FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic ß-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in ß-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

Compound A attenuates proinflammatory cytokine-induced endoplasmic reticulum stress in beta cells and displays beneficial therapeutic effects in a mouse model of autoimmune diabetes.

Type 1 diabetes (T1D) is characterized by an immune-mediated progressive destruction of the insulin-producing ß-cells. Proinflammatory cytokines trigger endoplasmic reticulum (ER) stress and subsequent insulin secretory deficiency in cultured ß-cells, mimicking the islet microenvironment in T1D. ß-cells undergo physiologic ER stress due to the high rate of insulin production and secretion under stimulated conditions. Severe and uncompensated ER stress in ß-cells is induced by several pathological mechanisms before onset and during T1D. We previously described that the small drug Compound A (CpdA), a selective glucocorticoid receptor (GR/NR3C1, nuclear receptor subfamily 3, group C, member 1) ligand with demonstrated inflammation-suppressive activity in vivo, is an effective modulator of effector T and dendritic cells and of macrophages, yet, in a GR-independent manner. Here, we focus on CpdA's therapeutic potential in T1D cellular and animal models. We demonstrate that CpdA improves the unfolded protein response (UPR) by attenuating ER stress and favoring the survival and function of ß-cells exposed to an environment of proinflammatory cytokines. CpdA administration to NODscid mice adoptively transferred with diabetogenic splenocytes (from diabetic NOD mice) led to a delay of disease onset and reduction of diabetes incidence. Histological analysis of the pancreas showed a reduction in islet leukocyte infiltration (insulitis) and preservation of insulin expression in CpdA-treated normoglycemic mice in comparison with control group. These new findings together with our previous reports justify further studies on the administration of this small molecule as a novel therapeutic strategy with dual targets (effector immune and ß-cells) during autoimmune diabetes.

Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 94 Is Essential for Proinsulin Handling.

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within ß-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in ß-cells selectively activated protein kinase R-like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.

SPARC is required for the maintenance of glucose homeostasis and insulin secretion in mice.

Obesity, metabolic syndrome, and type 2 diabetes, three strongly interrelated diseases, are associated to increased morbidity and mortality worldwide. The pathogenesis of obesity-associated disorders is still under study. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein expressed in many cell types including adipocytes, parenchymal, and non-parenchymal hepatic cells and pancreatic cells. Studies have demonstrated that SPARC inhibits adipogenesis and promotes insulin resistance; in addition, circulating SPARC levels were positively correlated with body mass index in obese individuals. Therefore, SPARC is being proposed as a key factor in the pathogenesis of obesity-associated disorders. The aim of this study is to elucidate the role of SPARC in glucose homeostasis. We show here that SPARC null (SPARC-/-) mice displayed an abnormal insulin-regulated glucose metabolism. SPARC-/- mice presented an increased adipose tissue deposition and an impaired glucose homeostasis as animals aged. In addition, the absence of SPARC worsens high-fat diet-induced diabetes in mice. Interestingly, although SPARC-/- mice on high-fat diet were sensitive to insulin they showed an impaired insulin secretion capacity. Of note, the expression of glucose transporter 2 in islets of SPARC-/- mice was dramatically reduced. The present study provides the first evidence that deleted SPARC expression causes diabetes in mice. Thus, SPARC deficient mice constitute a valuable model for studies concerning obesity and its related metabolic complications, including diabetes.

Interactions Between the Neuroendocrine System and T Lymphocytes in Diabetes.

It is well established that there is a fine-tuned bidirectional communication between the immune and neuroendocrine tissues in maintaining homeostasis. Several types of immune cells, hormones, and neurotransmitters of different chemical nature are involved as communicators between organs. Apart of being key players of the adaptive arm of the immune system, it has been recently described that T lymphocytes are involved in the modulation of metabolism of several tissues in health and disease. Diabetes may result mainly from lack of insulin production (type 1 diabetes) or insufficient insulin and insulin resistance (type 2 diabetes), both influenced by genetic and environmental components. Herein, we discuss accumulating data regarding the role of the adaptive arm of the immune system in the pathogenesis of diabetes; including the action of several hormones and neurotransmitters influencing on central and peripheral T lymphocytes development and maturation, particularly under the metabolic burden triggered by diabetes. In addition, we comment on the role of T-effector lymphocytes in adipose and liver tissues during diabetes, which together enhances pancreatic ß-cell stress aggravating the disease.

Pluripotent nontumorigenic multilineage differentiating stress enduring cells (Muse cells): a seven-year retrospective.

