Enterocyte Brush Border Research Articles

Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers.

In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another.

Myosin Vb Traffics P-Glycoprotein to the Apical Membrane of Intestinal Epithelial Cells

The xenobiotic efflux pump P-glycoprotein is highly expressed on the apical membrane of the gastrointestinal tract, where it regulates the levels of intracellular substrates. P-glycoprotein is altered in disease, but the mechanisms that regulate the levels of P-glycoprotein are still being explored. The molecular motor myosin Vb (Myo5b) traffics diverse cargo to the apical membrane of intestinal epithelial cells. We hypothesized that Myo5b was responsible for the delivery of P-glycoprotein to the apical membrane of enterocytes. We used multiple murine models that lack functional Myo5b or the myosin binding partner Rab11a to analyze P-glycoprotein localization. Pig and human tissue were analyzed to determine P-glycoprotein localization in the setting of MYO5B mutations. Intestinal organoids were used to examine P-glycoprotein trafficking and to assay P-glycoprotein function when MYO5 is inhibited. In mice lacking Myo5b or the binding partner Rab11a, P-glycoprotein was improperly trafficked and had decreased presence in the brush border of enterocytes. Immunostaining of a pig model lacking functional Myo5b and human biopsies from a patient with an inactivating mutation in Myo5b also showed altered localization of intestinal P-glycoprotein. Human intestinal organoids expressing the motorless MYO5B tail domain had colocalization with P-glycoprotein, confirming that P-glycoprotein was trafficked by MYO5B in human enterocytes. Inhibition of MYO5 in human intestinal cell lines and organoids resulted in decreased P-glycoprotein capacity. Additionally, inhibition of MYO5 in human colon cancer cells diminished P-glycoprotein activity and increased cell death in response to a chemotherapeutic drug. Collectively, these data demonstrate that Myo5b is necessary for the apical delivery of P-glycoprotein.

Rotavirus-mediated DGAT1 degradation: A pathophysiological mechanism of viral-induced malabsorptive diarrhea.

Gastroenteritis is among the leading causes of mortality globally in infants and young children, with rotavirus (RV) causing ~258 million episodes of diarrhea and ~128,000 deaths annually in infants and children. RV-induced mechanisms that result in diarrhea are not completely understood, but malabsorption is a contributing factor. RV alters cellular lipid metabolism by inducing lipid droplet (LD) formation as a platform for replication factories named viroplasms. A link between LD formation and gastroenteritis has not been identified. We found that diacylglycerol O-acyltransferase 1 (DGAT1), the terminal step in triacylglycerol synthesis required for LD biogenesis, is degraded in RV-infected cells by a proteasome-mediated mechanism. RV-infected DGAT1-silenced cells show earlier and increased numbers of LD-associated viroplasms per cell that translate into a fourfold-to-fivefold increase in viral yield (P < 0.05). Interestingly, DGAT1 deficiency in children is associated with diarrhea due to altered trafficking of key ion transporters to the apical brush border of enterocytes. Confocal microscopy and immunoblot analyses of RV-infected cells and DGAT1-/- human intestinal enteroids (HIEs) show a decrease in expression of nutrient transporters, ion transporters, tight junctional proteins, and cytoskeletal proteins. Increased phospho-eIF2α (eukaryotic initiation factor 2 alpha) in DGAT1-/- HIEs, and RV-infected cells, indicates a mechanism for malabsorptive diarrhea, namely inhibition of translation of cellular proteins critical for nutrient digestion and intestinal absorption. Our study elucidates a pathophysiological mechanism of RV-induced DGAT1 deficiency by protein degradation that mediates malabsorptive diarrhea, as well as a role for lipid metabolism, in the pathogenesis of gastroenteritis.

