Viral and cellular determinants of polarized trafficking of viral envelope proteins from insect-specific and insect-vectored viruses in insect midgut and salivary gland cells.

Systemic viral infection of insects typically begins with the primary infection of midgut epithelial cells (enterocytes) and subsequent transit of the progeny virus in an apical-to-basal orientation into the hemocoel. For insect-vectored viruses, an oppositely oriented process (basal-to-apical transit) occurs upon secondary infection of salivary glands and is necessary for virus transmission to non-insect hosts. To examine this inversely oriented virus transit in these polarized tissues, we assessed the intracellular trafficking of two model viral envelope proteins (baculovirus GP64 and vesicular stomatitis virus G) in the midgut and salivary gland cells of the model insect, Drosophila melanogaster. Using fly lines that inducibly express either GP64 or VSV G, we found that each protein, expressed alone, was trafficked basally in midgut enterocytes. In salivary gland cells, VSV G was trafficked apically in most but not all cells, whereas GP64 was consistently trafficked basally. We demonstrated that a YxxØ motif present in both proteins was critical for basal trafficking in midgut enterocytes but dispensable for trafficking in salivary gland cells. Using RNAi, we found that clathrin adaptor protein complexes AP-1 and AP-3, as well as seven Rab GTPases, were involved in polarized VSV G trafficking in midgut enterocytes. Our results indicate that these viral envelope proteins encode the requisite information and require no other viral factors for appropriately polarized trafficking. In addition, they exploit tissue-specific differences in protein trafficking pathways to facilitate virus egress in the appropriate orientation for establishing systemic infections and vectoring infection to other hosts. IMPORTANCE: Viruses that use insects as hosts must navigate specific routes through different insect tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Both insect pathogenic viruses and insect-vectored viruses must navigate through the polarized cells of the midgut epithelium to establish a systemic infection. In addition, insect-vectored viruses must also navigate through the polarized salivary gland epithelium for transmission. Thus, insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins (VSV G and baculovirus GP64) alone encode the signals necessary for the polarized trafficking associated with their respective life cycles. Using Drosophila as a model to examine tissue-specific polarized trafficking of these viral envelope proteins, we identified one of the virus-encoded signals and several host proteins associated with regulating the polarized trafficking in the midgut epithelium.

Effects of ∆-9 tetrahydrocannabinol on the small intestine altered by high fructose diet: A Histopathological study.

The consumption of fructose is increasing day by day. Understanding the impact of increasing fructose consumption on the small intestine is crucial since the small intestine processes fructose into glucose. ∆9-Tetrahydrocannabinol (THC), a key cannabinoid, interacts with CB1 and CB2 receptors in the gastrointestinal tract, potentially mitigating inflammation. Therefore, this study aimed to investigate the effects of the high-fructose diet (HFD) on the jejunum of rats and the role of THC consumption in reversing these effects. Experiments were conducted on Sprague-Dawley rats, with the experimental groups as follows: control (C), HFD, THC, and HFD + THC. The HFD group received a 10% fructose solution in drinking water for 12 weeks. THC groups were administered 1.5 mg/kg/day of THC intraperitoneally for the last four weeks. Following sacrification, the jejunum was evaluated for mucus secretion capacity. IL-6, JNK, CB2 and PCNA expressions were assessed through immunohistochemical analysis and the ultrastructural alterations via transmission electron microscopy. The results showed that fructose consumption did not cause weight gain but triggered inflammation in the jejunum, disrupted the cell proliferation balance, and increased mucus secretion in rats. Conversely, THC treatment displayed suppressed inflammation and improved cell proliferation balance caused by HFD. Ultrastructural examinations showed that the zonula occludens structures deteriorated in the HFD group, along with desmosome shrinkage. Mitochondria were found to be increased due to THC application following HFD. In conclusion, the findings of this research reveal the therapeutic potential of THC in reversing HFD-related alterations and provide valuable insights for clinical application.

How the Microbiota May Affect Celiac Disease and What We Can Do.

