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Autoimmunity is a complex and multifaceted process that contributes to widespread functional decline that affects multiple organs and tissues. The pandemic of autoimmune diseases, which are a global health concern, augments in both the prevalence and incidence of autoimmune diseases, including type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. The development of autoimmune diseases is phenotypically associated with gut microbiota-modulated features at the molecular and cellular levels. The etiology and pathogenesis of autoimmune diseases comprise the alterations of immune systems with the innate and adaptive immune cell infiltration into specific organs and the augmented production of proinflammatory cytokines stimulated by commensal microbiota. However, the relative importance and mechanistic interrelationships between the gut microbial community and the immune system during progression of autoimmune diseases are still not well understood. In this review, we describe studies on the profiling of gut microbial signatures for the modulation of immunological homeostasis in multiple inflammatory diseases, elucidate their critical roles in the etiology and pathogenesis of autoimmune diseases, and discuss the implications of these findings for these disorders. Targeting intestinal microbiome and its metabolomic associations with the phenotype of autoimmunity will enable the progress of developing new therapeutic strategies to counteract microorganism-related immune dysfunction in these autoimmune diseases.

Inflammatory colon diseases, which are a global health concern, include a variety of gastrointestinal tract disorders, such as inflammatory bowel disease and colon cancer. The pathogenesis of these colon disorders involves immune alterations with the pronounced infiltration of innate and adaptive immune cells into the intestines and the augmented expression of mucosal pro-inflammatory cytokines stimulated by commensal microbiota. Epidemiological studies during the past half century have shown that the proportion of obese people in a population is associated with the incidence and pathogenesis of gastrointestinal tract disorders. The advancement of understanding of the immunological basis of colon disease has shown that adipocyte-derived biologically active substances (adipokines) modulate the role of innate and adaptive immune cells in the progress of intestinal inflammation. The biomedical significance in immunological homeostasis of adipokines, including adiponectin, leptin, apelin and resistin, is clear. In this review, we highlight the existing literature on the effect and contribution of adipokines to the regulation of immunological homeostasis in inflammatory colon diseases and discuss their crucial roles in disease etiology and pathogenesis, as well as the implications of these results for new therapies in these disorders.

Defects in mucosal immune balance can lead to colonic diseases such as inflammatory bowel diseases and colorectal cancer. With the advancement of understanding for the immunological and molecular basis of colonic disease, therapies targeting transcription factors have become a potential approach for the treatment of colonic disease. To date, the biomedical significance of unique post-translational modifications on transcription factors has been identified, including phosphorylation, methylation, acetylation, ubiquitination, SUMOylation, and O-GlcNAcylation. This review focuses on our current understanding and the emerging evidence of how post-translational regulations modify transcription factors involved in the etiology and pathophysiology of colonic disease as well as the implications of these findings for new therapeutic approaches in these disorders.

Inflammatory bowel disease (IBD) is a chronic disorder manifested as Crohn’s disease (CD) and ulcerative colitis (UC) characterized by intestinal inflammation and involves a dysregulated immune response against commensal microbiota through the activation of CD4 T helper cells. T helper cell differentiation to effector or regulatory phenotypes is controlled by cytokine networks and transcriptional regulators. Distinct polarized T helper cells are able to alter their phenotypes to adapt to diverse and fluctuating physiological environments. T helper cells exhibit intrinsic instability and flexibility to express cytokines of other lineages or transdifferentiate from one T helper cell type to another in response to various perturbations from physiological cytokine milieu as a means of promoting local immunity in response to injury or ensure tissue homeostasis. Furthermore, functional plasticity and diversity of T helper cells are associated with pathogenicity and are critical for immune homeostasis and prevention of autoimmunity. In this review, we provide deeper insights into the combinatorial extrinsic and intrinsic signals that control plasticity and transdifferentiation of T helper cells and also highlight the potential of exploiting the genetic reprogramming plasticity of T helper cells in the treatment of IBD.

