Intestinal Protein Characterisation of SARS-CoV-2 Entry Molecules ACE2 and TMPRSS2 in Inflammatory Bowel Disease (IBD) and Fatal COVID-19 Infection

Abstract—The coronavirus SARS-CoV-2 contributes to morbidity and mortality mainly as a result of immune-pathology in the lungs. Recent data has shown multi-system involvement with widespread viral tropism. Here we present a detailed intestinal protein characterisation of SARS-Cov-2 entry molecules ACE2 and TMPRSS2 in patients with inflammatory bowel disease ([IBD]; ulcerative colitis [UC] and Crohn’s disease [CD]) with age- and sex-matched non-IBD controls, and in those with fatal COVID-19 infection. In our dataset, ACE2 and TMPRSS2 displayed a membrane enterocyte staining in the ileum (due to presence of brush border/microvilli) in contrast to a cytoplasmic pattern in the colon. We also showed a high ACE2/low TMPRSS2 expression pattern in the ileum with a reverse trend in the colon. In UC, colonic ACE2 and TMPRSS2 are cytoplasmic in nature, with significantly higher ACE2 staining intensity compared to non-IBD controls. In inflamed and unaffected IBD mucosa, ileal and colonic enterocyte ACE2 and TMPRSS2 expressions are not modified in the histologic presence of inflammation. We observed immune cells within the lamina propria that expressed ACE2 and TMPRSS2, at higher frequencies in IBD when compared to non-IBD controls. These were identified as plasma cells with multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) expression. We further analysed the gut histology of six fatal COVID-19 cases, with no difference in colonic and ileal ACE2/TMRPSS2 staining (compared to non-IBD controls) and identified ACE2 + lamina propria plasma cells. Of interest, in this COVID-19 cohort, there was no histologic evidence gut inflammation despite known evidence of viral tropism within the enterocytes. Our data provides evidence for tissue expression of entry molecules ACE2 and TMPRSS2 including a close apposition to plasma cells — both pointing towards a role of the gut in the antecedent immune response to SARS-CoV-2 infection.

Caracterización de un modelo murino de enterocolitis necrotizante

Introducción: La enterocolitis necrotizante (ECN) es la patología gastrointestinal de las más comunes y devastadoras en recién nacido con muy bajo peso al nacer (rango entre 500-1500g) y se caracteriza por inflamación y necrosis intestinal. Los objetivos de este estudio fueron desarrollar un modelo murino de ECN así como un modelo de sobreexpresión de proteínas en el intestino mediante la administración enteral mediante sonda de vectores lentivirales. Métodos: Para el modelo de ECN se utilizaron cepas de ratón C57BL6 y CD1 a los cuales se les trató por 6 veces cada dos horas con una dosis de anoxia con CO2 al 100% durante 10 o 7.5 minutos seguida una reoxigenación mediante hiperoxia al 95% por 5 minutos. Además, para activar el sistema inmune se administró LPS en las primeras dos dosis. Para la sobreexpresión de prolactina (PRL) en el intestino se administraron vectores lentivirales que sobreexpresan GFP (como control) o PRL por vía enteral a ratones CD1 en edades postnatales P2 y P3. Posteriormente se analizó la presencia de GFP y prolactina de las muestras de intestino mediante visualización por microscopia de fluorescencia y Western blot, respectivamente. Resultados: Se obtuvo una mortalidad del 45% y una eficiencia de desarrollo de ECN entre los animales vivos del 100% en ratones CD1 de edad postnatal P1, en contraste con la mortalidad de 85% y la eficiencia de desarrollo de ECN entre los animales vivos del 0% en ratones C57Bl6 de P1. En relación al modelo de sobreexpresión de proteínas en el intestino, se detectó GFP en el intestino de ratones administrados con 106 TU/ml vectores lentivirales para la sobreexpresión de GFP en el día P2 y evaluados 24 horas después. No se observó la sobreexpresión de PRL en el intestino de ratones administrados con 106 y 108 TU/ml vectores lentivirales para la sobreexpresión de PRL en los días P2 y P3 y evaluados 48 horas después. Conclusión: El modelo de ECN en ratones CD1 de P1 tuvo una efectividad del 100% a pesar de una mortalidad elevada. Además, se logró estandarizar el método para la sobreexpresión de proteínas en el intestino de ratones en P2 24 horas después de la administración de vectores lentivirales por la via enteral. La determinación de sobreexpresión de PRL en el intestino no fue conclusiva. Background: Necrotizating enterocolitis (NEC) is one of the most common and devastating gastrointestinal disease in newborns with very low weight birth (range among 500 -1500 g). NEC is characterized by intestinal inflammation and necrosis. Our aims of this study were to develop a NEC murine model and a intestinal protein expression model by means of enteral administration of lentiviral vectors. Method: For the NEC model were used C57BL6 and CD1 mice which were treated with anoxia with 100% CO2 for 10 or 7.5 minutes followed by 95% O2 for 5 minutes. This treatment was repeated six times with a 2 hours interval. Moreover, to activate the immune system, LPS was administrated orally in the first two doses. For the overexpression of prolactin (PRL) in the intestine, lentiviral vectors that overexpress GFP (as a control) or PRL were administered by orally to CD1 mice at postnatal ages P2 and P3. Then, the presence of GFP and prolactin in the intestine samples was analyzed by fluorescence microscopy and Western blot, respectively. Result: Mortality of 45% and a NEC development efficiency of 100% was obtained among live animals in CD1 mice of P1 postnatal age, in contrast to the mortality of 85% and development efficiency of 0% among live animals in C57BL6 mice of P1 age. In relation to the protein overexpression model in the intestine, GFP was detected in the mice gut administrated with 106 lentiviral vectors for the GFP overexpression on P2 evaluated 24 hours later. PRL overexpression was not observed in mice that received on day P1 postnatal 106 and 108 TU/ml of lentiviral vectors for the overexpression of PRL and evaluated on days P2 and P3. Conclusion: NEC model had an effectiveness of 100% in CD1 mice of 1 day of life, despite the high mortality. Moreover, a method for protein overexpression in the intestine was standardized. Lenviral vectors were orally administered 24 hours after birth and the expression of the protein was detected 24 hours later. Prolactin overexpression determination was not conclusive.

