Secretory Cell Differentiation Research Articles

Single Cell Analysis of Human Colonoids Exposed to Uranium-Bearing Dust.

Uranium exposure remains an important environmental legacy and physiological health concern, with hundreds of abandoned uranium mines located in the Southwestern United States largely impacting underserved indigenous communities. The negative effects of heavy metals on barrier permeability and inhibition of intestinal epithelial healing have been described; however, transcriptomic changes within the intestinal epithelial cells and impacts on lineage differentiation are largely unknown. Herein, we sought to determine the molecular and cellular changes that occur in the colon in response to uranium bearing dust (UBD) exposure. Human colonoids from three biologically distinct donors were acutely exposed to UBD then digested for single cell RNA sequencing to define the molecular changes that occur to specific identities of colonic epithelial cells. Validation in colonoids was assessed using morphological and imaging techniques. Human colonoids acutely exposed to UBD exhibited disrupted proliferation and hyperplastic differentiation of the secretory lineage cell, enteroendocrine cells (EEC). Single-cell RNA sequencing also showed more EEC subtypes present in UBD-exposed colonoids. These findings highlight the significance of crypt-based proliferative cells and secretory cell differentiation using human colonoids to model major colonic responses to uranium-bearing particulate dust exposure. https://doi.org/10.1289/EHP13855.

Epithelial Skiv2l deletion promotes aberrant secretory cell differentiation in the small intestine

Background: Inflammatory bowel disease (IBD) remains a global concern, with millions affected and incidence increasing. The etiology of IBD is multifaceted, but gene variants in epithelial and immune cells can contribute to its onset. Trichohepatoenteric syndrome (THES) is a form of monogenic very early onset IBD (VEO-IBD) that arises due to variants in SKIV2L and TTC37, which are components of the cytoplasmic RNA exosome involved in mRNA quality control. Patients with THES experience a decreased quality of life characterized by poor growth, intractable diarrhea, and immune defects. There are limited drugs approved by the FDA for treatment of IBD in children and those that have been approved are geared towards suppressing the immune system and have side effects. Therefore, there is a need for the development of therapeutics that target epithelial regeneration and repair. Understanding how genetic variants, such as those observed in THES, are involved in epithelial function is one avenue to improved therapies. The objective of the current study is to understand how the RNA exosome contributes to intestinal epithelial homeostasis by studying deletion of SKIV2L. Preclinical studies in mice revealed that global Skiv2l deletion resulted in increased mTOR signaling, and overactivated T cells, while B cell specific deletion of Skiv2l led to impaired B cell development. Ex vivo studies from our lab revealed that colonoids from THES patients exhibit decreased organoid formation effciency (OFE), suggesting that SKIV2L may have important roles in the intestinal stem cell self-renewal. We hypothesize that SKIV2L is required for intestinal epithelial cell growth and tissue function. Methods: We generated VillinCre;Skiv2l ( Skiv2l KO) mice with Skiv2l deletion in intestinal epithelial cells. Skiv2l WT and KO mice weights were monitored weekly, starting from ~2 weeks of age. Between 4 and 6 weeks of age, intestines were harvested for immunofluorescence, flow cytometry and OFE assays. Results and conclusion: Skiv2l KO mice experienced failure to thrive, were significantly smaller, and had shorter colons and small intestines compared to their WT counterparts. Immunofluorescence and flow cytometry revealed increased secretory cells in the small intestine of Skiv2l KO compared to WT mice, with significant accumulation of goblet cells in the crypt region in KO mice. Furthermore, Skiv2l KO mice had reduced OFE. These findings suggest that SKIV2L is essential for intestinal homeostasis and that aberrant secretory cell differentiation may underlie the decreased stem cell self-renewal observed in Skiv2l KO mice and THES patients. More broadly, these studies suggest an essential role for mRNA quality control in normal intestinal homeostasis. University of Pennsylvania, Institution of Regenerative Medicine; NIH R01DK124369. This is the full abstract presented at the American Physiology Summit 2024 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.