Glycosylation is a ubiquitous posttranslational modification of proteins that occurs in the endoplasmic reticulum/Golgi. N-glycans and mucin-type O-glycans are achieved via a series of glycohydrolase- and glycosyltransferase-mediated reactions. Glycosylation modulates immune responses by regulating thymocyte development and T helper cell differentiation. Autoimmune diseases result from an abnormal immune response by self-antigens and subsequently lead to the destruction of the target tissues. The modification of N-glycans has been studied in several animal models of T-cell-mediated autoimmune diseases. This review summarizes and highlights the modulatory effects of N-glycosylation in several autoimmune diseases, including multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and type 1 diabetes mellitus.

SUMOylation is involved in the development of several inflammatory diseases, but the physiological significance of SUMO-modulated c-Maf in autoimmune diabetes is not completely understood. Here, we report that an age-dependent attenuation of c-Maf SUMOylation in CD4+ T cells is positively correlated with the IL-21-mediated diabetogenesis in NOD mice. Using 2 strains of T cell-specific transgenic NOD mice overexpressing wild-type c-Maf (Tg-WTc) or SUMOylation site-mutated c-Maf (Tg-KRc), we demonstrated that Tg-KRc mice developed diabetes more rapidly than Tg-WTc mice in a CD4+ T cell-autonomous manner. Moreover, SUMO-defective c-Maf preferentially transactivated Il21 to promote the development of CD4+ T cells with an extrafollicular helper T cell phenotype and expand the numbers of granzyme B-producing effector/memory CD8+ T cells. Furthermore, SUMO-defective c-Maf selectively inhibited recruitment of Daxx/HDAC2 to the Il21 promoter and enhanced histone acetylation mediated by CREB-binding protein (CBP) and p300. Using pharmacological interference with CBP/p300, we illustrated that CBP30 treatment ameliorated c-Maf-mediated/IL-21-based diabetogenesis. Taken together, our results show that the SUMOylation status of c-Maf has a stronger regulatory effect on IL-21 than the level of c-Maf expression, through an epigenetic mechanism. These findings provide new insights into how SUMOylation modulates the pathogenesis of autoimmune diabetes in a T cell-restricted manner and on the basis of a single transcription factor.

Idiopathic membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis and the most common cause of idiopathic nephrotic syndrome in adult humans. A tumor necrosis factor α (TNF-α)-mediated inflammatory response via TNF receptor 1 (TNFR1) and TNFR2 has been proposed as a pathogenic factor. In this study, we assessed the therapeutic response to blocking TNF signaling in experimental MN. Murine MN was induced experimentally by cationic bovine serum albumin (cBSA); phosphate-buffered saline was used in control mice. In MN mice, TNF was inhibited by etanercept blocking of TNFR1/TNFR2 or the preligand assembly domain fusion protein (PLAD.Fc), a small fusion protein that can preferentially block TNFR1 signaling. Disease severity and possible mechanisms were assessed by analyzing the metabolic and histopathology profiles, lymphocyte subsets, immunoglobulin production, oxidative stress, and apoptosis. cBSA-induced MN mice exhibited typical nephrotic syndrome and renal histopathology. MN mice given etanercept or PLAD.Fc did not exhibit significant reduction of proteinuria, amelioration of glomerular lesions, or attenuation of immune complex deposition. Immune cell subsets, serum immunoglobulin levels, production of reactive oxygen species, and cell apoptosis in the kidney were not altered by TNF inhibition. By contrast, MN mice receiving etanercept or PLAD.Fc exhibited significantly decreased infiltration of immune cells into the kidney. These results show that the therapeutic effects of blocking TNFR1 and/or TNFR2 signaling in experimental MN are not clinically effective. However, TNF signaling inhibition significantly attenuated renal immune cell infiltration in experimental MN.

