Gut Tissue Research Articles

β-Catenin Drives Butyrophilin-like Molecule Loss and γδ T-cell Exclusion in Colon Cancer.

Intraepithelial lymphocytes (IEL) expressing γδ T-cell receptors (γδTCR) play key roles in elimination of colon cancer. However, the precise mechanisms by which progressing cancer cells evade immunosurveillance by these innate T cells are unknown. Here, we investigated how loss of the Apc tumor suppressor in gut tissue could enable nascent cancer cells to escape immunosurveillance by cytotoxic γδIELs. In contrast with healthy intestinal or colonic tissue, we found that γδIELs were largely absent from the microenvironment of both mouse and human tumors, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR interactions, were also downregulated in tumors. We then demonstrated that β-catenin activation through loss of Apc rapidly suppressed expression of the mRNA encoding the HNF4A and HNF4G transcription factors, preventing their binding to promoter regions of Btnl genes. Reexpression of BTNL1 and BTNL6 in cancer cells increased γδIEL survival and activation in coculture assays but failed to augment their cancer-killing ability in vitro or their recruitment to orthotopic tumors. However, inhibition of β-catenin signaling via genetic deletion of Bcl9/Bcl9L in either Apc-deficient or mutant β-catenin mouse models restored Hnf4a, Hnf4g, and Btnl gene expression and γδ T-cell infiltration into tumors. These observations highlight an immune-evasion mechanism specific to WNT-driven colon cancer cells that disrupts γδIEL immunosurveillance and furthers cancer progression.

TSG6 hyaluronan matrix remodeling dampens the inflammatory response during colitis

In response to tissue injury, changes in the extracellular matrix (ECM) can directly affect the inflammatory response and contribute to disease progression or resolution. During inflammation, the glycosaminoglycan hyaluronan (HA) becomes modified by tumor necrosis factor stimulated gene-6 (TSG6). TSG6 covalently transfers heavy chain (HC) proteins from inter-α-trypsin inhibitor (IαI) to HA in a transesterification reaction and is to date is the only known HC-transferase. By modifying the HA matrix, TSG6 generates HC:HA complexes that are implicated in mediating both protective and pathological responses. Inflammatory bowel disease (IBD) is a lifelong chronic disorder with well-described remodeling of the ECM and increased mononuclear leukocyte influx into the intestinal mucosa. Deposition of HC:HA matrices is an early event in inflamed gut tissue that precedes and promotes leukocyte infiltration. However, the mechanisms by which TSG6 contributes to intestinal inflammation are not well understood. The aim of our study was to understand how the TSG6 and its enzymatic activity contributes to the inflammatory response in colitis. Our findings indicate that inflamed tissues of IBD patients show an elevated level of TSG6 and increased HC deposition and that levels of HA strongly associate with TSG6 levels in patient colon tissue specimens. Additionally, we observed that mice lacking TSG6 are more vulnerable to acute colitis and exhibit an aggravated macrophage-associated mucosal immune response characterized by elevated pro-inflammatory cytokines and chemokines and diminished anti-inflammatory mediators including IL-10. Surprisingly, along with significantly increased levels of inflammation in the absence of TSG6, tissue HA levels in mice were found to be significantly reduced and disorganized, absent of typical “HA-cable” structures. Inhibition of TSG6 HC-transferase activity leads to a loss of cell surface HA and leukocyte adhesion, indicating that the enzymatic functions of TSG6 are a major contributor to stability of the HA ECM during inflammation. Finally, using biochemically generated HC:HA matrices derived by TSG6, we show that HC:HA complexes can attenuate the inflammatory response of activated monocytes. In conclusion, our data suggests that TSG6 exerts a tissue-protective, anti-inflammatory effect via the generation of HC:HA complexes that become dysregulated in IBD.

Major histocompatibility complex class IIα and IIβ of pufferfish (Takifugu obscurus): Identification and functional characterization in response to bacterial challenge.

Classical major histocompatibility complex (MHC) class II molecules play an essential role in immune system. In this study, MHC IIα (Pf-MHC IIα) and MHC IIβ (Pf-MHC IIβ) homology genes from pufferfish (Takifugu obscurus) were cloned and their functional characterization in response to bacterial challenge was identified. The nucleotide sequences of the open reading frames (ORFs) of pufferfish Pf-MHC IIα and Pf-MHC IIβ were 708 bp and 750 bp, encoding 235 aa and 249 aa, respectively. The structure of Pf-MHC IIα or Pf-MHC IIβ contained a signal peptide, an α1/β1 domain, an α2/β2 domain, a transmembrane region and a cytoplasmic region. Multiple sequence alignment and phylogenetic analysis showed that Pf-MHC IIα and Pf-MHC IIβ molecules had the highest similarity with Fugu rubripes (Takifugu rubripes). Cellular localization analysis indicated that the distribution of Pf-MHC IIα and Pf-MHC IIβ was in the lymphocyte membrane and cytoplasm. qRT-PCR results showed that Pf-MHC IIα and Pf-MHC IIβ expressed relatively high in skin, gills and gut. In addition, after stimulation challenge in vitro (lipopolysaccharide, or polyinosinic: polycytidylic acid) and in vivo (A. hydrophila), the mRNA expressions of Pf-MHC IIα and Pf-MHC IIβ were significantly up-regulated in lymphocytes and in tissues of skin, gills, gut and head kidney. Moreover, Pf-MHC IIα or Pf-MHC IIβ neutralization reduced the ability of A. hydrophila to induce the expressions of lymphocyte cytokines (TNF-α, IL-1β and IL-10). Overall, it is speculated that Pf-MHC IIα and Pf-MHC IIβ may play an important role in the host response against A. hydrophila in pufferfish.

