SARS-CoV-2 Spike protein triggers gut impairment since mucosal barrier to innermost layers: From basic science to clinical relevance.

Studies have reported the occurrence of gastrointestinal (GI) symptoms, primarily diarrhea, in COVID-19. However, the pathobiology regarding COVID-19 in the GI tract remains limited. This work aimed to evaluate SARS-CoV-2 Spike protein interaction with gut lumen in different experimental approaches. Here, we present a novel experimental model with the inoculation of viral protein in the murine jejunal lumen, in vitro approach with human enterocytes, and molecular docking analysis. Spike protein led to increased intestinal fluid accompanied by Cl- secretion, followed by intestinal edema, leukocyte infiltration, reduced glutathione levels, and increased cytokine levels [interleukin (IL)-6, tumor necrosis factor-α, IL-1ß, IL-10], indicating inflammation. Additionally, the viral epitope caused disruption in the mucosal histoarchitecture with impairment in Paneth and goblet cells, including decreased lysozyme and mucin, respectively. Upregulation of toll-like receptor 2 and toll-like receptor 4 gene expression suggested potential activation of local innate immunity. Moreover, this experimental model exhibited reduced contractile responses in jejunal smooth muscle. In barrier function, there was a decrease in transepithelial electrical resistance and alterations in the expression of tight junction proteins in the murine jejunal epithelium. Additionally, paracellular intestinal permeability increased in human enterocytes. Finally, in silico data revealed that the Spike protein interacts with cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride conductance (CaCC), inferring its role in the secretory effect. Taken together, all the events observed point to gut impairment, affecting the mucosal barrier to the innermost layers, establishing a successful experimental model for studying COVID-19 in the GI context.

Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis.

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.

Clinical investigation of intestinal fatty acid-binding protein (I-FABP) as a biomarker of SARS-CoV-2 infection.

OBJECTIVES: SARS-CoV-2 exhibits tropism for the gastrointestinal tract; however, lesions in enterocytes and their correlation with disease severity and patient prognosis are still unknown. METHODS: SARS-CoV-2 patients were enrolled in 5 medical centres in São Paulo, Brazil and their clinical characteristics and laboratory findings recorded. At admission, day 7 and day 14 of hospitalisation, plasma and urine samples were collected, and cytokine levels and intestinal fatty acid-binding protein (I-FABP) concentrations measured. RESULTS: COVID-19 patients displayed ≈48-, 74- and 125-fold increased urinary I-FABP levels at admission (n=283; P<0.001), day 7 (n=142; P<0.01) and day 14 (n=75; P<0.01) of hospitalisation. Critically ill patients and nonsurvivors showed higher I-FABP concentrations compared with patients with less severe illness. At admission, infected patients demonstrated enhanced production of plasma interferon (IFN)-γ and interleukin (IL)-6. The receiver operating characteristic curve suggested I-FABP as a biomarker for COVID-19 disease severity at admission (P<0.0001; Youden index=6.89; area under the curve=0.699). Patients with I-FABP ≥6.89 showed higher IL-6 and C-reactive protein levels (P<0.001) at admission and had a prolonged length of hospital stay. CONCLUSIONS: Our findings revealed damage to enterocytes in SARS-CoV-2 infection, which is associated with illness severity, poor prognosis and exacerbated inflammatory response.

Morphological and biochemical effects of food deprivation during the early development of Pacific red snapper Lutjanus peru.

We report the effects of food deprivation on the early development of Pacific red snapper Lutjanus peru during the first days of development. The point of no return (PNR) was determined using the feeding incidence after a delay in first feeding. The gradual deterioration of the larvae during food deprivation was recorded using morphometric, histological, enzymatic and biochemical analysis. The time to reach the PNR was 120 h after hatching. Morphologically, the total length, muscle height, head length, tail length and pectoral angle showed the biggest reductions and their growth coefficients changed significantly during food deprivation. Histologically, enterocyte height also was reduced significantly. The protein concentration and activities of the digestive enzymes trypsin, cathepsin-like and lipase showed a significant decrease; meanwhile, amylase activity remained constant during food deprivation. The concentration of total essential free amino acids (EFAAs) decreased significantly while that of the nonessential free amino acids (NEFAAs) remain stable during food deprivation. The most abundant EFAAs were lysine, leucine, isoleucine and valine; the most abundant NEFAAs were alanine, glycine and glutamate, suggesting a more prominent role as energy substrates. At the time of the PNR the concentration of almost all the free amino acids showed a significant decrease. Early food deprivation has a significant impact on the morphology and biochemical characteristics of L. peru. These results suggest that initial feeding of L. peru should begin within 3 days of yolk sac depletion to avoid the PNR. Further studies are necessary to confirm and validate the characters identified in this study as biomarkers of starvation under culture conditions and evaluate their possible utility in ichthyoplankton surveys.

