Thymic mimetic cells function beyond self-tolerance.

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

Effects of different coating materials on the morphological characteristics of chicken adenohypophyseal folliculo-stellate cells in vitro.

Chicken adenohypophyseal cells were cultured in plates coated with different materials, and their morphologies were examined to confirm the characteristics of chicken folliculo-stellate (FS) cells in vitro. The adenohypophyseal cells were dispersed with a collagenase/trypsin mixture in media and seeded in plates coated in either poly L-lysine (PLL), collagen, or laminin. After 7 days of culture, the cells were fixed and immunocytochemistry was performed. 5-Bromo-2'-deoxyuridine incorporation test indicated that the proliferation activity of the culture cells was different based on the coating materials, and it was higher in the collagen-coated plate than two other coating materials. Fluorescence immunocytochemistry was also performed using mixed antibodies against growth hormone, prolactin, luteinizing hormone ß-subunit, basic cytokeratin (bCK), and S100B. The culture cells on the PLL- and laminin-coated surfaces were round or oval in shape, and bCK-immunopositive FS cells were morphologically indistinguishable from endocrine cells. In the collagen-coated plate, many endocrine cells were round or oval in shape, but FS cells displayed a larger and flattened morphology. S100B-immunoreactions were localized in the nuclei of bCK-immunopositive FS cells. These results suggest that culturing the chicken adenohypophyseal cells in the collagen-coated plate enables the distinction of FS cells from endocrine cells.

Oral berberine ameliorates high-fat diet-induced obesity by activating TAS2Rs in tuft and endocrine cells in the gut.

BACKGROUND AND AIMS: Although oral berberine, a natural compound extracted from the Chinese herbal medicine curcumin, has low bioavailability, it is still effective in suppressing obesity; however, the underlying mechanism is unclear. Berberine can bind to bitter-taste receptors (TAS2Rs) in intestinal endocrine secretin tumor (STC-1) cells to promote glucagon-like peptide-1 (GLP-1) secretion. Notably, TAS2Rs also exist in the tuft cells of the gut. Therefore, this study aimed to explore whether the beneficial effect of oral berberine on obesity is dependent on bitter-taste signaling in the tuft cells of the gut. METHODS AND RESULTS: Standard chow diet (SCD) or high-fat diet (HFD) was administered to C57BL/6 mice, with or without berberine (100 mg/kg, 200 mg/kg, p. o.). The PLCß2 inhibitor U73122 was used to verify whether the anti-obesity effect of berberine was dependent on the bitter-taste signaling pathway. In this study, we observed that the oral administration of berberine alleviated HFD-induced obesity in mice that U73122 partially inhibited. Both in vivo and ex vivo, berberine upregulated the release of GLP-1, promoted the proliferation of tuft cells and secretion of IL-25 in obesity via the TAS2R signaling pathway. CONCLUSIONS: Oral berberine ameliorated HFD-induced obesity through the TAS2R-IL-25 signaling pathway in tuft cells in the gut. SIGNIFICANCE: We identified and functionally characterized the TAS2Rs and Gα-gustducin/Gß1γ13 signaling pathway utilized by tuft cells in response to oral berberine in obese mice and proposed a new mechanism underlying the anti-obesity effect of berberine.

[The role of the main risk factors and endocrine cells of the antrum of the stomach producing motilin in the occurrence of cholelithiasis].

AIM: To establish the role of the main risk factors and endocrine cells of the antrum of the stomach producing motilin (M-cells) in the occurrence of cholelithiasis. MATERIALS AND METHODS: The first group included 122 patients with cholelithiasis. The second group consisted of 30 healthy individuals who underwent medical examination. The groups were matched for gender and age. The work analyzed anamnestic, biochemical and anthropometric data. All patients underwent esophagogastroduodenoscopy with targeted biopsy of the mucous membrane from the antrum. Biopsies were subjected to cytological and immunohistochemical studies in order to verify Helicobacter pylori and estimate the number of M-cells. RESULTS: Patients with cholelithiasis more often belonged to the group of people of mental labor, had low physical activity, were committed to inappropriate nutrition and more often indicated the presence of aggravated heredity for cholelithiasis. Patients with gallstone disease had higher body mass index, waist volume, total cholesterol, low-density lipoprotein cholesterol, triglycerides, glucose, lower high-density lipoprotein cholesterol, H. pylori infection was more often verified and M-cell hypoplasia in the mucous membrane was established. stomach in comparison with the representatives of the second group. CONCLUSION: Our results suggest that certain external factors, nutritional characteristics of the metabolic syndrome components, hypoplasia of M-cells in the gastric mucosa are important factors in the formation of calculi in the gallbladder.