Single and combined effects of polyethylene microplastics and acetochlor on accumulation and intestinal toxicity of zebrafish (Danio rerio)

The co-exposure of microplastics (MPs) and other contaminants has aroused extensive attention, but the combined impacts of MPs and pesticides remain poorly understood. Acetochlor (ACT), a widely used chloroacetamide herbicide, has raised concerns for its potential bio-adverse effects. This study evaluated the influences of polyethylene microplastics (PE-MPs) for acute toxicity, bioaccumulation, and intestinal toxicity in zebrafish to ACT. We found that PE-MPs significantly enhanced ACT acute toxicity. Also, PE-MPs increased the accumulation of ACT in zebrafish and aggravate the oxidative stress damage of ACT in intestines. Exposure to PE-MPs or/and ACT causes mild damage to the gut tissue of zebrafish and altered gut microbial composition. In terms of gene transcription, ACT exposure triggered a significant increase in inflammatory response-related gene expressions in the intestines, while some pro-inflammatory factors were found to be inhibited by PE-MPs. This study provides a new perspective on the fate of MPs in the environment and on the assessment of the combined effects of MPs and pesticides on organisms.

#6846 CHRONIC CMV DIARRHOEA IN RENAL TRANPLANT RECIPIENTS - AN UNDERRECOGNISED SPECTRUM OF COMPARTMENTALISED GUT INVASIVE CMV DISEASE

Abstract Background and Aims Chronic diarrhoea in post-renal transplant recipients due to Cytomegalovirus (CMV) presents as Blood CMV PCR (polymerised chain reaction) positive chronic diarrhoea and Gut-invasive (rectal tissue PCR positive, blood PCR-negative) compartmentalised CMV. The available literature on compartmentalised gut CMV is scarce. The aim is to study the clinical presentation and outcome of patients with compartmentalized gut invasive CMV when compared with Blood CMV positive chronic diarrhea in renal transplant recipients. Method This is a retrospective single-centre follow-up study of patients with CMV disease who were transplanted between 2000 and 2020. Diagnostic variables like blood CMV PCR (polymerised chain reaction) positivity with constitutional symptoms, presence or absence of chronic diarrhoea, tissue PCR positivity and site of tissue biopsy were given harmony codes and 6 syndromes were identified – CMV diarrhoea (Blood PCR positive, presence of diarrhoea), compartmentalized gut invasive CMV (Blood PCR negative, presence of diarrhoea, rectal tissue quantitative CMV PCR positive), Acute CMV syndrome, CMV Esophagitis, CMV nephropathy, CMV cystitis. Out of which patients presenting with diarrhoea (first two groups) were included for analysis. The baseline descriptive statistical measures were carried out using analysis of variance. The time to CMV disease from transplant was computed by identifying the pattern of distribution at 10%, 50% and 90% between groups using reliability models. The time to death from diagnosis was analyzed by identifying the pattern by parametric log-logistic distribution. Survival analysis was done by a parametric reliability model identifying location, scale and threshold parameters, from which month-wise prediction of probabilities was found for survival. The proportion of adverse outcomes (relapse, death and graft loss) between the two groups were analyzed by one-way ANOVA and Tukey pairwise comparison. All the patients were followed up till death or last follow-up. Results Out of 2208 renal transplants done between 2000 and 2020, 118 (5.3%), patients developed CMV disease. Chronic diarrhoea was the presentation in 88 (57%), of which 47 had manifestations of CMV diarrhoea, and 41 had compartmentalized gut CMV. Induction agents used (CMV diarrhoea Vs compartmentalized gut CMV) were basiliximab (51% Vs 56%), ATG (27% Vs 17%), Grafalon (0% Vs,2.4%) and no induction (21%,24%). Only 31.9% and 26.8% received Valganciclovir prophylaxis for pre-specified indications respectively. The time to CMV diagnosis from transplant for 10%, 50% and 90% of patients (in months) was 12,63,154 for CMV diarrhoea;12,52 and 139 months for compartmentalized gut CMV. The mean duration (weeks) of diarrhoea was similar in both groups (7 Vs 7.3). Associated opportunistic infections causing diarrhoea were higher in compartmentalized gut CMV (28% Vs 46% p = 0.08); the commonest being cryptosporidium. Quantitative PCR in the blood ranged from 1245 to 2511345 copies/ml; whereas in gut tissue, it ranged from 1325 to 3517920/25 mg. The major histopathological finding in compartmentalized gut CMV was active inflammatory pathology in 34 (83%) patients. The probability of survival was significantly lower (72.6%; CI 60% to 83%) in CMV diarrhoea when compared with compartmentalized Gut CMV (87.2%; CI 76% to 94%) for initial 12 months (p<0.05); however at 5 years (56.2%, Vs 58.5%) and 10 years (48.5% Vs 42.7%) the survival was similar. Compartmentalized gut CMV was associated with a higher relapse rate (19.5%) when compared with CMV diarrhoea (6.4%) on follow-up (p = 0.06). The mortality (36% Vs 29%; p = 0.490) and graft loss (25% Vs 22%; p = 0.692) were similar between both the group. Conclusion Compartmentalized gut invasive CMV represents a larger proportion of chronic diarrhoeal illness with a higher relapse rate, which needs invasive gut tissue PCR analysis, despite negative blood PCR, for early diagnosis and management.