B lymphocyte-induced maturation protein-1 (Blimp-1) serves as a master regulator of the development and function of antibody-producing B cells. Given that its function in T lymphocytes has been identified within the past decade, we review recent findings with emphasis on its role in coordinated control of gene expression during the development, differentiation, and function of T cells. Expression of Blimp-1 is mainly confined to activated T cells and is essential for the production of interleukin (IL)-10 by a subset of forkhead box (Fox)p3+ regulatory T cells with an effector phenotype. Blimp-1 is also required to induce cell elimination in the thymus and critically modulates peripheral T cell activation and proliferation. In addition, Blimp-1 promotes T helper (Th) 2 lineage commitment and limits Th1, Th17 and follicular helper T cell differentiation. Furthermore, Blimp-1 coordinates with other transcription factors to regulate expression of IL-2, IL-21 and IL-10 in effector T lymphocytes. In CD8+ T cells, Blimp-1 expression is distinct in heterogeneous populations at the stages of clonal expansion, differentiation, contraction and memory formation when they encounter antigens. Moreover, Blimp-1 plays a fundamental role in coordinating cytokine receptor signaling networks and transcriptional programs to regulate diverse aspects of the formation and function of effector and memory CD8+ T cells and their exhaustion. Blimp-1 also functions as a gatekeeper of T cell activation and suppression to prevent or dampen autoimmune disease, antiviral responses and antitumor immunity. In this review, we discuss the emerging roles of Blimp-1 in the complex regulation of gene networks that regulate the destiny and effector function of T cells and provide a Blimp-1-dominated transcriptional framework for T lymphocyte homeostasis.

Background and aims: The aim of this study was to investigate the effect of a glucagon-like peptide-1 receptor agonist (GLP-1RA), exenatide, on clozapine-associated glucose dysregulation in mice. Materials and methods: We randomly separated B6 male mice into four groups (A to D). Mice in groups C and D received a daily oral dose of 13.5 mg/kg body weight of clozapine for 4 months. Mice in groups B and D received 1 μg of exenatide daily. The body weight and blood glucose before and 2 h after clozapine treatment were measured twice a week. Intraperitoneal glucose tolerance test (IPGTT) scores and the amount of daily food intake were recorded. The pancreases of the mice were removed for insulin content (PIC) measurement and histological examination after sacrifice. Results: The mean non-fasting blood glucose levels were not different, and the mean changes in blood glucose 2 h after oral clozapine were 0 ± 4, −40 ± 2, 25 ± 3, and −39 ± 2, in groups A to D, respectively. There was no significant difference in daily calorie intake or area under the curve of IPGTT among the four groups. At sacrifice, the PIC of mice treated with clozapine was significantly lower than that of the control mice, however the PIC was completely restored in the mice treated with exenatide. Histological examination of the pancreas revealed that exenatide treatment reversed the clozapine-induced apoptosis of islet cells. Conclusion: Our results provide preclinical evidence of a pharmaceutical role of GLP-1RA in managing glucose dysregulation in schizophrenic patients under long-term atypical antipsychotic treatments. Keywords: Clozapine, Exenatide, Glucose dysregulation, Beta cell, Apoptosis

In this study, we examined the combined effect of two recombinant immunomodulator proteins, cytotoxic T-lymphocyte antigen-4-Ig (CTLA4Ig) and interleukin-1 receptor antagonist (IL1ra), on the survival of subcutaneous mouse islet grafts with temperature-sensitive hydrogel, poly(N-isopropylacrylamide) (pNIPAAm)-chitosan-hyaluronic acid (CPNHA), and gelatin microspheres in an allogeneic mouse model. The phase-transition temperature for the CTLA4Ig-grafted CPNHA (CPNCHA) was 28 °C. An in vitro cytotoxicity assay and in vivo tissue reactions showed that both CPNCHA and the gelatin microspheres were biocompatible, biodegradable and nontoxic. The survival of the allogeneic islets was prolonged when the grafts were transplanted into a subcutaneous matrix composed of CPNCHA and gelatin microspheres containing IL1ra relative to those implanted in CPNHA containing gelatin microspheres without an immunomodulator. In conclusion, our results suggest that the temperature-sensitive hydrogel, CPNCHA, is a suitable subcutaneous matrix for islet transplantation, and grafted CTLA4Ig and encapsulated IL1ra reduce immune rejection and prolong the survival of the functioning allogeneic islets. The combined effect of cytotoxic T-lymphocyte antigen-4-Ig (CTLA4Ig) and interleukin-1 receptor antagonist (IL1ra) on the survival of subcutaneous mouse islet grafts with temperature-sensitive hydrogel, pNIPAAm-chitosan-hyaluronic acid (CPNHA), and gelatin microspheres in an allogeneic mouse model is examined. The survival of the allogeneic islets was prolonged when the grafts were transplanted into a subcutaneous matrix composed of CPN-CTLA4Ig-HA (CPNCHA) and gelatin microspheres containing IL1ra relative to those implanted in CPNHA containing gelatin microspheres without an immunomodulator.