Celiac disease (CeD) is an autoimmune disease with a strong association with human leukocyte antigen (HLA), characterized by the production of specific autoantibodies and immune-mediated enterocyte killing. CeD is a unique autoimmune condition, as it is the only one in which the environmental trigger is known: gluten, a storage protein present in wheat, barley, and rye. How and when the loss of tolerance of the intestinal mucosa to gluten occurs is still unknown. This event, through the activation of adaptive immune responses, enhances epithelial cell death, increases the permeability of the epithelial barrier, and induces secretion of pro-inflammatory cytokines, resulting in the transition from genetic predisposition to the actual onset of the disease. While the role of gastrointestinal infections as a possible trigger has been considered on the basis of a possible mechanism of antigen mimicry, a more likely alternative mechanism appears to involve a complex disruption of the gastrointestinal microbiota ecosystem triggered by infections, rather than the specific effect of a single pathogen on intestinal mucosal homeostasis. Several lines of evidence show the existence of intestinal dysbiosis that precedes the onset of CeD in genetically at-risk subjects, characterized by the loss of protective bacterial elements that both epigenetically and functionally can influence the response of the intestinal epithelium leading to the loss of gluten tolerance. We have conducted a literature review in order to summarize the current knowledge about the complex and in part still unraveled dysbiosis that precedes and accompanies CeD and present some exciting new data on how this dysbiosis might be prevented and/or counteracted. The literature search was conducted on PubMed.gov in the time frame 2010 to March 2024 utilizing the terms "celiac disease and microbiota", "celiac disease and microbiome", and "celiac disease and probiotics" and restricting the search to the following article types: Clinical Trials, Meta-Analysis, Review, and Systematic Review. A total of 364 papers were identified and reviewed. The main conclusions of this review can be outlined as follows: (1) quantitative and qualitative changes in gut microbiota have been clearly documented in CeD patients; (2) intestinal microbiota's extensive and variable interactions with enterocytes, viral and bacterial pathogens and even gluten combine to impact the inflammatory immune response to gluten and the loss of gluten tolerance, ultimately affecting the pathogenesis, progression, and clinical expression of CeD; (3) gluten-free diet fails to restore the eubiosis of the digestive tract in CeD patients, and also negatively affects microbial homeostasis; (4) new tools allowing targeted microbiota therapy, such as the use of probiotics (a good example being precision probiotics like the novel strain of B. vulgatus (20220303-A2) begin to show exciting potential applications.

Intestinal absorption of sphingosine: new insights on generated ceramide species using stable isotope tracing in vitro.

Dietary sphingomyelin (SM) has been reported to favorably modulate postprandial lipemia. Mechanisms underlying these beneficial effects on cardiovascular risk markers are not fully elucidated. Rodent studies showed that tritiated SM was hydrolyzed in the intestinal lumen into ceramides (Cer) and further to sphingosine (SPH) and fatty acids (FA) that were absorbed by the intestine. Our objective was to investigate the uptake and metabolism of SPH and/or tricosanoic acid (C23:0), the main FA of milk SM, as well as lipid secretion in Caco-2/TC7 cells cultured on semipermeable inserts. Mixed micelles (MM) consisting of different digested lipids and taurocholate were prepared without or with SPH, SPH and C23:0 (SPH+C23:0), or C23:0. Triglycerides (TG) were quantified in the basolateral medium, and sphingolipids were analyzed by tandem mass spectrometry. TG secretion increased 11-fold in all MM-incubated cells compared with lipid-free medium. Apical supply of SPH-enriched MM led to increased concentrations of total Cer in cells, and coaddition of C23:0 in SPH-enriched MM led to a preferential increase of C23:0 Cer and C23:0 SM. Complementary experiments using deuterated SPH demonstrated that SPH-d9 was partly converted to sphingosine-1-phosphate-d9, Cer-d9, and SM-d9 within cells incubated with SPH-enriched MM. A few Cer-d9 (2% of added SPH-d9) was recovered in the basolateral medium of (MM+SPH)-incubated cells, especially C23:0 Cer-d9 in (MM+SPH+C23:0)-enriched cells. In conclusion, present results indicate that MM enriched with (SPH+C23:0), such as found in postprandial micelles formed after milk SM ingestion, directly impacts sphingolipid endogenous metabolism in enterocytes, resulting in the secretion of TG-rich particles enriched with C23:0 Cer.

A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium.

Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

Air-liquid interface culture and modified culture medium promote the differentiation of human induced pluripotent stem cells into intestinal epithelial cells.

An in vitro system that evaluates pharmacokinetics in the small intestine is crucial for the development of oral drugs. We produced human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) with high drug metabolizing enzyme and drug transporter activities. However, the gene expression of our hiSIECs partially differed from that of the human small intestine, with low drug metabolizing enzyme activities. Therefore, we used air-liquid interface (ALI) culture and 5-aza-2'-deoxycytidine (5AZA)-free medium to generate hiSIECs (novel hiSIECs). Novel hiSIECs showed enhanced gene expression of drug metabolizing enzymes, such as cytochrome P450 (CYP)3A4, CYP2C9, CYP2C19, and carboxylesterase 2 that are highly expressed in the small intestine. In addition, the expression of genes involved in nutrient absorption-one of the major functions of the small intestine-also increased. The novel hiSIECs expressed ZO-1 and E-cadherin. Moreover, the novel hiSIECs exhibited a barrier function that allowed low lucifer yellow permeation. The novel hiSIECs showed high activities of CYP3A4, CYP2C9, and CYP2C19, which are abundantly expressed in the small intestine. In conclusion, the novel hiSIECs have great potential as an in vitro system to evaluate pharmacokinetics in the small intestine.

