Endotoxin deactivation by transient acidification.

Recombinant proteins are an important tool for research and therapeutic applications. Therapeutic proteins have been delivered to several cell types and tissues and might be used to improve the outcome of the cell transplantation. Recombinant proteins are propagated in bacteria, which will contaminate them with the lypopolysacharide endotoxin found in the outer bacterial membrane.

Prolactin supplementation to culture medium improves beta-cell survival.

Objectives: Recent studies demonstrated that prolactin (PRL) has beneficial effects on beta cells for islet transplantation. We examined the effect of human recombinant PRL (rhPRL) supplementation to the culture media to determine its potential use in the context of clinical islet transplantation.

Materials And Methods: Each human islet isolated from 14 deceased multiorgan donors was cultured in Miami modified media-1 supplemented with or without rhPRL (500 microg/L) for 48 hr.

ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic beta cell.

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human beta cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i), and hormone release in vitro, we show that human beta cells express ionotropic ATP receptors of the P2X(3) type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion.

Immune "tolerance profiles" in donor bone marrow infused kidney transplant patients using multiple ex vivo functional assays.

Ex vivo identification of donor-specific unresponsiveness in organ transplant recipients is important for immunosuppression (IS) minimization. We tested three groups of stable living, related-donor kidney transplant patients up to 11 years postoperatively, i.e.

Closing the circle of care with new firmware for diabetes: MyDiaBase+RxChecker.

Background: Satisfactory glycemic control, meeting American Diabetes Association recommendations, is difficult to achieve. Technologically, this is most likely because the circle of care is incomplete. Many have suggested that the introduction of information technology may remedy the situation.

Prescription checking device promises to resolve intractable hypoglycemia.

Background: Satisfactory glycemic control, meeting American Diabetes Association recommendations, is often accompanied by unsatisfactory hypoglycemia. The converse is also true. We hypothesize that this diabetes treatment dilemma may be resolved by repeated, objective, prescription checks.

Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T-cells.

Objective: To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients.

Research Design And Methods: We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays.

Lipotoxicity and decreased islet graft survival.

OBJECTIVE To evaluate if baseline serum lipids are associated with islet graft survival in type 1 diabetes islet transplant (ITx) recipients. RESEARCH DESIGN AND METHODS Baseline fasting lipid profile was collected from 44 ITx recipients. Comparisons were performed between subjects below and above the median values of each lipid fraction.

Long-term metabolic and hormonal effects of exenatide on islet transplant recipients with allograft dysfunction.

The initial success of islet transplantation (ITx) is followed by graft dysfunction (GDF) and insulin reintroduction. Exenatide, a GLP-1 agonist, increases insulin and decreases glucagon secretion and has potential for beta-cell regeneration. To improve functional islet mass, exenatide treatment was given to ITx recipients with GDF.

Immune tolerance induction by integrating innate and adaptive immune regulators.

A diversity of immune tolerance mechanisms have evolved to protect normal tissues from immune damage. Immune regulatory cells are critical contributors to peripheral tolerance. These regulatory cells, exemplified by the CD4(+)Foxp3(+) regulatory T (Treg) cells and a recently identified population named myeloid-derived suppressor cells (MDSCs), regulate immune responses and limiting immune-mediated pathology.

Nonalbumin proteinuria in islet transplant recipients.

The aim of this study was to evaluate the importance of nonalbumin-predominant proteinuria on kidney function (KF) after islet transplantation (ITx). Twenty-four-hour proteinuria and albuminuria were available in 27 recipients. KF was assessed by serum creatinine and estimated glomerular filtration rate (eGFR) was calculated by Modification of Diet in Renal Disease formula.

Insulin2 gene (Ins2) transcription by NOD bone marrow-derived cells does not influence autoimmune diabetes development in NOD-Ins2 knockout mice.

Insulin is a critical autoantigen for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. About 80% of NOD females and 30-40% of NOD males develop diabetes. However, Insulin2 (Ins2) knockout NOD mice develop autoimmune diabetes with complete penetrance in both sexes, at an earlier age, and have stronger autoimmune responses to insulin.

Nephrin is expressed on the surface of insulin vesicles and facilitates glucose-stimulated insulin release.

Objective: Nephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic beta-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in beta-cell function.

Research Design And Methods: Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling.

Quantitative assessment of islet cell products: estimating the accuracy of the existing protocol and accounting for islet size distribution.

