A novel antiviral formulation containing caprylic acid inhibits SARS-CoV-2 infection of a human bronchial epithelial cell model.

A novel proprietary formulation, ViruSAL, has previously been demonstrated to inhibit diverse enveloped viral infections in vitro and in vivo. We evaluated the ability of ViruSAL to inhibit SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infectivity, using physiologically relevant models of the human bronchial epithelium, to model early infection of the upper respiratory tract. ViruSAL potently inhibited SARS-CoV-2 infection of human bronchial epithelial cells cultured as an air-liquid interface (ALI) model, in a concentration- and time-dependent manner. Viral infection was completely inhibited when ViruSAL was added to bronchial airway models prior to infection. Importantly, ViruSAL also inhibited viral infection when added to ALI models post-infection. No evidence of cellular toxicity was detected in ViruSAL-treated cells at concentrations that completely abrogated viral infectivity. Moreover, intranasal instillation of ViruSAL to a rat model did not result in any toxicity or pathological changes. Together these findings highlight the potential for ViruSAL as a novel and potent antiviral for use within clinical and prophylactic settings.

Platelet activating factor receptor acts to limit colitis-induced liver inflammation.

Liver inflammation is a common extraintestinal manifestation in inflammatory bowel disease (IBD), yet, the mechanisms driving gut-liver axis inflammation remain poorly understood. IBD leads to a breakdown in the integrity of the intestinal barrier causing an increase in portal and systemic gut-derived antigens, which challenge the liver. Here, we examined the role of platelet activating factor receptor (PAFR) in colitis-associated liver damage using dextran sulfate sodium (DSS) and anti-CD40-induced colitis models. Both DSS and anti-CD40 models exhibited liver inflammation associated with colitis. Colitis reduced global PAFR protein expression in mouse livers causing an exclusive re-localization of PAFR to the portal triad. The global decrease in liver PAFR was associated with increased sirtuin 1 while relocalized PAFR expression was limited to Kupffer cells (KCs) and co-localized with toll-like receptor 4. DSS activated the NLRP3-inflammasome and increased interleukin (IL)-1ß in the liver. Antagonism of PAFR amplified the inflammasome response by increasing NLRP3, caspase-1, and IL-1ß protein levels in the liver. LPS also increased NLRP3 response in human hepatocytes, however, overexpression of PAFR restored the levels of NLPR3 and caspase-1 proteins. Interestingly, KCs depletion also increased IL-1ß protein in mouse liver after DSS challenge. These data suggest a protective role for PAFR-expressing KCs during colitis and that regulation of PAFR is important for gut-liver axis homeostasis.

A novel antiviral formulation inhibits a range of enveloped viruses.

Some free fatty acids derived from milk and vegetable oils are known to have potent antiviral and antibacterial properties. However, therapeutic applications of short- to medium-chain fatty acids are limited by physical characteristics such as immiscibility in aqueous solutions. We evaluated a novel proprietary formulation based on an emulsion of short-chain caprylic acid, ViroSAL, for its ability to inhibit a range of viral infections in vitro and in vivo. In vitro, ViroSAL inhibited the enveloped viruses Epstein-Barr, measles, herpes simplex, Zika and orf parapoxvirus, together with Ebola, Lassa, vesicular stomatitis and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) pseudoviruses, in a concentration- and time-dependent manner. Evaluation of the components of ViroSAL revealed that caprylic acid was the main antiviral component; however, the ViroSAL formulation significantly inhibited viral entry compared with caprylic acid alone. In vivo, ViroSAL significantly inhibited Zika and Semliki Forest virus replication in mice following the inoculation of these viruses into mosquito bite sites. In agreement with studies investigating other free fatty acids, ViroSAL had no effect on norovirus, a non-enveloped virus, indicating that its mechanism of action may be surfactant disruption of the viral envelope. We have identified a novel antiviral formulation that is of great interest for the prevention and/or treatment of a broad range of enveloped viruses, particularly those of the skin and mucosal surfaces.

Migration of Fasciola hepatica newly excysted juveniles is inhibited by high-mannose and oligomannose-type N-glycan-binding lectins.

