Frequencies of CD8 and DN MAIT Cells Among Children Diagnosed With Type 1 Diabetes Are Similar to Age-Matched Controls.

Mucosal-associated invariant T (MAIT) cells have been implicated in various forms of autoimmunity, including type 1 diabetes (T1D). Here, we tested the hypothesis that CD8 and double negative (DN) MAIT cell frequencies were altered among diagnosed T1D subjects compared to controls. To do this, we analyzed cryopreserved peripheral blood mononuclear cells (PBMCs) from age-matched T1D and control children using flow cytometry. We observed that CD8 and DN MAIT cell frequencies were similarly abundant between the two groups. We tested for associations between MAIT cell frequency and T1D-associated parameters, which could reveal a pathogenic role for MAIT cells in the absence of changes in frequency. We found no significant associations between CD8 and DN MAIT cell frequency and levels of islet cell autoantibodies (ICA), glutamate decarboxylase 65 (GAD65) autoantibodies, zinc transporter 8 (ZNT8) autoantibodies, and insulinoma antigen 2 (IA-2) autoantibodies. Furthermore, CD8 and DN MAIT cell frequencies were not significantly associated with time since diagnosis, c-peptide levels, HbA1c, and BMI. As we have examined this cohort for multiple soluble factors previously, we tested for associations between relevant factors and MAIT cell frequency. These could help to explain the broad range of MAIT frequencies we observed and/or indicate disease-associated processes. Although we found nothing disease-specific, we observed that levels of IL-7, IL-18, 25 (OH) vitamin D, and the ratio of vitamin D binding protein to 25 (OH) vitamin D were all associated with MAIT cell frequency. Finally, previous cytomegalovirus infection was associated with reduced CD8 and DN MAIT cells. From this evaluation, we found no connections between CD8 and DN MAIT cells and children with T1D. However, we did observe several intrinsic and extrinsic factors that could influence peripheral MAIT cell abundance among all children. These factors may be worth consideration in future experimental design.

Confirmation and Identification of Biomarkers Implicating Environmental Triggers in the Pathogenesis of Type 1 Diabetes.

Multiple environmental triggers have been proposed to explain the increased incidence of type 1 diabetes (T1D). These include viral infections, microbiome disturbances, metabolic disorders, and vitamin D deficiency. Here, we used ELISA to examine blood plasma from juvenile T1D subjects and age-matched controls for the abundance of several circulating factors relevant to these hypotheses. We screened plasma for sCD14, mannose binding lectin (MBL), lipopolysaccharide binding protein (LBP), c-reactive protein (CRP), fatty acid binding protein 2 (FABP2), human growth hormone, leptin, total adiponectin, high molecular weight (HMW) adiponectin, total IgG, total IgA, total IgM, endotoxin core antibodies (EndoCAbs), 25(OH) vitamin D, vitamin D binding protein, IL-7, IL-10, IFN-γ, TNF-α, IL-17A, IL-18, and IL-18BPa. Subjects also were tested for prevalence of antibodies targeting adenovirus, parainfluenza 1/2/3, Coxsackievirus, cytomegalovirus, Epstein-Barr virus viral capsid antigen (EBV VCA), herpes simplex virus 1, and Saccharomyces cerevisiae. Finally, all subjects were screened for presence and abundance of autoantibodies targeting islet cell cytoplasmic proteins (ICA), glutamate decarboxylase 2 (GAD65), zinc transporter 8 (ZNT8), insulinoma antigen 2 (IA-2), tissue transglutaminase, and thyroid peroxidase, while ß cell function was gauged by measuring c-peptide levels. We observed few differences between control and T1D subjects. Of these, we found elevated sCD14, IL-18BPa, and FABP2, and reduced total IgM. Female T1D subjects were notably elevated in CRP levels compared to control, while males were similar. T1D subjects also had significantly lower prevalence of EBV VCA antibodies compared to control. Lastly, we observed that c-peptide levels were significantly correlated with leptin levels among controls, but this relationship was not significant among T1D subjects. Alternatively, adiponectin levels were significantly correlated with c-peptide levels among T1D subjects, while controls showed no relationship between these two factors. Among T1D subjects, the highest c-peptide levels were associated with the lowest adiponectin levels, an indication of insulin resistance. In total, from our examination we found limited data that strongly support any of the hypotheses investigated. Rather, we observed an indication of unexplained monocyte/macrophage activation in T1D subjects judging from elevated levels of sCD14 and IL-18BPa. These observations were partnered with unique associations between adipokines and c-peptide levels among T1D subjects.