The diameter and sphericity of alginate-poly-L-lysine-alginate microcapsules, determined by the size and the shape of calcium alginate microspheres, affect their in vivo durability and biocompatibility and the results of transplantation. The commonly used air-jet spray method generates microspheres with a wider variation in diameter, larger sphere morphology, and evenly distributed encapsulated cells. In order to overcome these drawbacks, we designed a field effect microparticle generator to create a stable electric field to prepare microparticles with a smaller diameter and more uniform morphology. Using this electric field microparticle generator the encapsulated cells will be located at the periphery of the microspheres, and thus the supply of oxygen and nutrients for the encapsulated cells will be improved compared with the centrally located encapsulated cells in the air-jet spray method.

The diameter and sphericity of alginate-poly-L-lysine-alginate microcapsules, determined by the size and the shape of calcium alginate microspheres, affect their in vivo durability and biocompatibility and the results of transplantation. The commonly used air-jet spray method generates microspheres with a wider variation in diameter, larger sphere morphology, and evenly distributed encapsulated cells. In order to overcome these drawbacks, we designed a field effect microparticle generator to create a stable electric field to prepare microparticles with a smaller diameter and more uniform morphology. Using this electric field microparticle generator the encapsulated cells will be located at the periphery of the microspheres, and thus the supply of oxygen and nutrients for the encapsulated cells will be improved compared with the centrally located encapsulated cells in the air-jet spray method.

Glucosamine has immunomodulatory effects on autoimmune diseases. However, the mechanism(s) through which glucosamine modulates different T cell subsets and diseases remain unclear. We demonstrate that glucosamine impedes Th1, Th2, and iTreg but promotes Th17 differentiation through down-regulating N-linked glycosylation of CD25 and subsequently inhibiting its downstream Stat5 signaling in a dose-dependent manner. The effect of glucosamine on T helper cell differentiation was similar to that induced by anti-IL-2 treatment, further supporting an IL-2 signaling-dependent modulation. Interestingly, excess glucose rescued this glucosamine-mediated regulation, suggesting a functional competition between glucose and glucosamine. High-dose glucosamine significantly decreased Glut1 N-glycosylation in Th1-polarized cells. This finding suggests that both down-regulated IL-2 signaling and Glut1-dependent glycolytic metabolism contribute to the inhibition of Th1 differentiation by glucosamine. Finally, glucosamine treatment inhibited Th1 cells in vivo, prolonged the survival of islet grafts in diabetic recipients, and exacerbated the severity of EAE. Taken together, our results indicate that glucosamine interferes with N-glycosylation of CD25, and thereby attenuates IL-2 downstream signaling. These effects suggest that glucosamine may be an important modulator of T cell differentiation and immune homeostasis.

