Survivin Expression in Luminal Breast Cancer and Adjacent Normal Tissue for Immuno-Oncology Applications.

Survivin (BIRC5) is a tumor-associated antigen (TAA) overexpressed in various tumors but present at low to undetectable levels in normal tissue. Survivin is known to have a high expression in breast cancer (e.g., Ductal Carcinoma in situ (DCIS) and triple negative breast cancer). Previous studies have not compared survivin expression levels in DCIS tumor samples to levels in adjacent, normal breast tissue from the same patient. To ensure the effective use of survivin as a target for T cell immunotherapy of breast cancer, it is essential to ascertain the varying levels of survivin expression between DCIS tumor tissue samples and the adjacent normal breast tissue taken from the same patient simultaneously. Next-generation sequencing of RNA (RNA-seq) in normal breast tissue and tumor breast tissue from five women presenting with DCIS for lumpectomy was used to identify sequence variation and expression levels of survivin. The identity of both tumor and adjacent normal tissue samples were corroborated by histopathology. Survivin was overexpressed in human breast tissue tumor samples relative to the corresponding adjacent human normal breast tissue. Wild-type survivin transcripts were the predominant species identified in all tumor tissue sequenced. This study demonstrates upregulated expression of wild type survivin in DCIS tumor tissue versus normal breast tissue taken from the same patient at the same time, and provides evidence that developing selective cytotoxic T lymphocyte (CTL) immunotherapy for DCIS targeting survivin warrants further study.

New roles for dopamine D2 and D3 receptors in pancreatic beta cell insulin secretion.

Although long-studied in the central nervous system, there is increasing evidence that dopamine (DA) has important roles in the periphery including in metabolic regulation. Insulin-secreting pancreatic ß-cells express the machinery for DA synthesis and catabolism, as well as all five DA receptors. In these cells, DA functions as a negative regulator of glucose-stimulated insulin secretion (GSIS), which is mediated by DA D2-like receptors including D2 (D2R) and D3 (D3R) receptors. However, the fundamental mechanisms of DA synthesis, storage, release, and signaling in pancreatic ß-cells and their functional relevance in vivo remain poorly understood. Here, we assessed the roles of the DA precursor L-DOPA in ß-cell DA synthesis and release in conjunction with the signaling mechanisms underlying DA's inhibition of GSIS. Our results show that the uptake of L-DOPA is essential for establishing intracellular DA stores in ß-cells. Glucose stimulation significantly enhances L-DOPA uptake, leading to increased DA release and GSIS reduction in an autocrine/paracrine manner. Furthermore, D2R and D3R act in combination to mediate dopaminergic inhibition of GSIS. Transgenic knockout mice in which ß-cell D2R or D3R expression is eliminated exhibit diminished DA secretion during glucose stimulation, suggesting a new mechanism where D2-like receptors modify DA release to modulate GSIS. Lastly, ß-cell-selective D2R knockout mice exhibit marked postprandial hyperinsulinemia in vivo. These results reveal that peripheral D2R and D3R receptors play important roles in metabolism through their inhibitory effects on GSIS. This opens the possibility that blockade of peripheral D2-like receptors by drugs including antipsychotic medications may significantly contribute to the metabolic disturbances observed clinically.

The Radiolabeling of a Gly-Sar Dipeptide Derivative with Flourine-18 and Its Use as a Potential Peptide Transporter PET Imaging Agent.

: We have developed a novel fluorine-18 radiotracer, dipeptide 1, radiolabeled in two steps from mesylate 3. The initial radiolabeling is achieved in a short reaction time (10 min) and purified through solid-phase extraction (SPE) with modest radiochemical yields (rcy = 10 ± 2%, n = 5) in excellent radiochemical purity (rcp > 99%, n = 5). The de-protection of the tert-butyloxycarbonyl (Boc) and trityl group was achieved with mild heating under acidic conditions to provide 18F-tagged dipeptide 1. Preliminary analysis of 18F-dipeptide 1 was performed to confirm uptake by peptide transporters (PepTs) in human pancreatic carcinoma cell lines Panc1, BxPC3, and ASpc1, which are reported to express the peptide transporter 1 (PepT1) . Furthermore, we confirmed in vivo uptake of 18F-dipeptide tracer 1 using microPET/CT in mice harboring subcutaneous flank Panc1, BxPC3, and Aspc1 tumors. In conclusion, we have established the radiolabeling of dipeptide 1 with fluoride-18, and demonstrated its potential as an imaging agent which may have clinical applications for the diagnosis of pancreatic carcinomas.

