Endocrine Research Articles

PMON145 The Role of Pou3f4 in Pituitary Development and Disease

Abstract Mutations in the pituitary specific transcription factor Prophet of Pit-1 (PROP1) are the most common genetic etiology of combined pituitary hormone deficiency (CPHD), which affects 1 in 8,000 newborns worldwide. CPHD is associated with short stature, attributable to growth hormone (GH) deficiency and/or thyroid stimulating hormone (TSH) deficiency. Pathogenic lesions impair pituitary development and differentiation of endocrine cells. Prop1-mutant mice show increased expression of genes including SRY-Box Transcription Factor 21 (Sox21) and Pou Domain, Class 3, Transcription Factor 4 (Pou3f4). POU3F4 is expressed in the developing inner ear and restricts the proliferation and lineage potential of neural stem cells. Genetic defects cause non-syndromic X-linked deafness (DFNX2) in humans and mice. The function of POU3F4 in pituitary development is largely unknown and the implications of its overexpression in Prop1-mutant pituitaries remain unexplored. Therefore, we aim to address the role of Pou3f4 in pituitary development and determine the contributions of Pou3f4 upregulation to pituitary disease by utilizing Pou3f4-mutant mice as well as double mutant mice lacking both Prop1 and Pou3f4. Utilizing immunofluorescent analysis at critical timepoints during pituitary development, we have characterized the timeline for endogenous POU3F4 expression. Initial expression is detected at e11.5 in differentiating cells on the ventral side of Rathke's pouch and is restricted to the proliferating anterior lobe at e13.5, continuing postnatally, similar to previously published expression data. POU3F4 expression then decreases and becomes gradually restricted to the periluminal stem cell niche at e15.5 and p0. We further validated the upregulation of POU3F4 in histological sections of Prop1-mutant mice at e15 and p0, finding increased expression in the developing anterior lobe of mutants compared to wildtypes. Single-cell regulatory network inference and clustering (SCENIC) analyses, which utilize single-cell transcription factor co-expression with potential direct downstream targets that contain the corresponding transcription factors’ binding sequence to infer active transcription networks, predict increased activity of the Pou3f4 gene regulatory network in a subpopulation of Sox2-expressing stem cells in Prop1-mutant mice. Preliminary analyses indicate that Pou3f4 mutant have normal morphology at P1, together with grossly normal expression of Sox2 and Pou1f1. We are performing quantitative PCR to determine possible compensatory mechanisms in response to loss of Pou3f4 expression. To investigate whether any Prop1-mutant phenotypes, including dysmorphology and ectopic expression of Sox21 and Otog, are attributable to Pou3f4 upregulation, we are studying Pou3f4;Prop1 mice. We will determine the impacts of POU3F4 upregulation on cellular morphology, stem cell maintenance and endocrine cell differentiation. The mechanisms of action in the regulation of stem cell differentiation constitutes a gap in our current understanding of the pathogenesis of CPHD. These studies will provide novel insights into the mechanistic function of Pou3f4 during embryonic pituitary development and potential novel etiologies of CPHD in human patients. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

RF13 | PMON143 BRF1-Mediated Paracrine Signalling by a Subset of SOX2-Expressing Stem Cells is Required for Normal Development of the Stem Cell Compartment and Terminal Differentiation of Pituitary Committed Progenitors