Macrophages and the development and progression of non-alcoholic fatty liver disease.

The liver is the site of first pass metabolism, detoxifying and metabolizing blood arriving from the hepatic portal vein and hepatic artery. It is made up of multiple cell types, including macrophages. These are either bona fide tissue-resident Kupffer cells (KC) of embryonic origin, or differentiated from circulating monocytes. KCs are the primary immune cells populating the liver under steady state. Liver macrophages interact with hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells to maintain homeostasis, however they are also key contributors to disease progression. Generally tolerogenic, they physiologically phagocytose foreign particles and debris from portal circulation and participate in red blood cell clearance. However as immune cells, they retain the capacity to raise an alarm to recruit other immune cells. Their aberrant function leads to the development of non-alcoholic fatty liver disease (NAFLD). NAFLD refers to a spectrum of conditions ranging from benign steatosis of the liver to steatohepatitis and cirrhosis. In NAFLD, the multiple hit hypothesis proposes that simultaneous influences from the gut and adipose tissue (AT) generate hepatic fat deposition and that inflammation plays a key role in disease progression. KCs initiate the inflammatory response as resident immune effectors, they signal to neighbouring cells and recruit monocytes that differentiated into recruited macrophages in situ. Recruited macrophages are central to amplifying the inflammatory response and causing progression of NAFLD to its fibro-inflammatory stages. Given their phagocytic capacity and their being instrumental in maintaining tissue homeostasis, KCs and recruited macrophages are fast-becoming target cell types for therapeutic intervention. We review the literature in the field on the roles of these cells in the development and progression of NAFLD, the characteristics of patients with NAFLD, animal models used in research, as well as the emerging questions. These include the gut-liver-brain axis, which when disrupted can contribute to decline in function, and a discussion on therapeutic strategies that act on the macrophage-inflammatory axis.

A gut-isolated Enterococcus strain (HcM7) triggers the expression of antimicrobial peptides that aid resistance to nucleopolyhedrovirus infection of Hyphantria cunea larvae.

Commensal microorganisms are widely distributed in insect gut tissues and play important roles in to host nutrition, metabolism, reproductive regulation and, especially the immune functioning and tolerance to pathogens. Consequently, gut microbiota represent a promising resource for the development of microbial-based products for pest control and management. However, the interactions among host immunity, entomopathogen infections, and gut microbiota remain poorly understood for many arthropod pests. We previously isolated an Enterococcus strain (HcM7) from Hyphantria cunea larvae guts that increased the survival rates of larvae challenged with nucleopolyhedrovirus (NPV). Here, we further investigated whether this Enterococcus strain stimulates a protective immune response against NPV proliferation. Infection bioassays demonstrated that the re-introduction of the HcM7 strain to germ-free larvae pre-activated the expression of several antimicrobial peptides (particularly H. cunea gloverin 1, HcGlv1), resulting in the significant repression of virus replication in host guts and hemolymph, and consequently improved host survivorship after NPV infection. Furthermore, silencing of the HcGlv1 gene by RNA interference markedly enhanced the deleterious effects of NPV infection, revealing a role of this gut symbiont-induced gene in host defenses against pathogenic infections. These results show that some gut microorganisms can stimulate host immune systems, thereby contributing to resistance to entomopathogens. Furthermore, HcM7, as a functional symbiotic bacteria of H. cunea larvae, may be a potential target for increasing the effectiveness of biocontrol agents against this devastating pest.