Exocrine-Endocrine Crosstalk: The Influence of Pancreatic Cellular Communications on Organ Growth, Function and Disease.

Diabetes mellitus, a disease that affects nearly 536.6 million people worldwide, is characterized by the death or dysfunction of insulin-producing beta cells of the pancreas. The beta cells are found within the islets of Langerhans, which are composed of multiple hormone-producing endocrine cells including the alpha (glucagon), delta (somatostatin), PP (pancreatic polypeptide), and epsilon (ghrelin) cells. There is direct evidence that physical and paracrine interactions between the cells in the islet facilitate and support beta cell function. However, communication between endocrine and exocrine cells in the pancreas may also directly impact beta cell growth and function. Herein we review literature that contributes to the view that "crosstalk" between neighboring cells within the pancreas influences beta cell growth and function and the maintenance of beta cell health.

Expression of p11 and heteromeric TASK channels in mouse adrenal cortical cells and H295R cells.

TWIK-related acid-sensitive K+ (TASK) channels are thought to contribute to the resting membrane potential in adrenal cortical (AC) cells. However, the molecular identity of TASK channels in AC cells have not yet been elucidated. Thus, immunocytochemical and molecular biological approaches were employed to investigate the expression and intracellular distribution of TASK1 and TASK3 in mouse AC cells and H295R cells derived from human adrenocortical carcinoma. Immunocytochemical study revealed that immunoreactive materials were mainly located in the cytoplasm for TASK1 and at the cell periphery for TASK3 in mouse AC cells. A similar pattern of localization was observed when GFP-TASK1 and GFP-TASK3 were exogenously expressed in H295R cells. In addition, p11 that is known to suppress the endoplasmic reticulum exit of TASK1 was localized in the cytoplasm in mouse AC and H295R cells, but not in adrenal medullary cells. Proximity ligation assay (PLA) suggested formation of heteromeric TASK1-3 channels that were found predominantly in the cytoplasm and weakly at the cell periphery. A similar distribution was observed following exogenous expression of tandem TASK1-3 channels in H295R cells. When stimulated by angiotensin II, however, tandem TASK1-3 channels were present mainly in the cytoplasm in all H295R cells. In contrast to that in H295R cells, tandem channels were exclusively located at the cell periphery in all non-stimulated and exclusively in the cytoplasm in stimulated PC12 cells, respectively. From these results, we conclude that TASK1 proteins are present mainly in the cytoplasm and minimally at the cell periphery as a heteromeric channel with TASK3, whereas the majority of TASK3 is at the cell periphery as homomeric and heteromeric channels.

Endocrine cell micronests in an ovarian mucinous borderline tumor: a potential diagnostic pitfall for microinvasion.