Heightened Levels of Antimicrobial Response Factors in Patients With Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease leading to considerable disability over time. The disease can be characterized by the presence of multiple autoantibodies in the serum and synovial fluid. Microbial dysbiosis is proposed to play a role in the pathogenesis of RA. Increased systemic bacterial exposure leads to elevated levels of antimicrobial response factors (ARFs) in the circulation. In the present study, we tested whether RA patients have increased levels of ARFs by analyzing the levels of multiple ARFs in serum from RA patients and healthy age and sex-matched controls. The levels of soluble CD14 (sCD14), lysozyme, and CXCL16 were significantly elevated in RA patients compared to healthy controls. Lipopolysaccharide binding protein (LBP) levels remained unchanged in RA patients compared to healthy controls. A positive correlation of LBP with rheumatoid factor (RF) was also found in RA subjects. Interestingly, the levels of anti-endotoxin core antibodies (EndoCAb) IgM, total IgM, EndoCAb IgA, and total IgA were significantly elevated in RA patients compared to healthy controls. No significant changes in the levels of EndoCAb IgG and total IgG were observed in RA patients compared to healthy controls. Furthermore, lysozyme and CXCL16 levels were positively correlated with disease severity among RA subjects. Increases in the levels of several ARFs and their correlations with clinical indices suggest systemic microbial exposure in the RA cohort. Modulation of microbial exposure may play an important role in disease pathogenesis in individuals with RA.

Human Islet Response to Selected Type 1 Diabetes-Associated Bacteria: A Transcriptome-Based Study.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from destruction of pancreatic ß-cells. T1D subjects were recently shown to harbor distinct intestinal microbiome profiles. Based on these findings, the role of gut bacteria in T1D is being intensively investigated. The mechanism connecting intestinal microbial homeostasis with the development of T1D is unknown. Specific gut bacteria such as Bacteroides dorei (BD) and Ruminococcus gnavus (RG) show markedly increased abundance prior to the development of autoimmunity. One hypothesis is that these bacteria might traverse the damaged gut barrier, and their constituents elicit a response from human islets that causes metabolic abnormalities and inflammation. We have tested this hypothesis by exposing human islets to BD and RG in vitro, after which RNA-Seq analysis was performed. The bacteria altered expression of many islet genes. The commonly upregulated genes by these bacteria were cytokines, chemokines and enzymes, suggesting a significant effect of gut bacteria on islet antimicrobial and biosynthetic pathways. Additionally, each bacteria displayed a unique set of differentially expressed genes (DEGs). Ingenuity pathway analysis of DEGs revealed that top activated pathways and diseases included TREM1 signaling and inflammatory response, illustrating the ability of bacteria to induce islet inflammation. The increased levels of selected factors were confirmed using immunoblotting and ELISA methods. Our data demonstrate that islets produce a complex anti-bacterial response. The response includes both symbiotic and pathogenic aspects. Both oxidative damage and leukocyte recruitment factors were prominent, which could induce beta cell damage and subsequent autoimmunity.

Coordinated Induction of Antimicrobial Response Factors in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by dysregulated autoantibody production and complement activation leading to multi-organ damage. The disease is associated with increased intestinal permeability. In this study, we tested the hypothesis that SLE subjects have increased systemic exposure to bacteria. Since bacteria induce the expression of antimicrobial response factors (ARFs), we measured the levels of a series of clinically relevant ARFs in the plasma of SLE subjects. We found that levels of sCD14, lysozyme, and CXCL16 were significantly elevated in SLE subjects. A strong positive correlation was also observed between sCD14 and SELENA-SLEDAI score. Interestingly, the ratio of EndoCAb IgM:total IgM was significantly decreased in SLE and this ratio was negatively correlated with sCD14 levels. Although, there were no significant differences in the levels of lipopolysaccharide binding protein (LBP) and fatty acid binding protein 2 (FABP2), we observed significant positive correlations between lysozyme levels and sCD14, LBP, and FABP2. Moreover, galectin-3 levels also positively correlate with lysozyme, sCD14, and LBP. Since our SLE cohort comprised 43.33% males, we were able to identify gender-specific changes in the levels of ARFs. Overall, these changes in the levels and relationships between ARFs link microbial exposure and SLE. Approaches to reduce microbial exposure or to improve barrier function may provide therapeutic strategies for SLE patients.