Abstract Introduction Hormone-producing pituitary cell lineages are derived from a population of embryonic precursors expressing SOX2. These cells maintain multipotency into early postnatal life, acting as the resident population of pituitary stem cells (PSCs) and contributing extensively to all the endocrine cell lineages. In addition to this direct contribution to pituitary turnover, paracrine signalling from PSCs has been shown to be important for cell proliferation of neighbouring progenitors (PMC7803373). It is not known if SOX2+ PSCs are involved in the regulation of additional cell attributes during normal physiology and if there is functional heterogeneity among the SOX2+ PSC population. Experimental Methods We have carried out single-cell RNA-Sequencing of SOX2+ PSCs from Sox2Egfp/+ mouse pituitaries at three postnatal stages from P3 to P56 and used computational approaches to analyse their molecular signatures. A novel conditional mouse model expressing a constitutively active mutant form of the RNA binding factor BRF1 (R26stop-mBRF1) has been used to attenuate the expression of several cytokines and chemokines in SOX2+ cells embryonically and postnatally (PMC4589897). Results We show that the SOX2+ PSC population consists of three subgroups (SC1, SC2 and SC3). We reveal that SC1-SC2 express abundant cytokines and secreted factors, suggesting paracrine function. In contrast, SC3 is characterised by robust expression of Lef1, is identified as a committing PSC cluster, and its presence diminishes with age. Key markers of PSC clusters SC1-SC2 include the RNA binding factor BRF1. We show that BRF1 is highly expressed in PSCs and validate its expression by immunohistochemistry in both mouse and human pituitaries. Secondly, we show that the dysregulation of BRF1 in embryonic SOX2+ cells using the Hesx1-Cre driver (PMC3461924) results in pituitary hypoplasia and severe hypopituitarism due to a failure of the PIT1 and SF1 cell-lineage committed progenitors to terminally differentiate into hormone-producing cells. Additionally, there is a significant reduction of the stem cell compartment, manifested by lower numbers of SOX2/SOX9+ stem cells. This phenotype is recapitulated when using a Sox2-CreERT2 driver (PMID24094324). The differentiation failure can be rescued in vitro through co-culture of mutant cells with wild-type stem cells, as well as in vivo, in mutant pituitaries where activation of constitutively active BRF1 is restricted to few SOX2+ PSCs in a mosaic manner. Finally, we identify key ligands underlying this differentiation phenotype and demonstrate a partial restoration of terminal differentiation in the mutant, when cultured in the presence of these ligands. Conclusion We provide evidence indicating the presence of functionally distinct groups of SOX2+ pituitary stem cells and reveal a critical role for a PSC subset in the development of the stem cell compartment and in driving terminal differentiation of committed progenitors. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

GC/TOF-MS-based phytochemical analysis, in vitro antiproliferative effects, antioxidant and antibacterial activity of Sarcophyte sanguinea subsp. piriei (Hutch.) B. Hansen

Antibiotic resistance necessitates the need for continued search for new antimicrobial compounds. In this study, we report the antibacterial activity, cytotoxic, and antioxidant activity of Sarcophyte sanguinea subsp. piriei (Hutch.) B. The extracts were evaluated for antibacterial activity using the agar well diffusion, micro-dilution, minimum bactericidal concentrations (MBC) and lactate dehydrogenase (LDH) release assay. Furthermore, the cytotoxic effects of extracts were determined using MTT assay against Human Embryonic Kidney (HEK293), Human Breast Endocrine Cells (SKBR-3), Human Colorectal Carcinoma Cells (Caco-2) and Human Hepatocellular Carcinoma (HepG2) cells. The extracts were also assessed for antioxidant activity against 2,2-diphenyl-1-picryl hydrazyl (DPPH), 2–2′-Azino-di-[3-ethylbenzthiazoline sulfonate (ABTS), nitric oxide radicals and Fe2+ chelating assays. The methanol extract (MeOH) was subjected to GC-TOF-MS analysis. MeOH extract exhibited highest zone of inhibition (ZI) of 15.67 mm against Staphylococcus aureus. The extract further exhibited 67.0% inhibition against Escherichia coli in a lactate dehydrogenase (LDH) membrane damage assay. In the cytotoxicity assay, MeOH and DCM extracts had LC50 value of 237 µg/ml and 221 µg/ml against SKBR-3 cell line respectively. Gas-chromatography time-of-flight mass spectrometry (GC/TOF-MS) analysis of methanol extract indicated the presence of 5-aminoimidazole-4-carboxamide-1-ád-ribofuranosyl 5′-monophosphate (36.827%), 3-O-methyl-d-glucose (36.827%) and phthalic acid, di(oct‑3-yl) ester (8.161%). Even though the investigated plant is suggested to have anticancer activities by traditional healers, the extracts had little inhibition on selected cancerous cell lines. Given the good antibacterial and antioxidant activity of the extracts, the plant may act as an immune booster and prevent infections in immunosuppressed cancer patients. Detected compounds may be influence the observed biological activity of the plant.