Multilineage differentiating stress enduring (Muse) cells, discovered in the spring of 2010 at Tohoku University in Sendai, Japan, were quickly recognized by scientists as a possible source of pluripotent cells naturally present within mesenchymal tissues. Muse cells normally exist in a quiescent state, singularly activated by severe cellular stress in vitro and in vivo. Muse cells have the capacity for self-renewal while maintaining pluripotent cell characteristics indicated by the expression of pluripotent stem cell markers. Muse cells differentiate into cells representative of all three germ cell layers both spontaneously and under media-specific induction. In contrast to embryonic stem and induced pluripotent stem cells, Muse cells exhibit low telomerase activity, a normal karyotype, and do not undergo tumorigenesis once implanted in SCID mice. Muse cells efficiently home into damaged tissues and differentiate into specific cells leading to tissue regeneration and functional recovery as described in different animal disease models (i.e., fulminant hepatitis, muscle degeneration, skin ulcers, liver cirrhosis, cerebral stroke, vitiligo, and focal segmental glomerulosclerosis). Circulating Muse cells have been detected in peripheral blood, with higher levels present in stroke patients during the acute phase. Furthermore, Muse cells have inherent immunomodulatory properties, which could contribute to tissue generation and functional repair in vivo. Genetic studies in Muse cells indicate a highly conserved cellular mechanism as seen in more primitive organisms (yeast, Saccharomyces cerevisiae, Caenorhabditis elegans, chlamydomonas, Torpedo californica, drosophila, etc.) in response to cellular stress and acute injury. This review details the molecular and cellular properties of Muse cells as well as their capacity for tissue repair and functional recovery, highlighting their potential for clinical application in regenerative medicine.

Pluripotent Nontumorigenic Adipose Tissue-Derived Muse Cells have Immunomodulatory Capacity Mediated by Transforming Growth Factor-ß1.

Adult mesenchymal stromal cell-based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage-differentiating stress-enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse-AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse-AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse-AT cells grown as spheroids have a limited proliferation rate, a diameter of ∼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse-AT cells evidenced high stage-specific embryonic antigen-3 (SSEA-3) expression (∼60% of cells) after 7-10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA-3+ -Muse-AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide-stimulated macrophages and antigen-challenged T-cell assays, we have shown that Muse-AT cells have anti-inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. Muse-AT cells spontaneously gained transforming growth factor-ß1 expression that, in a phosphorylated SMAD2-dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T-box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse-AT cells that can potentially be harnessed as immunoregulators to treat immune-related disorders. Stem Cells Translational Medicine 2017;6:161-173.

GR-independent down-modulation on GM-CSF bone marrow-derived dendritic cells by the selective glucocorticoid receptor modulator Compound A.

Dendritic cells (DC) initiate the adaptive immune response. Glucocorticoids (GCs) down-modulate the function of DC. Compound A (CpdA, (2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride) is a plant-derived GR-ligand with marked dissociative properties. We investigated the effects of CpdA on in vitro generated GM-CSF-conditioned bone marrow-derived DC (BMDC). CpdA-exposed BMDC exhibited low expression of cell-surface molecules and diminution of the release of proinflammatory cytokines upon LPS stimulation; processes associated with BMDC maturation and activation. CpdA-treated BMDC were inefficient at Ag capture via mannose receptor-mediated endocytosis and displayed reduced T-cell priming. CpdA prevented the LPS-induced rise in pErk1/2 and pP38, kinases involved in TLR4 signaling. CpdA fully inhibited LPS-induced pAktSer473, a marker associated with the generation of tolerogenic DC. We used pharmacological blockade and selective genetic loss-of-function tools and demonstrated GR-independent inhibitory effects of CpdA in BMDC. Mechanistically, CpdA-mediated inactivation of the NF-κB intracellular signaling pathway was associated with a short-circuiting of pErk1/2 and pP38 upstream signaling. Assessment of the in vivo function of CpdA-treated BMDC pulsed with the hapten trinitrobenzenesulfonic acid showed impaired cell-mediated contact hypersensitivity. Collectively, we provide evidence that CpdA is an effective BMDC modulator that might have a benefit for immune disorders, even when GR is not directly targeted.

Effect of TGF-ß1 Stimulation on the Secretome of Human Adipose-Derived Mesenchymal Stromal Cells.

Adipose tissue is an attractive source of mesenchymal stromal cells (MSCs) owing to the relative ease of obtaining large volumes with more MSC abundance compared with other sources. Increasing evidence supports the fact that trophic factors secreted by MSCs play a pivotal therapeutic role. Several strategies in regenerative medicine use MSCs, mainly exploiting their immunosuppressive effect and homing capacity to sites of damage. Transforming growth factor-ß1 (TGF-ß1) is a pleiotropic cytokine that, depending on the cell niche, can display either anti-inflammatory or proinflammatory effects. TGF-ß1 expression increases in various tissues with damage, especially when accompanied by inflammation. Thus, we analyzed the effect of TGF-ß1 on the secretion by adipose-derived mesenchymal stromal cells (ASCs) of a panel of 80 cytokines/chemokines using an antibody array. To avoid a possible effect of fetal bovine serum (FBS) on ASCs secretion, we performed our analysis by culturing cells in FBS-free conditions, only supplemented with 0.1% of bovine serum albumin. We report the cytokine profile secreted by ASCs. We also found that TGF-ß1 exposure modulates 8 chemokines and 18 cytokines, including TGF-ß1 and -ß2, and other important cytokines involved in immunosuppression, allergic responses, and bone resorption.