Anti-human Interleukin(IL)-4 Clone 8D4-8 Cross-Reacts With Myosin-9 Associated With Apoptotic Cells and Should Not Be Used for Flow Cytometry Applications Querying IL-4 Expression.

Interleukin(IL)-4 is produced by T cells and other leukocytes and is a critical mediator of monocyte and B cell responses. During routine flow cytometry panel validation for the investigation of intracellular cytokines, we observed unique IL-4 expression patterns associated with the widely available monoclonal antibody 8D4-8. Namely, IL-4 (8D4-8) expression was observed in the absence of cellular activation and enhanced following staurosporine exposure. Mass spectrometry analysis of immunoprecipitates from peripheral blood lymphocytes (PBL) revealed that 8D4-8 cross-reacts with the ubiquitous cytoskeletal protein myosin-9. We confirmed these results by western blotting immunoprecipitates, using immunofluorescence among staurosporine-treated Caco-2 cells, and by surface-labeling PBL for 8D4-8 and myosin-9 and analyzing by flow cytometry. Although previously reported from several independent groups, we found no evidence to support the hypothesis that IL-4 is produced by apoptotic cells. Rather, this appears to have been myosin-9. Our data indicate clone 8D4-8 should not be used in the flow cytometric study of IL-4. Furthermore, our work calls for a reevaluation of previous flow cytometric studies that have used this clone for IL-4 analysis and highlights the importance of validation in antibody-based assays.

Current understanding of the role of gut dysbiosis in type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disorder that results from destruction of the insulin-producing pancreatic ß-cells. The disease mainly affects juveniles. Changes in the composition of the gut microbiota (dysbiosis) and changes in the properties of the gut barrier have been documented in T1D subjects. Because these factors affect immune system functions, they are likely to play a role in disease pathogenesis. However, their exact role is currently not fully understood and is under intensive investigation. In this article we discuss recent advancements depicting the role of intestinal dysbiosis on immunity and autoimmunity in T1D. We also discuss therapies aimed at maintaining a healthy gut barrier as prevention strategies for T1D.

Abnormal T Cell Frequencies, Including Cytomegalovirus-Associated Expansions, Distinguish Seroconverted Subjects at Risk for Type 1 Diabetes.

We analyzed T cell subsets from cryopreserved PBMC obtained from the TrialNet Pathway to Prevention archives. We compared subjects who had previously seroconverted for one or more autoantibodies with non-seroconverted, autoantibody negative individuals. We observed a reduced frequency of MAIT cells among seroconverted subjects. Seroconverted subjects also possessed decreased frequencies of CCR4-expressing CD4 T cells, including a regulatory-like subset. Interestingly, we found an elevation of CD57+, CD28-, CD127-, CD27- CD8 T cells (SLEC) among seroconverted subjects that was most pronounced among those that progressed to disease. The frequency of these SLEC was strongly correlated with CMV IgG abundance among seroconverted subjects, associated with IA-2 levels, and most elevated among CMV+ seroconverted subjects who progressed to disease. Combined, our data indicate discrete, yet profound T cell alterations are associated with islet autoimmunity among at-risk subjects.

Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL-18.

The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α- NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor.

Exploiting Expression of Hippo Effector, Yap, for Expansion of Functional Islet Mass.