Primary nonfunction (PNF) adversely impacts islet transplantation. In addition to determining whether sodium 4-phenylbutyrate (4-SPB), an anti-inflammatory agent, reduces PNF, this study investigates how 4-SPB affects PNF. Streptozotocin-induced diabetic C57BL/6 mice, that received 75 syngeneic islets underneath left subrenal space, were fed twice daily of either 4-SPB at 500 mg/kg body weight or isotonic saline (NaCl) from 2 days before through 7 days after transplantation. The graft was removed at days 3, 10 and 84 following transplantation. At 68 h following transplantation, serum levels of interleukin-1beta (IL-1beta) were 2.2+/-0.4 and 0.4+/-0.2 pmol/L (n=6, p0.005) for NaCl and 4-SPB groups, respectively. Graft genetic expression of IL-1beta was significantly suppressed in 4-SPB group (p0.01). At day 10, the blood glucose levels were 22.7+/-1.0 and 17.1+/-1.7 mmol/L (n=12, p0.05) and graft insulin contents (IC) were 35.0+/-8.3 and 107.6+/-29.7 pmol (n=12, p0.05) for NaCl and 4-SPB groups, respectively. Moreover, the 4-SPB group had a shorter temporary hyperglycemia (15+/-2, n=21 vs. 25+/-2 days, n=19, p=0.001) and a higher cumulative cure rate of diabetes (p0.001) than the NaCl group. In-vitro studies indicated that 4-SPB did not impact the islets function. These experimental results demonstrated that perioperative administration of 4-SPB decreased serum level and graft genetic expression of IL-1beta and attenuated PNF, which enhanced islet engraftment in a syngeneic transplantation mouse model.

Loofa sponge was investigated as a three-dimensional scaffold for stationary and perfusion culture of human hepatoblastoma cell line C3A/HepG2. In stationary culture, C3A/HepG2 cells in loofa cubes showed higher alpha-fetoprotein and albumin secretion rates than those in polyurethane foam (PU). To use loofa cylinders in a packed-bed reactor, immobilization of C3A/HepG2 cells by recirculating medium at 26 mL/min (superficial velocity = 51.7 cm/min) resulted in a cell loading density of 5.15 x 10(7) cells/cm(3)-loofa. This cell loading density is higher than values reported in the literature for packed-bed reactor intended for bioartificial liver. During 9 days of perfusion culture in the reactor, immobilized C3A/HepG2 showed steady synthesis of albumin with an average synthesis rate at 42.2 microg/10(6) cells/day. These experimental results and observations by SEM suggested that loofa sponge is a suitable scaffold for high-density culture of human hepatocyte cell line and the immobilized cells could express high levels of liver-specific functions.

Because the development of surface neogrowth composed mainly of macrophages and fibroblasts precedes the recurrence of hyperglycemia in treated diabetic animals, the pericapsular macrophages may adversely affect the graft function of i.p. alginate-poly-L-lysine-alginate (A-P-A) microencapsulated islets. In order to clarify the role of pericapsular macrophages on late islet xenograft dysfunction, we investigated whether 15-deoxyspergualin (15-DSG), a macrophage inhibitor, has a rescue effect on the recurrent hyperglycemia in streptozotocin-induced diabetic mice that had been treated with i.p. transplantation of A-P-A microencapsulated rat islets. The mean duration of normoglycemia (whole blood glucose level below 8.3 mmol/l) in streptozotocin-induced diabetic mice treated with implantation of about 2200-2400 of A-P-A microencapsulated rat islets was 75 days. When the blood glucose levels were higher than 11.1 mmol/l for two consecutive determinations, 15-DSG at a dose of 0.625 mg/kg body weight or isotonic sodium chloride solution (control group) was given daily s.c.. The blood glucose levels decreased significantly from 13.9 +/- 0.5 mmol/l to 11.0 +/- 1.3 mmol/l (n = 18, p < 0.05) at the fourth day and to 7.6 +/- 1.0 mmol/l (n = 18) at the 14th day of 15-DSG administration. That was not significantly different from the mean glycemic level during the normoglycemic period (7.6 +/- 1.0 vs. 7.0 +/- 1.7 mmol/l, n = 18, p = NS). Isotonic sodium chloride solution injections did not reduce glycemic levels of mice in the control group. As another control, 10 streptozotocin-induced diabetic mice were given the same daily doses of 15-DSG for 14 days. 15-DSG did not decrease the blood glucose levels of diabetic mice in the control group. We further studied the effect of 15-DSG on the expression of interleukin-1beta (IL-1beta) in peritoneal exudate mononuclear cells (PEMCs) using reverse transcription-polymerase chain reaction. It was found that the mRNA of IL-1beta was undetectable in PEMCs of 15-DSG-treated diabetic mice even after those cells were stimulated by lipopolysaccharides in vitro. Administration of 15-DSG at a daily dose of 0.625 mg/kg body weight from the 22nd to the 28th day after transplantation and 7 consecutive days every 3 weeks thereafter did not prolong graft survival of i.p. microencapsulated rat islets. Our data suggest that 15-DSG has a rescue effect when A-P-A microencapsulated islets have induced cellular overgrowth that threatens the survival of the graft. It is possible that the surface overgrowth composed of macrophages is involved in the pathophysiology of late failure of A-P-A microencapsulated xenogeneic islets.

