Establishment of CD8+ T Cell Thymic Central Tolerance to Tissue-Restricted Antigen Requires PD-1.

Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted Ag did not compromise tolerance. In this study, we determined that murine T cells that failed clonal deletion were rendered functionally impaired in the thymus. Programmed cell death protein 1 (PD-1) was induced in the thymus and was required to establish cell-intrinsic tolerance to tissue-restricted Ag in CD8+ thymocytes independently of clonal deletion. In bone marrow chimeras, tolerance was not observed in PD-L1-deficient recipients, but tolerance was largely maintained following adoptive transfer of tolerant thymocytes or T cells to PD-L1-deficient recipients. However, CRISPR-mediated ablation of PD-1 in tolerant T cells resulted in broken tolerance, suggesting different PD-1 signaling requirements for establishing versus maintaining tolerance. Finally, we showed that chronic exposure to high-affinity Ag supported the long-term maintenance of tolerance. Taken together, our study identifies a critical role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion. It also sheds light on potential mechanisms of action of anti-PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.

Feature-specific quantile normalization and feature-specific mean-variance normalization deliver robust bi-directional classification and feature selection performance between microarray and RNAseq data.

BACKGROUND: Cross-platform normalization seeks to minimize technological bias between microarray and RNAseq whole-transcriptome data. Incorporating multiple gene expression platforms permits external validation of experimental findings, and augments training sets for machine learning models. Here, we compare the performance of Feature Specific Quantile Normalization (FSQN) to a previously used but unvalidated and uncharacterized method we label as Feature Specific Mean Variance Normalization (FSMVN). We evaluate the performance of these methods for bidirectional normalization in the context of nested feature selection. RESULTS: FSQN and FSMVN provided clinically equivalent bidirectional model performance with and without feature selection for colon CMS and breast PAM50 classification. Using principal component analysis, we determine that these methods eliminate batch effects related to technological platforms. Without feature selection, no statistical difference was identified between the performance of FSQN and FSMVN of cross-platform data compared to within-platform distributions. Under optimal feature selection conditions, balanced accuracy was FSQN and FSMVN were statistically equivalent to the within-platform distribution performance in multivariable linear regression analysis. FSQN and FSMVN also provided similar performance to within-platform distributions as the number of selected genes used to create models decreases. CONCLUSIONS: In the context of generating supervised machine learning classifiers for molecular subtypes, FSQN and FSMVN are equally effective. Under optimal modeling conditions, FSQN and FSMVN provide equivalent model accuracy performance on cross-platform normalization data compared to within-platform data. Using cross-platform data should still be approached with caution as subtle performance differences may exist depending on the classification problem, training, and testing distributions.

MRI monitoring of transplanted neonatal porcine islets labeled with polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles in a mouse model.

Islet transplantation is a potential treatment option for certain patients with type 1 diabetes; however, it still faces barriers to widespread use, including the lack of tools to monitor islet grafts post-transplantation. This study investigates whether labeling neonatal porcine islets (NPI) with polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles (PVP-SPIO) affects their function, and whether this nanoparticle can be utilized to monitor NPI xenografts with magnetic resonance imaging (MRI) in a mouse model. In vitro, PVP-SPIO-labeled NPI in an agarose gel was visualized clearly by MRI. PVP-SPIO-labeled islets were then transplanted under the kidney capsules of immunodeficient nondiabetic and diabetic mice. All diabetic mice that received transplantation of PVP-SPIO-labeled islets reached normoglycemia. Grafts appeared as hypo-intense areas on MRI and were distinguishable from the surrounding tissues. Following injection of spleen cells from immunocompetent mice, normoglycemic recipient mice became diabetic and islet grafts showed an increase in volume, accompanied by a mixed signal on MRI. Overall, this study demonstrates that PVP-SPIO did not affect the function of NPI that PVP-SPIO-labeled islets were easily seen on MRI, and changes in MRI signals following rejection suggest a potential use of PVP-SPIO-labeled islets to monitor graft viability.

Anti-Mycobacterial Activity of Flavonoid and Pyrimidine Compounds.