Loss of pancreas ß-cell function is the precipitating factor in all forms of diabetes. Cell replacement therapies, such as islet transplantation, remain the best hope for a cure; however, widespread implementation of this method is hampered by availability of donor tissue. Thus, strategies that expand functional ß-cell mass are crucial for widespread usage in diabetes cell replacement therapy. Here, we investigate the regulation of the Hippo-target protein, Yes-associated protein (Yap), during development of the endocrine pancreas and its function after reactivation in human cadaveric islets. Our results demonstrate that Yap expression is extinguished at the mRNA level after neurogenin-3-dependent specification of the pancreas endocrine lineage, correlating with proliferation decreases in these cells. Interestingly, when a constitutively active form of Yap was expressed in human cadaver islets robust increases in proliferation were noted within insulin-producing ß-cells. Importantly, proliferation in these cells occurs without negatively affecting ß-cell differentiation or functional status. Finally, we show that the proproliferative mammalian target of rapamycin pathway is activated after Yap expression, providing at least one explanation for the observed increases in ß-cell proliferation. Together, these results provide a foundation for manipulating Yap activity as a novel approach to expand functional islet mass for diabetes regenerative therapy.

WS6 induces both alpha and beta cell proliferation without affecting differentiation or viability.

Agents that stimulate human pancreatic beta cell proliferation are needed to improve diabetes mellitus treatment. Recently, a small molecule, WS6, was observed to stimulate human beta cell proliferation. However, little is known about its other effects on human islets. To better understand the role of WS6 as a possible beta cell regenerative therapy, we carried out in-depth phenotypic analysis of WS6-treated human islets, exploring its effects on non-beta cell proliferation, beta cell differentiation, and islet cell viability. WS6 not only stimulated beta cell proliferation in cultured human islets (in agreement with previous reports), but also human alpha cell proliferation, indicating that WS6 is not a beta cell-specific mitogen. WS6 did not change the proportion of insulin-positive beta cells or the expression of beta cell-specific transcription factors, suggesting that WS6 does not alter beta cell differentiation, and WS6 had no effect on human islet cell apoptosis or viability. In conclusion, WS6 stimulates proliferation of both human beta and alpha cells while maintaining cellular viability and the beta cell differentiated phenotype. These findings expand the literature on WS6 and support the suggestion that WS6 may help increase human islet mass needed for successful treatment of diabetes.

Altered CD161 bright CD8+ mucosal associated invariant T (MAIT)-like cell dynamics and increased differentiation states among juvenile type 1 diabetics.

Type 1A diabetes (T1D) is believed to be caused by immune-mediated destruction of ß-cells, but the immunological basis for T1D remains controversial. Microbial diversity promotes the maturation and activation of certain immune subsets, including CD161 bright CD8+ mucosal associated invariant T (MAIT) cells, and alterations in gut mucosal responses have been reported in type 1 diabetics (T1Ds). We analyzed T cell populations in peripheral blood leukocytes from juvenile T1Ds and healthy controls. We found that proportion and absolute number of MAIT cells were similar between T1Ds and controls. Furthermore, while MAIT cell proportions increased with age among healthy controls, this trend was not observed among long-standing T1Ds. Additionally, the CD27- MAIT cell subset is significantly increased in T1Ds and positively correlated with HbA1c levels. However, after T1Ds are stratified by age, the younger group has significantly increased proportions of CD27- MAIT cells compared to age-matched controls, and this proportional increase appears to be independent of HbA1c levels. Finally, we analyzed function of the CD27- MAIT cells and observed that IL-17A production is increased in CD27- compared to CD27+ MAIT cells. Overall, our data reveal disparate MAIT cell dynamics between T1Ds and controls, as well as signs of increased MAIT cell activation in T1Ds. These changes may be linked to hyperglycemia and increased mucosal challenge among T1Ds.

Increased expression of IL-18 in the serum and islets of type 1 diabetics.

Type 1 diabetes (T1D) is a chronic disease characterized by autoimmune-mediated destruction of pancreatic insulin-producing beta cells. Interleukin (IL)-18 is a pro-inflammatory cytokine implicated in the pathogenesis of a number of inflammatory diseases. Here, we analyzed IL-18 levels in the plasma of juveniles with T1D. Compared to control subjects, IL-18 levels were significantly elevated in patients with T1D. On the other hand, levels of IL-18 binding protein (IL-18BP) and IL-37, two negative regulators of IL-18 function, remained unchanged when comparing T1D to control samples. Notably, however, although IL-18BP levels were not elevated, IL-18 and IL-18BP were found to be positively correlated in type 1 diabetics. Even so, free, unbound IL-18 remained significantly increased in diabetic patients. Additionally, correlation studies also revealed that IL-18 and IL-18BP are positively correlated with HbA1c levels in T1D patients, suggesting a potential link between IL-18 and metabolic control in these patients. Finally, we observed a significant increase in IL-18 protein expression within human pancreatic islet specimens collected from type 1 diabetics. These results further expand our knowledge of the role of IL-18 in T1D, may give insight into common pathogenic mechanisms associated with metabolic control in both T1D and T2D, and suggest that targeting this cytokine may improve therapeutic outcomes for T1D patients.