Abstract 5636: CUX1 transcriptionally regulates intestinal epithelial homeostasis

Abstract The small intestine digests and absorbs nutrients, a role made possible by specialized secretory and absorptive cells that constantly replenish from the intestinal stem cell niche. Disruption in intestinal epithelium homeostasis has been linked to pathologies including inflammatory bowel disease (IBD) and cancer. However, the processes regulating differentiation in healthy and disease setting are incompletely understood. CUX1 is a highly conserved homeodomain-containing transcription factor that is expressed in small intestinal crypts and has been previously shown to be essential in mice and Drosophila. CUX1 is upregulated upon inflammatory stress conditions, including intestinal damage and in IBD. In addition, CUX1 is predicted to be protective against IBD from genome-wide association studies. The aim of the current work is to determine the mechanistic role of CUX1 in secretory intestinal cells differentiation and epithelial homeostasis. To this end, we have generated transgenic mouse models that modulate CUX1 levels in an inducible manner. Mice with near-null CUX1 levels exhibit rapid weight loss requiring euthanasia while mice with ~50% residual CUX1 halt normal weight gain. Histologic examination of tissues from mice with CUX1 knock-down reveals a lack of mature secretory cells, including goblet and Paneth cells, the latter playing a supportive role to intestinal stem cells. Moreover, mouse intestinal tissues from our knock-down models demonstrated decreased proliferation with increased apoptosis. In transcriptome profiling, we observed downregulation of proliferation, intestinal stem cell, and digestive system developmental gene signatures and decreased levels of key secretory cell regulators, pointing towards a role for CUX1 in stem cell maintenance and differentiation. Indeed, reducing CUX1 levels decreased β-catenin protein levels, while Wnt/β-catenin signaling is required for the organization of the crypt-villus axis. Finally, CUX1 knock-down compromises the ability to form budding organoids, demonstrating the cell intrinsic role for CUX1 in stem cell differentiation. In ongoing studies, we are identifying CUX1 genome-wide binding targets and the epigenetic impact of CUX1 in intestinal epithelium cells. Our findings indicate that CUX1 is a critical regulator of intestinal epithelium homeostasis that has a role in differentiation and potential role in injury-induced de-differentiation in healthy as well as IBD patients. We propose that CUX1 regulates stem cell maintenance and crypt composition by regulating Wnt/β-catenin signaling and inducing fetal-like transcriptomic repair program shown to be coopted by colon cancers. Resolving an intricate network of transcriptional regulation of differentiation in the intestinal crypt holds the potential to successfully target inflammation-related damage in human intestines. Citation Format: Katarzyna Zawieracz, Ningfei An, Megan McNerney. CUX1 transcriptionally regulates intestinal epithelial homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5636.

Stem cell mTOR signaling directs region-specific cell fate decisions during intestinal nutrient adaptation.

The adult intestine is a regionalized organ, whose size and cellular composition are adjusted in response to nutrient status. This involves dynamic regulation of intestinal stem cell (ISC) proliferation and differentiation. How nutrient signaling controls cell fate decisions to drive regional changes in cell-type composition remains unclear. Here, we show that intestinal nutrient adaptation involves region-specific control of cell size, cell number, and differentiation. We uncovered that activation of mTOR complex 1 (mTORC1) increases ISC size in a region-specific manner. mTORC1 activity promotes Delta expression to direct cell fate toward the absorptive enteroblast lineage while inhibiting secretory enteroendocrine cell differentiation. In aged flies, the ISC mTORC1 signaling is deregulated, being constitutively high and unresponsive to diet, which can be mitigated through lifelong intermittent fasting. In conclusion, mTORC1 signaling contributes to the ISC fate decision, enabling regional control of intestinal cell differentiation in response to nutrition.

Constitutive expression of spliced X-box binding protein 1 inhibits dentin formation in mice.