Insulin Hexamer-Caged Gadolinium Ion as MRI Contrast-o-phore.

High-relaxivity protein-complexes of GdIII are being pursued as MRI contrast agents in hope that they can be used at much lower doses that would minimize toxic-side effects of GdIII release from traditional contrast agents. We construct here a new type of protein-based MRI contrast agent, a proteinaceous cage based on a stable insulin hexamer in which GdIII is captured inside a water filled cavity. The macromolecular structure and the large number of "free" GdIII coordination sites available for water binding lead to exceptionally high relaxivities per one GdIII ion. The GdIII slowly diffuses out of this cage, but this diffusion can be prevented by addition of ligands that bind to the hexamer. The ligands that trigger structural changes in the hexamer, SCN- , Cl- and phenols, modulate relaxivities through an outside-in signaling that is allosterically transduced through the protein cage. Contrast-o-phores based on protein-caged metal ions have potential to become clinical contrast agents with environmentally-sensitive properties.

Bioinformatic, Biochemical, and Immunological Mining of MHC Class I Restricted T Cell Epitopes for a Marburg Nucleoprotein Microparticle Vaccine.

The Marburg virus (MARV), the virus responsible for Marburg hemorrhagic fever (MHF), is considered a top-priority pathogen for vaccine development. Recent outbreaks in Equatorial Africa have highlighted the urgency of MARV because of its high fatality rate and historical concerns about potential weaponization. Currently, there are no licensed vaccines for MARV. Existing vaccine candidates rely on attenuated recombinant vesicular stomatitis virus carrying MARV glycoprotein (VSVΔG) or the chimpanzee replication-defective adenovirus 3 vector ChAd3-MARV. Although these platforms provide significant protection in animal models, they face challenges because of their limited thermal stability and the need for cold storage during deployment in resource-poor areas. An alternative approach involves using adjuvanted poly (lactic-co-glycolic acid) (PLGA) microparticles loaded with synthetic peptides representing MHC class I-restricted T cell epitopes. This vaccine platform has demonstrated effectiveness in protecting against SARS-CoV-2 and EBoV disease in animal models and has the advantage of not requiring cold storage and remaining stable at room temperature for over six months. This report outlines the design, manufacturing, and in vivo immunogenicity testing of PLGA microparticle human vaccines designed to prevent Marburg hemorrhagic fever.

Analysis of well-annotated next-generation sequencing data reveals increasing cases of SARS-CoV-2 reinfection with Omicron.

SARS-CoV-2 has extensively mutated creating variants of concern (VOC) resulting in global infection surges. The Omicron VOC reinfects individuals exposed to earlier variants of SARS-CoV-2 at a higher frequency than previously seen for non-Omicron VOC. An analysis of the sub-lineages associated with an Omicron primary infection and Omicron reinfection reveals that the incidence of Omicron-Omicron reinfections is occurring over a shorter time interval than seen after a primary infection with a non-Omicron VOC. Our analysis suggests that a single infection from SARS-CoV-2 may not generate the protective immunity required to defend against reinfections from emerging Omicron lineages. This analysis was made possible by Next-generation sequencing (NGS) of a Danish cohort with clinical metadata on both infections occurring in the same individual. We suggest that the continuation of COVID-19 NGS and inclusion of clinical metadata is necessary to ensure effective surveillance of SARS-CoV-2 genomics, assist in treatment and vaccine development, and guide public health recommendations.

Survivin (BIRC5) Peptide Vaccine in the 4T1 Murine Mammary Tumor Model: A Potential Neoadjuvant T Cell Immunotherapy for Triple Negative Breast Cancer: A Preliminary Study.