A mystery unraveled: nontumorigenic pluripotent stem cells in human adult tissues.

INTRODUCTION: Embryonic stem cells and induced pluripotent stem cells have emerged as the gold standard of pluripotent stem cells and the class of stem cell with the highest potential for contribution to regenerative and therapeutic application; however, their translational use is often impeded by teratoma formation, commonly associated with pluripotency. We discuss a population of nontumorigenic pluripotent stem cells, termed Multilineage Differentiating Stress Enduring (Muse) cells, which offer an innovative and exciting avenue of exploration for the potential treatment of various human diseases. AREAS COVERED: This review discusses the origin of Muse cells, describes in detail their various unique characteristics, and considers future avenues of their application and investigation with respect to what is currently known of adult pluripotent stem cells in scientific literature. We begin by defining cell potency, then discuss both mesenchymal and various reported populations of pluripotent stem cells, and finally delve into Muse cells and the characteristics that set them apart from their contemporaries. EXPERT OPINION: Muse cells derived from adipose tissue (Muse-AT) are efficiently, routinely and painlessly isolated from human lipoaspirate material, exhibit tripoblastic differentiation both spontaneously and under media-specific induction, and do not form teratomas. We describe qualities specific to Muse-AT cells and their potential impact on the field of regenerative medicine and cell therapy.

Primera Red Sudamericana de Biomedicina. Investigación, educación y biotecnología aplicadas a la salud / [First South american network of biomedical research. Education and biotechnology for health].

It is in our interest, in this brief manuscript, to report the creation of the first program of regional integration of a network of research institutes in Biomedicine belonging to members of the MERCOSUR countries. We discuss some of the foundations that gave sustenance to its creation and its objectives in the medium and long term. In addition, we consider the potential of the results of this program in the fields of applied medical research, education and biotechnology.

[First South american network of biomedical research. Education and biotechnology for health]. / Primera Red Sudamericana de Biomedicina. Investigación, educación y biotecnología aplicadas a la salud.

It is in our interest, in this brief manuscript, to report the creation of the first program of regional integration of a network of research institutes in Biomedicine belonging to members of the MERCOSUR countries. We discuss some of the foundations that gave sustenance to its creation and its objectives in the medium and long term. In addition, we consider the potential of the results of this program in the fields of applied medical research, education and biotechnology.

Primera Red Sudamericana de Biomedicina. Investigación, educación y biotecnología aplicadas a la salud. / [First South american network of biomedical research. Education and biotechnology for health].

It is in our interest, in this brief manuscript, to report the creation of the first program of regional integration of a network of research institutes in Biomedicine belonging to members of the MERCOSUR countries. We discuss some of the foundations that gave sustenance to its creation and its objectives in the medium and long term. In addition, we consider the potential of the results of this program in the fields of applied medical research, education and biotechnology.

Compound A, a dissociated glucocorticoid receptor modulator, inhibits T-bet (Th1) and induces GATA-3 (Th2) activity in immune cells.

BACKGROUND: Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively. RESULTS: Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively. CONCLUSIONS: Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders.

Disease-modifying immunotherapy for the management of autoimmune diabetes.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that destroys the insulin-secreting beta-cells of the pancreas. It is now possible to predict those candidates that will progress to T1D before the full onset of the disease. Prevention of uncontrollable autoimmunity against beta-cells in therapies for T1D is mandatory to preserve the beta-cell mass. Therefore, immunomodulatory strategies directed to inhibiting the activity of self-reactive T cell clones as well as induction of regulatory T cells would be beneficial for prevention of T1D or recurrence of beta-cell autoimmunity against islet cell allografts.

Curcumin inhibits the growth, induces apoptosis and modulates the function of pituitary folliculostellate cells.

