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.

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.

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.

Increased CD4/CD8 Lymphocyte ratio predicts favourable neoadjuvant treatment response in gastric cancer: A prospective pilot study.

BACKGROUND: Despite optimal neoadjuvant chemotherapy only 40% of gastric cancer tumours achieve complete or partial treatment response. In the absence of treatment response, neoadjuvant chemotherapy in gastric cancer contributes to adverse events without additional survival benefit compared to adjuvant treatment or surgery alone. Additional strategies and methods are required to optimize the allocation of existing treatment regimens such as FLOT chemotherapy (5-Fluorouracil, Leucovorin, Oxaliplatin and Docetaxel). Predictive biomarkers detected using immunohistochemistry (IHC) methods may provide useful data regarding treatment response. AIM: To investigate the utility of CD4, CD8, Galectin-3 and E-cadherin in predicting neoadjuvant FLOT chemotherapy tumour response in gastric adenocarcinoma. METHODS: Forty-three adult patients with gastric adenocarcinoma, of which 18 underwent neoadjuvant chemotherapy, were included in a prospective clinical cohort. Endoscopic biopsies were obtained from gastric cancer and normal adjacent gastric mucosa. Differences in expression of Galectin-3, E-cadherin, CD4+ and CD8+ molecules between tumours with and without treatment response to neoadjuvant chemotherapy were assessed with IHC. Treatment response was graded by clinical pathologists using the Tumour Regression Score according to the College of American Pathologists criteria. Treatment response was defined as complete or near complete tumour response, whereas partial or poor/no response was defined as incomplete. Digital IHC images were annotated and quantitatively assessed using QuPath 0.3.1. Biomarker expression between responsive and incomplete response tumours was assessed using a two-sided Wilcoxon test. Biomarker expression was also compared between normal and cancer tissue and between 15 paired tumour samples before and after chemotherapy. We performed a preliminary multivariate analysis and power analysis to guide future study. Statistical analyses were completed using R 4.1.2. RESULTS: The ratio between CD4+ and CD8+ lymphocytes was significantly greater in treatment responsive tumours (Wilcoxon, P = 0.03). In univariate models, CD4+/CD8+ ratio was the only biomarker that significantly predicted favourable treatment response (Accuracy 86%, P < 0.001). Using a glmnet multivariate model, high CD4+/CD8+ ratio and low Galectin-3 expression were the most influential variables in predicting a favourable treatment response. Analyses of paired samples found that FLOT chemotherapy also results in increased expression of CD4+ and CD8+ tumour infiltrating lymphocytes (Paired Wilcoxon, P = 0.002 and P = 0.008, respectively). Our power analysis suggests future study requires at least 35 patients in each treatment response group for CD8 and Galectin-3 molecules, whereas 80 patients in each treatment response group are required to assess CD4 and E-cadherin biomarkers. CONCLUSION: We demonstrate that an elevated CD4+/CD8+ Ratio is a promising IHC-based biomarker to predict favourable treatment response to FLOT neoadjuvant chemotherapy in locally advanced gastric cancer.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Characterization of immune responses of human PBMCs infected with Mycobacterium tuberculosis H37Ra: Impact of donor declared BCG vaccination history on immune responses and M. tuberculosis growth.

This study characterized the immune responses in early Mycobacterium tuberculosis (Mtb) H37Ra infection of human peripheral blood mononuclear cell (PBMC)-collagen matrix culture and the impact of Bacille Calmette-Guérin (BCG) vaccination history of donor PBMCs on the immune responses to Mtb infection. Aggregates of PBMCs were initially observed on day 3 and the size of aggregates continued to increase on day 8 post-infection, where macrophages and T cell subsets were identified to be present. Similarly, mycobacterial load progressively increased in infected PBMCs during the 8 days of culture but were significantly lower in infected PBMCs from BCG vaccinated (BCG+) donors compared to unvaccinated (BCG-) donors. The levels of INF-γ, TNF-α, IL-4, IL-6, IL-10 and IL-17 in the supernatants of Mtb-infected PBMCs peaked at day 3 and decreased on days 5 and 8. The levels of these cytokines except IL-10 were significantly lower in Mtb-infected PBMCs from BCG+ donors compared to infected PBMCs from BCG- donors. The percentages of activated naïve Th cells, activated effector memory Th cells and activated central memory Tc cells were significantly higher in Mtb-infected PBMCs compared to uninfected PBMCs at day 8 post-infection. Further, the proportion of activated central memory Tc cells was significantly higher in infected PBMCs from BCG+ donors compared to the BCG- donors. This study highlights the possibility that BCG vaccination may confound results that utilize human PBMCs to study Mtb infection.

Protective effect of cyanidin-3-O-glucoside on neonatal porcine islets.