Identificación del fenotipo de inestabilidad microsatelital en carcinoma colorrectal mediante el análisis de la expresión de proteínas reparadoras del ADN: Revisión narrativa / Identification of microsatellite instability phenotype in colorectal carcinoma through expression analysis of DNA mismatch repair proteins: Narrative review

El carcinoma colorrectal (CCR) es de las primeras causas de mortalidad del mundo, presentando Guatemala una incidencia anual de 7.4/millón de habitantes. El síndrome de Lynch se caracteriza clínicamente por un inicio temprano del CCR con lesiones causadas por alteraciones en genes que codifican proteínas reparadoras.Los microsatélites son regiones del ADN con una unidad repetitiva de uno o más nucleótidos y son susceptibles a errores durante la replicación de ADN de los enterocitos. Existe un sistema de reparación que corrige estos errores. Cuando las proteínas reparadoras de este sistema están mutadas o ausentes, dichos errores del ADN persisten. Estas proteínas reparadoras se expresan en el núcleo de las células colónicas normales y son detecta-bles utilizando estudios de inmunohistoquímica (IHQ). Los genes MLH1 y MSH2 pueden encontrarse mutados en el 90% de los casos de cáncer colorrectal y el resto corresponde a MSH6 y PMS2. Esta vía oncogénica se caracteriza por alteración del sistema de reparación de errores durante la replicación del ADN, controlado por los genes MMR (mismatch repair), principalmente MLH1, MSH2, MSH6 y PMS2. Se realizó una revisión extensa de la literatura en PubMed, Springer y JAMA, usando las palabras clave: fenotipo de CCR, Síndrome de Lynch e inestabilidad microsatelital, detectándose 55 artículos. El objetivo de esta revisión es describir la importancia de la identificación del fenotipo del CCR por medios de IHQ y de pruebas moleculares para el eficaz tratamiento con inmunoterapia anti-PD1/PD-L1.

Colorectal cancer (CRC) is one of the leading causes of mortality in the world. In Guatemala it's an important cause of morbidity (7.4 per million inhabitants). Lynch syndrome is clinically characterized by an early onset of nonpolyposis colorectal carcinoma, with multiple lesions and neoplasms. The syndrome is caused by mutations in genes encoding DNA mismatch repair proteins. The microsatellites are regions of the DNA that repeat between one or more nucleotides and are susceptible to errors during replication, these are corrected by a repair system, when genes are mutated, the errors persist. The genes encoding repair proteins are expressed in the nuclei of normal colonic cells which can be observed using immunohistochemical studies. The MLH1, MSH2 genes are found to be mutated in 90% of the cases and the rest corresponds to the MSH6 and PMS2 genes. This oncogenic pathway characteristically consists of an alteration in the DNA repair system that is controlled by mismatch repair genes (MMR). An extensive research was conducted on PubMed, Springer and JAMA, using the keyword: CRC phenotype, Lynch syndrome and microsatellite instability. 55 articles were found. This review«s objective is to understand the mechanisms of nonpolyposis colorectal cancer and the importance of identifying patients with a mutant phenotype as a predictive factor for the efficacy of the anti-PD1/PDL1 immunotherapy and for prognosis.

Premises among SARS-CoV-2, dysbiosis and diarrhea: Walking through the ACE2/mTOR/autophagy route.