A triple negative breast cancer model using the murine 4T1 tumor cell line was used to explore the efficacy of an adjuvanted survivin peptide microparticle vaccine using tumor growth as the outcome metric. We first performed tumor cell dose titration studies to determine a tumor cell dose that resulted in sufficient tumor takes but allowed multiple serial measurements of tumor volumes, yet with minimal morbidity/mortality within the study period. Later, in a second cohort of mice, the survivin peptide microparticle vaccine was administered via intraperitoneal injection at the study start with a second dose given 14 days later. An orthotopic injection of 4T1 cells into the mammary tissue was performed on the same day as the administration of the second vaccine dose. The mice were followed for up to 41 days with subcutaneous measurements of tumor volume made every 3-4 days. Vaccination with survivin peptides was associated with a peptide antigen-specific gamma interferon enzyme-linked immunosorbent spot response in the murine splenocyte population but was absent from the control microparticle group. At the end of the study, we found that vaccination with adjuvanted survivin peptide microparticles resulted in statistically significant slower primary tumor growth rates in BALB/c mice challenged with 4T1 cells relative to the control peptideless vaccination group. These studies suggest that T cell immunotherapy specifically targeting survivin might be an applicable neoadjuvant immunotherapy therapy for triple negative breast cancer. More preclinical studies and clinical trials are needed to explore this concept further.

Design, Synthesis, and Characterization of a Novel Fluoroprobe for Live Human Islet Cell Imaging of Serotonin 5-HT1A Receptor.

Mounting evidence suggests that the serotonin system serves in signal transmission to regulate insulin secretion in pancreatic islets of Langerhans. Among the 5-HT receptor subtype found in pancreatic islets, serotonin receptor 1A (5-HT1A ) demonstrates a unique ability to inhibit ß-cell insulin secretion. We report the design, synthesis, and characterization of two novel fluorescent probes for the 5-HT1A receptor. The compounds were prepared by conjugating the scaffold of the 5-HT1A receptor agonist 8-OH-DPAT with two fluorophores suitable for live-cell imaging. Compound 5a {5-(dimethylamino)-N-[5-[(8-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino]pentyl]naphtalen-1-sulfonammide} showed high affinity for the 5-HT1A receptor (Ki =1.8 nM). Fluoroprobe 5a was able to label the 5-HT1A receptor in pancreatic islet cell cultures in a selective manner, as the fluorescence emission was significantly attenuated by co-administration of the 5-HT1A receptor antagonist WAY-100635. Thus, fluoroprobe 5a showed useful properties to further characterize this unique receptor's role.

A Synthetic Peptide CTL Vaccine Targeting Nucleocapsid Confers Protection from SARS-CoV-2 Challenge in Rhesus Macaques.

BACKGROUND: Persistent transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has given rise to a COVID-19 pandemic. Several vaccines, conceived in 2020, that evoke protective spike antibody responses are being deployed in mass public health vaccination programs. Recent data suggests, however, that as sequence variation in the spike genome accumulates, some vaccines may lose efficacy. METHODS: Using a macaque model of SARS-CoV-2 infection, we tested the efficacy of a peptide-based vaccine targeting MHC class I epitopes on the SARS-CoV-2 nucleocapsid protein. We administered biodegradable microspheres with synthetic peptides and adjuvants to rhesus macaques. Unvaccinated control and vaccinated macaques were challenged with 1 × 108 TCID50 units of SARS-CoV-2, followed by assessment of clinical symptoms and viral load, chest radiographs, and sampling of peripheral blood and bronchoalveolar lavage (BAL) fluid for downstream analysis. RESULTS: Vaccinated animals were free of pneumonia-like infiltrates characteristic of SARS-CoV-2 infection and presented with lower viral loads relative to controls. Gene expression in cells collected from BAL samples of vaccinated macaques revealed a unique signature associated with enhanced development of adaptive immune responses relative to control macaques. CONCLUSIONS: We demonstrate that a room temperature stable peptide vaccine based on known immunogenic HLA class I bound CTL epitopes from the nucleocapsid protein can provide protection against SARS-CoV-2 infection in nonhuman primates.

Diabetes mellitus impairs tendon-bone healing after rotator cuff repair.