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated, which subsequently converts an unspliced X-box binding protein 1 (XBP1U) mRNA to a spliced mRNA that encodes a potent XBP1S transcription factor. XBP1S is essential for relieving ER stress and secretory cell differentiation. We previously established Twist2-Cre;Xbp1 CS/+ mice that constitutively expressed XBP1S in the Twist2-expressing cells as well as in the cells derived from the Twist2-expressing cells. In this study, we analyzed the dental phenotype of Twist2-Cre;Xbp1 CS/+ mice. We first generated a mutant Xbp1s minigene that corresponds to the recombinant Xbp1 Δ26 allele (the Xbp1 CS allele that has undergone Cre-mediated recombination) and confirmed that the Xbp1s minigene expressed XBP1S that does not require IRE1α activation in vitro. Consistently, immunohistochemistry showed that XBP1S was constitutively expressed in the odontoblasts and other dental pulp cells in Twist2-Cre;Xbp1 CS/+ mice. Plain X-ray radiography and µCT analysis revealed that constitutive expression of XBP1S altered the dental pulp chamber roof- and floor-dentin formation, resulting in a significant reduction in dentin/cementum formation in Twist2-Cre;Xbp1 CS/+ mice, compared to age-matched Xbp1 CS/+ control mice. However, there is no significant difference in the density of dentin/cementum between these two groups of mice. Histologically, persistent expression of XBP1S caused a morphological change in odontoblasts in Twist2-Cre;Xbp1 CS/+ mice. Nevertheless, in situ hybridization and immunohistochemistry analyses showed that continuous expression of XBP1S had no apparent effects on the expression of the Dspp and Dmp1 genes. In conclusion, these results support that sustained production of XBP1S adversely affected odontoblast function and dentin formation.

The Regulatory Network of CREB3L1 and Its Roles in Physiological and Pathological Conditions.

CREB3 subfamily belongs to the bZIP transcription factor family and comprises five members. Normally they are located on the endoplasmic reticulum (ER) membranes and proteolytically activated through RIP (regulated intramembrane proteolysis) on Golgi apparatus to liberate the N-terminus to serve as transcription factors. CREB3L1 acting as one of them transcriptionally regulates the expressions of target genes and exhibits distinct functions from the other members of CREB3 family in eukaryotes. Physiologically, CREB3L1 involves in the regulation of bone morphogenesis, neurogenesis, neuroendocrine, secretory cell differentiation, and angiogenesis. Pathologically, CREB3L1 implicates in the modulation of osteogenesis imperfecta, low grade fibro myxoid sarcoma (LGFMS), sclerosing epithelioid fibrosarcoma (SEF), glioma, breast cancer, thyroid cancer, and tissue fibrosis. This review summarizes the upstream and downstream regulatory network of CREB3L1 and thoroughly presents our current understanding of CREB3L1 research progress in both physiological and pathological conditions with special focus on the novel findings of CREB3L1 in cancers.

Amino acid transporter SLC7A5 regulates cell proliferation and secretary cell differentiation and distribution in the mouse intestine.

The intestine is critical for not only processing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell (IEC)-specific knockout (ΔIEC) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5ΔIEC reduces mTORC1 signaling. Surprisingly, adult Slc7a5ΔIEC intestinal crypts have increased cell proliferation but reduced mature Paneth cells. Goblet cells, the other major secretory cell type in the small intestine, are increased in the crypts but reduced in the villi. Analyses with scRNA-seq and electron microscopy have revealed dedifferentiation of Paneth cells in Slc7a5ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. Thus, SLC7A5 likely regulates secretory cell differentiation to affect stem cell niche and indirectly regulate cell proliferation.

Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.

Embryo-derive TNF promotes decidualization via fibroblast activation.