We evaluated the anti-mycobacterial effect of a flavonoid 5,7-dihydroxy-2-(4-hydroxyphenyl) 4H-chromen-4-one (1) and two pyrimidines, 4-hydroxy-2-dimethylamino-5-nitroso-6-aminopyrimidine (2) and 2-chloro-5-n-nonylpyrimidine (3) in vitro against Mycobacterium tuberculosis (M. tuberculosis, H37Ra) and Mycobacterium avium (M. avium), using a Microplate Alamar Blue Assay (MABA). The effects of the compounds 1-3 in combination with first- and second-line anti-TB drugs isoniazid, rifampicin, cycloserine, and clarithromycin on the growth of M. tuberculosis and M. avium were also evaluated in in vitro assays. As a single agent, compounds 1 and 2 exhibited modest activity while compound 3 was the most effective against M. tuberculosis and M. avium. When compounds 1-3 were evaluated at lower than 50% of their inhibitory concentrations in a two-drug combination with isoniazid or rifampicin, they showed additive to synergistic interactions. This inhibitory effect was improved when each of the three compounds was tested together in a three-drug combination with two of the first-line anti-TB drugs. Compounds 1-3 also demonstrated strong synergistic interaction in combination with cycloserine and clarithromycin in inhibiting the growth of M. tuberculosis and M. avium, respectively. This study demonstrated that compounds 1-3 have potential to be developed as effective anti-TB agents with combined use.

Impact of donor and prolonged cold ischemia time of neonatal pig pancreas on neonatal pig islet transplant outcome.

BACKGROUND: Genetically modified pigs (GMP) have been developed to alleviate the shortage of donors in human islet transplantation and rejection. In this study, we characterized and compared the islets from GalTKO, GalTKO/hCD46, GalTKO/hCD46/hCD39, and wild-type (WT) neonatal pigs. METHODS: Islets were isolated from GMP and WT pig pancreases that have been packaged with ice pack for at least 24 hours. The difference in gene expression and function of islets were evaluated by microarray analysis and transplantation of islets under the kidney capsule of streptozotocin-induced diabetic immune-deficient mice, respectively. Blood glucose levels of these mice were monitored weekly post-transplantation for >100 days, and islet grafts were collected and evaluated for the presence of endocrine cells. RESULTS: The genes involved in extracellular components, cell adhesion, glucose metabolism, and inflammatory response are differentially expressed between GMP and WT pig islets. Variation in the ability of pig islets in correcting the diabetic state of the mouse recipients appears to be dependent on the pig donor. In addition, prolonged cold ischemia time had a negative effect on the transplant outcome. All normoglycemic mice were able to respond well to glucose challenge despite the initial differences in the ability of islet transplants to reverse their diabetic state. Islet xenografts of normoglycemic mice contained abundant insulin- and glucagon-positive cells. CONCLUSION: The effect of GMP and WT neonatal pig islet transplants on hyperglycemia in mice appears to be dependent on the pig donor, and prolonged cold ischemia time negatively affects the neonatal pig islet transplant outcome.

Early immune mechanisms of neonatal porcine islet xenograft rejection.

BACKGROUND: Neonatal pigs have the potential to provide an inexhaustible source of islets for the treatment of type 1 diabetes. However, the immunological barriers to islet xenotransplantation still need to be overcome. A better understanding of the xeno-specific immune responses that are involved in neonatal porcine islet (NPI) xenotransplant rejection will help to facilitate the identification of new targets for anti-rejection therapies, and thus enable more specific targeting of the immune cells and molecules involved. METHODS: In this study, we examined the early events of NPI xenograft rejection in the absence of autoimmunity using an immune-competent B6 mouse transplant model. Immune cells were identified by immunohistochemistry and immune molecules were identified by reverse transcription-PCR and flow cytometry assays. RESULTS: Our results demonstrated that early events in NPI xenograft rejection are characterized by initial infiltration of innate immune cells such as macrophages (M1) and neutrophils. CONCLUSIONS: Targeting these cells, which appear early in the rejection process, may provide an opportunity to abort the rejection process prior to activation of T cells. One strategy could be the blockade of chemotactic signals associated with preferential recruitment of immune cells into the graft site. Collectively, our studies demonstrated that early recruitment of immune cells into graft site is controlled by chemotactic activities and suggest a potential target to prevent the early infiltration of immune cells within the graft. Our findings in this study will have significance in improving NPI xenograft acceptance and induce long-term xenograft survival.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Executive summary.