Totum-070, a Polyphenol-Rich Plant Extract, Prevents Hypercholesterolemia in High-Fat Diet-Fed Hamsters by Inhibiting Intestinal Cholesterol Absorption.

Atherosclerotic cardiovascular disease is the leading cause of mortality worldwide, and hypercholesterolemia is a central risk factor for atherosclerosis. This study evaluated the effects of Totum-070, a plant-based polyphenol-rich supplement, in hamsters with high-fat diet (HFD)-induced dyslipidemia. The molecular mechanisms of action were explored using human Caco2 enterocytes. Totum-070 supplementation reduced the total cholesterol (-41%), non-HDL cholesterol (-47%), and triglycerides (-46%) in a dose-dependent manner, compared with HFD. HFD-induced hepatic steatosis was also significantly decreased by Totum-070, an effect associated with the reduction in various lipid and inflammatory gene expression. Upon challenging with olive oil gavage, the post-prandial triglyceride levels were strongly reduced. The sterol excretion in the feces was increased in the HFD-Totum-070 groups compared with the HFD group and associated with reduction of intestinal cholesterol absorption. These effects were confirmed in the Caco2 cells, where incubation with Totum-070 inhibited cholesterol uptake and apolipoprotein B secretion. Furthermore, a microbiota composition analysis revealed a strong effect of Totum-070 on the alpha and beta diversity of bacterial species and a significant decrease in the Firmicutes to Bacteroidetes ratio. Altogether, our findings indicate that Totum-070 lowers hypercholesterolemia by reducing intestinal cholesterol absorption, suggesting that its use as dietary supplement may be explored as a new preventive strategy for cardiovascular diseases.

Histología del tubo digestivo de tres especies de pepino de mar Isostichopus badionotus, Isostichopussp.yStichopus hermanni (Aspidochirotida: Stichopodidae)

os pepinos de mar tienen un importante papel ecológico en el medio marino, ya que son capaces de alimentarse de la materia orgánica e inorgánica, con lo cual contribuyen a la oxigenación y la transferencia de energía en el ecosistema. En general, existe una falta de conocimiento de la morfología básica de especies nativas de pepino de mar y la función de los órganos vitales. El objetivo de este estudio fue describir la histología del tracto digestivo (DT) de tres especies de holotúridos de la bahía del Rodadero, Colombia. Treinta ejemplares de Isostichopus badionotus, Isostichopus sp. y Stichopus hermanni se capturaron y se sacrificaron por hipotermia. En el laboratorio se obtuvieron secciones del intestino anterior, medio y posterior y se fijaron en formalina (10 %) para su procesamiento histológico convencional. Además, algunas muestras fueron fijadas en glutaraldehído (3 %) para su inclusión en resinas y estudios en microscopía electrónica de alta resolución. Para las especies estudiadas, el TD es largo, lobulado, y se distribuye en la cavidad celómica; tiene al menos dos veces la longitud del cuerpo del pepino de mar. El TD presenta vellosidades revestidas por un epitelio ciliado columnar pseudoestratificado, que descansa sobre una membrana basal y una capa de fibras de colágeno. Se identificaron cuatro tipos de células: coelocmocitos, células cafes, enterocitos y células mucosas y las espículas fueron evidentes en todo el tejido del TD. La microscopía óptica mostró inclusiones alargadas de formación calcárea situadas esencialmente en el intestino grueso, "los cuerpos psamoma". Técnicas de microscopía de alta resolución y de microscopía electrónica mostraron células mucosas granulares cuya superficie apical contiene numerosas microvellosidades. La conformación tisular de los TD de I. badionotus, Isostichopus sp. y S. hermanni fueron similares. Se encontraron diferencias (p < 0.05) en el espesor del tejido submucosa intestinal de las especies estudiadas, que pueden estar ligadas a los hábitos específicos de alimentación de cada uno. La caracterización morfo-histológica del tracto digestivo del pepino de mar es una herramienta útil para entender su fisiología alimenticia.