Lrig1+ small intestinal progenitor cells require MYO5B for proper crypt proliferation and enterocyte maturation

Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea. MVID is caused by inactivating mutations in myosin Vb (MYO5B), a motor protein. Mice lacking MYO5B in their intestinal epithelium have demonstrated the importance of MYO5B in enterocyte brush border formation and epithelial cell differentiation. Additionally, the proliferative zone of the intestinal crypt of MYO5B-deficient mice is elongated compared to controls. We hypothesize that the progenitor cell-specific Cre driver under Lrig1 demonstrates crypt-specific effects caused by MYO5B loss. Lrig1-CreERT2;R26R-YFP and Myo5bfl/fl mice were crossbred to generate Lrig1-CreERT2;R26R-YFP;MYO5B fl/fl (Lrig1 ΔMYO5B ) mice. Adult Lrig1 ΔMYO5B mice and control littermates received tamoxifen injection at day 0. GI tissues were collected 2.5, 3, 4, and 5 days after tamoxifen administration for histological characterization. Lrig1 ΔMYO5B mice began experiencing significant weight loss 4 days after tamoxifen injection and lost 20% of their starting body weight on day 5. At day 5, the Lrig1 ΔMYO5B mouse colon was devoid of solid feces and their small intestine contained clear luminal contents, together suggesting a watery diarrheal phenotype. Enterocytes of Lrig1DMYO5B mice on day 5 contained abnormal cytoplasmic accumulation of PAS staining. Fewer goblet cells were apparent in intestinal sections from day 3 and 5 Lrig1 ΔMYO5B mice compared to controls. Crypts were elongated starting at day 3. Similarly, the PCNA+ epithelial region was expanded in Lrig1 ΔMYO5B mice, even reaching into the villi at day 5. The time course of the PCNA+ region expansion corresponded to the wave of MYO5B-loss beginning at the base of crypts. MYO5B expression was diminished in the crypts at day 3 and nearly absent from the epithelium by day 5. Interestingly, expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), the rate-limited enzyme of ketogenesis that is important for stem cell function, was expanded in the crypt at day 3 and 5 after tamoxifen. Sodium/glucose cotransporter 1 (SGLT1), which is present along the apical surface of mature enterocytes, was progressively diminished and mis-localized away from the apical surface after tamoxifen administration. Apical sodium-dependent bile acid transporter (ASBT), found in the distal small intestine, demonstrated a similar mis-localization away from the apical membrane in enterocytes.We established a novel mouse model that demonstrates progressive MYO5B loss in the intestinal epithelium, starting at the base of the crypt and reaching villi tips over the course of 5 days following Cre induction. This Lrig1 ΔMYO5B strain will allow for lineage tracing studies to better understand the effect of MYO5B loss on intestinal epithelial differentiation and progenitor function. NSF GFRP, NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Сучасні уявлення про значущість забезпечення цинком при інфекційній патології в дітей

Zinc (Zn) is an essential trace element that is critical for growth, development and maintenance of immune function. Zn is an indispensable trace element for humans, which is part of more than 20 metalloenzymes, including DNA and RNA polymerase, phosphatase, carbonic anhydrase and some others. The cycle of cell development requires this trace element. The permeability of cell membranes is also related to Zn. It has antioxidant properties and delays the apoptosis of peripheral cells. The formation of immunity depends on the body’s supply of Zn, and its deficiency causes atrophy of the thymic-lymphatic system. Zn is part of insulin, accelerates the regeneration of the mucous layer of the intestines, increases the activity of enzymes of the brush border of enterocytes, increases the level of secretory antibodies and the intensity of cellular immunity. The provision of this element cannot but affect the course of various infectious pathologies. Purpose - by analyzing modern literature data, to find out the importance of Zn provision in infectious pathologies in children. It is analyzed a review of the literature on the pathogenetic role of Zn in the diagnosis, pathogenesis and treatment of infectious diseases, based on a search for articles in the Science, Medline and PubMed databases published from January 2017 to June 2021. An analysis of current literature has shown that Zn is an important micronutrient involved in the regulation of innate and adaptive immune responses. Zn is necessary to ensure the barrier function of membranes. It is involved in the modulation of the pro-inflammatory response. Zn homeostasis is essential for many aspects of the immune system, including hematopoiesis, cell maturation and differentiation, cell cycle progression, and proper immune cell function. Consequently, Zn deficiency leads to cell-mediated immune dysfunctions. Such dysfunctions lead to deterioration of the body's response to bacterial infection. Zn is known to modulate antiviral immunity. Due to zinc, the production of interferon-α is activated and its antiviral activity is increased. Studies show that during the infectious process there is a decrease in the concentration of zinc in the blood plasma. Such changes, on the one hand, provide the needs of immune protection factors, and on the other hand, reduce the availability of the trace element for pathogens. Conclusions. Zn is one of the important trace elements that plays a leading role in maintaining homeostasis and is an integral component of the pathogenesis of various pathological conditions of infectious origin. The most studied is Zn deficiency in infectious diarrhea in children, but studies have been conducted in developing countries with limited resources. No works studying the prevalence of Zn deficiency in Ukraine were found. No conflict of interests was declared by the authors.