The International Xenotransplantation Association has updated its original "Consensus Statement on Conditions for Undertaking Clinical Trials of Porcine Islet Products in Type 1 Diabetes," which was published in Xenotransplantation in 2009. This update is timely and important in light of scientific progress and changes in the regulatory framework pertinent to islet xenotransplantation. Except for the chapter on "informed consent," which has remained relevant in its 2009 version, all other chapters included in the initial consensus statement have been revised for inclusion in this update. These chapters will not provide complete revisions of the original chapters; rather, they restate the key points made in 2009, emphasize new and under-appreciated topics not fully addressed in 2009, suggest relevant revisions, and communicate opinions that complement the consensus opinion. Chapter 1 provides an update on national regulatory frameworks addressing xenotransplantation. Chapter 2 a, previously Chapter 2, suggests several important revisions regarding the generation of suitable source pigs from the perspective of the prevention of xenozoonoses. The newly added Chapter 2b discusses conditions for the use of genetically modified source pigs in clinical islet xenotransplantation. Chapter 3 reviews porcine islet product manufacturing and release testing. Chapter 4 revisits the critically important topic of preclinical efficacy and safety data required to justify a clinical trial. The main achievements in the field of transmission of all porcine microorganisms, the rationale for more proportionate recipient monitoring, and response plans are reviewed in Chapter 5. Patient selection criteria and circumstances where trials of islet xenotransplantation would be both medically and ethically justified are examined in Chapter 6 in the context of recent advances in available and emerging alternative therapies for serious and potentially life-threatening complications of diabetes. It is hoped that this first update of the International Xenotransplantation Association porcine islet transplant consensus statement will assist the islet xenotransplant scientific community, sponsors, regulators, and other stakeholders actively involved in the clinical translation of islet xenotransplantation.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 3: Porcine islet product manufacturing and release testing criteria.

In the 2009 IXA consensus, the requirements for the quality and control of manufacturing of porcine islet products were based on the U.S. regulatory framework where the porcine islet products fall within the definition of somatic cell therapy under the statutory authority of the U.S. Food and Drug Administration (FDA). In addition, porcine islet products require pre-market approval as a biologic product under the Public Health Services Act and they meet the definition of a drug under the Federal Food, Drug, and Cosmetic Act (FD&C Act). Thus, they are subject to applicable provisions of the law and as such, control of manufacturing as well as reproducibility and consistency of porcine islet products, safety of porcine islet products, and characterization of porcine islet products must be met before proceeding to clinical trials. In terms of control of manufacturing as well as reproducibility and consistency of porcine islet products, the manufacturing facility must be in compliance with current Good Manufacturing Practices (cGMP) guidelines appropriate for the initiation of Phase 1/2 clinical trials. Sponsors intending to conduct a Phase 1/2 trial of islet xenotransplantation products must be able to demonstrate the safety of the product through the establishment of particular quality assurance and quality control procedures. All materials (including animal source and pancreas) used in the manufacturing process of the porcine islet products must be free of adventitious agents. The final porcine islet product must undergo tests for the presence of these adventitious agents including sterility, mycoplasma (if they are cultured), and endotoxin. Assessments of the final product must include the safety specifications mentioned above even if the results are not available until after release as these data would be useful for patient diagnosis and treatment if necessary. In addition, a plan of action must be in place for patient notification and treatment in case the sterility culture results are positive. In terms of the characterization of porcine islet products and product release criteria, the information on the porcine islet products should be acquired from a sample of the final product to be used for transplantation and must include the morphology of the islets, specific identity, purity, viability, and potency of the product. In addition, information on the quantity of the islet products should also be provided in a standardized fashion and this should be in terms of islet equivalents and/or cell numbers. The current consensus was created to provide guidelines that manufacturing facilities may find helpful in the manufacture of and the release criteria for porcine islet products including encapsulated islets and combined islet products. Our intent with the above recommendations is to provide a framework for individual porcine islet manufacturing facilities to ensure a high level of safety for the initiation of Phase 1/2 clinical trials on porcine islet xenotransplantation.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 1: update on national regulatory frameworks pertinent to clinical islet xenotransplantation.