An artificial LAMA2-GelMA hydrogel microenvironment for the development of pancreatic endocrine progenitors

The biomimetic pancreatic microenvironment improves the differentiation efficiency and function of human embryonic stem cell-derived β-cells (SC-β cells). Thus, a laminin subunit alpha 2-gelatin methacrylate (LAMA2-GelMA) hybrid hydrogel as a bionics carrier for the formation and maturation of endocrine lineage was developed in our research, based on pancreas proteomics analysis of postnatal mice. Pancreatic endocrine cells cultured on the hybrid hydrogel in vitro, which was composed of 0.5 μg/mL LAMA2 protein and 4% GelMA, the expression of transcription factors (TFs), including NKX6.1, NKX6.2, and NEUROD1 were upregulated. Single-cell transcriptomics was performed after LAMA2 knockdown during the early differentiation of pancreatic progenitor (PP) cells, a marked decrease in the forkhead box protein A2 (FOXA2+)/GATA-binding factor 6 (GATA6+) cluster was detected. Also, we clarified that as a receptor of LAMA2, integrin subunit alpha 7 (ITGA7) participated in Integrin-AKT signaling transduction and influenced the protein levels of FOXA2 and PDX1. In vivo experiments showed that, PP cells encapsulated in the LAMA2-GelMA hydrogel exhibited higher serum C-peptide levels compared to the GelMA and Matrigel groups in nude mice and reversed hyperglycemia more quickly in STZ-induced diabetic nude mice. Taken together, our findings highlighted the feasibility of constructing a pancreas-specific microenvironment based on proteomics and tissue engineering for the treatment of diabetes.

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

Background and aimsAlthough 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 resultsStandard 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 PLCβ2 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. ConclusionsOral berberine ameliorated HFD-induced obesity through the TAS2R-IL-25 signaling pathway in tuft cells in the gut. SignificanceWe identified and functionally characterized the TAS2Rs and Gα-gustducin/Gβ1γ13 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.

CAPN8 involves with exhausted, inflamed, and desert immune microenvironment to influence the metastasis of thyroid cancer.

Thyroid cancer (THCA) is the most prevalent malignant disease of the endocrine system, in which 5-year survival can attain about 95%, but patients with metastasis have a poor prognosis. Very little is known about the role of CAPN8 in the metastasis of THCA. In particular, the effect of CAPN8 on the tumor immune microenvironment (TIME) and immunotherapy response is unclear. Multiome datasets and multiple cohorts were acquired for analysis. Firstly, the expression and the prognostic value of CAPN8 were explored in public datasets and in vitro tumor tissues. Then, hierarchical clustering analysis was performed to identify the immune subtypes of THCA according to the expression of CAPN8 and the activities of related pathways. Subsequent analyses explored the different patterns of TIME, genetic alteration, DNA replication stress, drug sensitivity, and immunotherapy response among the three immune phenotypes. Finally, five individual cohorts of thyroid cancer were utilized to test the robustness and extrapolation of the three immune clusters. CAPN8 was found to be a significant risk factor for THCA with a markedly elevated level of mRNA and protein in tumor tissues. This potential oncogene could induce the activation of epithelial-mesenchymal transition and E2F-targeted pathways. Three subtypes were identified for THCA, including immune exhausted, inflamed, and immune desert phenotypes. The exhausted type was characterized by a markedly increased expression of inhibitory receptors and infiltration of immune cells but was much more likely to respond to immunotherapy. The immune desert type was resistant to common chemotherapeutics with extensive genomic mutation and copy number variance. The present study firstly explored the role of CAPN8 in the metastasis of THCA from the aspects of TIME. Three immune subtypes were identified with quite different patterns of prognosis, immunotherapy response, and drug sensitivity, providing novel insights for the treatment of THCA and helping understand the cross-talk between CAPN8 and tumor immune microenvironment.

PREVALENCE OF SOME ENDOCRINE DISORDERS IN GEORGIA UNDER THE CONDITIONS OF THE COVID-19 PANDEMIC

The purpose of the study was to review the spread of some endocrine disorders in Georgia under the conditions of the Covid-19 pandemic. The distribution of some diseases of the endocrine system between 2015 and 2021 was analyzed, such as diabetes mellitus - type I and II, as well as thyroid gland diseases - iodine-deficiency hypothyroidism and other forms of hypothyroidism, thyroiditis, thyrotoxicosis. It was determined that during the pandemic, the morbidity rate of these diseases was significantly (almost 2 times) lower than the values recorded before the pandemic. It should be noted that, most likely, these indicators cannot reflect the true number of patients, which is related to the isolation of the population and the disruption of registration in the conditions of the Covid-19 pandemic. Disorders of the endocrine system, nutrition and metabolism during the current pandemic have not yet been adequately studied. The association between endocrine, nutritional and metabolic disorders and covid-19 is mostly tentative and based on recent small-scale studies. In order to obtain reliable conclusions about the existence of a correlation relationship, it is necessary to continue research on the mentioned issue, in particular, to study the detection and frequency of cases of thyroid gland and pancreas dysfunction in groups of people infected with and vaccinated against Covid-19.

