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.

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.

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.

Investigation of facilitative urea transporters in the human gastrointestinal tract.

The symbiotic relationship between humans and their intestinal microbiome is supported by urea nitrogen salvaging. Previous studies have shown that colonic UT-B urea transporters play a significant role in this important physiological process. This current study investigated UT-A and UT-B urea transporter expression along the human gastrointestinal tract. Initial end-point PCR experiments determined that UT-A RNA was predominantly expressed in the small intestine, while UT-B RNA was expressed in stomach, small intestine, and colon. Using western blotting experiments, a strong 40-60 kDa UT-B signal was found to be abundant in both ileum and colon. Importantly, this signal was deglycosylated by PNGaseF enzyme treatment to a core protein of 30 kDa in both tissues. Further immunolocalization studies revealed UT-B transporter proteins were present at the apical membrane of the villi in the ileum, but predominantly at the basolateral membrane of the colonic surface epithelial cells. Finally, a blind scoring immunolocalization study suggested that there was no significant difference in UT-B abundance throughout the colon (NS, ANOVA, N = 5-21). In conclusion, this current study suggested UT-B to be the main human intestinal urea transporter. Intriguingly, these data suggested that the same UT-B isoform was present in all intestinal epithelial cells, but that the precise cellular location varied.

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.

Surgical Management of Patients with Neuroendocrine Neoplasms of the Appendix: Appendectomy or More.

BACKGROUND: Appendiceal neuroendocrine neoplasms (ANEN) are mostly indolent tumours treated effectively with simple appendectomy. However, controversy exists regarding the necessity of oncologic right hemicolectomy (RH) in patients with histologic features suggestive of more aggressive disease. We assess the effects of current guidelines in selecting the surgical strategy (appendectomy or RH) for the management of ANEN. Methods/Aims: This is a retrospective review of all ANEN cases treated over a 14-year period at 3 referral centres and their management according to consensus guidelines of the European and the North American Neuroendocrine Tumor Societies (ENETS and NANETS, respectively). The operation performed, the tumour stage and grade, the extent of residual disease, and the follow-up outcomes were evaluated. RESULTS: Of 14,850 patients who had appendectomies, 215 (1.45%) had histologically confirmed ANEN. Four patients had synchronous non-ANEN malignancies. One hundred and ninety-three patients had index appendectomy. Seventeen patients (7.9%) had lymph node metastases within the mesoappendix. Forty-nine patients underwent RH after appendectomy. The percentages of 30-day morbidity and mortality after RH were 2 and 0%, respectively. Twelve patients (24.5%) receiving completion RH were found to have lymph node metastases. Two patients had liver metastases, both of them synchronous. The median follow-up was 38.5 months (range 1-143). No patient developed disease recurrence. Five- and 10-year overall survival for all patients with ANEN as the only malignancy was both 99.05%. CONCLUSIONS: The current guidelines appear effective in identifying ANEN patients at risk of harbouring nodal disease, but they question the oncological relevance of ANEN lymph node metastases. RH might present an overtreatment for a number of patients with ANEN.

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 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.

Differential protein abundance of a basolateral MCT1 transporter in the human gastrointestinal tract.

Bacterially derived short chain fatty acids (SCFAs), such as butyrate, are vital in maintaining the symbiotic relationship that exists between humans and their gastrointestinal microbial populations. A key step in this process is the transport of SCFAs across colonic epithelial cells via MCT1 transporters. This study investigated MCT1 protein abundance in various human intestinal tissues. Initial RT-PCR analysis confirmed the expected MCT1 RNA expression pattern of colon > small intestine > stomach. Using surgical resection samples, immunoblot analysis detected higher abundance of a 45 kDa MCT1 protein in colonic tissue compared to ileum tissue (P < 0.001, N = 4, unpaired t-test). Importantly, MCT1 abundance was found to be significantly lower in sigmoid colon compared to ascending colon (P < 0.01, N = 8-11, ANOVA). Finally, immunolocalization studies confirmed MCT1 to be abundant in the basolateral membranes of surface epithelial cells of the ascending, transverse, and descending colon, but significantly less prevalent in the sigmoid colon (P < 0.05, N = 5-21, ANOVA). In conclusion, these data confirm that basolateral MCT1 protein abundance is correlated to levels of bacterially derived SCFAs along the human gastrointestinal tract. These findings highlight the importance of precise tissue location in studies comparing colonic MCT1 abundance between normal and diseased states.

Hepatic gateways.

The intestinal mucosal barrier contributes to homeostasis by limiting systemic dissemination of microbes and toxins while allowing nutrients to pass through to the systemic circulation. In a recent issue of Science, Spadoni et al. demonstrated a novel mechanism to enable this selectivity: the existence of a gut-vascular barrier (GVB) as indicated by a series of studies on the interaction between murine and human intestine with Salmonella typhimurium species . They showed that (i) enteroglial cells and pericytes in contact with endothelial cells (ECs) form the GVB (ii) Salmonella typhimurium can penetrate it by a mechanism dependent on the pathogenicity island (Spi) 2-encoded type III secretion system and on decreased ß-catenin dependent signaling in gut endothelial cells. Understanding the GVB may provide new insights into the regulation of the gut-liver axis.

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.

The effects of polyamines on human colonic mucosal function.