INTRODUCTION: Studies have demonstrated a significant decrease in skeletal mass, bone mineral density, and impaired fracture healing in the diabetic population. However, the effect of sustained hyperglycemia on tendon-to-bone healing is unknown. MATERIALS AND METHODS: Forty-eight male, Lewis rats underwent unilateral detachment of the supraspinatus tendon followed by immediate anatomic repair with transosseous fixation. In the experimental group (n = 24), diabetes was induced preoperatively via intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) and confirmed with both pre- and post-STZ injection intraperitoneal glucose tolerance tests (IPGTT). Animals were sacrificed at 1 and 2 weeks postoperatively for biomechanical, histomorphometric, and immunohistochemical analysis. Serum hemoglobin A1c (HbA1c) levels were measured at 2 weeks postoperatively. Statistical comparisons were performed using Student t tests with significance set at P < .05. RESULTS: IPGTT analysis demonstrated a significant impairment of glycemic control in the diabetic compared to control animals (P < .05). Mean HbA1c level at 2 weeks postoperatively was 10.6 ± 2.7% and 6.0 ± 1.0% for the diabetic and control groups, respectively (P < .05). Diabetic animals demonstrated significantly less fibrocartilage and organized collagen, and increased AGE deposition at the tendon-bone interface (P < .05). The healing enthesis of diabetic animals demonstrated a significantly reduced ultimate load-to-failure (4.79 ± 1.33 N vs 1.60 ± 1.67 N and 13.63 ± 2.33 N vs 6.0 ± 3.24 N for control versus diabetic animals at 1 and 2 weeks, respectively) and stiffness compared to control animals (P < .05). DISCUSSION: Sustained hyperglycemia impairs tendon-bone healing after rotator cuff repair in this rodent model. These findings have significant clinical implications for the expected outcomes of soft tissue repair or reconstructive procedures in diabetic patients with poor glycemic control.

PET Imaging of Pancreatic Dopamine D2 and D3 Receptor Density with 11C-(+)-PHNO in Type 1 Diabetes.

Type 1 diabetes mellitus (T1DM) has traditionally been characterized by a complete destruction of ß-cell mass (BCM); however, there is growing evidence of possible residual BCM present in T1DM. Given the absence of in vivo tools to measure BCM, routine clinical measures of ß-cell function (e.g., C-peptide release) may not reflect BCM. We previously demonstrated the potential utility of PET imaging with the dopamine D2 and D3 receptor agonist 3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol (11C-(+)-PHNO) to differentiate between healthy control (HC) and T1DM individuals. Methods: Sixteen individuals participated (10 men, 6 women; 9 HCs, 7 T1DMs). The average duration of diabetes was 18 ± 6 y (range, 14-30 y). Individuals underwent PET/CT scanning with a 120-min dynamic PET scan centered on the pancreas. One- and 2-tissue-compartment models were used to estimate pancreas and spleen distribution volume. Reference region approaches (spleen as reference) were also investigated. Quantitative PET measures were correlated with clinical outcome measures. Immunohistochemistry was performed to examine colocalization of dopamine receptors with endocrine hormones in HC and T1DM pancreatic tissue. Results: C-peptide release was not detectable in any T1DM individuals, whereas proinsulin was detectable in 3 of 5 T1DM individuals. Pancreas SUV ratio minus 1 (SUVR-1) (20-30 min; spleen as reference region) demonstrated a statistically significant reduction (-36.2%) in radioligand binding (HCs, 5.6; T1DMs, 3.6; P = 0.03). Age at diagnosis correlated significantly with pancreas SUVR-1 (20-30 min) (R2 = 0.67, P = 0.025). Duration of diabetes did not significantly correlate with pancreas SUVR-1 (20-30 min) (R2 = 0.36, P = 0.16). Mean acute C-peptide response to arginine at maximal glycemic potentiation did not significantly correlate with SUVR-1 (20-30 min) (R2 = 0.57, P = 0.05), nor did mean baseline proinsulin (R2 = 0.45, P = 0.10). Immunohistochemistry demonstrated colocalization of dopamine D3 receptor and dopamine D2 receptor in HCs. No colocalization of the dopamine D3 receptor or dopamine D2 receptor was seen with somatostatin, glucagon, or polypeptide Y. In a separate T1DM individual, no immunostaining was seen with dopamine D3 receptor, dopamine D2 receptor, or insulin antibodies, suggesting that loss of endocrine dopamine D3 receptor and dopamine D2 receptor expression accompanies loss of ß-cell functional insulin secretory capacity. Conclusion: Thirty-minute scan durations and SUVR-1 provide quantitative outcome measures for 11C-(+)-PHNO, a dopamine D3 receptor-preferring agonist PET radioligand, to differentiate BCM in T1DM and HCs.