Physiological levels of adrenaline fail to stop pancreatic beta cell activity at unphysiologically high glucose levels.

Adrenaline inhibits insulin secretion from pancreatic beta cells to allow an organism to cover immediate energy needs by unlocking internal nutrient reserves. The stimulation of α2-adrenergic receptors on the plasma membrane of beta cells reduces their excitability and insulin secretion mostly through diminished cAMP production and downstream desensitization of late step(s) of exocytotic machinery to cytosolic Ca2+ concentration ([Ca2+]c). In most studies unphysiologically high adrenaline concentrations have been used to evaluate the role of adrenergic stimulation in pancreatic endocrine cells. Here we report the effect of physiological adrenaline levels on [Ca2+]c dynamics in beta cell collectives in mice pancreatic tissue slice preparation. We used confocal microscopy with a high spatial and temporal resolution to evaluate glucose-stimulated [Ca2+]c events and their sensitivity to adrenaline. We investigated glucose concentrations from 8-20 mM to assess the concentration of adrenaline that completely abolishes [Ca2+]c events. We show that 8 mM glucose stimulation of beta cell collectives is readily inhibited by the concentration of adrenaline available under physiological conditions, and that sequent stimulation with 12 mM glucose or forskolin in high nM range overrides this inhibition. Accordingly, 12 mM glucose stimulation required at least an order of magnitude higher adrenaline concentration above the physiological level to inhibit the activity. To conclude, higher glucose concentrations stimulate beta cell activity in a non-linear manner and beyond levels that could be inhibited with physiologically available plasma adrenaline concentration.

ZBTB20 Regulates Prolactin Expression and Lactotrope Function in Adult Mice.

Lactotropes are prolactin (PRL)-secreting endocrine cells in the anterior pituitary. We have established the zinc finger protein ZBTB20 as an essential transcription factor for lactotrope specification, the disruption of which results in complete loss of lactotropes in mice. However, the potential role of ZBTB20 in mature lactotropes remains unclear. Here we demonstrate that ZBTB20 acts as a critical cell-autonomous regulator for PRL expression in mature lactotropes in adult mice. Via a CRISPR/Cas9 approach, we first generated a tamoxifen-inducible Prl-CreER knockin mouse line that could efficiently mediate gene recombination specifically in lactotropes. Conditional deletion of the Zbtb20 gene specifically in mature lactotropes at adulthood led to a substantial decrease in PRL levels both in the pituitary and in plasma, without significant alterations of lactotrope relative density in the pituitary from male or female mice. Furthermore, conditional disruption of Zbtb20 in adult female mice did not significantly change pregnancy-elicited lactotrope expansion, but caused an impaired mammary gland expansion and lactation due to the PRL defect. Thus, our data point to an important role of ZBTB20 in regulating PRL expression and lactotrope function at adulthood.

Integration of genomic analysis and transcript expression of ABCC8 and KCNJ11 in focal form of congenital hyperinsulinism.

The focal form of CHI is caused by an autosomal recessive pathogenic variant affecting the paternal homologue of genes ABCC8 or KCNJ11 and a second somatic event specifically occurring in the affected islet of Langerhans. The approach of this study was to integrate the genetic changes occurring in pancreatic focal lesions of CHI at the genomic and transcriptional level. Patients receiving therapeutic surgery and with proven ABCC8 or KCNJ11 pathogenic variants were selected and analyzed for loss of heterozygosity (LOH), changes in copy number and uniparental disomy (UPD) on the short am of chromosome 11 by molecular microarray analysis and methylation-specific MLPA. Gene expression was analyzed by RT-PCR and Massive Analysis of cDNA Ends (MACE). Both genes, ABCC8 and KCNJ11, are located in proximity to the Beckwith-Wiedemann (BWS) imprinting control region on chromosome 11p15. Somatic paternal uniparental isodisomy (UPD) at chromosome 11p was identified as second genetic event in focal lesions resulting in LOH and monoallelic expression of the mutated ABCC8/KCNJ11 alleles. Of five patients with samples available for microarray analysis, the breakpoints of UPD on chromosome 11p were different. Samples of two patients were analyzed further for changes in gene expression. Profound downregulation of growth suppressing genes CDKN1 and H19 was detected in focal lesions whereas growth promoting gene ASCL2 and pancreatic transcription factors of the endocrine cell lineage were upregulated. Paternal UPD on the short arm of chromosome 11 appears to be the major second genetic event specifically within focal lesions of CHI but no common breakpoint for UDP can be delineated. We show for the first time upregulation of growth promoting ASCL2 (achaete-scute homolog 2) suggestive of a driving factor in postnatal focal expansion in addition to downregulation of growth suppressing genes CDKN1C and H19.