The ability to consistently and reliably assess the total number and the size distribution of isolated pancreatic islet cells from a small sample is of crucial relevance for the adequate characterization of islet cell preparations used for research or transplantation purposes. Here, data from a large number of isolations were used to establish a continuous probability density function describing the size distribution of human pancreatic islets. This function was then used to generate a polymeric microsphere mixture with a composition resembling those of isolated islets, which, in turn, was used to quantitatively assess the accuracy, reliability, and operator-dependent variability of the currently utilized manual standard procedure of quantification of islet cell preparation.

Validation of methodologies for quantifying isolated human islets: an Islet Cell Resources study.

Background: Quantification of islet mass is a crucial criterion for defining the quality of the islet product ensuring a potent islet transplant when used as a therapeutic intervention for select patients with type I diabetes.

Methods: This multi-center study involved all eight member institutions of the National Institutes of Health-supported Islet Cell Resources Consortium. The study was designed to validate the standard counting procedure for quantifying isolated, dithizone-stained human islets as a reliable methodology by ascertaining the accuracy, repeatability (intra-observer variability), and intermediate precision (inter-observer variability).

Small-molecule costimulatory blockade: organic dye inhibitors of the CD40-CD154 interaction.

Costimulatory blockade is one of the most promising therapeutic targets in autoimmune diseases as well as in transplant recipients, and inhibition of the cluster of differentiation (CD)40-CD154 interaction, which is required for T cell activation and development of an effective immune response, is particularly promising in islet transplant recipients. Here, we report the ability of several small-molecule organic dyes to concentration dependently inhibit this interaction with IC(50) values in the low-micromolar range. They were found to be considerably more active in inhibiting this interaction than the tumor necrosis factor (TNF)-R1-TNF-alpha or B cell-activating factor (BAFF)-R-BAFF interaction, which are members of the same family.

Incidence of insulin-requiring diabetes in the US military.

Aims/hypothesis: The aim of the study was to determine age- and race-related, and overall incidence rates of insulin-requiring diabetes in adults in the US military.

Methods: Electronic records for admissions to US military and Tricare hospitals during 1990-2005 and visits to military clinics during 2000-2005 were identified using the Career History Archival Medical and Personnel System at the Naval Health Research Center, San Diego, CA, USA. Population data were obtained from the Defense Manpower Data Center and Defense Medical Epidemiology Database.

Clinical use of fructosamine in islet transplantation.

Many islet transplant recipients have medical conditions that could interfere with the accuracy of HbA1c measurements (e.g., anemia/dapsone use).

Organic dyes as small molecule protein-protein interaction inhibitors for the CD40-CD154 costimulatory interaction.

It is becoming increasingly clear that small molecules can often act as effective protein-protein interaction (PPI) inhibitors, an area of increasing interest for its many possible therapeutic applications. We have identified several organic dyes and related small molecules that (i) concentration-dependently inhibit the important CD40-CD154 costimulatory interaction with activities in the low micromolar (microM) range, (ii) show selectivity toward this particular PPI, (iii) seem to bind on the surface of CD154, and (iv) concentration-dependently inhibit the CD154-induced B cell proliferation. They were identified through an iterative activity screening/structural similarity search procedure starting with suramin as lead, and the best smaller compounds, the main focus of the present work, achieved an almost 3-fold increase in ligand efficiency (DeltaG(0)/nonhydrogen atom = 0.

Type 2 diabetes mellitus phenotype and graft survival after islet transplantation.

Background: Body fat accumulation decreases insulin sensitivity. It has being associated with earlier onset of type 1 diabetes mellitus (DM) and islet graft failure. The aim of this study was to evaluate whether insulin resistance, characterized by risk factors for type 2 DM, can predict islet graft survival in type 1 DM islet transplant (ITx) recipients.

Oxygen: a master regulator of pancreatic development?

Beyond its role as an electron acceptor in aerobic respiration, oxygen is also a key effector of many developmental events. The oxygen-sensing machinery and the very fabric of cell identity and function have been shown to be deeply intertwined. Here we take a first look at how oxygen might lie at the crossroads of at least two of the major molecular pathways that shape pancreatic development.

Effects of pancreas cold ischemia on islet function and quality.

We used a rat model of pancreas cold preservation to assess its effects on islets. Glands were surgically retrieved and stored in University of Wisconsin (UW) solution for 3 hours (Short) or 18 hours (Long) cold ischemia time (CIT). Islet yield was significantly lower in the Long-CIT than the Short-CIT group, as well as islet recovery after overnight culture (P < .