Fasciola hepatica has both zoonotic importance and high economic impact in livestock worldwide. After ingestion by the definitive host, the Newly Excysted Juveniles (NEJ) penetrate the intestine before reaching the peritoneal cavity. The role of some NEJ-derived proteins in invasion has been documented, but the role of NEJ glycans or lectin-binding receptors during initial infection in the gut is still unknown. To address these questions, the migration of NEJ through rat intestine was recorded at 30 min intervals up to 150 min by two ex vivo methods. Firstly, jejunal sheets were challenged with NEJ incubated with biotinylated lectins. Secondly, untreated NEJ were incubated with distal jejunum pre-treated with lectins. Both Concanavalin A (ConA) and Galanthus nivalis (GNL), which recognize mannose-type N-glycans, significantly inhibited NEJ migration across the jejunum. Most of the lectins bound to the tegument and oral sucker of the NEJ, but only ConA and GNL maintained this interaction over 150 min. None of the lectins examined significantly reduced NEJ migration when pre-incubated with jejunal sheets, suggesting that host glycans might not be essential for initial binding/recognition of the gut by NEJ. Agents capable of blocking mannose-type N-glycans on the NEJ tegument may have potential for disrupting infection.

Development of a versatile in vitro method for understanding the migration of Fasciola hepatica newly excysted juveniles.

Fasciola hepatica is a parasitic trematode that causes serious losses to livestock producers, and also zoonotic disease. The limitations of chemotherapy for the control of fasciolosis have led to significant interest in the development of vaccines to protect cattle and sheep from infection. However, relatively few studies have concentrated on the mechanisms of invasion of the gut by newly excysted juvenile liver flukes (NEJ) and the host response triggered by this event. The aim of this work was to develop an in vitro model to study invasion by NEJ, while also reducing the requirement for challenge infections of experimental animals. Fasciola hepatica metacercariae were excysted in vitro and placed into compartments containing rat distal jejunal sheets. Variations in incubation medium, chamber size and incubation temperature were used to identify optimal conditions for NEJ migration across the gut. Histological examination showed increased migration until 120 min post-incubation. The use of RPMI, without gassing at 39 °C, as the incubation medium was found to be optimal, with 40·5% of NEJ migrating after 150 min. This study describes a readily-reproducible method for studying the migration of F. hepatica NEJ within the definitive host. It will be useful for identifying potential drug and vaccine targets.

A whey protein hydrolysate promotes insulinotropic activity in a clonal pancreatic ß-cell line and enhances glycemic function in ob/ob mice.

Whey protein hydrolysates (WPHs) represent novel antidiabetic agents that affect glycemia in animals and humans, but little is known about their insulinotropic effects. The effects of a WPH were analyzed in vitro on acute glucose-induced insulin secretion in pancreatic BRIN-BD11 ß cells. WPH permeability across Caco-2 cell monolayers was determined in a 2-tiered intestinal model. WPH effects on insulin resistance were studied in vivo following an 8-wk oral ingestion (100 mg/kg body weight) by ob/ob (OB-WPH) and wild-type mice (WT-WPH) compared with vehicle control (OB and WT groups) using a 2 × 2 factorial design, genotype × treatment. BRIN-BD11 cells showed a robust and reproducible dose-dependent insulinotropic effect of WPH (from 0.01 to 5.00 g/L). WPH bioactive constituents were permeable across Caco-2 cell monolayers. In the OB-WPH and WT-WPH groups, WPH administration improved glucose clearance after a glucose challenge (2 g/kg body weight), as indicated by differences in the area under curves (AUCs) (P ≤ 0.05). The basal plasma glucose concentration was not affected by WPH treatment in either genotype. The plasma insulin concentration was lower in the OB-WPH than in the OB group (P ≤ 0.005) but was similar between the WT and WT-WPH groups; the interaction genotype × treatment was significant (P ≤ 0.005). Insulin release from pancreatic islets isolated from the OB-WPH group was greater (P ≤ 0.005) than that from the OB group but did not differ between the WT-WPH and WT groups; the interaction genotype × treatment was not significant. In conclusion, an 8-wk oral administration of WPH improved blood glucose clearance, reduced hyperinsulinemia, and restored the pancreatic islet capacity to secrete insulin in response to glucose in ob/ob mice. Hence, it may be useful in diabetes management.

Chloride-led disruption of the intestinal mucous layer impedes Salmonella invasion: evidence for an 'enteric tear' mechanism.