ea cucumbers have an important ecological role in the marine environment because they are able to process organic and inorganic matter, which contributes to the oxygenation and energy transfer in the ecosystem.In general, there is a lack of knowledge on the basic morphology of native species of sea cucumber and the function of vital organs. The aim of this study was to describe the histology of the digestive tract (DT) of three species of holothuroids from Rodadero Bay, Colombia. Thirty specimens of Isostichopus badionotus, Isostichopus sp. and Stichopus hermanni were obtained and sacrificed by hypothermia. In the laboratory, sections of foregut, midgut and hindgut were obtained and fixed in formalin (10%) for later conventional histological processes; besides, some samples were fixed in glutaraldehyde (3%) for their inclusion in resins and studies in high resolution and electron microscopy. For the studied species, the DT is long, folded, and is distributed in the coelomic cavity; it has at least twice the length of the sea cucumber body. The DT presents villi lined by a columnar pseudostratified ciliated epithelium, which rests on a basement membrane and a layer of collagen fibers. Four types of cells were identified: coelocmocytes, brown cells, enterocytes and mucous cells, and the spicules were evident throughout the digestive tract tissue. Light microscopy showed elongated inclusions of calcareous formation located essentially in the hindgut, "the psamoma bodies". We observed granular mucous cells with an apical surface with numerous microvilli. The histology of the DT of I. badionotus, Isostichopus sp. and S. hermanni were found to be similar, but we found differences (p<0.05) in the thickness of the intestinal submucosa tissue, which can be tied to specific feeding habits of each species. Characterization of the morphohistology of the digestive tract of sea cucumber is a useful tool to understand their feeding physiology. Rev. Biol. Trop. 63 (4): 1021-1033. Epub 2015 December 01.

Intraepithelial lymphocytes in the duodenal mucosa of meat quails receiving diets containing or not containing probiotic and different levels of crude protein / Linfócitos intraepiteliais na mucosa do duodeno de codornas de corte recebendo dietas contendo ou não probiótico e diferentes níveis de proteína bruta / Linfocitos intraepiteliales en la mucosa duodenal de codornices de abate recibiendo dietas con o sin probiótico y diferentes niveles de proteína bruta

The aim of this study is to evaluate the effect of probiotic supplementation associated to different levels of crude protein (CP) on the count of intraepithelial lymphocytes (IELs) in the duodenum of meat quails. A total of 2304 quails were distributed in a completely randomized design in a 2 x 4 factorial scheme (with and without probiotic and four levels of CP - 15, 20, 25 and 30%), with two replicates per treatment in two experimental periods, in a total of 32 experimental units. At seven days old, two quails from each experimental unit were euthanized to harvest the duodenum segment. Semi-serial 7-µm histological sections were obtained, which were subsequently stained with hematoxylin-eosin to perform the IEL count. For the calculation, first 2500 epithelial cells were counted from the mucosa of each animal, and then the IELs present between these cells were counted, with the results expressed in amounts of IELs/100 epithelial cells. No differences were found in the IEL count among the treatments. Under the experimental conditions, it can be concluded that the use of probiotic associated to different levels of CP supplementation does not alter the IEL count in the duodenum of meat quails.

O objetivo deste trabalho foi avaliar o efeito da suplementação de probiótico associado com diferentes níveis de proteína bruta (PB) sobre a contagem de linfócitos intraepiteliais (LIEs) no duodeno de codornas de corte. Um total de 2304 codornas foram distribuídas em um delineamento inteiramente casualizado em esquema fatorial 2 x 4 (com e sem probiótico e quatro níveis de PB ­ 15, 20, 25 e 30%), com duas repetições por tratamento em dois períodos experimentais, totalizando 32 unidades experimentais. Aos sete dias de idade, duas codornas de cada unidade experimental foi eutanasiada para colheita do segmento do duodeno. Cortes histológicos semi-seriados de sete µm foram obtidos, os quais foram posteriormente corados com hematoxilina-eosina para realização da contagem de LIEs. Para o cálculo, primeiro foram contados 2500 células epiteliais da mucosa de cada animal, e então os LIEs presentes entre essas células foram contados e os resultados expressos como quantidade de LIEs /100 células epiteliais. Não foram encontradas diferenças entre os tratamentos na contagem de LIEs. Nas condições em que o experimento foi desenvolvido, conclui-se que o uso de probiótico associado a diferentes níveis de PB não altera a contagem de LIEs do duodeno de codornas de corte.

