Gene Expression and Profiling of Human Islet Cell Subtypes

The endocrine pancreas contains multiple cell types co-localized into clusters called the islets of Langerhans. The predominant cell types include alpha and beta cells, which produce glucagon and insulin, respectively. The regulated release of these hormones maintains whole body glucose homeostasis, essential to prevent complications from diabetes (e.g. blindness, kidney failure, and cardiovascular disease). In type 1 diabetes, an autoimmune reaction destroys the beta cells and patients must monitor their blood sugar levels and inject insulin in order to maintain euglycemia. In type 2 diabetes, the beta cells fail to produce sufficient insulin to overcome the individual’s decreased insulin sensitivity. Most studies to date have focused on whole islets, which are very heterogeneous. Recent focus has shifted to studying the individual islet cell subsets (i.e. alpha, beta, delta, PP, and other cell types). Unlike immunological cells, surface molecule reagents do not yet exist to specifically distinguish beta from alpha cells. We have successfully isolated pure populations of insulin producing beta cells and glucagon producing alpha cells by using intracellular hormone staining and fluorescence activated cell sorting. We present data that describe the ratio of beta cells to alpha cells across gender, age, and BMI. Further, we have characterized the miRNA profiles of alpha and beta cells and have begun to investigate the unique gene expression patterns of the two cell types. By developing the ability to profile multiple characteristics of alpha and beta cells, we hope to determine how gene, miRNA, and protein profiles change under environmental conditions that lead to beta cell failure, and others that may promote beta cell health or stimulate beta cell growth and proliferation

The average magnetic field draping and consistent plasma properties of the Venus magnetotail

A new technique has been developed to determine the average structure of the Venus magnetotail (in the range from −8 Rv to −12 Rv) from the Pioneer Venus magnetometer observations. The spacecraft position with respect to the cross-tail current sheet is determined from an observed relationship between the field-draping angle and the magnitude of the field referenced to its value in the nearby magnetosheath. This allows us statistically to remove the effects of tail flapping and variability of draping for the first time and thus to map the average field configuration in the Venus tail. From this average configuration we calculate the cross-tail current density distribution and J × B forces. Continuity of the tangential electric field is utilized to determine the average variations of the X-directed velocity which is shown to vary from −250 km/s at −8 Rv to −470 km/s at −12 Rv. From the calculated J × B forces, plasma velocity, and MHD momentum equation the approximate plasma acceleration, density, and temperature in the Venus tail are determined. The derived ion density is approximately ∼0.07 p+/cm³ (0.005 O+/cm³) in the lobes and ∼0.9 p+/cm³ (0.06 O+/cm³) in the current sheet, while the derived approximate average plasma temperature for the tail is ∼6×106 K for a hydrogen plasma or ∼9×107 K for an oxygen plasma

Gene Expression Profiling of Islet Cell Subtypes

Abstract Pancreatic endocrine cells are co-located into clusters called the islets of Langerhans that are comprised of glucagon producing alpha cells, insulin secreting beta cells, somatostatin generating delta cells, and other cell types. Type 1 diabetes results from an autoimmune process in which autoreactive T cells destroy the insulin producing beta cells, requiring the patient to inject insulin to regulate their blood glucose levels. Thus far, attempts to cure diabetes via islet transplantation have been limited by insufficient donor supply, inconsistent isolated islet quality, continued autoimmunity, alloimmune rejection, and limited beta cell regeneration. Diabetes research has focused on preventing the autoimmune response, promoting stem cell to beta cell differentiation, and defining the factors that influence beta cell proliferation. Islet research, in turn, has been limited to whole islet studies since, isolating the islet cell subtypes has not been possible. Using a method recently developed for mouse islet cells (Pechhold et al. Nat Biotechnol. 2009 Nov; 27(11):1038-42), that uses intracellular hormone staining and flow cytometry, we are able to sort human islets into populations uniquely expressing glucagon, insulin, or somatostatin. Further, we have developed a human gene array to measure candidate gene expression using a quantitative nuclease protection assay (qNPA). This technique uses 50 base oligomers that specifically recognize RNA from each gene of interest, overcoming limitations caused by the harsh conditions required for intracellular staining. We report gene expression analysis for specific hormones and transcription factors expressed in each islet cell population. We are further modifying this technique to study nonhuman primate islets, and investigate the specific proteome and miRNA profiles for individual islet cell populations. The goal of these studies is to characterize the genetic differences between the islet cell populations and understand which factors control beta cell regeneration and proliferation. We have shown that we can purify adult human islets into individual cellular populations. This is the first step in understanding the genetic and environmental components that regulate increased beta cell proliferation and beta cell mass. In the absence of full-length mRNA for RT-PCR or next generation sequencing, the qNPA technique provides candidate gene expression profiles for these cells