Longitudinal noninvasive PET-based beta cell mass estimates in a spontaneous diabetes rat model.

Diabetes results from an absolute or relative reduction in pancreatic beta cell mass (BCM) leading to insufficient insulin secretion and hyperglycemia. Measurement of insulin secretory capacity is currently used as a surrogate measure of BCM. However, serum insulin concentrations provide an imprecise index of BCM, and no reliable noninvasive measure of BCM is currently available. Type 2 vesicular monoamine transporters (VMAT2) are expressed in human islet beta cells, as well as in tissues of the CNS. [11C]Dihydrotetrabenazine ([11C]DTBZ) binds specifically to VMAT2 and is a radioligand currently used in clinical imaging of the brain. Here we report the use of [11C]DTBZ to estimate BCM in a rodent model of spontaneous type 1 diabetes (the BB-DP rat). In longitudinal PET studies of the BB-DP rat, we found a significant decline in pancreatic uptake of [11C]DTBZ that anticipated the loss of glycemic control. Based on comparison of standardized uptake values (SUVs) of [11C]DTBZ and blood glucose concentrations, loss of more than 65% of the original SUV correlated significantly with the development of persistent hyperglycemia. These studies suggest that PET-based quantitation of VMAT2 receptors provides a noninvasive measurement of BCM that could be used to study the pathogenesis of diabetes and to monitor therapeutic interventions.

11C-dihydrotetrabenazine PET of the pancreas in subjects with long-standing type 1 diabetes and in healthy controls.

UNLABELLED: Type 2 vesicular monoamine transporter (VMAT2), found in the brain, is also expressed by beta-cells of the pancreas in association with insulin. Preclinical experiments suggested that (11)C-dihydrotetrabenazine PET-measured VMAT2 binding might serve as a biomarker of beta-cell mass. We evaluated the feasibility of (11)C-dihydrotetrabenazine PET quantification of pancreatic VMAT2 binding in healthy subjects and patients with long-standing type 1 diabetes. METHODS: (11)C-Dihydrotetrabenazine PET was performed on 6 patients and 9 controls. VMAT2 binding potential (BP(ND)) was estimated voxelwise by using the renal cortex as reference tissue. As an index of total pancreatic VMAT2, the functional binding capacity (the sum of voxel BP(ND) x voxel volume) was calculated. Pancreatic BP(ND), functional binding capacity, and stimulated insulin secretion measurements were compared between groups. RESULTS: The pancreatic mean BP(ND) was decreased in patients (1.86 +/- 0.05) to 86% of control values (2.14 +/- 0.08) (P = 0.01). In controls, but not in patients, BP(ND) correlated with stimulated insulin secretion (r(2) = 0.50, P = 0.03). The average functional binding capacity was decreased by at least 40% in patients (P = 0.001). The changes in functional binding capacity and BP(ND) were less than the near-complete loss of stimulated insulin secretion observed in patients (P = 0.001). CONCLUSION: These results suggest that (11)C-dihydrotetrabenazine PET allows quantification of VMAT2 binding in the human pancreas. However, BP(ND) and functional binding capacity appear to overestimate beta-cell mass given the near-complete depletion of beta-cell mass in long-standing type 1 diabetes, which may be due to higher nonspecific binding in the pancreas than in the renal cortex.

A rodent model of metabolic surgery for study of type 2 diabetes and positron emission tomography scanning of beta cell mass.