Recently, a new coronavirus (SARS-CoV-2) was discovered in China. Due to its high level of contagion, it has already reached most countries, quickly becoming a pandemic. Although the most common symptoms are related to breathing problems, SARS-CoV-2 infections also affect the gastrointestinal tract culminating in inflammation and diarrhea. However, the mechanisms related to these enteric manifestations are still not well understood. Evidence shows that the SARS-CoV-2 binds to the angiotensin-converting enzyme receptor 2 (ACE2) in host cells as a viral invasion mechanism and can infect the lungs and the gut. Other viruses have already been linked to intestinal symptoms through binding to ACE2. In turn, this medical hypothesis article conjectures that the ACE2 downregulation caused by the SARS-CoV-2 internalization could lead to decreased activation of the mechanistic target of mTOR with increased autophagy and lead to intestinal dysbiosis, resulting in diarrhea. Besides that, dysbiosis can directly affect the respiratory system through the lungs. Although there are clues to other viruses that modulate the ACE2/gut/lungs axis, including the participation of autophagy and dysbiosis in the development of gastrointestinal symptoms, there is still no evidence of the ACE2/mTOR/autophagy pathway in SARS-CoV-2 infections. Thus, we propose that the new coronavirus causes a change in the intestinal microbiota, which culminates in a diarrheal process through the ACE2/mTOR/autophagy pathway into enterocytes. Our assumption is supported by premises that unregulated intestinal microbiota increases the susceptibility to other diseases and extra-intestinal manifestations, which can even cause remote damage in lungs. These putative connections lead us to suggest and encourage future studies aiming at assessing the aforementioned hypothesis and regulating dysbiosis caused by SARS-CoV-2 infection, in order to confirm the decrease in lung injuries and the improvement in the prognosis of the disease.

Effects of paralytic shellfish toxins on the middle intestine of Oncorhynchus mykiss: Glutathione metabolism, oxidative status, lysosomal function and ATP-binding cassette class C (ABCC) proteins activity.

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.

Morphological bases for intestinal paracellular absorption in bats and rodents.

Flying mammals present unique intestinal adaptations, such as lower intestinal surface area than nonflying mammals, and they compensate for this with higher paracellular absorption of glucose. There is no consensus about the mechanistic bases for this physiological phenomenon. The surface area of the small intestine is a key determinant of the absorptive capacity by both the transcellular and the paracellular pathways; thus, information about intestinal surface area and micro-anatomical structure can help explain differences among species in absorptive capacity. In order to elucidate a possible mechanism for the high paracellular nutrient absorption in bats, we performed a comparative analysis of intestinal villi architecture and enterocyte size and number in microchiropterans and rodents. We collected data from intestines of six bat species and five rodent species using hematoxylin and eosin staining and histological measurements. For the analysis we added measurements from published studies employing similar methodology, making in total a comparison of nine species each of rodents and bats. Bats presented shorter intestines than rodents. After correction for body size differences, bats had ~41% less nominal surface area (NSA) than rodents. Villous enhancement of surface area (SEF) was ~64% greater in bats than in rodents, mainly because of longer villi and a greater density of villi in bat intestines. Both taxa exhibited similar enterocyte diameter. Bats exceeded rodents by ~103% in enterocyte density per cm2 NSA, but they do not significantly differ in total number of enterocytes per whole animal. In addition, there is a correlation between SEF and clearance per cm2 NSA of L-arabinose, a nonactively transported paracellular probe. We infer that an increased enterocyte density per cm2 NSA corresponds to increased density of tight junctions per cm2 NSA, which provides a partial mechanistic explanation for understanding the high paracellular absorption observed in bats compared to nonflying mammals.

Morphological characterization of the adherence and invasion of Streptococcus agalactiae to the intestinal mucosa of tilapia Oreochromis sp.: An in vitro model.

Streptococcosis in tilapia Oreochromis sp. is possibly the most important bacterial disease for fish production worldwide. In Colombia, streptococcosis is caused by Streptococcus agalactiae (GBS), but in other countries, Streptococcus iniae is also involved. Prevention of streptococcosis is required and must be addressed for economic, social, international trade and public health reasons. This research used an in vitro culture of tilapia intestine to detail the intestinal mucosal response once the pathogen contacts the epithelium. We show that S. agalactiae sheds off its capsule to adhere to the epithelium. The bacterium adheres as a single individuum, in groups or in chains and is able to divide on the apical border of enterocytes. GBS adheres at and invades exclusively through the apical portion of the intestinal folds, using the transepithelial route. Once within the cytoplasm of enterocytes, the bacteria continue to divide. On the basolateral side of the epithelium, the microorganisms leave the cells to reach the propria and travel through the microcirculation. No evidence of an immuno-inflammatory reaction or goblet cell response in the epithelium or the lamina propria was seen during the process of adherence and invasion of the pathogen.

[Role of the enterocyte in type 2 diabetes mellitus associated dyslipidemia]. / Rol del enterocito en la dislipidemia de la diabetes mellitus tipo 2.