Niclosamide exposure disrupts antioxidant defense, histology, and the liver and gut transcriptome of Chinese soft-shelled turtle (Pelodiscus sinensis)

Niclosamide (NIC) is the only commercially available molluscicide for controlling schistosomiasis, and its negative effects on aquatic animals had been frequently reported in recent years. However, the toxicity mechanism of NIC on the Chinese soft-shelled turtle (Pelodiscus sinensis) have not yet been investigated. Therefore, juvenile turtles were exposed to 0 (control group), 10 (low NIC, L), and 50 (high NIC, H) μg/L NIC for 120 h and our results demonstrated that NIC exposure induced severe pathological changes in the liver of P. sinensis. And the typical symptom included edema, nuclear migration and deformation, and vacuolization. Compared with the liver, the NIC exposure did not cause significant damage in the gut tissue. In addition, the DHE staining demonstrated that the ROS production of liver and gut increased with the increase in concentration of NIC. The activities of antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) was inhibited with increased malondialdehyde (MDA) content, indicating that the antioxidant defense was significantly perturbed. Further, the transcriptome sequencing and was applied to evaluate the underlying toxicity mechanisms of NIC exposure in liver and gut of P. sinensis. Pathway enrichment showed that the disorder of lipid metabolism and innate immune regulation, including Toll-like receptors (TLRs), tumor necrosis factor (TNF), lectins, and complement and coagulation cascades, were toxicological properties of NIC on P. sinensis. Overall, the current study provides valuable information to understand the toxic effect of NIC on Chinese soft-shelled turtle.

Valorization of the insect waste as a source of dietary protein in replacing the fishmeal protein for the cage reared Pangasianodon hypophthalmus: An approach to search the alternate non-conventional feed resource of animal origin

A ninety days experiment was carried-out to evaluate the effect of dietary replacement of fishmeal (FM) (locally available dried non-defatted Coilia dussumieri V.) by graded inclusion levels of non-defatted non-mulberry Indian tropical tasar silkworm spent pupae (Antheraea mylitta D.) on growth performance, feed consumption, digestive enzyme response, body chemical composition, and intestinal cellular micro-morphological status of the reared striped catfish (Pangasianodon hypophthalmus S.). Five iso-nitrogenous (280 g protein kg-1) and iso-energic (19 MJ kg-1) extruded floating feeds were formulated by gradually replacing the FM with NDSWP (non-defatted silkworm pupae) at 0%, 25%, 50%, 75%, and 100% (on protein to protein basis) levels and the diets are designated as NDSWP0, NDSWP25, NDSWP50, NDSWP75, and NDSWP100, respectively. The contribution of calculated pupae crude protein was 0%, 4.75%, 9.50%, 14.25%, and 19% out of the total protein (28%) content in the respective diets. The experiment was executed in the floating net-cage located at the Maithon reservoir, India which is a field research station of ICAR-CIFRI, Barrackpore, Kolkata, India. The designated diets were randomly allocated to 10 numbers of experimental cages (each 5 ×5 ×3 m), in triplicates stocked with 2475 numbers (stocking density at 33 numbers per m3) of striped catfish in each replication and fed daily twice at 08.00 h and 16.00 h. The initial stocking size of the striped catfish fingerling was 16.56 ± 0.08 g. At the termination of the feeding experiment, the final body weight (g), individual weight gain (g), weight gained (%), feed conversion ratio (FCR), specific growth rate (SGR), and protein efficiency ratio (PER) found to be non-significantly different (p < 0.05) up to 50% protein replacement level (NDSWP50) in comparison to the control (NDSWP0). Among the biological indices, hepatosomatic index (HSI) exhibited a non-significant difference stretching to NDSWP50 treatment group with a distinct significant difference between NDSWP25 and NDSWP100 (p < 0.05), when, an inverse trend was recorded in relation to increasing pupae meal in the diets. Among the digestive enzymes, relatively higher protease enzyme activity was observed in the diet fed at NDSWP50 compared to the rest of the treatment groups, although, it was statistically non-significant (p > 0.05). Amylase and lipase enzyme activities recorded a significant difference among the treatment groups (p < 0.05). A higher amount of fish muscle crude protein was noticed in the NDSWP50 treated group than in the rest of the treatment groups. The lipid content of fish muscle indicated its higher content over the initial and exhibited an inverse relationship with moisture and ash content at the end of the experiment. Histo-morphology of gut tissue confirms the suitability of feeding pupae protein incorporated diet with the gradual replacement of fishmeal protein. The findings of the current feeding trial suggests that the inclusion of non-defatted non-mulberry silkworm pupae protein at 50% level of fishmeal protein substitution without compromising the growth performance, nutrient utilization, conversion of feed, and other biological indices, and economics of diet.

Gut-Associated Lymphatic Tissue in Food-Restricted Rats: Influence of Refeeding and Probiotic Supplementation.