Enteropathy endoscopic findings in small intestine in patients with severe COVID-19

Background and aim: Small intestine study is a challenge in the field of clinical research. However, capsule endoscopy is a safe and minimally invasive alternative. During the COVID-19 pandemic, this endoscopic procedure acquires added value. Although SARS-CoV-2 has mainly respiratory manifestations, gastrointestinal symptoms are observed in 30% of cases. ACE-2 used by SARS-CoV-2 to infect cells is highly expressed in the brush border of enterocytes. This enzyme transports essential amino acids in the small intestine and regulates intestinal inflammation through antimicrobial peptides production. Here we describe the endoscopic changes in the mucosa of the small intestine secondary to severe SARS-CoV-2 infection in patients with and without gastrointestinal symptoms. Methods: We performed a prospective and observational study in hospitalized patients with severe COVID-19 disease. Capsule endoscopy and detection of SARS-CoV-2 RNA in feces were performed in each participant. Each capsule was reviewed separately by two trained endoscopists. Results: Twenty individuals were enrolled. Diarrhea was observed in 78% of participants. In 45% of capsule endoscopy studies, changes characterized by shortening of villi, atrophy or denuded areas accompanied by red spots or patches, hyperemia or edema were found. Individuals showing these changes in the intestinal mucosa were positive for SARS-CoV-2 in stool. Conclusions: Here we provide evidence of macroscopic changes along the small intestine in COVID-19 patients. We proposed the term of COVID-19 enteropathy to denote involvement of the mucosa and in the villi of the small intestine in SARS-CoV-2 infected individuals. Longitudinal follow-up will be valuable for a better understanding of COVID-19 enteropathy.

An adherent-invasive Escherichia coli-colonized mouse model to evaluate microbiota-targeting strategies in Crohn's disease.

Adherent-invasive Escherichia coli (AIEC) were investigated for their involvement in the induction/chronicity of intestinal inflammation in Crohn's disease (CD). AIEC gut establishment is favoured by overexpression of the glycoprotein CEACAM6 in the ileal epithelium. We generated a transgenic mouse model, named 'Vill-hCC6', in which the human CEACAM6 gene was under the control of the villin promoter, conditioning expression in the small intestine. We demonstrated that CEACAM6 is strongly expressed in the small intestine mucosa and is correlated with numerous glycosylations displayed at the brush border of enterocytes. Ex vivo, the AIEC-enterocyte interaction was enhanced by CEACAM6 expression and necessitated the presence of the bacterial adhesive factor FimH. Finally, AIEC bacteria preferentially persisted in a FimH-dependent manner in the ileal mucosa of Vill-hCC6 mice compared to wild-type mice. This preclinical model opens new perspectives in the mechanistic study of the AIEC pathobiont and represents a valuable tool to evaluate the efficacy of new strategies to eliminate AIEC implanted in the ileal mucosa, such as phages, inhibitory and/or anti-virulence molecules, or CRISPR-based strategies targeting virulence or fitness factors of AIEC bacteria.

Effects of Cadmium Exposure on Gut Villi in Danio rerio.

In aquatic organisms, cadmium exposure occurs from ovum to death and the route of absorption is particularly wide, being represented by skin, gills and gastrointestinal tract, through which contaminated water and/or preys are ingested. It is known that cadmium interferes with the gut; however, less information is available on cadmium effects on an important component of the gut, namely goblet cells, specialized in mucus synthesis. In the present work, we studied the effects of two sublethal cadmium concentrations on the gut mucosa of Danio rerio. Particular attention was paid to changes in the distribution of glycan residues, and in metallothionein expression in intestinal cells. The results show that cadmium interferes with gut mucosa and goblet cells features. The effects are dose- and site-dependent, the anterior gut being more markedly affected than the midgut. Cadmium modifies the presence and/or distribution of glycans in the brush border and cytoplasm of enterocytes and in the goblet cells’ cytoplasm and alters the metallothionein expression and localization. The results suggest a significant interference of cadmium with mucosal efficiency, representing a health risk for the organism in direct contact with contamination and indirectly for the trophic chain.