Wnt4 is heterogeneously activated in maturing β-cells to control calcium signaling, metabolism and function

Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic β-cells. β-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between β-cells via gap junctions. Here, we identify the importance of signaling between β-cells via the extracellular signal WNT4. We show heterogeneity in Wnt4 expression, most strikingly in the postnatal maturation period, Wnt4-positive cells, being more mature while Wnt4-negative cells are more proliferative. Knock-out in adult β-cells shows that WNT4 controls the activation of calcium signaling in response to a glucose challenge, as well as metabolic pathways converging to lower ATP/ADP ratios, thereby reducing insulin secretion. These results reveal that paracrine signaling between β-cells is important in addition to gap junctions in controling insulin secretion. Together with previous reports of WNT4 up-regulation in obesity our observations suggest an adaptive insulin response coordinating β-cells.

Biological and Therapeutic Implications of the Tumor Microenvironment in Pituitary Adenomas.

Pituitary adenomas (PAs) are neoplasms derived from the endocrine cells of the anterior pituitary gland. Most frequently, they are benign tumors, but may sometimes display an aggressive course, and in some cases metastasize. Their biology, including their wide range of behavior, is only partly understood. In terms of therapeutic targeting, most PAs are easily treated with available medical treatments, surgery, and sometimes radiotherapy. Nevertheless, gonadotroph adenomas lack medical therapeutic options, and treatment of aggressive PAs and pituitary carcinomas remains challenging. Here, we present an overview of the implications of the tumor microenvironment in PAs, reviewing its composition and function, as well as published cases that have been treated thus far using tumor microenvironment-targeting therapies. Additionally, we discuss emerging views, such as the concept of nonangiogenic tumors, and present perspectives regarding treatments that may represent future potential therapeutic options. Tumor-infiltrating lymphocytes, tumor-associated macrophages, folliculostellate cells, tumor-associated fibroblasts, angiogenesis, as well as the extracellular matrix and its remodeling, all have complex roles in the biology of PAs. They have been linked to hormone production/secretion, size, invasion, proliferation, progression/recurrence, and treatment response in PAs. From a therapeutic perspective, immune-checkpoint inhibitors and bevacizumab have already shown a degree of efficacy in aggressive PAs and pituitary carcinomas, and the use of numerous other tumor microenvironment-targeting therapies can be foreseen. In conclusion, similar to other cancers, understanding the tumor microenvironment improves our understanding of PA biology beyond genetics and epigenetics, and constitutes an important tool for developing future therapies.

Comorbidity and multimorbidity in patients with chronic hepatitis C

The aim of the study was to analyze comorbidity in patients who underwent a course of antiviral therapy for chronic hepatitis C with direct antiviral drugs.Materials and methods: 288 patients diagnosed with chronic hepatitis C with stages of liver fibrosis from F1 to F4 according to the METAVIR scale, various genotypes, who underwent antiviral therapy with direct antiviral drugs in the period from 2018 to 2021, were under observation, with an analysis of comorbid and multimorbid diseases.Results of the study: most often in these patients, regardless of gender and antiviral therapy regimen, lesions of the gastrointestinal tract were registered in the form of various inflammatory diseases, as well as non-alcoholic fatty liver disease. In second place in terms of frequency of occurrence, diseases of the endocrine system were diagnosed, primarily due to the development of metabolic syndrome, as well as damage to the thyroid gland, as an extrahepatic manifestation of chronic HCV infection. Slightly less marked diseases of the cardiovascular system. A relationship was found between the number of comorbid diseases and the body mass index, with an increase in which the stage of liver fibrosis progresses.Conclusions: The frequency of comorbidity and multimorbidity in patients with chronic HCV infection is significantly higher than in uninfected patients. Patients with chronic hepatitis C with advanced stages of liver fibrosis (F3 and F4 according to the MEVIR scale) with overweight and obesity of varying severity are characterized by the highest incidence of comorbidities. A variety of extrahepatic manifestations of chronic hepatitis C determine the polymorbid profile of the patient.