Decidualization is a process in which endometrial stromal fibroblasts differentiate into specialized secretory decidual cells and essential for the successful establishment of pregnancy. The underlying mechanism during decidualization still remains poorly defined. Because decidualization and fibroblast activation share similar characteristics, this study was to examine whether fibroblast activation is involved in decidualization. In our study, fibroblast activation-related markers are obviously detected in pregnant decidua and under in vitro decidualization. ACTIVIN A secreted under fibroblast activation promotes in vitro decidualization. We showed that arachidonic acid released from uterine luminal epithelium can induce fibroblast activation and decidualization through PGI2 and its nuclear receptor PPARδ. Based on the significant difference of fibroblast activation-related markers between pregnant and pseudopregnant mice, we found that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium. Fibroblast activation is also detected under human in vitro decidualization. Similar arachidonic acid-PGI2-PPARδ-ACTIVIN A pathway is conserved in human endometrium. Collectively, our data indicate that embryo-derived TNF promotes CPLA2α phosphorylation and arachidonic acid release from luminal epithelium to induce fibroblast activation and decidualization.

FEATURES OF THE MICROSTRUCTURE OF THE LEAVES OF Fatsia japonica (THUNB.) DECNE. ET PLANCH. (Araliaceae)

The internal structure of leaf of the F. japonica that is introduced in Georgia as a source of biologically active substances, was studied, and its microstructural diagnostic signs were determined. F. japonica leaf glabrous, bilateral, dorsoventral, hypostomatic structure. The basal cells of the abaxial and adaxial epidermis of the leaf are non-stitched, curvilinear and curved-walled, the cuticular mosaic of the lower epidermis is wavy. The stomatal apparatus of the Japanese fatsia leaf is anisocytic, the stomatal gap is fusiform, and the location of the stomata is chaotic. A reverse-collateral conductive bundle is imprinted in the pulp of the leaf. In the structural units of all morphological organs of the plant leaf, differentiation of calcium oxalate druse crystals and secretory cells is noticeable. The tissue of the main vein of the leaf of F. japonica is polygonal, the conducting system both in the vein and in the petiole is of fibro-vascular, open-collateral type, but in the main vein bundles are asymmetrical, while in the petiole they are symmetrical and closely interconnected. The  xylem of the main vein and petiole of F. japonica leaf’s has differentiated bulbs arranged in longitudinal rows. Xylem conductive vessels with spiral, ring and mesh thickenings of the shell are observed lengthwise the petiole.

Absence of gut microbiota impairs depletion of Paneth cells but not goblet cells in germ-free Atoh1lox/lox VilCreERT2 mice.

Mouse atonal homolog 1 (Math1/Atoh1) is a basic helix-loop-helix transcription factor important for the differentiation of secretory cells within the intestinal epithelium. The analysis of Paneth depletion efficiency on Math1lox/loxVilCreERT2 (Math1ΔIEC) mice treatment with tamoxifen in the presence or absence of intestinal microbiota showed a failure on Paneth cell depletion in germ-free mice as compared with specific pathogen-free (SPF) mice. However, goblet cells were efficiently depleted in Math1ΔIEC germ-free mice. The gene expression of Math1 was significantly reduced in the ileum of germ-free Math1ΔIEC mice 5 days after tamoxifen injection as compared with germ-free control, but its protein expression was still detectable in the nuclei of epithelial cells in the crypts. Germ-free mice showed low proliferative ileal crypts and apoptotic cells that were mainly detected in the tip of the villus, consistent with a slow turnover rate of epithelial cells. Although Paneth cells were not depleted in germ-free Math1ΔIEC mice for the first 7 wk after the last tamoxifen injection, far already from the 5 days time-laps observed in SPF conditions, an incomplete depletion of Paneth cells was observed 14 wk after the last tamoxifen injection. Colonization of germ-free mice restored the phenotype observed in SPF mice, highlighting the regulatory role of gut microbes in our model. We conclude that absence of intestinal microbiota in Math1ΔIEC mice is associated with reduced epithelial cell renewal and delays the depletion of preexisting Paneth cells.NEW & NOTEWORTHY Cre-lox system is a powerful and widely used research tool developed to understand the specific role of genes. It allows to control the spatial and temporal expression of genes in experimental models. Several limitations including toxicity of Cre recombinase or incomplete excision of floxed loci have been reported in the past. To date, this is the first research study reporting that gut microbes also influence the expected phenotype of Paneth cell depletion in the genetically modified Math1lox/loxVilCreERT2 mouse model.