METHODS: The intestinal epithelial layer can switch from a net absorptive state to one of net secretion in the presence of luminal toxins and pathogens. This suggests an innate defence role for regulated secretion of mucus, electrolytes and water. We hypothesised that chloride-led fluid secretion across the mucus-covered intestinal epithelium alters barrier properties by influencing the overlying mucous-gel layer. RESULTS: We demonstrated that chloride-led disruption of the epithelial-associated mucus-gel covering HT29-MTX-E12 (E12) human colonic epithelial monolayers, a goblet-cell like line derived from parental HT29 cells, resulted in reduction of associated mucus as well as a reduction in mucous-gel density and barrier properties. Changes in epithelial secretory state were accompanied by increased water transport, and the resulting loss of gel integrity reduced Salmonella typhimurium invasion of epithelia in both E12 monolayers and of isolated rat ileal mucosae. However, neither chloride secretion nor mucus disruption altered numbers of adhering bacteria. CONCLUSION: These data suggest a role for chloride led fluid secretion in the shedding of the adherent mucous-gel layer, possibly as a rate-limiting innate defence mechanism, and offer evidence for "enteric tears" in intestinal host defence.

The Vibrio parahaemolyticus Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis.

BACKGROUND: Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined. RESULTS: V. parahaemolyticus deployed its TTSS1 to induce activation of the JNK, p38 and ERK MAPK in human epithelial cells. VP1680 was identified as the TTSS1 effector protein responsible for MAPK activation in Caco-2 cells and the activation of JNK and ERK by this protein was important in induction of host cell death. V. parahaemolyticus actively induced IL-8 secretion in a response mediated by TTSS1. A role for VP1680 and for the ERK signalling pathway in the stimulation of IL-8 production in epithelial cells by V. parahaemolyticus was established. Interestingly, TTSS2 inhibited IL-8 mRNA transcription at early stages of interaction between the bacterium and the cell. CONCLUSIONS: This study demonstrated that V. parahaemolyticus activates the three major MAPK signalling pathways in intestinal epithelial cells in a TTSS1-dependent manner that involves the TTSS1 effector VP1680. Furthermore VP1680 and JNK and ERK activation were needed for maximal cytotoxicity of the bacterium. It was shown that V. parahaemolyticus is a strong inducer of IL-8 secretion and that induction reflects a balance between the effects of TTSS1 and TTSS2. Increases in IL-8 secretion were mediated by TTSS1 and VP1680, and augmented by ERK activation. These results shed light on the mechanisms of bacterial pathogenesis mediated by TTSS and suggest significant roles for MAPK signalling during infection with V. parahaemolyticus.

Drug delivery systems in domestic animal species.

Delivery of biologically active agents to animals is often perceived to be the poor relation of human drug delivery. Yet this field has a long and successful history of species-specific device and formulation development, ranging from simple approaches and devices used in production animals to more sophisticated formulations and approaches for a wide range of species. While several technologies using biodegradable polymers have been successfully marketed in a range of veterinary and human products, the transfer of delivery technologies has not been similarly applied across species. This may be due to a combination of specific technical requirements for use of devices in different species, inter-species pharmacokinetic, pharmacodynamic and physiological differences, and distinct market drivers for drug classes used in companion and food-producing animals. This chapter reviews selected commercialised and research-based parenteral and non-parenteral veterinary drug delivery technologies in selected domestic species. Emphasis is also placed on the impact of endogenous drug transporters on drug distribution characteristics in different species. In vitro models used to investigate carrier-dependent transport are reviewed. Species-specific expression of transporters in several tissues can account for inter-animal or inter-species pharmacokinetic variability, lack of predictability of drug efficacy, and potential drug-drug interactions.

Cyclooxygenase-2 mRNA expression in equine nonglandular and glandular gastric mucosal biopsy specimens obtained before and after induction of gastric ulceration via intermittent feed deprivation.

OBJECTIVE: To measure the expression of cyclooxygenase-2 (COX-2) mRNA in gastric biopsy specimens serially obtained from horses before, during, and after an 8-day intermittent feed-deprivation trial and to investigate the mucosal location of COX-2. ANIMALS: 9 mixed-breed horses for retrieval of gastric biopsy specimens and 16 additional horses for immunohistochemical analysis. PROCEDURES: Gastric biopsy specimens were obtained from 6 horses; 3 of these horses and 3 more participated in an intermittent feed-deprivation trial 9 weeks later. A quantitative PCR assay was used to determine the amount of COX-2 mRNA in biopsy specimens from nonulcerated and ulcerated gastric mucosa. Immunohistochemical staining of specimens by use of a polyclonal anti-COX-2 antibody was performed on full-thickness postmortem gastric biopsy specimens. RESULTS: COX-2 mRNA was expressed in all glandular gastric mucosal specimens but was only detectable in nonglandular mucosal specimens when ulceration was present or during ulcer healing. Positive staining for COX-2 was present in 12 of 14 nonulcerated glandular mucosal sections. Although such staining was weak or absent in nonulcerated nonglandular sections, stronger staining was evident in regenerating epithelium at the rims of erosions and ulcers. CONCLUSIONS AND CLINICAL RELEVANCE: COX-2 was constitutively present in equine glandular gastric mucosa, although its contribution to mucosal protection remains unclear. Our finding of COX-2 mRNA expression in ulcer margins during healing may support a role for the products of this enzyme in mucosal repair. The potential roles of COX-2 should be considered when COX-2-selective inhibitors are prescribed for horses with gastric ulcers.