The occurrence of endocrine cell micronests in ovarian tumors is rarely reported. To our knowledge, there are only three prior cases reported to date: one occurring in an ovarian mucinous cystadenoma, one in an ovarian mucinous cystadenofibroma, and another in an ovarian mucinous carcinoma with a predominant borderline component. This is a 27-year-old woman that presented with a one-month history of abdominal pain and fullness. Imaging studies revealed a large multiloculated cystic and solid mass measuring 23 cm occupying the majority of the pelvis and abdomen concerning for a primary ovarian malignancy. The patient underwent a right salpingo-oophorectomy with appendectomy. Histologic sections from the ovary showed a multiloculated, cystic and focally solid mass lined by gastrointestinal-type mucinous epithelium with variable degrees of proliferation accounting for greater than 10% of the tumor. In addition to the mucinous epithelial component, there were several foci of bland, monotonous epithelioid cells arranged in solid nests with focal tubular/acinar formation within the fibrous septa and mucinous epithelium. Immunohistochemical studies showed that these cells were positive for cytokeratin, EMA, and synaptophysin, while negative for inhibin. The Ki-67 proliferation index was low (<1%). The presence of endocrine cell nests associated with an ovarian mucinous neoplasm is a rare phenomenon. Whether this represents preservation of endocrine cells in the context of epithelial degeneration or an independent neoplastic component is unclear. Progression related to this endocrine cell proliferation is unlikely and the recognition of this phenomenon holds more diagnostic value than prognostic significance, as it could be confused with microinvasion or sex cord stromal elements.

Increased Expression of Viral Sensor MDA5 in Pancreatic Islets and in Hormone-Negative Endocrine Cells in Recent Onset Type 1 Diabetic Donors.

The interaction between genetic and environmental factors determines the development of type 1 diabetes (T1D). Some viruses are capable of infecting and damaging pancreatic ß-cells, whose antiviral response could be modulated by specific viral RNA receptors and sensors such as melanoma differentiation associated gene 5 (MDA5), encoded by the IFIH1 gene. MDA5 has been shown to be involved in pro-inflammatory and immunoregulatory outcomes, thus determining the response of pancreatic islets to viral infections. Although the function of MDA5 has been previously well explored, a detailed immunohistochemical characterization of MDA5 in pancreatic tissues of nondiabetic and T1D donors is still missing. In the present study, we used multiplex immunofluorescence imaging analysis to characterize MDA5 expression and distribution in pancreatic tissues obtained from 22 organ donors (10 nondiabetic autoantibody-negative, 2 nondiabetic autoantibody-positive, 8 recent-onset, and 2 long-standing T1D). In nondiabetic control donors, MDA5 was expressed both in α- and ß-cells. The colocalization rate imaging analysis showed that MDA5 was preferentially expressed in α-cells. In T1D donors, we observed an increased colocalization rate of MDA5-glucagon with respect to MDA5-insulin in comparison to nondiabetic controls; such increase was more pronounced in recent-onset with respect to long-standing T1D donors. Of note, an increased colocalization rate of MDA5-glucagon was found in insulin-deficient-islets (IDIs) with respect to insulin-containing-islets (ICIs). Strikingly, we detected the presence of MDA5-positive/hormone-negative endocrine islet-like clusters in T1D donors, presumably due to dedifferentiation or neogenesis phenomena. These clusters were identified exclusively in donors with recent disease onset and not in autoantibody-positive nondiabetic donors or donors with long-standing T1D. In conclusion, we showed that MDA5 is preferentially expressed in α-cells, and its expression is increased in recent-onset T1D donors. Finally, we observed that MDA5 may also characterize the phenotype of dedifferentiated or newly forming islet cells, thus opening to novel roles for MDA5 in pancreatic endocrine cells.

Characterization and reduction of non-endocrine cells accompanying islet-like endocrine cells differentiated from human iPSC.

The differentiation of pancreatic endocrine cells from human pluripotent stem cells has been thoroughly investigated for their application in cell therapy against diabetes. Although non-endocrine cells are inevitable contaminating by-products of the differentiation process, a comprehensive profile of such cells is lacking. Therefore, we characterized non-endocrine cells in iPSC-derived pancreatic islet cells (iPIC) using single-cell transcriptomic analysis. We found that non-endocrine cells consist of (1) heterogeneous proliferating cells, and (2) cells with not only pancreatic traits but also liver or intestinal traits marked by FGB or AGR2. Non-endocrine cells specifically expressed FGFR2, PLK1, and LDHB. We demonstrated that inhibition of pathways involving these genes selectively reduced the number of non-endocrine cells in the differentiation process. These findings provide useful insights into cell purification approaches and contribute to the improvement of the mass production of endocrine cells for stem cell-derived cell therapy for diabetes.