Gastric Stem Cell Biology and Helicobacter pylori Infection.

Helicobacter pylori colonizes the human gastric mucosa and persists lifelong. An interactive network between the bacteria and host cells shapes a unique microbial niche within gastric glands that alters epithelial behavior, leading to pathologies such as chronic gastritis and eventually gastric cancer. Gland colonization by the bacterium initiates aberrant trajectories by inducing long-term inflammatory and regenerative gland responses, which involve various specialized epithelial and stromal cells. Recent studies using cell lineage tracing, organoids and scRNA-seq techniques have significantly advanced our knowledge of the molecular "identity" of epithelial and stromal cell subtypes during normal homeostasis and upon infection, and revealed the principles that underly stem cell (niche) behavior under homeostatic conditions as well as upon H. pylori infection. The activation of long-lived stem cells deep in thegastric glands has emerged as a key prerequisite of H. pylori-associated gastric site-specific pathologies such as hyperplasia in the antrum, and atrophy or metaplasia in the corpus, that are considered premalignant lesions. In addition to altering the behaviour of bona fide stem cells, injury-driven de-differentiation and trans-differentation programs, such as "paligenosis", subsequently allow highly specialized secretory cells to re-acquire stem cell functions, driving gland regeneration. This plastic regenerative capacity of gastric glands is required to maintain homeostasis and repair mucosal injuries. However, these processes are co-opted in the context of stepwise malignant transformation in chronic H. pylori infection, causing the emergence, selection and expansion of cancer-promoting stem cells.

Deep Crypt Secretory Cell Differentiation in the Colonic Epithelium Is Regulated by Sprouty2 and Interleukin 13

Deep crypt secretory (DCS) cells are a critical component of the colonic stem cell niche. However, the regulatory mechanisms controlling DCS cell numbers and function are not well understood. Sprouty2 is an inflammation-responsive regulator of intracellular signaling that influences colonic secretory cell numbers in colitis via an epithelial-stromal interleukin (IL)33/IL13 signaling loop. Here, we tested the hypothesis that IL13, induced by epithelial Sprouty2 down-regulation, promotes DCS cell differentiation and function. Distal colons from mice with an intestinal epithelial-specific Sprouty2 deletion (Spry2ΔIE) and littermate controls were analyzed by in situ hybridization for Reg4+ DCS cells. Single-cell RNA sequencing and immunostaining were used to identify DCS cell-derived host defense peptides (HDPs) and localization of IL13 and IL13 receptor; bulk RNA sequencing and quantitative polymerase chain reaction were used to quantify changes in expression of identified HDPs. Cytokine-treated colonoids were assessed for DCS cells. A requirement for an IL33/IL13 signaling loop in the regulation of DCS cells was assessed invivo using IL13 null mice. Reg4+ DCS cell numbers were increased 2-fold in distal colons of Spry2ΔIE mice with a concomitant overall increase in DCS cell marker expression (Reg4, Spink4, and Agr2). Single-cell transcriptomics showed the HDP Retnlb/Resistin Like Beta (RELMβ) is highly enriched in DCS cells. Retnlb/RELMβ expression was increased in Spry2ΔIE colons. IL13, but not IL33, induced Reg4 and Retnlb expression in colonic epithelial organoids, and IL33-mediated expansion of the DCS cell population invivo was dependent on IL13, which was expressed predominantly by type II innate lymphoid cells in the colonic mucosa. Sprouty2 limits colonic DCS cell differentiation through suppression of IL13 signaling. At homeostasis, DCS cells are marked by high levels of the HDP RELMβ. Loss of epithelial Sprouty2 activates type II innate lymphoid cells to release IL13, promoting expansion of the DCS cell population and increased colonic RELMβ levels.

R-spondin/YAP axis promotes gastric oxyntic gland regeneration and Helicobacter pylori-associated metaplasia in mice.