In type 2 diabetes mellitus there is an overproduction of chylomicron in the postprandial state that is associated with increased cardiovascular risk. Current evidence points out a leading role of enterocyte in dyslipidemia of type 2 diabetes mellitus, since it increases the production of apolipoprotein B-48 in response to a raise in plasma free fatty acids and glucose. The chylomicron metabolism is regulated by many factors apart from ingested fat, including hormonal and metabolic elements. More recently, studies about the role of gut hormones, have demonstrated that glucagon-like peptide-1 decreases the production of apolipoprotein B-48 and glucagon-like peptide-2 enhances it. Insulin acutely inhibits intestinal chylomicron production in healthy humans, whereas this acute inhibitory effect on apolipoprotein B-48 production is blunted in type 2 diabetes mellitus. Understanding these emerging regulators of intestinal chylomicron secretion may offer new mechanisms of control for its metabolism and provide novel therapeutic strategies focalized in type 2 diabetes mellitus postprandial hyperlipidemia with the reduction of cardiovascular disease risk.

Rol del enterocito en la dislipidemia de la diabetes mellitus tipo 2 / Role of the enterocyte in type 2 diabetes mellitus associated dyslipidemia

En la diabetes mellitus tipo 2 el aumento en la producción de quilomicrón en el estado post-prandial se asocia a mayor riesgo cardiovascular. La evidencia actual posiciona al enterocito como actor principal en la dislipemia de la diabetes mellitus tipo 2 debido a que aumenta la producción de apolipoproteína B-48 en respuesta a una elevación de ácidos grasos libres y glucosa. El metabolismo del quilomicrón se encuentra regulado por múltiples factores independientes además de la ingesta de grasa alimentaria. Entre estos factores se destacan las hormonas intestinales, como el péptido similar al glucagón tipo 1 que disminuye la producción de apolipoproteína B-48 y el péptido similar al glucagón tipo 2 que la aumenta. Por otro lado, la insulina inhibe de forma aguda la producción de quilomicrón en el sujeto sano mientras que en la diabetes mellitus tipo 2, este efecto está ausente. La comprensión de los factores reguladores emergentes de la secreción de quilomicrón permite vislumbrar nuevos mecanismos de control en su metabolismo y aportar estrategias terapéuticas focalizadas en la hiperlipidemia posprandial en la diabetes mellitus tipo 2 con la reducción del riesgo cardiovascular.

In type 2 diabetes mellitus there is an overproduction of chylomicron in the postprandial state that is associated with increased cardiovascular risk. Current evidence points out a leading role of enterocyte in dyslipidemia of type 2 diabetes mellitus, since it increases the production of apolipoprotein B-48 in response to a raise in plasma free fatty acids and glucose. The chylomicron metabolism is regulated by many factors apart from ingested fat, including hormonal and metabolic elements. More recently, studies about the role of gut hormones, have demonstrated that glucagon-like peptide-1 decreases the production of apolipoprotein B-48 and glucagon-like peptide-2 enhances it. Insulin acutely inhibits intestinal chylomicron production in healthy humans, whereas this acute inhibitory effect on apolipoprotein B-48 production is blunted in type 2 diabetes mellitus. Understanding these emerging regulators of intestinal chylomicron secretion may offer new mechanisms of control for its metabolism and provide novel therapeutic strategies focalized in type 2 diabetes mellitus postprandial hyperlipidemia with the reduction of cardiovascular disease risk.

Microtubules regulate brush border formation.

Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.

Impact of chronic and acute inflammation on extra- and intracellular iron homeostasis.

Inflammation has a major impact on iron homeostasis. This review focuses on acute and chronic inflammation as it affects iron trafficking and, as a result, the availability of this essential micronutrient to the host. In situations of microbial infection, not only the host is affected but also the offending microorganisms, which, in general, not only require iron for their own growth but have evolved mechanisms to obtain it from the infected host. Key players in mammalian iron trafficking include several types of cells important to iron acquisition, homeostasis, and hematopoiesis (enterocytes, hepatocytes, macrophages, hematopoietic cells, and in the case of pregnancy, placental syncytiotrophoblast cells) and several forms of chaperone proteins, including, for nonheme iron, the transport protein transferrin and the intracellular iron-storage protein ferritin, and for heme iron, the chaperone proteins haptoglobin and hemopexin. Additional key players are the cell membrane-associated iron transporters, particularly ferroportin (FPN), the only protein known to modulate iron export from cells, and finally, the iron-regulatory hormone hepcidin, which, in addition to having antibacterial activity, regulates the functions of FPN. Interestingly, the impact of infection on iron homeostasis differs among pathogens whose mode of infection is mainly intracellular or extracellular. Understanding how inflammation affects each of these processes may be crucial for understanding how inflammation affects iron status, indicators of iron sufficiency, and iron supplementation during inflammation and how it may potentially result in a beneficial or detrimental impact on the host.