Anorexia nervosa (AN) is a severe and often chronic eating disorder that leads to alterations in the gut microbiome, which is known to influence several processes, such as appetite and body weight regulation, metabolism, gut permeability, inflammation, and gut-brain interactions. Using a translational activity-based anorexia (ABA) rat model, this study examined the effect of chronic food starvation, as well as multistrain probiotic supplementation and refeeding, on the structure of the gut and gut-associated lymphatic tissue (GALT). Our results indicated that ABA had an atrophic influence on intestinal morphology and increased the formation of GALT in the small bowel and colon. Higher formation of GALT in ABA rats appeared to be reversible upon application of a multistrain probiotic mixture and refeeding of the starved animals. This is the first time that increased GALT was found following starvation in the ABA model. Our results underscore a potential role of gut inflammatory alterations in the underlying pathophysiology of AN. Increased GALT could be linked to the gut microbiome, as probiotics were able to reverse this finding. These results emphasize the role of the microbiome-gut-brain axis in the pathomechanisms of AN and point to probiotics as potentially beneficial addendum in the treatment of AN.

Cardiovascular Brain Circuits.

The cardiovascular system is hardwired to the brain via multilayered afferent and efferent polysynaptic axonal connections. Two major anatomically and functionally distinct though closely interacting subcircuits within the cardiovascular system have recently been defined: The artery-brain circuit and the heart-brain circuit. However, how the nervous system impacts cardiovascular disease progression remains poorly understood. Here, we review recent findings on the anatomy, structures, and inner workings of the lesser-known artery-brain circuit and the better-established heart-brain circuit. We explore the evidence that signals from arteries or the heart form a systemic and finely tuned cardiovascular brain circuit: afferent inputs originating in the arterial tree or the heart are conveyed to distinct sensory neurons in the brain. There, primary integration centers act as hubs that receive and integrate artery-brain circuit-derived and heart-brain circuit-derived signals and process them together with axonal connections and humoral cues from distant brain regions. To conclude the cardiovascular brain circuit, integration centers transmit the constantly modified signals to efferent neurons which transfer them back to the cardiovascular system. Importantly, primary integration centers are wired to and receive information from secondary brain centers that control a wide variety of brain traits encoded in engrams including immune memory, stress-regulating hormone release, pain, reward, emotions, and even motivated types of behavior. Finally, we explore the important possibility that brain effector neurons in the cardiovascular brain circuit network connect efferent signals to other peripheral organs including the immune system, the gut, the liver, and adipose tissue. The enormous recent progress vis-à-vis the cardiovascular brain circuit allows us to propose a novel neurobiology-centered cardiovascular disease hypothesis that we term the neuroimmune cardiovascular circuit hypothesis.

The ecology of viruses in urban rodents with a focus on SARS-CoV-2

ABSTRACT Wild animals are naturally infected with a range of viruses, some of which may be zoonotic. During the human COVID pandemic there was also the possibility of rodents acquiring SARS-CoV-2 from people, so-called reverse zoonoses. To investigate this, we sampled rats (Rattus norvegicus) and mice (Apodemus sylvaticus) from urban environments in 2020 during the human COVID-19 pandemic. We metagenomically sequenced lung and gut tissue and faeces for viruses, PCR screened for SARS-CoV-2, and serologically surveyed for anti-SARS-CoV-2 Spike antibodies. We describe the range of viruses that we found in these two rodent species. We found no molecular evidence of SARS-CoV-2 infection, though in rats we found lung antibody responses and evidence of neutralization ability that are consistent with rats being exposed to SARS-CoV-2 and/or exposed to other viruses that result in cross-reactive antibodies.