The stomach corpus epithelium is organized into anatomical units that consist of glands and pits. Mechanisms that control the cellular organization of corpus glands and enable their recovery upon injury are not well understood. R-spondin 3 (RSPO3) is a WNT-signaling enhancer that regulates stem cell behavior in different organs. Here, we investigated the function of RSPO3 in the corpus during homeostasis, upon chief and/or parietal cell loss, and during chronic Helicobacter pylori infection. Using organoid culture and conditional mouse models, we demonstrate that RSPO3 is a critical driver of secretory cell differentiation in the corpus gland toward parietal and chief cells, while its absence promoted pit cell differentiation. Acute loss of chief and parietal cells induced by high dose tamoxifen - or merely the depletion of LGR5+ chief cells - caused an upregulation of RSPO3 expression, which was required for the initiation of a coordinated regenerative response via the activation of yes-associated protein (YAP) signaling. This response enabled a rapid recovery of the injured secretory gland cells. However, in the context of chronic H. pylori infection, the R-spondin-driven regeneration was maintained long term, promoting severe glandular hyperproliferation and the development of premalignant metaplasia.

Study of the leukocyte profile in the mammary gland of lactating rats under the influence of oxytocin

During lactation, the structure of the organ and the composition of lymphoid cells change in the mammary gland. The change in the cells of the leukocyte series indicates a deep relationship between the immune system and the lactation process. The functioning of the mammary gland is under the influence of pituitary hormones and sex hormones, which change the cytological structure of the organ and the content of leukocytes in it. The studies were carried out on lactating rats. Were formed, one control group and two experimental groups of animals, 5 in each. Oxytocin was injected intraperitoneally at a dose of 0.1 ml to rats of the 2nd and 3rd experimental groups. The structure of the mammary gland and the composition of leukocyte cells were studied before the influence of oxytocin, 5 and 10 minutes after the administration of the hormone. The mammary glands were dissected and impression smears were prepared. The smears were stained and lymphoid cells were counted using optics. Under the influence of the hormones of the adenohypophysis, during the lactation period, the glandular tissue in the mammary gland replaces the adipose tissue. Insulin, growth hormone are able to increase the rate of DNA synthesis, stimulate mitotic activity, due to which the cell mass of the developing organ increases. With the development of structural components characteristic of a differentiated secretory cell, the structure of the alveolus is formed. Along with the differentiation of secretory cells in the alveolus, the formation of the functional activity of the myoepithelium occurs. A slight increase in the number of lymphocytes in the preparations of the control group of rats was revealed. Under the influence of the neurohypophysis hormone, the composition of immunocompetent blood cells changed. The introduction of intraperitoneal oxytocin, after 5 minutes, stimulated a significant increase in the number of lymphocytes, compared with the control group, from 16,20±3.42 to 37,80±2.63 (p<0.001). By the 10th minute, on the contrary, there was a significant decrease to 5,40±1.48 cells (p<0.001). Monocytes and a small number of neutrophils were also established. The lactation and pre-lactation periods directly affect the functioning of the immune system, changes in the composition of its cells in the mammary gland and their migration through the hemato-milk barrier into colostrum.

Comparison of Androgen Receptor Expression in Patients with Benign Prostate Hyperplasia and Adenocarcinoma Prostate

Introduction: Benign prostate hyperplasia is a proliferation of stromal cells and epithelial cells which results an enlargement of the prostate gland and can cause obstruction of the urinary tract. Adenocarcinoma prostate is an invasive carcinoma consisting of a neoplasm of prostate epithelial cells with secretory cell differentiation. Abnormal growth of the prostate gland like benign lesions and malignant lesions are generally affected by androgen receptor (AR). The aim of this research is to analyse the comparison of AR expression in benign prostate hyperplasia and adenocarcinoma prostate.
 Methods: The design of this research is analiytical study with cross sectional approach. The samples are 18 cases of benign prostate hyperplasia and 20 cases of adenocarcinoma prostate. The samples are observed only once and at a time.
 Results: The average age of the adenocarcinoma prostate case was 63.95 ± 12.03 years (age range 33-84 years) while in the benign prostate hyperplasia case, the average age was 66.78 ± 9.49 (age range 54–89 years). There are no different about the expression of AR in benign prostate hyperplasia and adenocarcinoma prostate used Allred scores. There was a weak and negative correlation between Gleason score and AR expression in the adenocarcinoma prostate case group (p=0.237).
 Conclusion: There are no differences related to the expression of AR between the case of adenocarcinoma prostate and benign prostatic hyperplasia.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B.subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B.subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B.subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B.subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B.subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