BACKGROUND: Type 2 diabetes mellitus is a worldwide healthcare problem with major socioeconomic implications. Metabolic surgical procedures have been shown to improve diabetes, but the mechanism of action is poorly understood. The Goto-Kakizaki (GK) rodent is a type 2 diabetic animal model that is ideally situated for studying the effect of surgery on diabetes; however, the operative mortality is high. The aim of this study was to describe the operative technique, improvements in perioperative management, and the technique of micro-positron emission tomography (PET) scanning of the beta-cell mass in GK rodents. METHODS: A total of 53 GK rats were divided into 1 of 3 operative groups: sham, sleeve gastrectomy, and duodenojejunal bypass. A subset of animals underwent micro-PET scanning with [11C]-dihydrotetrabenazine to determine the vesicular monoamine transporter 2 binding index, an indicator of beta-cell mass. RESULTS: The 30-day mortality in the sham and sleeve gastrectomy rodents was 0; however, 2 sleeve gastrectomy rodents developed enterocutaneous fistula and 1 developed an abscess. In the duodenojejunal bypass group, the initial mortality rate was close to 90%; however, refinements in the surgical technique and perioperative management (fluids, antibiotics, pain control) lowered the mortality rate to 60%. The surgical technique is discussed in detail. [11C]-Dihydrotetrabenazine uptake in the pancreas was demonstrated on micro-PET scanning in the sham and duodenojejunal bypass rodents. CONCLUSION: Intensive medical management in the perioperative period and attention to the operative technique lowered the mortality. [11C]-Dihydrotetrabenazine micro-PET scanning is a feasible method for assessing the beta-cell mass in GK rodents and could prove to be an important modality for evaluating beta-cell performance in type 2 diabetes.

A novel optical tracer for VMAT2 applied to live cell measurements of vesicle maturation in cultured human ß-cells.

The islet ß-cells integrate external signals to modulate insulin secretion to better regulate blood glucose levels during periods of changing metabolic demand. The vesicular monoamine transporter type 2 (VMAT2), an important regulator of CNS neurotransmission, has an analogous role in the endocrine pancreas as a key control point of insulin secretion, with additional roles in regulating ß-cell differentiation and proliferation. Here we report on the synthesis and biological characterisation of a fluorescent ligand for VMAT2 suitable for live cell imaging. Staining for VMAT2 and dopamine in live ß-cell cultures show colocalisation in specific vesicles and reveal a heterogeneous population with respect to cell size, shape, vesicle number, size, and contents. Staining for VMAT2 and zinc ion, as a surrogate for insulin, reveals a wide range of vesicle sizes. Immunohistochemistry shows larger ß-cell vesicles enriched for proinsulin, whereas smaller vesicles predominantly contain the processed mature insulin. In ß-cell cultures obtained from nondiabetic donors, incubation at non-stimulatory glucose concentrations promotes a shift in vesicle diameter towards the more mature insulin vesicles at the expense of the larger immature insulin secretory vesicle population. We anticipate that this probe will be a useful reagent to identify living ß-cells within complex mixtures for further manipulation and characterisation.

A role for foregut tyrosine metabolism in glucose tolerance.

OBJECTIVE: We hypothesized that DA and L-DOPA derived from nutritional tyrosine and the resultant observed postprandial plasma excursions of L-DOPA and DA might affect glucose tolerance via their ability to be taken-up by beta cells and inhibit glucose-stimulated ß-cell insulin secretion. METHODS: To investigate a possible circuit between meal-stimulated 3,4-dihydroxy-L-phenylalanine (L-DOPA) and dopamine (DA) production in the GI tract and pancreatic ß-cells, we: 1) mapped GI mucosal expression of tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC); 2) measured L-DOPA and DA content of GI mucosal tissues following meal challenges with different L-tyrosine (TYR) content, 3) determined whether meal TYR content impacts plasma insulin and glucose excursions; and 4) characterized postprandial plasma excursions of L-DOPA and DA in response to meal tyrosine content in rodents and a population of bariatric surgery patients. Next, we characterized: 1) the metabolic transformation of TYR and L-DOPA into DA in vitro using purified islet tissue; 2) the metabolic transformation of orally administrated stable isotope labeled TYR into pancreatic DA, and 3) using a nuclear medicine technique, we studied endocrine beta cells in situ release and binding of DA in response to a glucose challenge. RESULTS: We demonstrate in rodents that intestinal content and circulatory concentrations L-DOPA and DA, plasma glucose and insulin are responsive to the tyrosine (TYR) content of a test meal. Intestinal expression of two enzymes, Tyrosine hydroxylase (TH) and Aromatic Amino acid Decarboxylase (AADC), essential to the transformation of TYR to DA was mapped and the metabolism of metabolism of TYR to DA was traced in human islets and a rodent beta cell line in vitro and from gut to the pancreas in vivo. Lastly, we show that ß cells secrete and bind DA in situ in response to glucose stimulation. CONCLUSIONS: We provide proof-of-principle evidence for the existence of a novel postprandial circuit of glucose homeostasis dependent on nutritional tyrosine. DA and L-DOPA derived from nutritional tyrosine may serve to defend against hypoglycemia via inhibition of glucose-stimulated ß-cell insulin secretion as proposed by the anti-incretin hypothesis.