Effect of a Diet Supplemented with Sphingomyelin and Probiotics on Colon Cancer Development in Mice.

Previous studies have reported that dietary sphingomyelin could inhibit early stages of colon cancer. Lactic acid–producing bacteria have also been associated with an amelioration of cancer symptoms. However, little is known about the potential beneficial effects of the combined administration of both sphingomyelin and lactic acid–producing bacteria. This article analyzes the effect of a diet supplemented with a combination of the probiotics Lacticaseibacillus casei and Bifidobacterium bifidum (108 CFU/ml) and sphingomyelin (0.05%) on mice with 1,2-dimethylhydrazine (DMH)-induced colon cancer. Thirty-six BALB/c mice were divided into 3 groups: one healthy group (group C) and two groups with DMH-induced cancer, one fed a standard diet (group D) and the other fed a diet supplemented with sphingomyelin and probiotics (DS). The number of aberrant crypt foci, marker of colon cancer development, was lower in the DS. The dietary supplementation with the synbiotic reversed the cancer-induced impairment of galactose uptake in enterocyte brush–border–membrane vesicles. These results confirm the beneficial effects of the synbiotic on the intestinal physiology of colon cancer mice and contribute to the understanding of the possible mechanisms involved.

Microbiota and Its Impact on the Immune System in COVID-19-A Narrative Review.

The microbiota is of interest for the development of a therapeutic strategy against SARS-CoV-2 coronavirus disease 2019 (COVID-19) due to its impact on the host immune system. Proven communications of the gut microbiota with the pulmonary microbiota (gut–lung axis) and the pathway of neural connections between the gut and brain (gut–brain axis) may be important in the face of the pandemic. SARS-CoV-2 was shown to affect almost all organs because of the presence of a host receptor known as angiotensin converting enzyme 2 (ACE2). The ACE2 receptor is mainly present in the brush border of intestinal enterocytes, ciliary cells, and type II alveolar epithelial cells in the lungs. The transport function of ACE2 has been linked to the ecology of gut microbes in the digestive tract, suggesting that COVID-19 may be related to the gut microbiota. The severity of COVID-19 may be associated with a number of comorbidities, such as hypertension, diabetes, obesity, and/or old age; therefore, attention is also paid to multiple morbidities and the modulation of microbiota through comorbidities and medications. This paper reviews the research in the context of the state of the intestinal microbiota and its impact on the cells of the immune system during the SARS-CoV-2 pandemic.

B0AT1 Amino Acid Transporter Complexed With SARS-CoV-2 Receptor ACE2 Forms a Heterodimer Functional Unit: In Situ Conformation Using Radiation Inactivation Analysis.

The SARS-CoV-2 receptor, angiotensin-converting enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared with all other human tissues. Enterocyte ACE2 is coexpressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2:B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2:B0AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on radiation target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size molecular weight 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2:B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2:B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut–lung axis approaches to understanding COVID-19. These findings advance understanding the physiology of B0AT1 interaction with ACE2 in the gut, and thereby contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul postacute sequelae of SARS-CoV-2.

Revisiting Iron Metabolism, Iron Homeostasis and Iron Deficiency Anemia.