Vaginal Birth Alters Gut Transcriptome of Neonatal Pigs

Background: Women at risk of preterm birth are routinely administered prenatal doses of glucocorticoids to facilitate lung maturation and improve pulmonary outcomes in preterm infants. Additionally, preterm infants are often administered low doses of glucocorticoids postnatally for prevention of bronchopulmonary dysplasia. However, there is limited understanding about the impact of perinatal glucocorticoid exposure on the gut and liver in preterm infants. In pigs, there is a spike in plasma cortisol in newborns during parturition. We previously showed in pigs that plasma cortisol levels positively correlate with plasma fibroblast growth factor 19 (FGF19), a gut hormone that negatively regulates hepatic bile acid synthesis. FGF19 is secreted by epithelial cells predominantly in the distal ileum in response to bile acids. However, in vaginally-born pigs, FGF19 mRNA levels were higher in the proximal jejunum. This led us to hypothesize that exposure to glucocorticoids in the perinatal period has a profound impact on the transcription of genes involved in the development of gut and liver. Objective: The objective of this study was to characterize the impact of spontaneous vaginal birth and the exposure to elevated cortisol on the transcriptome in gut and liver tissue of neonatal pigs Methods: Two sows underwent a cesarean section on gestation day 114 (“Term”; term=115 day), two sows underwent a cesarean section on gestation day 105 (“Preterm”) and two additional sows were allowed to farrow at term gestational age (“Vaginal”). Small intestine tissue and plasma was collected from newborn pigs (n= 10 Preterm; n=8 Term; n=6 Vaginal). RNA-seq analysis was performed on intestinal samples mapped to the latest Sus scrofa Sscrofa11.1 reference genome (NCBI). Results: Cortisol levels were statistically highest in the vaginally-born pigs (139 ng/mL), followed by the Term (101 ng/mL) and lowest in the Preterm (61 ng/mL) pigs. Gene Ontology (GO) analysis showed significant enrichment in pathways of upregulated genes involved in response to toxic substances, stress, detoxification and inorganic substances in the jejunum of Vaginal pigs compared to Preterm and Term pigs. KEGG analysis of the proximal jejunum indicated enrichment in pathways of upregulated genes in steroid hormone biosynthesis, bile secretion, TNF signaling and IL-17 Vaginal, compared to Term and Preterm pigs. The same pattern was not the same in the distal ileum where GO analysis showed pathway enrichment of upregulated genes in oxidation-reduction processes, and transition metal ion binding in Vaginal compared to Term and Preterm pigs. KEGG analysis of the distal ileum identified pathway enrichment of upregulated genes in complement and coagulation, bile acid synthesis and secretion, fatty acid metabolism, and amino acid metabolism in Vaginal pigs. Conclusion: Genes in metabolic and cellular signaling pathways associated with cortisol are upregulated in vaginally-born pigs. This work was supported in part by federal funds from the USDA, Agricultural Research Service under Cooperative Agreement Number 3092-51000-060-01, and grants from the National Institutes of Health Grant DK-094616 (D.G.B), and the Texas Medical Center Digestive Diseases Center (NIH Grant P30 DK-56338). Caitlin Vonderohe was supported by T32 DK007664 and Greg Guthrie was supported by K01 DK129408. 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.

Gut immunopathological signatures in Rag1 hypomorphic mice

Inflammatory bowel disease (IBD) has been reported in ~15% of patients with partial RAG deficiency. To investigate the pathophysiology of intestinal disease, we studied the interplay of T-cell immune dysregulation and microbiome abnormalities in a mouse model (Rag1w/w) homozygous fora hypomorphic Rag1 mutation (R972W) whose orthologue variant (R975W) has been reported in patients. Analysis of gut tissue in Rag1w/w mice revealed severe spontaneous colitis with lymphocytic infiltrates, which become histologically evident after weaning. To assess the relative contribution of genetic and environmental factors on the pathogenesis of gut inflammation, we performed flow cytometry and single-cell RNA-seq of lamina propria (LP) T cells and microbiome studies of fecal specimens in Rag1w/w and Rag1+/+ mice. Rag1w/w mice showed skewing towards a Th1/Th17 phenotype in CD4+ cells infiltrating the LP. Analysis of the fecal microbiota composition revealed a severe restriction of microbial diversity in Rag1w/w mice, with progressive depletion of three genera of firmicutes (Lachnospiraceae, Oscillibacter, Intestinimonas) producing butyrate. Principal component analysis of metabolomics data of fecal specimens clustered Rag1w/w separately from Rag1+/+ mice, with a severe defect in the amount of butyrate in the former. Consistent with an important role of butyrate in facilitating priming of IL-10 producing Tr1 cells, Il10 expression was markedly reduced in CD4+ LP T-cells from Rag1w/w compared to Rag1+/+ mice. Furthermore, scRNAseq of LP CD4+ T-cells revealed reduced presence of Tr1 cells in Rag1w/w mice, indicating that microbiome abnormalities contribute to the intestinal immune dysregulation. Treatment of Rag1w/w mice with vedolizumab (aiming at blocking gut homing of pathogenic T cells) had no effect on intestinal inflammation and on restriction of microbiome diversity. By contrast, both use of broad-spectrum antibiotics and bone marrow transplantation (BMT) significantly improved the inflammation and normalized the number of LP CD4+ cells. However, the LP CD4+ T cells of antibiotic-treated mice retained a strong Th1/Th17 signature, whereas only BMT rescued the phenotype, and induced normalization of microbiome diversity and restoration in the relative abundance of firmicutes. These data provide mechanistic insights into the pathophysiology of IBD associated with partial RAG deficiency, and confirm a curative role of bone marrow transplantation.

Stomach-derived human insulin-secreting organoids restore glucose homeostasis.

Gut stem cells are accessible by biopsy and propagate robustly in culture, offering an invaluable resource for autologous cell therapies. Insulin-producing cells can be induced in mouse gut, but it has not been possible to generate abundant and durable insulin-secreting cells from human gut tissues to evaluate their potential as a cell therapy for diabetes. Here we describe a protocol to differentiate cultured human gastric stem cells into pancreatic islet-like organoids containing gastric insulin-secreting (GINS) cells that resemble β-cells in molecular hallmarks and function. Sequential activation of the inducing factors NGN3 and PDX1-MAFA led human gastric stem cells onto a distinctive differentiation path, including a SOX4High endocrine and GalaninHigh GINS precursor, before adopting β-cell identity, at efficiencies close to 70%. GINS organoids acquired glucose-stimulated insulin secretion in 10 days and restored glucose homeostasis for over 100 days in diabetic mice after transplantation, providing proof of concept for a promising approach to treat diabetes.