Relationship between pancreatic vesicular monoamine transporter 2 (VMAT2) and insulin expression in human pancreas.

Vesicular monoamine transporter 2 (VMAT2) is expressed in pancreatic beta cells and has recently been proposed as a target for measurement of beta cell mass in vivo. We questioned, (1) What proportion of beta cells express VMAT2? (2) Is VMAT2 expressed by other pancreatic endocrine or non-endocrine cells? (3) Is the relationship between VMAT2 and insulin expression disturbed in type 1 (T1DM) or type 2 diabetes (T2DM)? Human pancreas (7 non-diabetics, 5 T2DM, 10 T1DM) was immunostained for insulin, VMAT2 and other pancreatic hormones. Most beta cells expressed VMAT2. VMAT2 expression was not changed by the presence of diabetes. In tail of pancreas VMAT2 immunostaining closely correlated with insulin staining. However, VMAT2 was also expressed in some pancreatic polypeptide (PP) cells. Although VMAT2 was not excluded as a target for beta cell mass measurement, expression of VMAT2 in PP cells predicts residual VMAT2 expression in human pancreas even in the absence of beta cells.

Novel hypoglycemic dihydropyridones serendipitously discovered from O- versus C-alkylation in the synthesis of VMAT2 antagonists.

Vesicular monoamine transporter type 2 (VMAT2) is a newly emerging target for both diagnostic and therapeutic applications in diabetes mellitus. In pursuit of novel VMAT2 antagonists, we identified a potent hypoglycemic agent with a novel dihydropyridone scaffold. Several analogs were designed and synthesized. A preliminary structure activity relationship (SAR) showed that the dihydropyridone scaffold is required for the activity.

State of the Art: Role of the Dendritic Cell in Induction of Allograft Tolerance.

Despite decades of research, the induction and maintenance of long-term allograft tolerance without immunosuppression remains an elusive goal in the field of solid organ and cell transplantation. Immunosuppressive medications frequently prevent or minimize acute cellular rejection but have failed to halt antidonor antibody production and chronic organ rejection. Past efforts aimed at promoting lasting allograft tolerance have focused primarily on peripheral T-cell depletion, augmentation of regulatory T cells, or induction via simultaneous hematopoietic stem cell transplantation and facilitation of donor chimerism. So far, none of these methods have led to consistently safe, feasible and long lasting donor organ acceptance. Over the course of the past 4 decades, the study of a unique population of antigen-presenting cells known as dendritic cells has shown promise for breaking new ground in achieving indefinite allograft survival without immunosuppression and its associated adverse effects. In this review, we discuss the discovery and early investigations of dendritic cells and chronicle some of the key studies demonstrating their role in transplantation, particularly in indirect allorecognition, the immunologic pathway thought to drive chronic rejection and perhaps tolerance induction.

Effect of interferon alpha on MHC class II gene expression in ex vivo human islet tissue.

Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing beta-cells of the islets of Langerhans. One still open question is where naive islet-reactive T cells encounter antigens and become stimulated. In this report we have re-examined the expression of MHC class II (MHCII) genes in human islets to further explore the possibility that non-professional antigen presenting cells (APCs) within islets contribute to autoimmunity. Since development of T1D has been linked to viral infections, we also studied ex-vivo MHCII expression in response to interferon-alpha (IFNalpha) in islet tissue and in different APCs. The findings are: first, MHCII genes expression in human islets is linked with the expression of the class II transactivator isoform transcribed from the promoter IV, similar to that described in non-professional APCs. Second, there is IFNalpha-mediated lineage-specific regulation of MHCII genes expression, seen as a decrease in the accumulation of MHCII transcripts in pancreatic islets opposite to an increase in dendritic cells and B-lymphoblastoid cell lines. Third, there is allele-specific regulation of the HLA-DQA1 gene by IFNalpha in islet tissue. These findings may begin to explain the molecular events that create favorable conditions for organ-specific autoimmunity and explain the incomplete penetrance of T1D susceptibility alleles.