Toxoplasma gondii infection causes structural changes in the jejunum of rats infected with different inoculum doses.

AIM: To evaluate the mucosal tunic and submucosal plexus of the jejunum of rats infected with different inoculum doses of Toxoplasma gondii. MAIN METHODS: Rats were infected with different inoculum doses (50, 500, 1000 and 5000 oocysts) of the T. gondii for 30days, while a control group (CG) received saline solution. Blood and feces were collected before euthanasia for analysis of blood and fecal leukocytes (LEs). Histological analysis of the mucosa, submucosa, villi, crypts and enterocytes were performed. Goblet cells, intraepithelial lymphocytes (IELs) and Paneth cells were quantified. Immunohistochemistry was used to assess enteroendocrine serotonergic (5HT-IR) cells, proliferative cells (PCNA+) and mast cells. Whole mounts were obtained to determine the total submucosal neurons by Giemsa staining and metabolically active neurons (NADH-d+), nitrergic neurons (NADPH-d+) and glial cells (S100). KEY FINDINGS: An increase in blood LEs was observed 30days post-infection (dpi). Fecal LEs were more abundant in the feces in all infected groups at 21 dpi when compared to the CG. The number of IELs, sulfomucin-producing goblet cells, Paneth cells, PCNA+ cells and mast cells increased, whereas the number of 5HT-IR cells decreased. The jejunal architecture was altered, with atrophy of the mucosa, submucosa, villi and crypts. The number of total submucosal neurons decreased, but the NADPH-d+ subpopulation increased. SIGNIFICANCE: The results show how chronic toxoplasmic infection affects the tissue and cellular composition of the rat jejunum. These structural changes tend to intensify with the inoculum dose, demonstrating the importance of the parasitic load on intestinal alterations.

FABP1 knockdown in human enterocytes impairs proliferation and alters lipid metabolism.

Fatty Acid-Binding Proteins (FABPs) are abundant intracellular proteins that bind long chain fatty acids (FA) and have been related with inmunometabolic diseases. Intestinal epithelial cells express two isoforms of FABPs: liver FABP (LFABP or FABP1) and intestinal FABP (IFABP or FABP2). They are thought to be associated with intracellular dietary lipid transport and trafficking towards diverse cell fates. But still their specific functions are not well understood. To study FABP1's functions, we generated an FABP1 knockdown model in Caco-2 cell line by stable antisense cDNA transfection (FABP1as). In these cells FABP1 expression was reduced up to 87%. No compensatory increase in FABP2 was observed, strengthening the idea of differential functions of both isoforms. In differentiated FABP1as cells, apical administration of oleate showed a decrease in its initial uptake rate and in long term incorporation compared with control cells. FABP1 depletion also reduced basolateral oleate secretion. The secreted oleate distribution showed an increase in FA/triacylglyceride ratio compared to control cells, probably due to FABP1's role in chylomicron assembly. Interestingly, FABP1as cells exhibited a dramatic decrease in proliferation rate. A reduction in oleate uptake as well as a decrease in its incorporation into the phospholipid fraction was observed in proliferating cells. Overall, our studies indicate that FABP1 is essential for proper lipid metabolism in differentiated enterocytes, particularly concerning fatty acids uptake and its basolateral secretion. Moreover, we show that FABP1 is required for enterocyte proliferation, suggesting that it may contribute to intestinal homeostasis.

Lawsonia intracellularis in Pigs: Progression of Lesions and Involvement of Apoptosis.

The purpose of this study was to follow the progression of gross and histologic lesions and apoptosis events in Lawsonia intracellularis-infected enterocytes through the course of the disease, proliferative enteropathy (PE). Thirty 5-week-old pigs were divided into 2 groups: 20 challenged and 10 control animals. Groups of 3 pigs, 2 challenged and 1 control, were euthanized at 1, 3, 5, 8, 11, 15, 19, 24, 29, and 35 days after inoculation. Complete necropsies were performed with gross evaluation. Tissue samples from different sites of the gastrointestinal tract and other visceral organs were collected for routine histologic staining and for immunohistochemistry (IHC) for L. intracellularis. In addition, caspase-3, terminal deoxyuridine nick-end labeling assay, and electron microscopy were performed in ileum samples. Macroscopic and histologic lesions suggestive of PE were first detected 11 days after infection and continued through day 24. L. intracellularis antigen was first detected in the intestine by IHC on day 5 after inoculation, and the bacterium was first detected by transmission electron microscopy on day 15. Positive IHC staining for [L. intracellularis] and enterocyte proliferation, but no gross lesion, were detected on day 29. All 3 pigs euthanized on day 35 were grossly and histologically normal and IHC negative. Hyperplastic crypts in challenge pigs had more apoptotic cells on days 15, 19, and 24 postinfection ( P < .05) compared to control pigs. Our results demonstrated the progression of lesions and infection by L. intracellularis and that inhibition of enterocyte apoptosis is not involved in the pathogenesis of proliferative enteropathy.

