic effects mediated by SigA are likely associated with the degradation of epithelial fodrin. Consistent with our data, this study also demonstrates that SigA cleaves intracellular fodrin in situ, causing its redistribution within cells. These results strongly implicate SigA in altering the cytoskeleton during the pathogenesis of shigellosis. On the basis of these findings, cleavage of fodrin is a novel mechanism of cellular intoxication for a Shigella toxin. Furthermore, information regarding immunogenicity to SigA in infected patients is lacking. We studied the immune response of SigA from day 28 post-challenge serum of one volunteer from S. flexneri 2a challenge studies. Our results demonstrate that SigA is immunogenic following infection with S. flexneri 2a.ConclusionsThis work shows that SigA binds to epithelial HEp-2 cells as well as being able to induce fodrin degradation in vitro and in situ, further extending its documented role in the pathogenesis of Shigella infections.

Rotavirus Induces a Biphasic Enterotoxic and Cytotoxic Response in Human‐Derived Intestinal Enterocytes, Which Is Inhibited by Human Immunoglobulins

The mechanisms of diarrhea due to rotavirus infection in humans are not fully understood; no specific therapy is available, but orally administered human serum immunoglobulins are effective in blocking stool output. We aimed to investigate the effect of rotavirus on ion transport and the role of NSP4 in human-derived enterocytes, and to test the efficacy of human serum immunoglobulin in a model of rotavirus infection. Soon after infection, rotavirus induces active chloride secretion in enterocytes. This effect is evident before viral replication leads to cell damage and correlates with NSP4 production. Inhibition of NSP4 prevents the early secretory phase but not cell damage. Incubation with human serum immunoglobulin blocks both ion secretion and cell damage. Rotavirus exerts an early NSP4-dependent ion secretion and subsequent tissue damage. The combined enterotoxic and cytotoxic effects may be responsible for the increased severity of diarrhea due to rotavirus infection, and both are counteracted by human serum immunoglobulin.