Iron deficiency anemia (IDA) is one of the commonest clinical scenarios especially in children, women of childbearing age, and elders. The crux of this review was revisiting iron homeostasis, mechanism of iron absorption, causes, laboratory diagnosis, and management of IDA. This narrative review is compiled after a relevant literature search from electronic databases including, but not limited to, Google, Google Scholar, PMC, PubMed, Science Direct, and Scopus. The key words used for searching relevant literature include iron, iron deficiency, iron deficiency anemia, iron metabolism, hepcidin, transferrin, causes of iron deficiency anemia, and laboratory diagnosis of iron deficiency anemia. Reference hema-tology books were also consulted. According to the published literature, about one mg of iron is required daily which equals its loss, although the iron requirement increases in pregnancy and lactating mothers. Dietary non heme iron (oxidized Fe3+) is reduced to the ferrous (Fe2+) form by ferrireductase present in the brush border of duodenal enterocytes. Ferrous iron is absorbed in the brush border of duodenal enterocytes through a carrier protein, divalent metal transporter 1 (DMT1). Heme iron is absorbed by the duodenal enterocytes through a mechanism that is not well understood or a receptor yet to be discovered. Transferrin receptor helps in the internalization of iron in the cells. Hepcidin acts as a gatekeeper and controls iron absorption by enterocytes and macrophages. IDA may be caused by decreased intake of iron, increased iron requirements or loss of iron from the body. Iron deficiency anemia is the most common nutritional anemia that affects large numbers of people in developed as well as in developing countries. It is estimated that approximately 2 billion people around the world have IDA. Microcytosis with marked reduction in serum iron, decreased % saturation of transferrin, low ferritin, and reduced or even undetectable hepcidin are the laboratory features of IDA. In addition, total iron binding capacity and soluble transferrin receptors increase significantly in IDA. Management of IDA is incomplete if the underlying cause is not ruled out and left untreated.

Marine-Derived Chitosan Nanoparticles Improved the Intestinal Histo-Morphometrical Features in Association with the Health and Immune Response of Grey Mullet (Liza ramada).

Marine-derived substances are known for their beneficial influences on aquatic animals’ performances and are recommended to improve intestinal health, immunity, and anti-oxidative status. The present study investigates the role of chitosan nanoparticles on the intestinal histo-morphometrical features in association with the health and immune response of Grey Mullet (Liza ramada). Chitosan nanoparticles are included in the diets at 0, 0.5, 1, and 2 g/kg and introduced to fish in a successive feeding trial for eight weeks. The final body weight (FBW), weight gain (WG), and specific growth rate (SGR) parameters are significantly increased while feed conversion ratio (FCR) decreases by chitosan nanoparticles compared to the control (p < 0.05). The morphometric analysis of the intestines reveals a significant improvement in villus height, villus width, and the number of goblet cells in chitosan-treated groups in a dose-dependent manner. Additionally, there is a positive correlation between the thickness of the enterocyte brush border and the chitosan dose, referring to an increasing absorptive activity. Histologically, the intestinal wall of Grey Mullet consists of four layers; mucosa, sub-mucosa, tunica muscularis (muscular layers), and serosa. The histological examination of the L. ramada intestine shows a normal histo-morphology. The epithelial layer of intestinal mucosa is thrown into elongated finger-like projections, the intestinal villi. The values of hemoglobin, hematocrit, red blood cells (RBCs), total protein (TP), albumin, and globulin are significantly increased in fish fed 1, and 2 g/kg of chitosan nanoparticles compared to fish fed 0 and 0.5 g/kg (p < 0.05). The highest levels of TP and albumin are observed in fish fed 1 g/kg diet (p < 0.05). The lysozyme activity and phagocytic index are significantly enhanced by feeding chitosan nanoparticles at 0.5, 1, and 2 g/kg, whereas the phagocytic activity is improved in fish fed 1 and 2 g/kg (p < 0.05). The highest lysozyme activity and phagocytic index are observed in fish fed 1 g/kg. SOD is significantly activated by feeding chitosan nanoparticles at 1 g/kg. Simultaneously, glutathione peroxidase (GPx) and catalase (CAT) activities also are enhanced by feeding chitosan at 1 and 2 g/kg, compared to fish fed 0 and 0.5 g/kg (p < 0.05). The highest GPx and CAT activities are observed in fish fed 1 g/kg (p < 0.05). Conversely, the malondialdehyde (MDA) levels are decreased by feeding chitosan at 1 and 2 g/kg, with the lowest being in fish fed 1 g/kg (p < 0.05). To summarize, the results elucidate that L. ramada fed dietary chitosan nanoparticles have a marked growth rate, immune response, and anti-oxidative response. These improvements are attributed to the potential role of chitosan nanoparticles in enhancing intestinal histo-morphometry and intestinal health. These results soundly support the possibility of using chitosan nanoparticles at 1–2 g/kg as a feasible functional supplement for aquatic animals.