Islet xenotransplantation represents an attractive solution to overcome the shortage of human islets for use in type 1 diabetes. The wide-scale application of clinical islet xenotransplantation, however, requires that such a procedure takes place in a specifically and tightly regulated environment. With a view to promoting the safe application of clinical islet xenotransplantation, a few years ago the International Xenotransplantation Association (IXA) published a Consensus Statement that outlined the key ethical and regulatory requirements to be satisfied before the initiation of xenotransplantation studies in diabetic patients. This earlier IXA Statement also documented a disparate regulatory landscape among different geographical areas. This situation clearly fell short of the 2004 World Health Assembly Resolution WHA57.18 that urged Member States "to cooperate in the formulation of recommendations and guidelines to harmonize global practices" to ensure the highest ethical and regulatory standards on a global scale. In this new IXA report, IXA members who are active in xenotransplantation research in their respective geographic areas herewith briefly describe changes in the regulatory frameworks that have taken place in the intervening period in the various geographic areas or countries. The key reassuring take-home message of the present report is that many countries have embraced the encouragement of the WHO to harmonize the procedures in a more global scale. Indeed, important regulatory changes have taken place or are in progress in several geographic areas that include Europe, Korea, Japan, and China. Such significant regulatory changes encompass the most diverse facets of the clinical application of xenotransplantation and comprise ethical aspects, source animals and product specifications, study supervision, sample archiving, patient follow-up and even insurance coverage in some legislations. All these measures are expected to provide a better care and protection of recipients of xenotransplants but also a higher safety profile to xenotransplantation procedures with an ultimate net gain in terms of international public health.

In vitro exposure of pig neonatal isletlike cell clusters to human blood.

BACKGROUND: Pig islet grafts have been successful in treating diabetes in animal models. One remaining question is whether neonatal pig isletlike cell clusters (NICC) are resistant to the early loss of islets from the instant blood-mediated inflammatory reaction (IBMIR). METHODS: Neonatal isletlike cell clusters were harvested from three groups of piglets-(i) wild-type (genetically unmodified), (ii) α1,3-galactosyltransferase gene-knockout (GTKO)/CD46, and (iii) GTKO/CD46/CD39. NICC samples were mixed with human blood in vitro, and the following measurements were made-antibody binding; complement activation; speed of islet-induced coagulation; C-peptide; glutamic acid decarboxylase (GAD65) release; viability. RESULTS: Time to coagulation and viability were both reduced in all groups compared to freshly drawn non-anticoagulated human blood and autologous combinations, respectively. Antibody binding to the NICC occurred in all groups. CONCLUSIONS: Neonatal isletlike cell clusters were subject to humoral injury with no difference associated to their genetic characteristics.

Individual Survival Distributions Generated by Multi-Task Logistic Regression Yield a New Perspective on Molecular and Clinical Prognostic Factors in Gastric Adenocarcinoma.

Recent advances in our understanding of gastric cancer biology have prompted a shift towards more personalized therapy. However, results are based on population-based survival analyses, which evaluate the average survival effects of entire treatment groups or single prognostic variables. This study uses a personalized survival modelling approach called individual survival distributions (ISDs) with the multi-task logistic regression (MTLR) model to provide novel insight into personalized survival in gastric adenocarcinoma. We performed a pooled analysis using 1043 patients from a previously characterized database annotated with molecular subtypes from the Cancer Genome Atlas, Asian Cancer Research Group, and tumour microenvironment (TME) score. The MTLR model achieved a 5-fold cross-validated concordance index of 72.1 ± 3.3%. This model found that the TME score and chemotherapy had similar survival effects over the entire study time. The TME score provided the greatest survival benefit beyond a 5-year follow-up. Stage III and Stage IV disease contributed the greatest negative effect on survival. The MTLR model weights were significantly correlated with the Cox model coefficients (Pearson coefficient = 0.86, p < 0.0001). We illustrate how ISDs can accurately predict the survival time for each patient, which is especially relevant in cases of molecular subtype heterogeneity. This study provides evidence that the TME score is principally associated with long-term survival in gastric adenocarcinoma. Additional external validation and investigation into the clinical utility of this ISD model in gastric cancer is an area of future research.