Detection of high biliary and fecal viral loads in patients with chronic hepatitis C virus infection.

BACKGROUND: The life cycle of the hepatitis C virus (HCV) is closely associated with lipid metabolism. Recently, NPC1L1 (a cholesterol transporter) has been reported to function as an HCV receptor. This receptor is expressed in the hepatocyte canalicular membrane and in the intestine; serving as a key transporter for the cholesterol enterohepatic cycle. OBJECTIVES: We hypothesized that HCV might have a similar cycle, so we aimed to study the presence of HCV in bile and stools of infected patients. MATERIALS AND METHODS: Blood, feces, and duodenal bile samples were collected from patients infected with HCV. The biliary viral load was normalized to the bile salt concentration of each sample and the presence of HCV core protein was also evaluated. A total of 12 patients were recruited. HCV RNA was detected in the bile from ten patients. RESULTS: The mean viral load was 2.5log10IU/60mg bile salt. In the stool samples, HCV RNA was detected in ten patients (mean concentration 2.7log10IU/g of feces). CONCLUSIONS: HCV RNA is readily detectable and is present at relatively high concentrations in the bile and stool samples of infected patients. This may be relevant as a source of infection in men who have sex with men. Biliary HCV secretion may perhaps play a role in the persistence of viral infection via an enterohepatic cycle of the virus or intrahepatic spread.

Lithocholic acid: a new emergent protector of intestinal calcium absorption under oxidant conditions.

LCA and 1,25(OH)2D3 are vitamin D receptor ligands with different binding affinity. The secosteroid stimulates intestinal Ca2+ absorption. Whether LCA alters this process remains unknown. The aim of our work was to determine the effect of LCA on intestinal Ca2+ absorption in the absence or presence of NaDOC, bile acid that inhibits the cation transport. The data show that LCA by itself did not alter intestinal Ca2+ absorption, but prevented the inhibitory effect of NaDOC. The concomitant administration of LCA avoided the reduction of intestinal alkaline phosphatase activity caused by NaDOC. In addition, LCA blocked a decrease caused by NaDOC on gene and protein expression of molecules involved in the transcellular pathway of intestinal Ca2+ absorption. The oxidative stress and apoptosis triggered by NaDOC were abrogated by LCA co-treatment. In conclusion, LCA placed in the intestinal lumen protects intestinal Ca2+ absorption against the inhibitory effects caused by NaDOC. LCA avoids the reduction of the transcellular Ca2+ movement, apparently by blocking the oxidative stress and apoptosis triggered by NaDOC, normalizing the gene and protein expression of molecules involved in Ca2+ movement. Therefore, LCA might become a possible treatment to improve intestinal calcium absorption under oxidant conditions.

Dynamic regulation of extracellular ATP in Escherichia coli.

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

Escherichia albertii, a novel human enteropathogen, colonizes rat enterocytes and translocates to extra-intestinal sites.

Diarrhea is the second leading cause of death of children up to five years old in the developing countries. Among the etiological diarrheal agents are atypical enteropathogenic Escherichia coli (aEPEC), one of the diarrheagenic E. coli pathotypes that affects children and adults, even in developed countries. Currently, genotypic and biochemical approaches have helped to demonstrate that some strains classified as aEPEC are actually E. albertii, a recently recognized human enteropathogen. Studies on particular strains are necessary to explore their virulence potential in order to further understand the underlying mechanisms of E. albertii infections. Here we demonstrated for the first time that infection of fragments of rat intestinal mucosa is a useful tool to study the initial steps of E. albertii colonization. We also observed that an E. albertii strain can translocate from the intestinal lumen to Mesenteric Lymph Nodes and liver in a rat model. Based on our finding of bacterial translocation, we investigated how E. albertii might cross the intestinal epithelium by performing infections of M-like cells in vitro to identify the potential in vivo translocation route. Altogether, our approaches allowed us to draft a general E. albertii infection route from the colonization till the bacterial spreading in vivo.