Single-cell RNA-seq transcriptomic landscape of human and mouse islets and pathological alterations of diabetes

Single-cell RNA sequencing has paved the way for delineating the pancreatic islet cell atlas and identifying hallmarks of diabetes. However, pathological alterations of type 2 diabetes (T2D) remain unclear. We isolated pancreatic islets from control and T2D mice for single-cell RNA sequencing (scRNA-seq) and retrieved multiple datasets from the open databases. The complete islet cell landscape and robust marker genes and transcription factors of each endocrine cell type were identified. GLRA1 was restricted to beta cells, and beta cells exhibited obvious heterogeneity. The beta subcluster in the T2D mice remarkably decreased the expression of Slc2a2, G6pc2, Mafa, Nkx6-1, Pdx1, and Ucn3 and had higher unfolded protein response (UPR) scores than in the control mice. Moreover, we developed a Web-based interactive tool, creating new opportunities for the data mining of pancreatic islet scRNA-seq datasets. In conclusion, our work provides a valuable resource for a deeper understanding of the pathological mechanism underlying diabetes.

Proteomic Profiling of Intra-Islet Features Reveals Substructure-Specific Protein Signatures

Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from studying the whole pancreas to isolated whole islets, to partial islet sections, and now to islet substructures isolated from within the islet. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection and field asymmetric waveform ion mobility spectrometry, we present an in-depth investigation of protein profiles specific to features within the islet. These features include the islet-acinar interface vascular tissue, inner islet vasculature, isolated endocrine cells, whole islet with vasculature, and acinar tissue from around the islet. Compared to interface vasculature, unique protein signatures observed in the inner vasculature indicate increased innervation and intra-islet neuron-like crosstalk. We also demonstrate the utility of these data for identifying localized structure-specific drug–target interactions using existing protein/drug binding databases.

Is ABO Compatibility Important in Islet Transplantation?

One of the current criteria for clinical islet allotransplantation is ABO blood group compatibility between donor and recipient.1 The distribution of ABH antigens in human pancreata was first examined >40 y ago, revealing expression in acinar and vascular cells but not in islets.2 This underlined the importance of ABO matching in whole pancreas transplantation, but the situation in islet transplantation is more complicated. Although islets in situ appear to be ABH-negative,2 islets prepared for transplant are invariably contaminated with exocrine tissue including acinar cells. Furthermore, it is not known whether the islet isolation and transplantation process itself influence ABH expression. It is therefore somewhat surprising that ABH expression in islet preparations before and after transplant has not been characterized until now. In this issue of Transplantation, Verhoeff et al3 present a detailed immunohistochemical and flow cytometric survey of ABH antigen levels in human pancreatic tissue, isolated human clinical-grade islets, and islets derived from human embryonic stem cells (hESCs). After confirming that ABH antigens are present on acinar cells but not on endocrine and ductal cells in the pancreas, the authors demonstrate that islet preparations consist primarily of ABH-negative endocrine cells but are significantly contaminated with ABH-positive acinar cells. When islets were transplanted into immunodeficient rats, the endocrine component was ABH-negative at 24 wk, whereas remnant acinar tissue stained positive. Pancreatic endocrine progenitors derived from hESCs were ABH-negative and remained so after in vivo maturation following transplantation into rats, although this was not unexpected given that the hESC donor was blood group O. The human-to-immunodeficient rat islet xenograft model used in the latter part of the study does have some limitations. First, the kidney subcapsular site does not accurately reflect the clinical scenario, in which islets are transplanted intraportally.1 Subcapsular delivery co-localizes the islets with acinar tissue fragments and potentially exposes the islets to “bystander” injury and inflammation when anti-ABH antibodies attack the acinar cells. In contrast, intraportal delivery disperses islets and acinar fragments widely throughout the liver microvasculature, which probably minimizes co-localization and thus reduces the likelihood of bystander effects. Furthermore, subcapsular but not intraportal delivery shields the graft from exposure to anti-ABH IgM antibodies. Although these differences are not relevant in the immunodeficient rat model, they may well be relevant in the clinical setting. Second, the immunodeficient rat model does not allow investigation of the importance of the in vivo anti-ABH immune response to islet grafts. I have worked with one of the authors (L.J.W.) to develop a mouse model of anti-A antibody-mediated rejection of transgenic blood group A-expressing cardiac grafts.4 However, these A-transgenic donor mice are unsuitable for examining anti-A–mediated islet graft rejection because expression of the antigen is endothelial cell-restricted and absent on islets. Humanized mouse models may be more appropriate, as suggested by the authors. The take-home message of this interesting paper is that ABO matching is indeed likely to be important in islet allotransplantation—as is the case for HLA matching5—but may become less so if islet purification efficiency improves sufficiently or if hESC-derived islets are used. On the former point, the authors speculate that the restriction of surface expression of ABH antigens to acinar tissue (starkly illustrated by Figure 6A in the paper) provides an opportunity to enrich the purity of A, B, or AB islets by some form of negative selection. Although practicality at scale may be an issue, this is an intriguing proposition that is worthy of further consideration.