THE CORRELATION BETWEEN IMMUNOHISTOCHEMICAL EXPRESSION OF CLUSTER OF DIFFERENTIATION 5 (CD5) AND STROMAL TUMOR INFILTRATING LYMPHOCYTES (STILS) GRADE IN PROSTATE ADENOCARCINOMA

Background: Adenocarcinoma of the prostate is an invasive carcinoma, a neoplasm of prostate epithelial cells with secretory cell differentiation. Histomorphology is glandular. The cells are sheets or single cells with neoplasia, basal cells are not seen in this carcinoma. Tumor-Infiltrating Lymphocytes (TILs) are lymphocytes that migrate to tumor cells or peritumoral cells. In a malignancy, T lymphocyte infiltration can be found, indicating an immune response to malignancy. The cluster of Differentiation 5 (CD5) is a transmembrane receptor that plays a role in regulating the function and development of T cells. Objective: To determine the correlation between Immunohistochemical expression of Cluster of Differentiation 5 (CD5) and the Stromal Tumor-Infiltrating Lymphocytes (sTILs) grade in Prostate Adenocarcinoma. Material and method: This study was conducted on slides of 32 prostate adenocarcinoma patients. Each slide was stained with hematoxylin and eosin (H&E), and the grade of STILs was observed on the slide. Furthermore, CD5 immunohistochemical staining was carried out to see and analyze whether the appearance was positive or negative. Findings: Among 32 specimens of prostate adenocarcinoma patients, it was found 10 mild STILs, 22 moderate STILs and no severe STILs on hematoxylin and eosin (HE) staining, CD5 immunohistochemical staining was obtained 16 positive expressions and 16 negative expressions. Conclusion: There is no correlation between immunohistochemical expression of Cluster of Differentiation 5 (CD5) and the grade of stromal Tumor-Infiltrating Lymphocyte (sTILS) in prostate adenocarcinoma. .