Secretome of Adipose Tissue as the Key to Understanding the Endocrine Function of Adipose Tissue.

The prevalence of obesity has reached pandemic levels and is becoming a serious health problem in developed and developing countries. Obesity is associated with an increased prevalence of comorbidities that include type II diabetes, cardiovascular diseases and some cancers. The recognition of adipose tissue as an endocrine organ capable of secreting adipokines that influence whole-body energy homeostasis was a breakthrough leading to a better molecular understanding of obesity. Of the adipokines known to be involved in the regulation of energy metabolism, very few are considered central regulators of insulin sensitivity, metabolism and energy homeostasis, and the discovery and characterization of new adipocyte-derived factors are still ongoing. Proteomics techniques, such as liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry, have proven to be useful tools for analyzing the secretory function of adipose tissue (the secretome), providing insights into molecular events that influence body weight. Apart from the identification of novel proteins, the considerable advantage of this approach is the ability to detect post-translational modifications that cannot be predicted in genomic studies. In this review, we summarize recent efforts to identify novel bioactive secretory factors through proteomics.

Interleukin-6 is dispensable in pituitary normal development and homeostasis but needed for pituitary stem cell activation following local injury.

Recently, we discovered that the cytokine interleukin-6 (IL-6) acts as a pituitary stem cell-activating factor, both when administered in vivo and when added to stem cell organoid cultures in vitro. Moreover, its expression, predominantly localized in the gland's stem and mesenchymal cells, promptly increases following damage in the adult pituitary, leading to stem-cell proliferative activation. Given these findings that IL-6 is involved in pituitary stem cell regulation, we addressed the question whether the cytokine has an impact on the pituitary phenotype during active phases of the gland's remodeling, in particular embryonic development and neonatal maturation, as well as during homeostasis at adulthood and aging, all unknown today. Using the IL-6 knock-out (KO) mouse model, we show that IL-6 is dispensable for pituitary embryonic and neonatal endocrine cell development, as well as for hormonal cell homeostasis in adult and aging glands. The findings match the absence of effects on the stem cell compartment at these stages. However, using this IL-6 KO model, we found that IL-6 is needed for the acute stem-cell proliferative activation reaction upon pituitary injury. Intriguingly, regeneration still occurs which may be due to compensatory behavior by other cytokines which are upregulated in the damaged IL-6 KO pituitary, although at lower but prolonged levels, which might lead to a delayed (and less forceful) stem cell response. Taken together, our study revealed that IL-6 is dispensable for normal pituitary development and homeostasis but plays a key role in the prompt stem cell activation upon local damage, although its presence is not essentially needed for the final regenerative realization.

Renewing an old interest: Pituitary folliculostellate cells.

Anterior pituitary folliculostellate (FS) cells, first described almost 50 years ago, have a wide range of functions with respect to supporting and coordinating endocrine cell function, in particular through paracrine and gap junction-mediated signalling. Our previous studies identified the morphological organisation of FS cells, which mediates coordinated calcium activity throughout the homotypic FS network and allows signalling across the whole pituitary gland. It is also clear that FS cells can modify endocrine output and feedback on pituitary axes over a range of timescales. Recently, several studies have defined FS cells as a source of anterior pituitary endocrine cell renewal, which has resulted in a renaming of FS cells as "Sox2+ve stem cells". Here, we highlight the broader potential of the FS cell population in fine-tuning and coordinating pituitary axes function. In addition, we identify a need for: the definition of the possible subtypes of FS cell and their relationship with the stem cell population; the potential role of FS cells in pulsatile hormone secretion and coordination of heterotypic cell networks; and the roles that FS cells may play in both early-life programming of pituitary axes and in memory, or anticipation, of demand. Further studies of FS cells may demonstrate the fundamental importance of this cell type and its potential as a therapeutic target to correct pituitary gland dysfunction, one of which is stem cell therapy. Clearly, a thorough understanding of all of these interactions and relationships of FS and endocrine cells is required whatever therapeutic use is suggested by their various roles.