Murine and Human Gastric Tissue Establishes Organoids after 48 Hours of Cold Ischemia Time during Shipment.

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank's Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose-response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation.

Yield, cell composition, and function of islets isolated from different ages of neonatal pigs.

The yield, cell composition, and function of islets isolated from various ages of neonatal pigs were characterized using in vitro and in vivo experimental models. Islets from 7- and 10-day-old pigs showed significantly better function both in vitro and in vivo compared to islets from 3- and 5-day-old pigs however, the islet yield from 10-day-old pigs were significantly less than those obtained from the other pigs. Since islets from 3-day-old pigs were used in our previous studies and islets from 7-day-old pigs reversed diabetes more efficiently than islets from other groups, we further evaluated the function of these islets post-transplantation. B6 rag-/- mouse recipients of various numbers of islets from 7-day-old pigs achieved normoglycemia faster and showed significantly improved response to glucose challenge compared to the recipients of the same numbers of islets from 3-day-old pigs. These results are in line with the findings that islets from 7-day-old pigs showed reduced voltage-dependent K+ (Kv) channel activity and their ability to recover from post-hypoxia/reoxygenation stress. Despite more resident immune cells and immunogenic characteristics detected in islets from 7-day-old pigs compared to islets from 3-day-old pigs, the combination of anti-LFA-1 and anti-CD154 monoclonal antibodies are equally effective at preventing the rejection of islets from both age groups of pigs. Collectively, these results suggest that islets from various ages of neonatal pigs vary in yield, cellular composition, and function. Such parameters may be considered when defining the optimal pancreas donor for islet xenotransplantation studies.

Cyanidin-3-O-Glucoside improves the viability of human islet cells treated with amylin or Aß1-42 in vitro.

Islet transplantation is being considered as an alternative treatment for type 1 diabetes. Despite recent progress, transplant recipients continue to experience progressive loss of insulin independence. Cyanidin-3-O-Glucoside (C3G) has shown to be protective against damage that may lead to post-transplant islet loss. In this study, human islets cultured with or without C3G were treated with human amylin, Aß1-42, H2O2, or rapamycin to mimic stresses encountered in the post-transplant environment. Samples of these islets were collected and assayed to determine C3G's effect on cell viability and function, reactive oxygen species (ROS), oxidative stress, amyloid formation, and the presence of inflammatory as well as autophagic markers. C3G treatment of human islets exposed to either amylin or Aß1-42 increased cell viability (p<0.01) and inhibited amyloid formation (p<0.01). A reduction in ROS and an increase in HO-1 gene expression as well as in vitro islet function were also observed in C3G-treated islets exposed to amylin or Aß1-42, although not significantly. Additionally, treatment with C3G resulted in a significant reduction in the protein expression of inflammatory markers IL-1ß and NLRP3 (p<0.01) as well as an increase in LC3 autophagic marker (p<0.05) in human islets treated with amylin, Aß1-42, rapamycin, or H2O2. Thus, C3G appears to have a multi-faceted protective effect on human islets in vitro, possibly through its anti-oxidant property and alteration of inflammatory as well as autophagic pathways.

Mouse pancreatic islets are resistant to indoleamine 2,3 dioxygenase-induced general control nonderepressible-2 kinase stress pathway and maintain normal viability and function.