α-Synuclein membrane-binding in the central and enteric nervous system in Parkinson’s disease (P3-11.002)

<h3>Objective:</h3> The goal is to understand whether changes in α-Synuclein (αSyn) membrane-binding can be detected in the enteric nervous system in Parkinson’s disease (PD) using a novel mouse model and human tissue. <h3>Background:</h3> αSyn aggregates are the main components of Lewy bodies found in both the brain and in the enteric nervous system of PD patients. Pathological changes and accompanying neurodegeneration precede diagnosis by years, with changes in gastrointestinal motility among the earliest prodromal symptoms of PD, pointing to the gut as a potential starting point for pathology. <h3>Design/Methods:</h3> We hypothesize that changes in αSyn membrane-binding occur first in the gut, and are a marker of early pathology. We characterized motor and gastrointestinal function in a mouse model expressing human αSyn under all its regulatory elements, thus enabling human-like spatiotemporal expression of αSyn. We also examined expression of αSyn in the brain and gut in these mice, as well as in post-mortem brain tissue and colon tissue collected during routine screening colonoscopy in subjects with PD. <h3>Results:</h3> We showed that our mouse model demonstrates typical motor impairments, as well as impairments of gastrointestinal motility. We previously demonstrated that membrane-bound αSyn is protected from aggregation, while cytosolic, soluble αSyn has a propensity to aggregate. We now show reduced membrane-binding of αSyn in brain and gut in our mouse model as well as in PD cortex. In preliminary results from human PD gut tissue, compared to healthy controls, we see a trend towards decreased membrane-binding of αSyn in the colon as well as changes in post-translational modifications that are known to affect membrane-binding. <h3>Conclusions:</h3> Changes in in αSyn membrane-binding can be detected in mouse and human tissue. This study represents the first biochemical assessment of αSyn in the gut, and suggests that changes in αSyn membrane-binding can serve as a biomarker of disease. <b>Disclosure:</b> Dr. Gao has nothing to disclose. Mr. Briano has nothing to disclose. Ms. Komer has nothing to disclose. Dr. Crawford has received personal compensation for serving as an employee of seres. Dr. Crawford has received personal compensation for serving as an employee of merck. Dr. Crawford has received personal compensation for serving as an employee of ferring. Dr. Crawford has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for seres. Dr. Crawford has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for ferring. Dr. Crawford has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for seres. Dr. Crawford has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for merck. Dr. Crawford has received stock or an ownership interest from seres. Dr. Crawford has received intellectual property interests from a discovery or technology relating to health care. Dr. Henchcliffe has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Amneal. Dr. Henchcliffe has received personal compensation in the range of $0-$499 for serving as a Consultant for MedExcelCap. Dr. Henchcliffe has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Bayer. Dr. Henchcliffe has stock in Axent Biosciences Inc. The institution of Dr. Henchcliffe has received research support from Weston Brain Institute. The institution of Dr. Henchcliffe has received research support from Blue Rock Therapeutics. Dr. Lee has nothing to disclose. Prof. Burré has received research support from NIH. Prof. Burré has received research support from Michael J. Fox Foundation. Prof. Burré has received personal compensation in the range of $500-$4,999 for serving as a Reviewer with NIH.

Intestinal epigenotype of Atlantic salmon (Salmo salar) associates with tenacibaculosis and gut microbiota composition

It remains a challenge to obtain the desired phenotypic traits in aquacultural production of Atlantic salmon, and part of the challenge might come from the effect that host-associated microorganisms have on the fish phenotype. To manipulate the microbiota towards the desired host traits, it is critical to understand the factors that shape it. The bacterial gut microbiota composition can vary greatly among fish, even when reared in the same closed system. While such microbiota differences can be linked to diseases, the molecular effect of disease on host-microbiota interactions and the potential involvement of epigenetic factors remain largely unknown.The aim of this study was to investigate the DNA methylation differences associated with a tenacibaculosis outbreak and microbiota displacement in the gut of Atlantic salmon. Using Whole Genome Bisulfite Sequencing (WGBS) of distal gut tissue from 20 salmon, we compared the genome-wide DNA methylation levels between uninfected individuals and sick fish suffering from tenacibaculosis and microbiota displacement. We discovered >19,000 differentially methylated cytosine sites, often located in differentially methylated regions, and aggregated around genes.The 68 genes connected to the most significant regions had functions related to the ulcerous disease such as epor and slc48a1a but also included prkcda and LOC106590732 whose orthologs are linked to microbiota changes in other species. Although the expression level was not analysed, our epigenetic analysis suggests specific genes potentially involved in host-microbiota interactions and more broadly it highlights the value of considering epigenetic factors in efforts to manipulate the microbiota of farmed fish.