Intestinal Inflammation Modulates the Expression of ACE2 and TMPRSS2 and Potentially Overlaps With the Pathogenesis of SARS-CoV-2–related Disease

Background and AimsThe presence of gastrointestinal symptoms and high levels of viral RNA in the stool suggest active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within enterocytes.MethodsHere, in multiple, large cohorts of patients with inflammatory bowel disease (IBD), we have studied the intersections between Coronavirus Disease 2019 (COVID-19), intestinal inflammation, and IBD treatment.ResultsA striking expression of ACE2 on the small bowel enterocyte brush border supports intestinal infectivity by SARS-CoV-2. Commonly used IBD medications, both biologic and nonbiologic, do not significantly impact ACE2 and TMPRSS2 receptor expression in the uninflamed intestines. In addition, we have defined molecular responses to COVID-19 infection that are also enriched in IBD, pointing to shared molecular networks between COVID-19 and IBD.ConclusionsThese data generate a novel appreciation of the confluence of COVID-19– and IBD-associated inflammation and provide mechanistic insights supporting further investigation of specific IBD drugs in the treatment of COVID-19. Preprint doi: https://doi.org/10.1101/2020.05.21.109124

Occult Colonic Perforation in a Patient With Coronavirus Disease 2019 After Interleukin-6 Receptor Antagonist Therapy.

BackgroundInterleukin-6 blockade (IL-6) has become a focus of therapeutic investigation for the coronavirus disease 2019 (COVID-19).MethodsWe report a case of a 34-year-old with COVID-19 pneumonia receiving an IL-6 receptor antagonist (IL-6Ra) who developed spontaneous colonic perforation. This perforation occurred despite a benign abdominal exam and in the absence of other known risk factors associated with colonic perforation.ResultsExamination of the colon by electron microscopy revealed numerous intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions abutting the microvilli of the colonic mucosa. Multiplex immunofluorescent staining revealed the presence of the SARS-CoV-2 spike protein on the brush borders of colonic enterocytes that expressed angiotensin-converting enzyme 2. However, no viral particles were observed within the enterocytes to suggest direct viral injury as the cause of colonic perforation.ConclusionsThese data and absence of known risk factors for spontaneous colonic perforation implicate IL-6Ra therapy as the potential mediator of colonic injury in this case. Furthermore, this report provides the first in situ visual evidence of the virus in the colon of a patient presenting with colonic perforation adding to growing evidence that intact infectious virus can be present in the stool.

A cryptic sequence targets the adhesion complex scaffold ANKS4B to apical microvilli to promote enterocyte brush border assembly

Nutrient-transporting enterocytes interact with their luminal environment using a densely packed collection of apical microvilli known as the brush border. Assembly of the brush border is controlled by the intermicrovillar adhesion complex (IMAC), a protocadherin-based complex found at the tips of brush border microvilli that mediates adhesion between neighboring protrusions. ANKS4B is known to be an essential scaffold within the IMAC, although its functional properties have not been thoroughly characterized. We report here that ANKS4B is directed to the brush border using a noncanonical apical targeting sequence that maps to a previously unannotated region of the scaffold. When expressed on its own, this sequence targeted to microvilli in the absence of any direct interaction with the other IMAC components. Sequence analysis revealed a coiled-coil motif and a putative membrane-binding basic-hydrophobic repeat sequence within this targeting region, both of which were required for the scaffold to target and mediate brush border assembly. Size-exclusion chromatography of the isolated targeting sequence coupled with in vitro brush border binding assays suggests that it functions as an oligomer. We further show that the corresponding sequence found in the closest homolog of ANKS4B, the scaffold USH1G that operates in sensory epithelia as part of the Usher complex, lacks the inherent ability to target to microvilli. This study further defines the underlying mechanism of how ANKS4B targets to the apical domain of enterocytes to drive brush border assembly and identifies a point of functional divergence between the ankyrin repeat-based scaffolds found in the IMAC and Usher complex.