S1910 A Rare Metastatic Lesion in the Pancreas Presenting With Biliary Obstruction

Introduction: Pancreatic malignancies can originate from either endocrine or non-endocrine cells of the pancreas. Ductal carcinoma is the most common type of non-endocrine tumor, of which adenocarcinomas constitute the most common pancreatic malignancy. Squamous cell carcinoma (SCC) is the other ductal tumor that is very rare as the pancreas is generally devoid of squamous cells. Primary SCC of the pancreas is thought to arise from the squamous metaplasia of the ductules secondary to chronic inflammatory conditions, however, metastasis remains the most common etiology. We present a patient with history of recurrent poorly differentiated SCC of the cervix complicated by pancreatic head metastasis and biliary obstruction. Case Description/Methods: A 46-year-old female diagnosed with poorly differentiated SCC of the cervix stage IIB, status post multiple rounds of chemotherapy over a period of 4 years, was started on a new medication – Tisotumab. Four months following the initiation of this medication, patient experienced abdominal pain and was noted to have a lipase level of 1160 U/L. She was diagnosed with acute pancreatitis and treated accordingly. Subsequent labs showed continued up trending of her liver chemistry, withalkaline phosphatase of 520 U/L and total bilirubin of 9.7 mg/dl. A CT abdomen performed revealed dilation of both common bile duct (CBD) and pancreatic duct (PD) with subtle hypodense area in the head of pancreas. Endoscopic ultrasound (EUS) was performed which revealed a 3 cm x 2 cm ill-defined hypoechoic and heterogenous mass in the pancreas head (Figure). Fine needle biopsy of the lesion revealed SCC, most likely metastatic from the cervix. ERCP was performed and a 10mm x 8cm fully covered metal CBD stent was placed with normalization of liver enzymes. Palliative immunotherapy with Keytruda is currently being pursued. Discussion: Metastasis to the pancreas is an uncommon clinical condition and accounts for 0.5-5% of all pancreatic malignancies. Because of the rarity, primary sources should always be excluded. Only a handful of reports of uterine and cervical cancer metastasis to pancreas have been reported, most of them being adenocarcinoma. We report a very rare case of SCC cervix metastasizing to the pancreas. Unfortunately, curative surgery is not an option with metastatic disease, and chemoradiation can be used palliatively. The median survival is generally poor with median rates of 4.8% and 1% at 1-yr and 5-yrs, respectively.Figure 1.: A - EUS image showing dilated CBD measuring up to x mm. B – EUS image demonstrating a 3 x 2 cm hypoechoic pancreatic head mass. C– H&E stain showing neoplastic clusters (black arrows) of metastatic SCC.