Esta investigación buscó evaluar el efecto de suplementos probióticos asociados a diferentes niveles de proteína bruta (PB) sobre el contaje de linfocitos intraepiteliales (LIEs) del duodeno de codornices de abate. Un total de 2304 codornices fueron distribuidas en un delineamiento enteramente casualizado en esquema factorial 2x4 (con y sin probiótico y cuatro niveles de PB ­ 15, 20, 25 y 30%), con dos repeticiones por tratamiento, en dos períodos experimentales, totalizando 32 unidades experimentales. A los siete días de edad, dos codornices de cada unidad experimental sufrieron eutanasia, para colecta de segmento del duodeno. Se obtuvo cortes histológicos semi seriados con sete µm, que fueron posteriormente colorados con hematoxilina-eosina para realización de contaje de LIEs. Para el cálculo, primeramente se contó 2500 células del epitelio de la túnica mucosa de cada animal, y en seguida los LIEs presentes entre esas células, siendo los resultados expresos en cantidad de LIEs/100 células epiteliales. No hubo diferencias en el contaje de LIEs en función de los tratamientos. En las condiciones en que el experimento se realizó, se puede concluir que el uso de probiótico asociado a diferentes niveles de suplementos de PB no altera el contaje de LIEs en el duodeno de codornices de abate.

Immunohistochemical studies on duodenum, spleen and liver in mice: distribution of ferroportin and prohepcidin in an inflammation model / Estudios inmunohistoquímicos en duodeno, bazo e hígado de ratón: distribución de ferroportina y prohepcidina en un modelo de inflamación

Duodenum, spleen and liver have a crucial role in iron balance on the whole organism and are the major sites of Ferroportin (FPN) expression. Specific regulations between FPN and hepcidin are responsible for changes seen in physiopathological conditions such as inflammation. We studied in vivo effects of turpentine oil-induced acute inflammation on FPN expression, and its relation with prohepcidin and iron mobilization. Immunohistochemical procedures were performed using rabbit anti-mouse FPN and prohepcidin antibodies with goat-labeled polymer-HRP anti-rabbit (DAB) as secondary antibody. Plasma and tissular iron were also studied. Our results showed a notable expression and redistribution of duodenal FPN to basolateral membrane in turpentine-treated mice, compared with supranuclear and the weak basolateral expression observed in healthy mice. Red pulp macrophages of healthy mice showed FPN-hemosiderin co-localization, compared with turpentine-treated mice which showed lack of FPN. In liver of healthy mice, FPN was seen in Kupffer cells, whereas in turpentine-treated mice decreased. In addition, we observed an increment of hepatic pro-hepcidin with a significant hypoferremia. Our findings demonstrated that acute inflammation induced a differential distribution of FPN, showing a cell type specific response. In macrophages, increased hepatic prohepcidin induced degradation of FPN, resulting in hypoferremia. In enterocytes, the redistribution observed of duodenal FPN reflects a different regulation in this tissue. The observed response of the proteins studied may be part of a cyclical pattern of systemic effects of acute inflammation on mouse tissue.

El duodeno, bazo e hígado desempeñan un rol clave en el balance de Fe del organismo y son los mayores sitios de expresión de ferroportina (FPN). Regulaciones específicas entre FPN y hepcidina son las responsables de los cambios observados en condiciones fisiopatológicas como la inflamación. Nuestro objetivo fue estudiar los efectos in vivo de la inflamación aguda inducida con turpentina sobre la expresión de FPN y su relación con prohepcidina y la movilización de hierro. Los procedimientos inmunohistoquímicos fueron desarrollados utilizando anticuerpos anti FPN y prohepcidina de ratón, desarrollados en conejo y un polímero conjugado con anticuerpos secundarios anti conejo desarrollado en cabra (HRP-DAB). Se evaluaron los niveles de Fe plasmático y tisular. Nuestros resultados mostraron una clara expresión y redistribución de FPN duodenal hacia la membrana basolateral en ratones tratados con turpentina, con respecto a la expresión perinuclear y leve expresión basolateral observada en ratón sano. Macrófagos de la pulpa roja esplénica mostraron co-localización de FPN y hemosiderina, comparado con la ausencia de expresión en ratón tratado con turpentina. En hígado de ratón sano, se observó expresión de FPN en células de Kupffer, mientras que en ratón tratado con turpentina la expresión fue menos evidente. Además, observamos un aumento en la expresión de prohepcidina hepática con una hipoferremia significativa. Nuestros resultados demostraron que la inflamación aguda indujo una distribución diferencial de FPN, mostrando una respuesta específica del tipo celular. En macrófagos, el aumento de prohepcidina hepática indujo degradación de FPN, resultando en hipoferremia. En enterocitos, la redistribución observada de FPN duodenal, refleja una regulación diferente en este tejido. La respuesta observada de las proteínas estudiadas podría ser parte de un patrón cíclico de efectos sistémicos de la inflamación aguda en tejidos murinos.

Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.