Estrogen and its role in gastrointestinal health and disease.

INTRODUCTION: While the concept of a role of estrogen in gastrointestinal (in particular, colonic) malignancy has generated excitement in recent years, no review has examined the role of this potent and omnipresent steroid hormone in physiological states or its contribution to the development of benign pathological processes. Understanding these effects (and mechanisms therein) may provide a platform for a deeper understanding of more complex disease processes. METHODS: A literature search was conducted using the PubMed database and the search terms were "estrogen," "estrogen AND gastrointestinal tract," "estrogen AND colon," "estrogen AND esophagus," "estrogen AND small intestine," "estrogen AND stomach," "estrogen AND gallbladder," and "estrogen AND motility." Bibliographies of extracted studies were further cross-referenced. In all, 136 full-text articles were selected for review. A logical organ-based approach was taken to enable extraction of data of clinical relevance and meaningful interpretation thereof. Insight is provided into the hypotheses, theories, controversies, and contradictions generated over the last five decades by extensive investigation of estrogen in human, animal, and cell models using techniques as diverse as autoradiographic studies of baboons to human population analysis. CONCLUSIONS: Effects from esophagus through to the colon and rectum are summarized in this first concise collection of data pertaining to estrogenic actions in gastrointestinal health and disease. Mechanisms of these actions are discussed where possible. Undoubtedly, this hormone exerts many actions yet to be elucidated, and its potential therapeutic applications remain, as yet, largely unexplored.

Practical classes: a platform for deep learning? Overall context in the first-year veterinary curriculum.

The aim of this study is to evaluate the many practical formats that support the first-year veterinary curriculum. These practical classes are diverse in content and style. They include laboratory-based formats, classes involving live animals and cadavers, classes conducted using computer-aided learning tools, study groups, and information technology training. This preliminary study examines ratings for these practical classes, but also relates these ratings to students' approaches to study with the aim of understanding how a deep learning approach manifests itself in the practical setting. The diverse behaviors and attitudes to practical classes are also evaluated in the light of the approaches to study. A questionnaire that evaluated (1) a total of 24 practical classes, (2) the 52-item Approaches to Study Inventory, and (3) 13 behaviors within and attitudes to practical classes was distributed to 69 first-year veterinary students in their final term. Practical classes that involved live animals and cadavers were rated most positively by this group of students. These ratings, however, did not correlate significantly with the deep or surface learning score. The majority of practical classes where the ratings were found to be associated with deep and surface learning were laboratory-based, although overall these practical classes tended to be rated lower than those involving animals. Ratings did not correlate significantly with the strategic approach. A number of behaviors and attitudes to practical classes were also found to be positively and significantly (p=0.0001) associated with the deep learning approach. This preliminary study indicates that this cohort of veterinary students has an overall positive perception of practical classes that permit contact with live animals or cadavers. Although the perception of laboratory-type practical classes was lower overall, the ratings for these practical classes appeared to be influenced by their deep and surface learning scores. We hypothesize that these approaches influence student engagement with and appreciation of laboratory-type classes, but not of classes involving live animals or cadavers. This would suggest that a different "type" of learning is taking place in these different contexts.

Increased intestinal permeability in rats subjected to traumatic frontal lobe percussion brain injury.