Butyrate driven raft disruption trots off enteric pathogen invasion: possible mechanism of colonization resistance

The gut microbiome derived short chain fatty acids perform multitude of functions to maintain gut homeostasis. Here we studied how butyrate stymie enteric bacterial invasion in cell using a simplistic binary model. The surface of the mammalian cells is enriched with microdomains rich in cholesterol that are known as rafts and act as entry points for pathogens. We showed that sodium butyrate treated RAW264.7 cells displayed reduced membrane cholesterol and less cholera-toxin B binding coupled with increased membrane fluidity compared to untreated cells indicating that reduced membrane cholesterol caused disruption of lipid rafts. The implication of such cellular biophysical changes on the invasion of enteric pathogenic bacteria was assessed. Our study showed, in comparison to untreated cells, butyrate-treated cells significantly reduced the invasion of Shigella and Salmonella, and these effects were found to be reversed by liposomal cholesterol treatment, increasing the likelihood that the rafts' function against bacterial invasion. The credence of ex vivo studies found to be in concordance in butyrate fed mouse model as evident from the significant drift towards a protective phenotype against virulent enteric pathogen invasion as compared to untreated mice. To produce a cytokine balance towards anti-inflammation, butyrate-treated mice produced more of the gut tissue anti-inflammatory cytokine IL-10 and less of the pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ. In histological studies of Shigella infected gut revealed a startling observation where number of neutrophils infiltration was noted which was correlated with the pathology and was essentially reversed by butyrate treatment. Our results ratchet up a new dimension of our understanding how butyrate imparts resistance to pathogen invasion in the gut.

Essential oils from Acacia nilotica (Fabales: Fabaceae) seeds: May have insecticidal effects?

In the present study Acacia nilotica seed derived essential oils were tested against Spodoptera litura, Tenebrio molitor, Oxycarenus hyalinipennis, and Aphis fabae, as well as their effects on non-target species Eudrilus eugeniae and Artemia salina at 24 h post treatment. The seed essential oil produced insecticidal activity against A. fabae (LC50 = 41.679, LC90 = 75.212 μl/mL), O. hyalinipennis (LC50 = 37.629, LC90 = 118.485 μl/mL), T. molitor (LC50 = 56.796, LC90 = 201.912 μl/mL), and S. litura (LC50 = 62.215, LC90 = 241.183 μl/mL). Essential oils do not cause a remarkable effect on E. eugeniae and A. salina cytotoxicity. The essential oils produced a lower effect on Artemia salina (LC50 = 384.382, LC90 = 1341.397 μl/mL) and no lethal effects were observed on E. eugeniae. The histopathological evaluation showed no sub-lethal effects of essential oils on earthworm gut tissues. GC-MS analysis results revealed that the major chemical constituent was hexadecane (19.560%) and heptacosane (17.214%) and FT-IR analysis revealed the presence of alkanes and alkyles, aromatics, and amides functional groups that may be involved in insecticidal activity. Overall, the results showed that the seed derived essential oil has excellent insecticidal action against major agricultural insect pests and may therefore offer an environmentally benign alternative to conventional insecticide.

Comparative analysis of temporal variation of heavy metal accumulation by two sea urchin species from a harbour region, including pre and post COVID 19 lock down period

Sea urchins are marine invertebrates belonging to phylum Echinodermata, recognized as relevant biological tool for assessing environmental pollution. In the present study, we assessed the bioaccumulation potential of different heavy metals by two sea urchin species, Stomopneustes variolaris Lamarck, 1816 and Echinothrix diadema Linnaeus, 1758, collected from a harbour region, along the south west coast of India, during four different sampling periods for 2 years, from the same sea urchin bed. Heavy metals like Pb, Cr, As, Cd, Co, Se, Cu, Zn, Mn and Ni were analysed from water, sediment and different body parts of sea urchins, such as shell, spine, tooth, gut and gonad. The sampling periods also included the pre and post COVID 19 lockdown period during which the harbour activities were closed. The bio-water accumulation factor (BWAF), bio-sediment accumulation factor (BSAF) and the metal content/test weight index (MTWI) were calculated, in order to compare the bioaccumulation of metals by both the species. The results showed that S. variolaris had higher bioaccumulation potential than E. diadema, for metals like Pb, As, Cr, Co and Cd especially in the soft body parts like gut and gonad. The hard parts of S. variolaris like shell, spine, and tooth also accumulated more Pb, Cu, Ni and Mn than E. diadema. Following the lockdown period, there was a decline in the concentration of all heavy metals in water, whereas in sediment, Pb, Cr, and Cu levels were reduced. The gut and gonad tissues of both the urchins showed a decrease in the concentration of most of the heavy metals following the lockdown phase and no significant reduction was observed in the hard parts. This study reveals the use of S. variolaris as an excellent bioindicator of heavy metal contamination in the marine environment which can be employed for coastal monitoring programs.