Electrogenic ion transport in human colon is a surrogate marker for colonic mucosal function, and may be manipulated by a variety of hormonal, neural, immune and paracrine mediators. Polyamines are present in vast quantities in the colonic lumen and appear to be integral to cellular function. This study explores some of the mechanisms of polyamine action on colonic tissue through study of their effects on differential secretory pathways, as well as examining their actions on intracellular cAMP and Ca(2+) accumulation. Human colonic mucosa was mounted in Ussing chambers and treated with polyamines (spermine, spermidine and putrescine) with changes in ion transport recorded. In separate experiments colonic crypts were treated with polyamines and intracellular cAMP levels determined by ELISA and intracellular calcium concentrations were quantified by fluorescent imaging. Polyamines at physiological concentrations (1mM) exert no effects on basal mucosal chloride secretion or transepithelial electrical resistance. Polyamines inhibit electrogenic ion secretion as stimulated by forskolin (cAMP-mediated), but not carbachol (Ach-mediated). All the polyamines used in this study inhibited intracellular cAMP accumulation, according to potency (spermine>spermidine>putrescine). Spermine increased intracellular Ca(2+) in a PKC-dependent manner, likely due to its effects on the extracellular calcium-sensing receptor (CaSR). Polyamines act to prevent cAMP-mediated Cl(-) hypersecretion in the colon, acting through CaSR to inhibit PKC-mediated [Ca(2+)]i release from intracellular stores.

Filiform Polyposis Secondary to Colonic Tuberculosis Presenting as Acute Colo-Colonic Intussusception.

Filiform polyposis represents a rare but recognised manifestation on the varied spectrum of histopathology in colonic tuberculosis. We report a case of filiform polyposis secondary to colonic tuberculosis presenting as colo-colonic intussusception diagnosed on an abdominal computed tomography (CT) scan. The patient required urgent hemicolectomy and defunctioning ileostomy. Examination of the resected bowel lesions revealed filiform polyposis. Induced sputum samples from the patient grew Mycobacterium tuberculosis. The patient recovered well from the surgery and received treatment for tuberculosis. At last follow-up, he was awaiting the reversal of his ileostomy. The protean nature of histological findings in colonic tuberculosis and other current diagnostic challenges are discussed. The importance of maintaining a high index of suspicion for colonic tuberculosis and instituting early treatment is highlighted in this case.

Gnotobiotic Human Colon Ex Vivo.

BACKGROUND: A novel emulsion with efficacy as an agent for eliminating biofilms was selected. The aim of this study was to examine efficacy and effect of a formulation of ML:8 against commensal bacteria harvested from ex vivo human colonic tissues. METHODS: Mucosal sheets, obtained at the time of surgery, were exposed for 2 minutes to one of four solutions: Krebs-Hensleit (KH) solution, saline (NaCl; 0.9%), povidone iodine (1%), or ML:8 (2%); n = 4. Lumenal surfaces were swabbed for culture under aerobic or anaerobic conditions. Following treatment, each sheet was mounted in Ussing chambers and voltage clamped. Tissues were challenged with carbachol. Permeability coefficient (Papp) was determined using mannitol fluxes. At the end of each experiment, tissues were examined histologically. RESULTS: Similar colony forming units grew in aerobic and anaerobic conditions in both control and NaCl treated tissues. Iodine reduced and ML:8 virtually abolished viable bacteria. Basal electrophysiological parameters were not different between treatments. Transepithelial electrical resistance values did not differ between groups. All tissues responded to carbachol, although this was attenuated in iodine treated tissue. Papp values were slightly elevated in all treated tissues but this did not reach significance. Histopathological assessment revealed no overt damage to tissues. CONCLUSION: Brief exposure to ML:8 reduced culturable bacterial burden from human intestinal tissues harvested at the time of surgical resection. Such gnotobiotic tissues retain structural and functional integrity. This is a novel approach to reduce bacterial burden.

Translocation of Vibrio parahaemolyticus across an in vitro M cell model.

Consumption of Vibrio parahaemolyticus via contaminated shellfish results in inflammatory gastroenteritis characterised by severe diarrhoea, nausea and stomach cramps. This study investigated the translocation of V. parahaemolyticus across a Peyer's patch M cell-like Caco-2/Raji B co-culture model system, as M cells represent a primary site of infection for many pathogenic bacteria. Vibrio parahaemolyticus translocated across co-culture monolayers in higher numbers as compared to Caco-2 monolayers. Moreover, the bacteria induced a greater disruption of the transepithelial resistance in M cell-like co-cultures than in Caco-2 monocultures. Virulence factors associated with this pathogen include two type three secretion systems (TTSS-1 and TTSS-2). TTSS-1 had no effect on translocation efficiency, with TTSS-2 exhibiting a modest enhancing effect. ERK activity was required for optimal translocation 1 h postinfection, however, neither ERK nor the JNK and p38 MAPK were required at 2 h pi. Additionally, TER disruption in response to bacterial infection occurred independently of the TTSS and MAPK activation. It was concluded that V. parahaemolyticus causes TER disruption of M cell-like co-cultures and translocates in high numbers across the M cell-like co-culture monolayer. These data implicate M cells as important sites for V. parahaemolyticus invasion across the intestinal epithelium during infection.

Activation of AMPK inhibits cholera toxin stimulated chloride secretion in human and murine intestine.

Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.

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.