BACKGROUND: Dysfunction of the gastrointestinal tract is a common occurrence after traumatic brain injury (TBI). We hypothesized that increased intestinal permeability may result from a precisely controlled percussion injury to the exposed brains of anesthetized rats and that such an effect could be assessed in vitro using excised intestinal mucosae mounted in Ussing chambers. METHODS: After craniotomy over the left medial prefrontal cortex on anesthetized rats, neurotrauma was produced using a pneumatically driven impactor on the exposed brain. Control rats were subjected to identical procedures but did not receive an impact. Muscle-stripped rat intestinal ileal and colonic segments were mounted in Ussing chambers within 30 minutes of death. Transepithelial electrical resistance (TEER) and the apparent permeability coefficient (Papp) of [C]-mannitol were recorded from intestinal tissue for 120 minutes. Histopathologic analysis was also performed to determine any gross morphologic changes in the intestine. RESULTS: Ileal and colonic mucosae showed no differences in TEER in ileum or colon of TBI rats compared with controls. The Papp of mannitol was significantly increased in ilea from rats previously exposed to TBI compared with controls. Histologic analysis showed gross changes to 50% of the ileal but not the colonic sections from TBI rats. CONCLUSION: TBI results in significantly reduced ileal barrier function, most likely mediated by open tight junctions. For patients with acute head injury, this may have implications for subsequent oral absorption of nutrients. Systemic delivery of luminal endotoxins may contribute to multiple organ failure.

Orphan Nuclear Receptors in Colorectal Cancer.

Colorectal cancer is one of the most common cancers worldwide, with an overall increased incidence annually. Despite improvements in treatment and surveillance, almost 50% develop recurrent and/or distant disease. Unknown cellular processes are the fundamental cause for treatment failure and metastatic disease. The interplay of chronic inflammation and carcinogenesis is well established. Recent work has highlighted the role of nuclear receptors and co-regulators in the inflammation to carcinogenesis process. Orphan nuclear receptors have been shown to be involved in numerous cellular processes, including both at a transcriptional and a non-genomic level. There is a significant emphasis to identify ligands that will interact and modify these nuclear receptors, with the long-term aim of developing novel pharmaceutical therapies. The identification of orphan nuclear receptor ligands will also help increase our current understanding of their role in cellular signaling, by enabling manipulation of these receptors. This review aims to provide a brief overview of some key orphan nuclear receptors which may be involved in colorectal cancer.

Early onset airway obstruction in response to organic dust in the horse.

Equine recurrent airway obstruction (RAO) has been used as a naturally occurring model of human asthma. However, it is unknown whether there is an early-phase response in RAO. The aim of this study was to determine whether exposure to organic dust induces immediate changes in lung function in RAO-affected horses, which could be mediated by airway mast cells. Six RAO-affected horses in remission and six control horses were challenged with hay-straw dust suspension by nebulization. Total respiratory resistance at 1 Hz, measured by forced oscillation, was increased from 0.62 +/- 0.09 cmH(2)O.l(-1).s (mean +/- SE) to 1.23 +/- 0.20 cmH(2)O.l(-1).s 15 min after nebulization in control horses (P = 0.023) but did not change significantly in the RAO group. Total respiratory reactance at 1 Hz (P = 0.005) was significantly lower in the control horses (-0.77 +/- 0.07 cmH(2)O.l(-1).s) than in the RAO group (-0.49 +/- 0.04 cmH(2)O.l(-1).s) 15 min after nebulization. Bronchoalveolar lavage fluid (BALF) histamine concentration was significantly elevated 10 and 20 min postnebulization in control horses but not in RAO horses. Minimum reactance at 1 Hz in the early postnebulization period significantly correlated with both prechallenge BALF mast cell numbers (r = -0.65, P = 0.02) and peak BALF histamine concentration postnebulization (r = -0.61, P = 0.04). In conclusion, RAO horses, unlike human asthmatic patients, do not exhibit an early-phase response. However, healthy control horses do demonstrate a mild but significant early (<20 min) phase response to inhaled organic dust. This response may serve to decrease the subsequent dose of dust inhaled and as such provide a protective mechanism, which may be compromised in RAO horses.

Development of a Non-Aqueous Dispersion to Improve Intestinal Epithelial Flux of Poorly Permeable Macromolecules.

Intestinal permeation enhancers (PEs) offer an attractive strategy to enable oral peptide administration. However, optimal presentation of peptide and PE from solid-dosage forms is offset by slow dissolution rates in the small intestine, which reduces the likelihood that the PE can reach the threshold concentration for sufficient permeability enhancement. The purpose of this study was to design a PE-based liquid dispersion that can improve intestinal permeation of macromolecules across Caco-2 monolayers and isolated rat/human intestinal mucosae mounted in Ussing chambers. An enhancer screen in monolayers based on permeability (TEER, Papp [14C]-mannitol) and cytotoxicity (MTT assay) initially identified methyl 10-hydroxydecanoate (10-OHC10CH3) as a candidate. 10-OHC10CH3 (20 mM) increased the Papp of fluorescent dextran of 4 kDa (FD4) (167-fold), 10 kDa (FD10) (429-fold), and 40 kDa (FD40) (520-fold) across monolayers. Blends of 10-OHC10CH3 with low molecular weight PEGs (0.2-1 kDa) formed liquid dispersions in which enhancement capacity across monolayers of 10-OHC10CH3 was increased over 10-OHC10CH3 alone in the order PEG200 < PEG400 < PEG600 < PEG1000. Finally, a 1:5 ratio of 10-OHC10CH3 (10-20 mM)/PEG600 (50-100 mM) increased the Papp of [14C]-mannitol across rat and human intestinal mucosae. This study highlights the potential future role for non-aqueous, PE-based liquid dispersions in oral delivery of macromolecules.