DYSREGULATED GATA2 AND GATA6 TRANSCRIPTION FACTOR EXPRESSION IN PATIENTS WITH ADENOMYOSIS: IMPLICATIONS FOR IMPAIRED ENDOMETRIAL RECEPTIVITY

GATA binding protein 2 and 6 (GATA2 and GATA6) are zinc-finger transcription factors involved in proliferation of hematopoietic and endocrine cell lineages, and in cell differentiation and organogenesis, respectively, which are important mechanisms in early pregnancy. Binding of GATA2 to the progesterone receptor (PGR) gene promoter induces PGR expression. GATA2 also interacts with PGR and contributes to PGR-mediated transcriptional activity. Gata2 deficient mice are infertile due to an impaired uterine response associated with lower PGR levels and attenuated progesterone signaling and defective decidualization.

S2242 Taking Caution at Road’s End: Incidental Finding of Appendiceal Goblet Cell Tumor on Colonoscopy

Introduction: Appendiceal cancer is uncommon and rarely diagnosed on colonoscopy. We present a case of incidentally discovered goblet cell adenocarcinoma of the appendix and its subsequent management. Case Description/Methods: 81-year-old male presented to GI clinic for persistent epigastric pain and reflux. Symptoms had been worsening for several years and were refractory to medical therapy. He denied unintentional weight loss, nausea, melena, or a family history of colorectal cancer. He had 9 colonoscopies between 1999 and 2016 for rectal bleeding due to hemorrhoids and diverticulosis, with a single 2mm TA removed in 2016 and 5-year follow-up advised. Repeat EGD and colonoscopy were scheduled, during which the appendiceal orifice (AO) had a “heaped-up” appearance and was biopsied with cold forceps. Histology revealed goblet cell adenocarcinoma (GCA.) CT of the abdomen and pelvis showed a mildly enlarged appendix, no LAD, and no evidence of metastasis. The patient underwent a laparoscopic right hemicolectomy. Pathology clarified that carcinoma extended from appendix to mesoappendix and cecum with extensive lymphovascular and perineural invasion. Resected margins and 19 resected lymph nodes were negative for malignancy and ultimately staged pT4aN0 (IIB). Colonoscopy 1 year later revealed healthy ileocolonic anastomosis without signs of polyps or other masses (Figure). Discussion: Appendiceal cancer is categorized into 2 types: epithelial and neuroendocrine. GCA histologically has features of both. Incidence is rare, reportedly 0.05/100,000/year. It is usually an incidental finding following appendectomy for acute appendicitis and uncommonly found via colonoscopy. The AO is a key endoscopic landmark that should be identified on all colonoscopies to ensure completeness. Endoscopists should have a low threshold to biopsy abnormal-appearing tissue surrounding the AO. While no established risk factors for GCA, cases report an association with schistosomiasis and caucasian proclivity. Clinical diagnosis is difficult as presentations range from abdominal pain to appendicitis. Swift work-up and therapy are essential as appendiceal GCA can aggressively spread nodally or intraperitoneally. While evidence-based guidelines are not available, current management is similar to that of colon cancer with hemicolectomy for localized disease, adjuvant chemotherapy based on the surgical pathology, and post-treatment surveillance. We hope this case highlights the rarity of appendiceal GCA and the need for evidence-based guidelines.Figure 1.: A) Appendiceal orifice with “heaped-up” appearance B) Healthy ileocolonic anastamosis C) Clusters of goblet-like mucinous cells with invasion into and through the lamina propria (10X) D) High power view (20X) of cohesive groups of goblet-like mucinous cells. Nuclear atypia is mild and mitotic figures are inconspicuous E) Cytokeratin AE1/AE3 highlights the colonic mucosa and the infiltrative goblet-cell tumor (10X) F) Chromogranin highlights scattered endocrine cells (10X).

Development of the human pancreas and its exocrine function.

The pancreas has both endocrine and exocrine function and plays an important role in digestion and glucose control. Understanding the development of the pancreas, grossly and microscopically, and the genetic factors regulating it provides further insight into clinical problems that arise when these processes fail. Animal models of development are known to have inherent issues when understanding human development. Therefore, in this review, we focus on human studies that have reported gross and microscopic development including acinar-, ductal-, and endocrine cells and the neural network. We review the genes and transcription factors involved in organ formation using data from animal models to bridge current understanding where necessary. We describe the development of exocrine function in the fetus and postnatally. A deeper review of the genes involved in pancreatic formation allows us to describe the development of the different groups (proteases, lipids, and amylase) of enzymes during fetal life and postnatally and describe the genetic defects. We discuss the constellation of gross anatomical, as well as microscopic defects that with genetic mutations lead to pancreatic insufficiency and disease states.