Islet transplantation is a promising treatment for diabetes. However, it faces several challenges including requirement of systemic immunosuppression. Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. Local expression of IDO in bystander fibroblasts suppresses islet allogeneic immune response in vitro. The aim of the present study was to investigate the impact of IDO on viability and function of mouse islets embedded within IDO-expressing fibroblast-populated collagen scaffold. Mouse islets were embedded within collagen matrix populated with IDO adenovector-transduced or control fibroblasts. Proliferation, insulin content, glucose responsiveness, and activation of general control nonderepressible-2 kinase stress-responsive pathway were then measured in IDO-exposed islets. In vivo viabilities of composite islet grafts were also tested in a syngeneic diabetic animal model. No reduction in islet cells proliferation was detected in both IDO-expressing and control composites compared to the baseline rates. Islet functional studies showed normal insulin content and secretion in both preparations. In contrast to lymphocytes, general control nonderepressible-2 kinase pathway was not activated in islets cocultured with IDO-expressing fibroblasts. When transplanted to diabetic mice, syngeneic IDO-expressing composite islet grafts were functional up to 100 days tested. These findings collectively confirm normal viability and functionality of islets cocultured with IDO-expressing cells and indicate the feasibility of development of a functional nonrejectable islet graft.

Evaluation of the effect of T regulatory cell depletion and donor BCG vaccination on Mycobacterium tuberculosis H37Ra infection using an in vitro model of human PBMC infection.

This study evaluated the effect of T regulatory cells (Treg cells) and the impact of BCG vaccination history of donors using an in vitro model of Mycobacterium tuberculosis H37Ra infection of peripheral blood mononuclear cells (PBMCs). PBMCs from donors with or without prior BCG vaccination were depleted of Treg cells (PBMCs-Tregs) or not depleted with Treg cells (PBMCs + Tregs) were infected up to 8 days with Mtb H37Ra. Cell aggregates were smaller in PBMCs-Tregs compared to PBMCs + Tregs at day 8 post-infection. Mtb CFUs were higher in the PBMCs-Tregs compared to PBMCs + Tregs at days 3, 5 and 8. The levels of IL-17, IFN-γ (at days 3 and 5), and TNF-α and IL-6 (at day 3) were lower in PBMCs-Tregs compared to PBMCs + Tregs. In contrast, the levels of IL-10 and IL-4 cytokines were higher at day 3 in PBMCs-Tregs compared to PBMCs + Tregs. BCG vaccination status of donors had no impact on the mycobacterial culture, level of cytokines and immune cell populations. This study shows that depletion of Tregs in human PBMCs infected with Mtb H37Ra in vitro leads to a shift from a Th1 to a Th2 cytokine rich environment that supports the survival of Mtb in this model.

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.

Prolonged survival of microencapsulated neonatal porcine islet xenografts in immune-competent mice without antirejection therapy.

Several studies have demonstrated that in vitro culture of islets prolonged islet graft survival in immune-competent mice without administration of antirejection drugs. However, we recently showed that in vitro cultured microencapsulated neonatal porcine islets (NPI) were rejected in immune-competent mice not receiving antirejection therapy. The aim of this study was to determine whether culture of microencapsulated NPI in vivo could promote long-term survival of microencapsulated NPI in immune-competent mice without administration of antirejection drugs. Microencapsulated NPI that were cultured in vitro for 7 and 50 days or transplanted initially in immune-deficient C.B.-17 SCID-BEIGE mice for 100 days (in vivo cultured) were characterized and transplanted into streptozotocin-induced diabetic immune-competent BALB/c mice. Day 50 in vitro cultured and day 100 in vivo cultured microencapsulated NPI showed significantly higher insulin and DNA content, indicating maturation of NPI compared to day 7 in vitro cultured microencapsulated NPI. Interestingly, in vivo cultured microencapsulated NPI expressed lower levels of porcine antigens compared to day 7 and day 50 in vitro cultured microencapsulated NPI. Transplantation of day 7 in vitro cultured microencapsulated NPI did not reverse diabetes in immune-competent BALB/c mouse recipients. In contrast, transplantation of day 50 in vitro cultured and in vivo cultured microencapsulated NPI into diabetic immune-competent BALB/c mice resulted in the immediate reversal of hyperglycemia within 2 days posttransplantation. However, all recipients of day 50 in vitro cultured microencapsulated NPI eventually rejected their grafts by day 15 posttransplantation, while 6 of 10 BALB/c mouse recipients of in vivo cultured microencapsulated NPI maintained normoglycemia for 100 days posttransplantation. These results show that in vivo culture of NPI in immune-deficient mice results in the modulation of NPI, which allows for their long-term survival in immune-competent mice without antirejection therapy.