Process parameter development for the scaled generation of stem cell-derived pancreatic endocrine cells.

Diabetes is a debilitating disease characterized by high blood glucose levels. The global prevalence of this disease has been projected to reach 700 million adults by the year 2045. Type 1 diabetes represents about 10% of the reported cases of diabetes. Although islet transplantation can be a highly effective method to treat type 1 diabetes, its widespread application is limited by the paucity of cadaveric donor islets. The use of pluripotent stem cells as an unlimited cell source to generate insulin-producing cells for implant is a promising alternative for treating diabetes. However, to be clinically relevant, it is necessary to manufacture these stem cell-derived cells at sufficient scales. Significant advances have been made in differentiation protocols used to generate stem cell-derived cells capable of reversing diabetes in animal models and for testing in clinical trials. We discuss the potential of both stem cell-derived pancreatic progenitors and more matured insulin-producing cells to treat diabetes. We discuss the need for rigorous bioprocess parameter optimization and identify some critical process parameters and strategies that may influence the critical quality attributes of the cells with the goal of facilitating scalable manufacturing of human pluripotent stem cell-derived pancreatic endocrine cells.

The role of endocrine hormones in the pathogenesis of adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a common spinal deformity characterized by changes in the three-dimensional structure of the spine. It usually initiates during puberty, the peak period of human growth when the secretion of numerous hormones is changing, and it is more common in females than in males. Accumulating evidence shows that the abnormal levels of many hormones including estrogen, melatonin, growth hormone, leptin, adiponectin and ghrelin, may be related to the occurrence and development of AIS. The purpose of this review is to provide a summary and critique of the research published on each hormone over the past 20 years, and to highlight areas for future study. It is hoped that the presentation will help provide a better understanding of the role of endocrine hormones in the pathogenesis of AIS.

Case Report: Severe Hypocalcemic Episodes Due to Autoimmune Enteropathy.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare monogenic disorder, associated with endocrine deficiencies and non-endocrine involvement. Gastrointestinal (GI) manifestations appear in approximately 25% of patients and are the presenting symptom in about 10% of them. Limited awareness among pediatricians of autoimmune enteropathy (AIE) caused by destruction of the gut endocrine cells in APECED patients delays diagnosis and appropriate therapy. We describe an 18-year-old female presenting at the age of 6.10 years with hypoparathyroidism, oral candidiasis and vitiligo. The clinical diagnosis of APECED was confirmed by sequencing the autoimmune regulator-encoding (AIRE) gene. Several characteristics of the disease-Hashimoto's thyroiditis, Addison's disease, diabetes mellitus type 1 and primary ovarian insufficiency-developed over the years. She had recurrent episodes of severe intractable hypocalcemia. Extensive GI investigations for possible malabsorption, including laboratory analyses, imaging and endoscopy with biopsies were unremarkable. Revision of the biopsies and chromogranin A (CgA) immunostaining demonstrated complete loss of enteroendocrine cells in the duodenum and small intestine, confirming the diagnosis of AIE. Management of hypocalcemia was challenging. Only intravenous calcitriol maintained calcium in the normal range. Between hypocalcemic episodes, the proband maintained normal calcium levels, suggesting a fluctuating disease course. Repeated intestinal biopsy revealed positive intestinal CgA immunostaining. The attribution of severe hypocalcemic episodes to AIE emphasizes the need for increased awareness of this unique presentation of APECED. The fluctuating disease course and repeated intestinal biopsy showing positive CgA immunostaining support a reversible effect of GI involvement. CgA immunostaining is indicated in patients with APECED for whom all other investigations have failed to reveal an explanation for the malabsorption.

HNRNPA2B1 regulates tamoxifen- and fulvestrant-sensitivity and hallmarks of endocrine resistance in breast cancer cells.

Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant disease remain unresolved. Previously we demonstrated that expression of the RNA binding protein and N6-methyladenosine (m6A) reader HNRNPA2B1 (A2B1) is higher in LCC9 and LY2 tamoxifen (TAM)-resistant ERα breast cancer cells relative to parental TAM-sensitive MCF-7 cells. Here we report that A2B1 protein expression is higher in breast tumors than paired normal breast tissue. Modest stable overexpression of A2B1 in MCF-7 cells (MCF-7-A2B1 cells) resulted in TAM- and fulvestrant- resistance whereas knockdown of A2B1 in LCC9 and LY2 cells restored TAM and fulvestrant, endocrine-sensitivity. MCF-7-A2B1 cells gained hallmarks of TAM-resistant metastatic behavior: increased migration and invasion, clonogenicity, and soft agar colony size, which were attenuated by A2B1 knockdown in MCF-7-A2B1 and the TAM-resistant LCC9 and LY2 cells. MCF-7-A2B1, LCC9, and LY2 cells have a higher proportion of CD44+/CD24-/low cancer stem cells (CSC) compared to MCF-7 cells. MCF-7-A2B1 cells have increased ERα and reduced miR-222-3p that targets ERα. Like LCC9 cells, MCF-7-A2B1 have activated AKT and MAPK that depend on A2B1 expression and are growth inhibited by inhibitors of these pathways. These data support that targeting A2B1 could provide a complimentary therapeutic approach to reduce acquired endocrine resistance.

Architecture of the Pancreatic Islets and Endocrine Cell Arrangement in the Embryonic Pancreas of the Grass Snake (Natrix natrix L.). Immunocytochemical Studies and 3D Reconstructions.

During the early developmental stages of grass snakes, within the differentiating pancreas, cords of endocrine cells are formed. They differentiate into agglomerates of large islets flanked throughout subsequent developmental stages by small groups of endocrine cells forming islets. The islets are located within the cephalic part of the dorsal pancreas. At the end of the embryonic period, the pancreatic islet agglomerates branch off, and as a result of their remodeling, surround the splenic "bulb". The stage of pancreatic endocrine ring formation is the first step in formation of intrasplenic islets characteristics for the adult specimens of the grass snake. The arrangement of endocrine cells within islets changes during pancreas differentiation. Initially, the core of islets formed from B and D cells is surrounded by a cluster of A cells. Subsequently, A, B, and D endocrine cells are mixed throughout the islets. Before grass snake hatching, A and B endocrine cells are intermingled within the islets, but D cells are arranged centrally. Moreover, the pancreatic polypeptide (PP) cells are not found within the embryonic pancreas of the grass snake. Variation in the proportions of different cell types, depending on the part of the pancreas, may affect the islet function-a higher proportion of glucagon cells is beneficial for insulin secretion.

Patient-derived scaffolds as a drug-testing platform for endocrine therapies in breast cancer.

Three-dimensional cell culture platforms based on decellularised patient-based microenvironments provide in vivo-like growth conditions allowing cancer cells to interact with intact structures and components of the surrounding tissue. A patient-derived scaffold (PDS) model was therefore evaluated as a testing platform for the endocrine therapies (Z)-4-Hydroxytamoxifen (4OHT) and fulvestrant as well as the CDK4/6-inhibitor palbociclib, monitoring the treatment responses in breast cancer cell lines MCF7 and T47D adapted to the patient-based microenvironments. MCF7 cells growing in PDSs showed increased resistance to 4OHT and fulvestrant treatment (100- and 20-fold) compared to 2D cultures. Quantitative PCR analyses of endocrine treated cancer cells in PDSs revealed upregulation of pluripotency markers further supported by increased self-renewal capacity in sphere formation assays. When comparing different 3D growth platforms including PDS, matrigel, gelatin sponges and 3D-printed hydrogels, 3D based cultures showed slightly varying responses to fulvestrant and palbociclib whereas PDS and matrigel cultures showed more similar gene expression profiles for 4OHT treatment compared to the other platforms. The results support that the PDS technique maximized to provide a multitude of smaller functional PDS replicates from each primary breast cancer, is an up-scalable patient-derived drug-testing platform available for gene expression profiling and downstream functional assays.