Detection of CD8+ T cell Responses in Individuals with Long-term Type 1 Diabetes and Generation of Human CD8+ T Cell Lines Specific to Islet-associated Autoantigens

Type 1 diabetes (T1D) is an autoimmune disease characterized by the activation of lymphocytes against insulin-producing β-cells in the pancreas. In humans, CD8+ T cells are predominantly found in sites of insulitis and are considered to be one of the main drivers of β-cell destruction, thus indicating the need to analyze the frequency and function of these autoreactive CD8+ T cells. Peripheral blood mononuclear cells (PBMC) from individuals with long-term T1D were stained ex vivo for T cell surface markers and HLA-A2 pentamers containing known islet-associated epitopes to determine if there are autoreactive CD8+ T cells circulating in the periphery. All T1D donors tested had at least one detectable autoreactive CD8 T cell population and the frequencies of these autoantigen-specific T cells were comparable to previously published data from T1D individuals. We then developed a method of establishing CD8 T cell lines by co-culturing negatively isolated CD8 T cells and peptide-pulsed monocyte-derived dendritic cells from the PBMC of one T1D donor (A*02:01, A*33:01, B*14:02, B*40:01, DRB1*01:02, DRB1*04:04). We expanded a CD8 T cell line specific to the preproinsulin peptide PPI15-24. This cell line produced IFN-γ and expressed CD107a in the presence of PPI15-24-pulsed target cells, but not to an unrelated peptide or media alone. Using a similar approach, we were able to generate CD8 T cell lines from the same T1D donor that were cytotoxic to target cells pulsed with the autoantigens glutamic acid decarboxylase peptide (GAD65114-123) and islet-specific glucose-6-phosphatase catalytic subunit-related protein peptide (IGRP265-273). These autoreactive T cell lines can be utilized in in vivo assays using humanized mouse models to further understand the mechanism of β-cell destruction and disease progression. Studying the functionalities of these autoreactive T cells will also provide insights into identifying immune correlates to better assess both novel and existing immunotherapeutic strategies for T1D

alpha Cell Function and Gene Expression Are Compromised in Type 1 Diabetes

Many patients with type 1 diabetes (T1D) have residual beta cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual beta cells and alpha cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant beta cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D alpha cells was markedly reduced, and these cells had alterations in transcription factors constituting alpha and beta cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of alpha-to-beta cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia

Human islets expressing HNF1A variant have defective beta cell transcriptional regulatory networks

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained beta cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1alpha (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment

Pancreatic Islet Transplantation

Islet transplantation offers hope to many patients with diabetes, who envision a life free of glucose checks and insulin injections. What are the barriers to its widespread implementation

Hospital Nurse Burnout: A Continuing Problem

RNs are a critically important component of the U.S. healthcare system. RN burnout – the feeling of exhaustion from working long hours without rest – is a real concern, having been reported in many hospitals. We examine the background, causes and consequences of burnout among RNs in U.S. hospitals, in order to identify solutions to this problem. Findings indicate that Burnout Syndrome in RNs can be analyzed in terms of four clusters of characteristics: individual, management, organizational, and work. The consequences of burnout include increased RN turnover rates, poor job performance, and threats to patient safety. RN burnout in hospitals negatively impacts the quality of care, patient safety, and the functioning of staff workers in the healthcare industry