Monotherapy with anti-LFA-1 monoclonal antibody promotes long-term survival of rat islet xenografts.

Previously we demonstrated that anti-LFA-1 monoclonal (mAb) could promote long-term survival of discordant porcine islet xenografts in mice. The aim of this study, therefore, was to determine whether a shortterm administration of anti-LFA-1 mAb would promote long-term survival of concordant rat islet xenografts in mice, and whether combining short-term administration of anti-LFA-1 mAb therapy with an immunosuppressive drug, rapamycin, would facilitate islet xenograft survival. Streptozotocin-induced diabetic BALB/c mice were transplanted with 500 Wistar-Furth rat islets under the kidney capsule and were either left untreated or treated with short-term administration of rapamycin (0.2 mg/kg) alone, anti-LFA-1 mAb (0.2 mg/ dose) alone, or a combination of rapamycin and anti-LFA-1 mAb using the same doses. All untreated mice rejected their grafts by 24 days posttransplantation with a mean graft survival time of 18.8 +/- 2.5 days posttransplantation (n = 5). All mice treated with rapamycin alone had prolonged islet graft survival but eventually rejected their islet grafts by 81 days posttransplantation. In contrast, the majority of the mice (27/ 28) treated with anti-LFA-1 mAb alone maintained long-term normoglycemia (>100 days). Rapamycin in combination with anti-LFA-1 mAb proved equally effective with 29 of 30 mice maintaining normoglycemia for more than 100 days posttransplantation. Low levels of mouse anti-rat antibodies, as well as a decrease in the degree of mononuclear cell infiltration of the islet graft, closely correlated with long-term islet xenograft survival. These results demonstrate that monotherapy with anti-LFA-1 mAb is highly effective in promoting long-term survival of rat islet xenografts and that combination of anti-LFA-1 mAb with rapamycin does not facilitate nor abrogate the induction of long-term xenograft survival by anti-LFA-1 mAb therapy in BALB/c mice. Our study indicates that immunomodulation through mAb therapy could form a significant component of future antirejection therapies in clinical islet xenotransplantation.

Comparison of successful and unsuccessful islet/Sertoli cell cotransplant grafts in streptozotocin-induced diabetic mice.

Sertoli cells (SC) protect islet allografts from immune destruction in diabetic rodents. In this study, we examined the difference between successful and rejected islet/SC cografts in order to further improve this procedure for optimal extension of islet allograft survival. We cotransplanted 500 BALB/c islets with 1-8 million BALB/c SC under the kidney capsule of diabetic BALB/c, C3H-HeJ, and C57BL/6 mice. Cotransplantation of islets with up to 8 million SC was not detrimental to long-term islet graft function in syngeneic mice. However, large numbers of SC were detrimental to islet graft survival in allogeneic mice with the optimal dose for cotransplantation of 4 or 1 million SC in C3H-HeJ or C57BL/6 mice, respectively. Examination of successful grafts, from euglycemic recipients, revealed the presence of SC arranged in tubule structures with islets surrounding these tubules. Cellular infiltrate in successful grafts revealed CD4 T cells and macrophages along the periphery and within the grafts, and very few CD8 T cells. Conversely, examination of unsuccessful grafts, harvested from hyperglycemic recipients at the time of rejection, revealed the presence of SC arranged randomly with islets adjacent to the Sertoli cells, when present, and massive CD4 and CD8 T cell as well as macrophage cell infiltration. Prolongation of islet allograft survival appeared to be a function of SC transplant mass and recipient genetic background. A consequence of long-term graft acceptance is the formation of SC tubule structures, which may be an additional requirement for optimal protection of islet allografts.