Intestinal Microbiota Play an Important Role in the Treatment of Type I Diabetes in Mice With BefA Protein

More and more studies have shown that the intestinal microbiota is the main factor in the pathogenesis of type 1 diabetes mellitus (T1DM). Beta cell expansion factor A (BefA) is a protein expressed by intestinal microorganisms. It has been proven to promote the proliferation of β-cells and has broad application prospects. However, as an intestinal protein, there have not been studies and reports on its application in diabetes and its mechanism of action. In this study, a T1DM model induced by multiple low-dose STZ (MLD-STZ) injections was established, and BefA protein was administered to explore its therapeutic effect in T1DM and the potential mechanism of intestinal microbiota. BefA protein significantly reduced the blood glucose, maintained the body weight, and improved the glucose tolerance of the mice. At the same time, the BefA protein significantly increased the expression of ZO-1, Occludin, and significantly reduced the expression of TLR-4, Myd88, and p-p65/p65. BefA protein significantly reduced the relative expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. In addition, our high-throughput sequencing shows for the first time that the good hypoglycemic effect of BefA protein is strongly related to the increase in the abundance of the beneficial gut bacteria Lactobacillus, Bifidobacterium and Oscillospria and the decrease in the abundance of the opportunistic pathogen Acinetobacter. Our group used animal models to verify the hypoglycemic effect of BefA protein, and first explored the potential mechanism of intestinal microbiota in BefA protein treatment.

Cysteine-Rich Intestinal Protein 1 Served as an Epithelial Ovarian Cancer Marker via Promoting Wnt/β-Catenin-Mediated EMT and Tumour Metastasis

Objective. To explore the expression, functions, and the possible mechanisms of cysteine-rich intestinal protein 1 (CRIP1) in epithelial ovarian cancer. Methods. Using open microarray datasets from The Cancer Genome Atlas (TCGA), we identified the tumorigenic genes in ovarian cancer. Then, we detected CRIP1 expression in 26 pairs of epithelial ovarian cancer tissue samples by immunohistochemistry (IHC) and performed a correlation analysis between CRIP1 and the clinicopathological features. In addition, epithelial ovarian cancer cell lines A2780 and OVCAR3 were used to examine CRIP1 expression by western blot and qRT-PCR. Various cell function experiments related to tumorigenesis were performed including the CCK8 assay, EdU, Annexin V-FITC/PI apoptosis assay, wound healing, and Transwell assay. In addition, the expression of epithelial-mesenchymal transition (EMT) markers was detected by western blot to illustrate the relationship between CRIP1 and EMT. Furthermore, KEGG pathway enrichment analysis and western blot were conducted to reveal the signaling pathways in which CRIP1 is involved in ovarian cancer pathogenesis. Results. CRIP1 was identified as an oncogene from the TCGA database. The IHC score demonstrated that the CRIP1 protein was expressed at a higher level in tumours than in tumour-adjacent tissues and was associated with a higher pathological stage, grade, and positive lymphatic metastasis. In cell models, CRIP1 was overexpressed in serous epithelial ovarian cancer. Cell function experiments showed that the knockdown of CRIP1 did not significantly affect cell proliferation or apoptosis but could exert an inhibitory effect on cell migration and invasion, and also induce changes in EMT markers. Furthermore, KEGG pathway enrichment analysis and western blot showed that CRIP1 could induce ovarian cancer cell metastasis through activation of the Wnt/β-catenin pathway. Conclusion. This study is the first to demonstrate that CRIP1 acts as an oncogene and may promote tumour metastasis by regulating the EMT-related Wnt/β-catenin signaling pathway, suggesting that CRIP1 may be an important biomarker for ovarian cancer metastasis and progression.