Inhibition of autophagic turnover in ß-cells by fatty acids and glucose leads to apoptotic cell death.

Autophagy, a cellular recycling process responsible for turnover of cytoplasmic contents, is critical for maintenance of health. Defects in this process have been linked to diabetes. Diabetes-associated glucotoxicity/lipotoxicity contribute to impaired ß-cell function and have been implicated as contributing factors to this disease. We tested the hypothesis that these two conditions affect ß-cell function by modulating autophagy. We report that exposure of ß-cell lines and human pancreatic islets to high levels of glucose and lipids blocks autophagic flux and leads to apoptotic cell death. EM analysis showed accumulation of autophagy intermediates (autophagosomes), with abundant engulfed cargo in palmitic acid (PA)- or glucose-treated cells, indicating suppressed autophagic turnover. EM studies also showed accumulation of damaged mitochondria, endoplasmic reticulum distention, and vacuolar changes in PA-treated cells. Pulse-chase experiments indicated decreased protein turnover in ß-cells treated with PA/glucose. Expression of mTORC1, an inhibitor of autophagy, was elevated in ß-cells treated with PA/glucose. mTORC1 inhibition, by treatment with rapamycin, reversed changes in autophagic flux, and cell death induced by glucose/PA. Our results indicate that nutrient toxicity-induced cell death occurs via impaired autophagy and is mediated by activation of mTORC1 in ß-cells, contributing to ß-cell failure in the presence of metabolic stress.

CD28⁻ CD8⁺ T cells are significantly reduced and correlate with disease duration in juveniles with type 1 diabetes.

Type 1 diabetes (T1D) is a chronic disease caused by autoimmune destruction of insulin-producing pancreatic ß-cells. T1D is typically diagnosed in children, but information regarding immune cell subsets in juveniles with T1D is scarce. Therefore, we studied various lymphocytic populations found in the peripheral blood of juveniles with T1D compared to age-matched controls (ages 2-17). One population of interest is the CD28(-) CD8(+) T cell subset, which are late-differentiated cells also described as suppressors. These cells are altered in a number of disease states and have been shown to be reduced in adults with T1D. We found that the proportion of CD28(-) cells within the CD8(+) T cell population is significantly reduced in juvenile type 1 diabetics. Furthermore, this reduction is not correlated with age in T1D juveniles, although a significant negative correlation between proportion CD28(-) CD8(+) T cells and age was observed in the healthy controls. Finally, correlation analysis revealed a significant and negative correlation between the proportion of CD28(-) CD8(+) T cells and T1D disease duration. These findings show that the CD28(-) CD8(+) T cell population is perturbed following onset of disease and may prove to be a valuable marker for monitoring the progression of T1D.

TLR signaling controls lethal encephalitis in WNV-infected brain.

Toll-like receptors (TLRs) are known to be activated in Central Nervous System (CNS) viral infections and are recognized to be a critical component in innate immunity. Several reports state a role for particular TLRs in various CNS viral infections. However, excessive TLR activation was previously reported by us in correlation with a pathogenic, rather than a protective, outcome, in a model of SIV encephalitis. Here we aimed at understanding the impact of TLR-mediated pathways by evaluating the early course of pathogenesis in the total absence of TLR signaling during CNS viral infections. We utilized a mouse model of sublethal West Nile virus (WNV) infection. WNV is an emerging neurotropic flavivirus, and a significant global cause of viral encephalitis. The virus was peripherally injected into animals that simultaneously lacked two key adapter molecules of TLR signaling, MyD88 and TRIF. On day 2 pi (post infection), MyD88/Trif-/- mice showed an increased susceptibility to WNV infection, and revealed an impairment in innate immune cytokines, when compared to wild type mice (WT). By day 6 pi, there was an increase in viral burden and robust expression of inflammatory cytokines as well as higher cell infiltration into the CNS in MyD88/Trif-/-, when compared to infected WT. A drastic increase in microglia activation, astrogliosis, and inflammatory trafficking were also observed on day 6 pi in MyD88/Trif-/-. Our observations show a protective role for TLR signaling pathways in preventing lethal encephalitis at early stages of WNV infection.

Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers.

Deregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents.

TGF-ß superfamily member Nodal stimulates human ß-cell proliferation while maintaining cellular viability.

In an effort to expand human islets and enhance allogeneic islet transplant for the treatment of type 1 diabetes, identifying signaling pathways that stimulate human ß-cell proliferation is paramount. TGF-ß superfamily members, in particular activin-A, are likely involved in islet development and may contribute to ß-cell proliferation. Nodal, another TGF-ß member, is present in both embryonic and adult rodent islets. Nodal, along with its coreceptor, Cripto, are pro-proliferative factors in certain cell types. Although Nodal stimulates apoptosis of rat insulinoma cells (INS-1), Nodal and Cripto signaling have not been studied in the context of human islets. The current study investigated the effects of Nodal and Cripto on human ß-cell proliferation, differentiation, and viability. In the human pancreas and isolated human islets, we observed Nodal mRNA and protein expression, with protein expression observed in ß and α-cells. Cripto expression was absent from human islets. Furthermore, in cultured human islets, exogenous Nodal stimulated modest ß-cell proliferation and inhibited α-cell proliferation with no effect on cellular viability, apoptosis, or differentiation. Nodal stimulated the phosphorylation of mothers against decapentaplegic (SMAD)-2, with no effect on AKT or MAPK signaling, suggesting phosphorylated SMAD signaling was involved in ß-cell proliferation. Cripto had no effect on human islet cell proliferation, differentiation, or viability. In conclusion, Nodal stimulates human ß-cell proliferation while maintaining cellular viability. Nodal signaling warrants further exploration to better understand and enhance human ß-cell proliferative capacity.

Methamphetamine administration targets multiple immune subsets and induces phenotypic alterations suggestive of immunosuppression.

Methamphetamine (Meth) is a widely abused stimulant and its users are at increased risk for multiple infectious diseases. To determine the impact of meth on the immune system, we utilized a murine model that simulates the process of meth consumption in a typical addict. Our phenotypic analysis of leukocytes from this dose escalation model revealed that meth affected key immune subsets. Meth administration led to a decrease in abundance of natural killer (NK) cells and the remaining NK cells possessed a phenotype suggesting reduced responsiveness. Dendritic cells (DCs) and Gr-1(high) monocytes/macrophages were also decreased in abundance while Gr-1(low) monocytes/macrophages appear to show signs of perturbation. CD4 and CD8 T cell subsets were affected by methamphetamine, both showing a reduction in antigen-experienced subsets. CD4 T cells also exhibited signs of activation, with increased expression of CD150 on CD226-expressing cells and an expansion of KLRG1(+), FoxP3(-) cells. These results exhibit that meth has the ability to disrupt immune homeostasis and impact key subsets of leukocytes which may leave users more vulnerable to pathogens.

Hippo signaling regulates pancreas development through inactivation of Yap.

The mammalian pancreas is required for normal metabolism, with defects in this vital organ commonly observed in cancer and diabetes. Development must therefore be tightly controlled in order to produce a pancreas of correct size, cell type composition, and physiologic function. Through negative regulation of Yap-dependent proliferation, the Hippo kinase cascade is a critical regulator of organ growth. To investigate the role of Hippo signaling in pancreas biology, we deleted Hippo pathway components in the developing mouse pancreas. Unexpectedly, the pancreas from Hippo-deficient offspring was reduced in size, with defects evident throughout the organ. Increases in the dephosphorylated nuclear form of Yap are apparent throughout the exocrine compartment and correlate with increases in levels of cell proliferation. However, the mutant exocrine tissue displays extensive disorganization leading to pancreatitis-like autodigestion. Interestingly, our results suggest that Hippo signaling does not directly regulate the pancreas endocrine compartment as Yap expression is lost following endocrine specification through a Hippo-independent mechanism. Altogether, our results demonstrate that Hippo signaling plays a crucial role in pancreas development and provide novel routes to a better understanding of pathological conditions that affect this organ.