The polyphenol curcumin (diferuloylmethane) is the active componenet of the spice plant Curcuma longa and has been shown to exert multiple actions on mammalian cells. We have studied its effect on folliculostellate (FS) TtT/GF mouse pituitary cells, representative of a multifunctional, endocrine inactive cell type of the anterior pituitary. Proliferation of TtT/GF cells was inhibited by curcumin in a monolayer cell culture and in the colony formation assay in soft agar. Fluorescence-activated cell-sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G(2)/M accompanied by inhibition of cyclin D(1) protein expression. Curcumin had a small effect on necrosis of TtT/GF cells, but it mainly stimulated apoptosis as demonstrated by FACS analysis (Annexin V-fluorescein isothiocyannate/7-aminoactinomycin D staining). Curcumin-induced apoptosis involved suppression of Bcl-2, stimulation of cleaved caspase-3 and induction of DNA fragmentation. Functional studies on FS cell-derived compounds showed that curcumin inhibited mRNA synthesis and release of angiogenic vascular endothelial growth factor-A (VEGF-A). Immune-like functions of FS cells were impaired since curcumin downregulated Toll-like receptor 4, reduced nuclear factor-kappaB expression and suppressed bacterial endotoxin-induced interleukin-6 (IL-6) secretion. The inhibitory action of curcumin on VEGF-A and IL-6 production was also found in primary rat pituitary cell cultures, in which FS cells are the only source of these proteins. The observed effects of curcumin on FS cell growth, apoptosis and functions may have therapeutic consequences for the intrapituitary regulation of hormone production and release as well as for pituitary tumor pathogenesis.

Suppression of autoimmune diabetes by soluble galectin-1.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the beta-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic beta-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of beta-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4(+) T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause beta-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed beta-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or beta-cell toxicity in NOD mice, the use of gal-1 to control beta-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D.

Molecular transduction mechanisms of cytokine-hormone interactions: role of gp130 cytokines.

Highly sophisticated mechanisms confer on the immune system the capacity to respond with a certain degree of autonomy. However, the final outcome of an immune response depends on the interaction of the immune system with other systems. The immune and neuroendocrine systems have an intimate cross-communication that makes possible a satisfactory response to environmental changes. Part of this interaction occurs through cytokines and steroid hormones. The last step of this cross-talk is the molecular level. As a model of interaction, this review focuses on the gp130 cytokine family. These cytokines, as well as their receptors, are expressed in pituitary cells. They regulate hormone production as well as growth of pituitary cells. During acute or chronic inflammation or infection, systemic, hypothalamic and hypophyseal gp130 cytokines act on anterior pituitary cells, integrating the neuroendocrine-immune response. Disruptions of these pathways may lead not only to abnormal growth of pituitary cells but also to immune disorders, for which, based on recent findings, targeting these cytokines might be a novel therapeutic approach.

Glucocorticoids in the regulation of transcription factors that control cytokine synthesis.

The interaction at different levels between intracellular signals elicited by cytokines and activated glucocorticoid receptors (GR) is essential for the regulation of immune responses. We describe different levels of interaction between glucocorticoids and cytokines which result in the induction or repression of gene transcription. These include the regulation of cytokine receptor expression, the molecular cross-talk between the GR and transcription factors (TFs) activated by cytokine signaling, the interaction with several signaling pathways and also posttranslational modifications of both GR and TFs. Also, an overview of the implications of chromatin remodeling in this interplay is discussed. The complexity of the intricate network involved in the interaction between GR and TFs is pivotal for the final outcome of cytokines biological action.

Dendritic cells expressing transgenic galectin-1 delay onset of autoimmune diabetes in mice.

Type 1 diabetes (T1D) is a disease caused by the destruction of the beta cells of the pancreas by activated T cells. Dendritic cells (DC) are the APC that initiate the T cell response that triggers T1D. However, DC also participate in T cell tolerance, and genetic engineering of DC to modulate T cell immunity is an area of active research. Galectin-1 (gal-1) is an endogenous lectin with regulatory effects on activated T cells including induction of apoptosis and down-regulation of the Th1 response, characteristics that make gal-1 an ideal transgene to transduce DC to treat T1D. We engineered bone marrow-derived DC to synthesize transgenic gal-1 (gal-1-DC) and tested their potential to prevent T1D through their regulatory effects on activated T cells. NOD-derived gal-1-DC triggered rapid apoptosis of diabetogenic BDC2.5 TCR-transgenic CD4+ T cells by TCR-dependent and -independent mechanisms. Intravenously administered gal-1-DC trafficked to pancreatic lymph nodes and spleen and delayed onset of diabetes and insulitis in the NODrag1(-/-) lymphocyte adoptive transfer model. The therapeutic effect of gal-1-DC was accompanied by increased percentage of apoptotic T cells and reduced number of IFN-gamma-secreting CD4+ T cells in pancreatic lymph nodes. Treatment with gal-1-DC inhibited proliferation and secretion of IFN-gamma of T cells in response to beta cell Ag. Unlike other DC-based approaches to modulate T cell immunity, the use of the regulatory properties of gal-1-DC on activated T cells might help to delete beta cell-reactive T cells at early stages of the disease when the diabetogenic T cells are already activated.