Influence of Bentonite Nanoclay on the State of the Rat Intestine Protective Barrier in the Experiment

Bentonite nanoclays are widely used in the food industry as technological aids, that is, adsorbents in the purification of vegetable oils and in the production of beverages. Permitted food additives include sodium, potassium and calcium aluminosilicates (E554–E556), bentonite (E558), and kaolin (E559). The data on the oral toxicity of nanoclays (NCs) are few and contradictory. It is known that in vitro native and organomodified NCs exhibit cytotoxicity against both normal and transformed animal and human cells. Due to high ion-exchange and adsorption capacity, NCs can interfere with the absorption of trace elements in the gastrointestinal tract, in particular zinc. Nanoclays can affect the composition and activity of intestinal microbiocenosis. In an experiment on the subacute 92-day oral toxicity of bentonite NCs for Wistar rats it was shown that NCs at a dose of 100 mg/kg bw (body weight) prevent the permeation of chicken ovalbumin macromolecules through the intestinal barrier, which were not detected in the blood of all tested rats 3 h after its intragastric administration at a dose of 2000 mg/kg bw. In the same animals, a significant increase in absolute body weight was noted. The results of microscopic examination of the mucous membrane of the small intestine showed an increase in the secretion of the intestinal mucus with the formation of a dense mucous barrier at an NC dose of 10 mg/kg bw or more. The formation of this barrier already occurred with the localization over the brush border of enterocytes with an increase in the number of goblet cells at a dose of 10 mg/kg bw. At a dose of 100 mg/kg bw, together with a sharp increase in the number of these cells, a merged layer of the dense mucus was formed, sticking together the villi in the apical part. At the lowest of the studied NC doses (1 mg/kg bw), an increase in the desquamation of enterocytes in the apical part of the villi was observed. Morphometric analysis of the villus length and crypt depth showed that the villus/crypt ratio decreased under NC consumption within 32–34% compared with the control regardless of the dose of the nanomaterial. NC consumption in the entire range of the studied doses had a negative effect on the morphofunctional state of the villi and crypts of the rat ileum, while excessive mucus secretion at an NC dose of 100 mg/kg bw can disrupt parietal digestion and affect the barrier and immunoregulatory functions of the intestine.

Non‐CTR1 mechanisms of intestinal copper absorption determined with the Caco2 cell culture model

Much is still unknown about how dietary copper is absorbed by the small intestine. The current view is that Cu(II) is reduced to Cu(I) by reductases in the brush border of enterocytes, and then taken up by copper transporter 1 (CTR1), the only as yet identified copper uptake transporter in mammals. Uptake of Cu(I) by CTR1 is inhibited by Ag(I) and can thus be used to assess the extent to which CTR1 contributes to copper uptake. Using Caco2 cell monolayers with tight junctions grown in bicameral chambers, we determined that, whether given as Cu(I) or Cu(II), only about one third of copper uptake was inhibited by an excess of Ag(I). So CTR1 is not the only copper uptake system in enterocytes. Our goals were thus to document the contributions of CTR1 and a potential chloride‐dependent process (reported by Zimnicka et al. [1]) to copper absorption, and to explore whether cellular iron status or the ability to reduce Cu(II) impacts these processes. Caco2 cell monolayers were grown on collagen‐coated filters in Transwell plates until trans‐epithelial electrical resistance reached or exceeded 250 Ohms. Rates of Cu uptake were measured by following accumulation of radioactive Cu over 60 min in cells and the basal medium (equivalent to blood fluid) after application of 67Cu‐labeled Cu(II)‐nitrilotriacetate (5 uM) to the apical surface, and were calculated in terms of % dose/h/mg cell protein. Ascorbate (1 mM) was added to produce Cu(I). Control uptake medium contained all chloride salts. The contribution of CTR1 to uptake was measured by the fall in uptake rate due to 50–100 uM Ag(I). Cu(II) reduction was inhibited by an excess of Fe(III). Chloride dependent uptake was determined by the fall in uptake rate when sulfate was substituted for chloride. Effects of substituting other halide ions for chloride were also recorded. Cellular Fe status was varied by preincubating monolayers for 3 days with 5 uM ferric ammonium citrate, or depleting them with desferrioxamine. Levels of mRNA for CTR1, DMT1, and reductases (STEAPs 2 and 3) were determined by qPCR, relative to 18S rRNA. We found that excess Ag(I) decreased rates of Cu(I) and Cu(II) uptake about 30%. The same was true when sulfate was substituted for chloride. Silver and sulfate effects were additive. Substituting bromide for chloride made no difference, while iodide substitution doubled uptake rates. Fe pretreatment reduced expression of CTR1 and DMT1 mRNAs but had little effect on rates of copper uptake. Fe deficiency greatly enhanced expression not only of DMT1 but also of CTR1 mRNA, and increased rates of copper uptake as well. Inhibition of Cu/Fe reductases with excess Fe(III) did not reduce rates of Cu(II) uptake. These results indicate that at least in this intestinal model, CTR1 and a halide‐dependent uptake process each account for about one third of Cu uptake; that CTR1 expression is enhanced by iron deficiency – which would explain increased uptake of copper in that condition; and that copper can be absorbed not only in the Cu(I) state but also as Cu(II).