Oxidative stress is a major cause of islet injury and dysfunction during isolation and transplantation procedures. Cyanidin-3-O-glucoside (C3G), which is present in various fruits and vegetables especially in Chinese bayberry, shows a potent antioxidant property. In this study, we determined whether C3G could protect neonatal porcine islets (NPI) from reactive oxygen species (H2O2)-induced injury in vitro and promote the function of NPI in diabetic mice. We found that C3G had no deleterious effect on NPI and that C3G protected NPI from damage induced by H2O2 Significantly higher hemeoxygenase-1 (HO1) gene expression was detected in C3G-treated NPI compared to untreated islets before and after transplantation (P < 0.05). Western blot analysis showed a significant increase in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K/Akt) proteins in C3G-treated NPI compared to untreated islets. C3G induced the nuclear translocation of nuclear erythroid 2-related factor 2 (NRF2) and the significant elevation of HO1 protein. Recipients of C3G-treated NPI with or without C3G-supplemented drinking water achieved normoglycemia earlier compared to recipients of untreated islets. Mice that received C3G-treated islets with or without C3G-supplemented water displayed significantly lower blood glucose levels at 5-10 weeks post-transplantation compared to mice that received untreated islets. Mice that received C3G-treated NPI and C3G-supplemented drinking water had significantly (P < 0.05) lower blood glucose levels at 7 and 8 weeks post-transplantation compared to mice that received C3G-treated islets. These findings suggest that C3G has a beneficial effect on NPI through the activation of ERK1/2- and PI3K/AKT-induced NRF2-mediated HO1 signaling pathway.

Low energy X-ray (grenz ray) treatment of purified islets prior to allotransplant markedly decreases passenger leukocyte populations.

Grenz rays, or minimally penetrating X-rays, are known to be an effective treatment of certain recalcitrant immune-mediated skin diseases, but their use in modulating allograft rejection has not been tested. We examined the capacity of grenz ray treatment to minimize islet immunogenicity and extend allograft survival in a mouse model. In a preliminary experiment, 1 of 3 immunologically intact animals demonstrated long-term acceptance of their grenz ray treated islet allograft. Further experiments revealed that 28.6% (2 of 7) grenz ray treated islet allografts survived >60 d. A low dose of 20Gy, was important; a 4-fold increase in radiation resulted in rapid graft failure, and transplanting a higher islet mass did not alter this outcome. To determine whether increased islet allograft survival after grenz treatment would be masked by immunosuppression, we treated the recipients with CTLA-4 Ig, and found an additive effect, whereby 17.5% more animals accepted the graft long-term versus those with CTLA-4 Ig alone. Cell viability assays verified that islet integrity was maintained after treatment with 20Gy. As well, through splenocyte infiltration analysis, donor CD4+ T cell populations 24-hours after transplant were decreased by more than16-fold in recipients receiving irradiated islets compared with control. Donor CD8+ T cell populations, although less prevalent, decreased in all treatment groups compared with control. Our results suggest that brief treatment of isolated islets with low energy grenz rays before allotransplantation can significantly reduce passenger leukocytes and promote graft survival, possibly by inducing donor dendritic cells to differentiate toward a tolerogenic phenotype.

Understanding the pathophysiology of the human TB lung granuloma using in vitro granuloma models.

Tuberculosis remains a major human health threat that infects one in three individuals worldwide. Infection with Mycobacterium tuberculosis is a standoff between host and bacteria in the formation of a granuloma. This review will introduce a variety of bacterial and host factors that impact individual granuloma fates. The authors describe advances in the development of in vitro granuloma models, current evidence surrounding infection and granuloma development, and the applicability of existing in vitro models in the study of human disease. In vitro models of infection help improve our understanding of pathophysiology and allow for the discovery of other potential models of study.

Trans-11 vaccenic acid improves insulin secretion in models of type 2 diabetes in vivo and in vitro.

SCOPE: Trans-11 vaccenic acid (VA) is a fatty acid produced by ruminants entering the human food supply through meat and dairy products, which appears not to have the health risks associated with industrially produced trans-fatty acids. In this study, we investigated the effect of VA on insulin secretion in vivo in rats and in vitro in human and rat islets after diabetogenic insult. METHODS AND RESULTS: Hyperglycemic clamp showed that VA dietary supplementation for 8 weeks significantly increased glucose turnover in rats with type 2 diabetes (T2D), accompanied by an elevated plasma C-peptide concentration, indicating improved insulin secretion. The ß-cell area and proliferation rate were higher in T2D+VA than T2D group. Isolated islets from T2D+VA rats had higher glucose-stimulated insulin secretion (GSIS) than T2D group. In vitro, VA treatment for 24 and 48 h significantly enhanced GSIS in rat and human islets after diabetogenic challenges. The mRNA expression of G-protein-coupled receptor 40 (GPR40) and regenerating islet-derived 1α (REG-1α) were consistently increased by VA in both rat and human islets. CONCLUSION: These results indicate that VA may improve insulin secretion and growth of islets in T2D, at least partly by altering GPR40 and REG-1α mRNA expression.

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.