Intestinal Protein Characterisation of SARS-CoV-2 Entry Molecules ACE2 and TMPRSS2 in Inflammatory Bowel Disease (IBD) and Fatal COVID-19 Infection

The coronavirus SARS-CoV-2 contributes to morbidity and mortality mainly as a result of immune-pathology in the lungs. Recent data has shown multi-system involvement with widespread viral tropism. Here we present a detailed intestinal protein characterisation of SARS-Cov-2 entry molecules ACE2 and TMPRSS2 in patients with Inflammatory Bowel Disease ([IBD]; Ulcerative Colitis [UC] and Crohn’s disease [CD]) with age- and sex-match non-IBD controls; and in those with fatal COVID19 infection. Our study showed that IBD gut inflammation did not influence ACE2 and TRPMSS2 expression. Of interest, colonic protein expression of ACE2 and TRPMSS2 are cytoplasmic distinct to the membranous pattern in the ileum. We observed a significant increase in immune cells within the lamina propria in UC and CD that expressed ACE2 and TRMPSS2 when compared to non-IBD controls. These were identified as plasma cells with multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) expression. In six fatal COVID19 cases, there was no gut inflammation despite evidence of viral tropism within the enterocytes. Our data provides evidence for tissue expression of entry molecules ACE2 and TMPRSS2 including a close apposition to plasma cells – both pointing towards a role of the gut in the immune response to SARS-CoV-2 infection.

The level of intestinal protein binding fatty acids in children with autism spectrum disorders, depending on the style of nutrition

Introduction. Autism spectrum disorders are a widespread pathology, but the etiological factors of their occurrence have not yet been established. The aim of the study was to compare the levels of I-FABP in children with autism spectrum disorders, depending on the adherence to a gluten-free diet. Materials and methods. The study included 36 patients with autism committed to a gluten-free diet for more than six months and 49 patients with autism spectrum disorders who did not have dietary restrictions. There are no statistically significant age differences between the groups. The patients were evaluated and compared with the level of intestinal protein binding fatty acids. Results. Children using diet therapy were found to have significantly lower intestinal permeability. In particular, the average level of I-FABP in children with autism spectrum disorders followed a gluten-free diet is 156.20 ± 102.16 pg/ml, with non-compliance with diet therapy. The average value of the index is diagnosed at the level of 528.26 ± 255.95 pg/ml because the protein binding fatty acids are a marker of small intestine permeability. Children with autism spectrum disorders without adherence to a gluten-free diet show intestinal permeability higher than that of their peers with autism, who long exclude gluten from the diet. Conclusions. Further research is needed to assess the significance of the intestinal fatty acid-binding protein in the etiology and clinical presentation of various diseases, particularly in autism spectrum disorders. Before using diet therapy, all patients with autism need to consult a gastroenterologist to objectively assess the need for its compliance and dynamic monitoring of its effectiveness in case of use.

Enhanced Intestinal Protein Fermentation Correlates With Severe Psychiatric Symptoms of Schizophrenia

Emerging findings highlighted the associations of mental illness to nutrition and dysbiosis in the intestinal microbiota, but the underlying mechanisms, especially in schizophrenia (SZ), remain unclarified. Here we conducted a case-control study of SZ by performing gut metagenome, fecal and plasma non-targeted metabolome, short-, medium-, and long-chain fatty acids, and targeted metabolite analysis. The results uncovered an apparent contradiction in SZ patients between inadequate protein intake and protein-fermentation-dominated intestinal microbial metabolism, which shifted from carbohydrate fermentation and protein synthesis in healthy conditions. Moreover, the extent of protein fermentation represented as the abundance of related enzymes and fecal levels of related products, usually nitrogenous and neurologically active, correlated with the severity of psychiatric symptoms. These findings provide a previously uncharacterized pathophysiological process in SZ related to the dysbiosis of gut microbiota and dysregulation in macronutrient metabolism, highlighting the importance of nutrition care and the potentials for developing microbiota-targeted therapeutics in SZ.

Intestinal Fatty Acid Binding Protein as the Promising Marker of Small Intestine Permeability

The Article provides the literature data on the possibilities to use the intestinal protein binding the fatty acids as the diagnostic marker for the intestinal injuries of different etiology. Aspects of the marker use for the celiac disease are considered. The information on the results of use of the various diagnostic techniques for the celiac disease compared with the definition of the intestinal protein binding the fatty acids is presented.