A110 DGAT1 MUTATION OF LIPID TRAFFICKING ASSOCIATED WITH A RARE CASE OF CONGENITAL DIARRHEA

Abstract Background Congenital diarrheas and enteropathies (CODEs) are rare and cause severe chronic diarrhea in children under 2 years of age.1 Current genomic research has identified several monogenic defects resulting in epithelial disorders of nutrient and electrolyte transport, enzymes function, metabolism and trafficking. Aims To discuss a rare case of congential diarrhea. Methods Case Report. Results A 10-month-old Ashkenazi Jewish boy was referred to Pediatric Gastroenterology for loose stools and failure to thrive. Born at 27 weeks, he was a NICU inpatient for his first 3 months of life. Explosive loose stools were first noticed after discharge and treated as cow’s milk protein allergy. At aged 10 months he was admitted to his local hospital with irritability, abdominal distension, foul smelling loose stools and developmental delay. Despite an intake of 130kcal/kg/day he failed to gain weight (weight and length &amp;lt; 3rd centile). His abdomen was distended but there was no organomegaly on examination. On transfer to Sick Kids he underwent extensive work up with low albumin 24 g/L but normal CBC, amylase, lipase and IgA and anti-TTG negative. The lipid profile showed hypertriglyceridemia 2.98 mmol/L (normal &amp;lt;0.85), low HDL 0.81 mmol/L but normal total cholesterol. Low apoliprotein A1 0.79 g/L (0.92 - 1.96), low apoliprotein B 0.54 g (0.59 - 1.46) and vitamin E deficiency 9.6 umol/L (14.5 - 33.0). Normal fecal elastase (&amp;gt; 500 ug/g stool). Upper GI endoscopy showed duodenal villi blunting with non-specific chronic inactive duodenitis on histopathology. Due to ongoing poor weight gain he commenced TPN. A repeat endoscopy four weeks after admission revealed patchy villous blunting, crypt hyperplasia and enterocyte vacuolization. However, ultrastructural electron microscopy examination showed normal enterocyte brush border with well-formed microvilli with no inclusions or vesicular bodies. With a differential diagnosis of chylomicron retention disorder, abetalipoproteinemia or hypolipoproteinemia he was commenced on a low-fat diet with MCT feeds. A 72-hour fecal fat collection on this diet was within normal range 5.4 mmol/day (2–7). One year later his weight is now on 3rd centile and his albumin levels have normalized. However, triglycerides remain elevated. Whole exome sequencing identified two pathogenic variants in the DGAT1 gene; c.629_631delCCT, pSer210del and c751 + 2T&amp;gt;C, IVSB+2T&amp;gt;C. Conclusions Diacylglycerol acyltransferase (DGAT)1 catalyzes triglyceride biosynthesis.2 Loss of function DGAT1 gene mutations are described in several cases studies3,4 characterized by electrolyte transport–related diarrhea, protein-losing enteropathy and growth failure. Our cases adds to the understanding of the phenotypic and histological spectrum of DGAT1 mutations. Management strategies currently focus on growth with dietary lipid restriction, while substituting fat-soluble vitamins and essential fatty acids. Funding Agencies CAG

The autoimmune targets in IPEX are dominated by gut epithelial proteins

The autoimmune targets in IPEX are dominated by gut epithelial proteins