Multi-drug resistance in Salmonella enterica: efflux mechanisms and their relationships with the development of chromosomal resistance gene clusters.

Bacterial drug resistance represents one of the most crucial problems in present day antibacterial chemotherapy. Of particular concern to public health is the continuing worldwide epidemic spread of Salmonella enterica serovar Typhimurium phage type DT104 harbouring a genomic island called Salmonella genomic island I (SGI-1). This island contains an antibiotic gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides and tetracyclines. These resistance genes are assembled in a mosaic pattern, indicative of several independent recombinational events. The mobility of SGI-1 coupled with the ability of various antibiotic resistance genes to be integrated and lost from the chromosomal resistance locus allows for the transfer of stable antibiotic resistance to most of the commonly used antibiotics and adaptation to new antibiotic challenges. This, coupled with the incidence of increasing fluoroquinolone resistance in these strains increases the risk of therapeutic failure in cases of life-threatening salmonellosis. Fluoroquinolone resistance has largely been attributed to mutations occurring in the genes coding for intracellular targets of these drugs. However, efflux by the AcrAB-TolC multi-drug efflux pump has recently been shown to directly contribute to fluoroquinolone resistance. Furthermore, the resistance to chloramphenicol-florfenicol and tetracyclines in DT104 isolates, is due to interaction between specific transporters for these antibiotics encoded by genes mapping to the SGI-1 and the AcrAB-TolC tripartite efflux pump. The potential for the use of efflux pump inhibitors to restore therapeutic efficacy to fluoroquinolones and other antibiotics offers an exciting developmental area for drug discovery.

Rat, ovine and bovine Peyer's patches mounted in horizontal diffusion chambers display sampling function.

Freshly excised rat, ovine and bovine ileal Peyer's patch (PP) and non-Peyer's patch tissues (NPP) were mounted in modified horizontal polyethylene diffusion chambers with a range of window areas. Rat tissue was initially used to establish that barrier function and histology were maintained for up to 60 min. Horse-radish peroxidase (HRP) fluxes and S. Typhimurium adherence and invasion were significantly higher in rat PP over NPP. Particle uptake was shown to be a rapid, energy-, time-, and size-dependent process, occurring more readily in PP than NPP tissue in each species. In a kinetic analysis, particles were localized initially in the follicle-associated epithelium and then in the dome region. For NPP uptake, particles were initially localized to villous epithelium, and were then detected in the crypts and lamina propria. Electrophysiological parameters including pharmacologically-stimulated inward short-circuit current responses were determined in isolated PP and NPP from each species mounted under identical conditions in Ussing chambers. In conclusion, comparative functional and histological characteristics of PP from several species were demonstrated in horizontal diffusion chambers. Horizontal diffusion chambers are therefore a useful in vitro model in which a range of functions including transport of particulate formulations by PP may be examined.

Toxigenic C. difficile induced inflammatory marker expression by human intestinal epithelial cells is asymmetrical.

Clostridium difficile infection of the intestinal epithelium and consequent pseudomembranous colitis is an important cause of morbidity and mortality. Pathogenesis has been ascribed exclusively to toxin production. Using in vitro models of human intestinal epithelial layers, we show that exposure to toxigenic C. difficile upregulates epithelial expression of IL-8 and ICAM-1, two molecules important in neutrophil chemoattraction and adhesion and subsequent inflammation. IL-8 production was also stimulated by toxin-containing supernatants. C difficile induced IL-8 release was inhibited by specific antiserum. Increased ICAM-1 expression only occurred after basolateral exposure to C. difficile while apical exposure had no effect on ICAM-1 expression. However, transepithelial electrical resistance was impaired by apical exposure to bacterial suspensions. We suggest that apical exposure to C. difficile induces changes in epithelial layer integrity which allows the bacteria and/or the toxin access to the basolateral compartment where pathogenic inflammatory mechanisms are activated.