The ErbB3 Receptor Restricts Bmi1 to Regulate Paneth Cells

ErbB3 is a RTK family member known to regulate proliferation and growth in the intestinal epithelium. Previous work by our lab determined that targeted Erbb3 deletion from the intestinal epithelium resulted in precocious Paneth cell development and an expanded population in mice. This was associated with increased expression of intestinal stem cell (ISC) markers. Conversely, ileal enteroids treated with the ErbB3 ligand neuregulin (NRG)‐1β had significantly reduced expression of ISC markers, including Lgr5 and Bmi1. These data suggest that ErbB3 restricts secretory cell differentiation and stemness in the intestinal crypt. However, the underlying mechanisms are largely unknown. BMI1 is a proto‐oncogene and member of the PRC complex. Bmi1+ intestinal epithelial cells represent a slow‐cycling ISC population that can regenerate the epithelium following injury and loss of Lgr5+ ISCs. Recent evidence determined that Bmi1+ ISCs are enriched in enteroendocrine markers and can give rise to goblet cells, suggesting BMI1 may be critical for differentiation of secretory lineages. We hypothesized that ErbB3 restricts secretory cell differentiation through regulation of BMI1.AimsTo define ErbB3‐dependent regulation of Bmi1 and determine if ErbB3 restriction of Paneth cell development and differentiation is through suppression of Bmi1.MethodsMucosal scrapings were collected from Vil‐Cre;Erbb3flox/flox mice and Erbb3flox/flox littermate controls for protein and gene expression analysis. Ileal enteroids and HT‐29 colorectal adenocarcinoma cells were treated with vehicle DMSO, the PI3K inhibitor LY294002 (10 μM), the MEK inhibitor U0126 (5 μM), the BMI1 inhibitor PTC‐209 (1 μM), and/or recombinant NRG‐1β (10 ng/mL). Enteroids and cell monolayers were harvested and prepared for gene expression analysis. RT‐qPCR for Bmi1 and Lyz1 was performed relative to Hprt as a loading control. Statistical analysis by one‐way ANOVA was conducted using Prism 9.ResultsConsistent with previous data, BMI1 protein and gene expression were significantly increased in small and large intestinal mucosal scrapings from Vil‐Cre;Erbb3flox/flox mice versus littermate controls. To determine if Bmi1 expression is sensitive to PI3K/Akt or MAPK signaling downstream of ErbB3, HT‐29 cells were treated with inhibitors followed by NRG‐1β after 1 hour, then harvested 24 hours later. PI3K and MAPK inhibition significantly increased Bmi1 expression. NRG‐1β treatment was able to overcome MAPK inhibition to reduce BMI1 expression. Similarly, in ileal enteroids, PI3K and MAPK blockade increased Bmi1 expression and NRG‐1β treatment reversed this effect. To determine whether BMI1 regulates expression of the Paneth cell marker LYZ1, HT‐29 cells were treated with the BMI1 inhibitor PTC‐209. BMI1 inhibition decreased LYZ1 expression.ConclusionsOur data demonstrate that ErbB3 regulates Bmi1 expression through PI3K/Akt and MAPK signaling in both human and mouse intestinal cells. Furthermore, BMI1 activity promotes LYZ1 expression. Together, these results support our hypothesis that ErbB3 regulates secretory cell differentiation through BMI1 in the intestinal epithelium.

Alpha‐ketoglutarate promotes goblet cell differentiation and alters metabolome in a mouse model of colitis

BackgroundAlpha‐ketoglutarate (aKG) is a key intermediate of the tricarboxylic acid (TCA) cycle. Previously, we demonstrated that aKG supplementation mitigates inflammation, enhances epithelial barrier integrity, and promotes oxidative phosphorylation in dextran sulfate sodium (DSS)‐induced colitis of mice. Intestinal epithelial homeostasis is regulated by cell energetics and metabolites from the microbiome. Given the importance of the mucosal layer in maintaining gut epithelial barrier function and microbiome composition, we hypothesized that dietary aKG facilitates the differentiation of secretory lineage epithelial cells and improves the gut metabolome.MethodsC57BL/6 male mice received drinking water with or without 1% aKG for three weeks, when mice were subjected to DSS induction for 7 days followed by 7 days recovery. The GC‐MS analysis was used to profile the cecal metabolomic changes due to aKG supplementation. In addition, colonic tissues were sampled for both biological and histochemical analyses.ResultsGC‐MS metabolomics revealed separation of the cecal metabolites between control (CON) and aKG supplemented (aKG) mice. In total, the contents of 40 metabolites were found to be different between CON and aKG groups. These metabolites include amino acids, fatty acids, and the B vitamin, niacin, succinic acid (butanedioic acid), the conjugate acid of succinate. Putrescine, a polyamine produced by intestinal microbiota and methysuccinic acid were among the metabolites with the largest fold change increased by dietary aKG. Amino acids including essential amino acids and those involved in the TCA cycle such as threonine and aspartic acid were elevated in the aKG group, of which, glycine displayed a 10‐fold increase. On the other hand, ornithine, an intermediate of the urea cycle, and amide products such as oleamide and urea were significantly decreased in aKG group. Pathway analysis revealed that the urea cycle, along with and arginine and proline metabolism and glutathione metabolism were altered due to aKG supplementation. Furthermore, aKG supplementation increased goblet cells based on Alcian blue staining, associated with increased mRNA expression of Mucin 2 and goblet cell differentiation markers Trefoil factor 3 and Krüppel‐like factor 4.ConclusionTogether, dietary aKG enhances intestinal epithelial differentiation to favor goblet cell differentiation, which correlates with improvement in metabolome.