Two positively charged derivatives of poly(ethylene glycol) (PEG) were coated onto alginate-poly( l -lysine)-alginate (A-P-A) microcapsules by allowing them to interact them with the negatively charged alginate on the capsule surface. The polymers are methoxypolyoxyethylene amine (PEGA1) and polyoxyethylene bis(amine) (PEGA2), which contain charged amine groups at one or both ends, respectively, with PEG as the backbone. The coating of the microcapsules with PEG-amine resulted in a much smoother capsule surface than A-P-A microcapsule surfaces as examined under a scanning electron microscope. The diffusivity of bovine serum albumin into the microcapsules remained the same after PEGA1 coating. But the diffusivity decreased to less than one-fifth that in A-P-A microcapsules coated with PEGA2. The biocompatibility of the microcapsules also improved as investigated by an in vivo study. Microcapsules were implanted in the peritoneal cavity of BALB c mice and retrieved 120 d after implantation. The fibrotic action against A-P-A microcapsules was severe and the capsules retrieved by peritoneal lavage aggregated into clusters. In contrast, the surface-modified capsules were free-flowing and free of cell overgrowth. Secretion of insulin from rat islets within A-P-A-PEGA microcapsules responded well to changes in glucose concentration in a static glucose test. Intraperitoneal transplantation of the microencapsulated islets into streptozotocin-induced diabetic mice could maintain normal blood glucose levels in test animals for up to 200 d without immunosuppression.

To study the role of a peroxisome proliferator-activated receptor agonist, rosiglitazone, on islet engraftment, streptozotocin-induced diabetic C57BL/6 mice were fed daily rosiglitazone (2.4 mg/kg) for 9 and 31 days starting 2 days before transplantation with 75 and 150 syngeneic islets, respectively. After receiving 75 islets and 9 days of rosiglitazone, half of the treated diabetic mice became normoglycemic at 4 weeks, while none were normoglycemic among those mice that did not receive treatment. After transplanting 150 islets and receiving 31 days of rosiglitazone, 80% of the treated diabetic mice became normoglycemic while the incidence was only 25% for the controls. The insulin content of the islet grafts in the rosiglitazone groups was 0.8 times (75-islet group) and 1.3 times (150-islet group) higher than that of control mice. The insulin content of pancreatic remnants did not differ significantly among all groups. An in vitro study revealed that the glucose-stimulated insulin secretion and insulin content of cultured islets was not different in the presence versus absence of 4.5 or 22.5 micromol/L rosiglitazone. In vitro study revealed that rosiglitazone inhibited the lipopolysaccharide-induced secretion of interleukin-1 beta and interferon-gamma from peritoneal exudate cells. In conclusion, our data suggest that short-term administration of rosiglitazone enhances islet engraftment.

Objective The soluble preligand assembly domain (PLAD) of tumour necrosis factor receptor 1 (TNFR1) interferes with receptor trimerisation to block downstream signalling, and mediates Th17 suppression. We explored the therapeutic potential of recombinant PLAD.Fc protein on a spontaneous experimental colitis. Design A T-cell-specific BLIMP-1 knockout mouse model with mixed Th1/Th17 responses, resembling human Crohn9s disease (CD) was established, and its colitogenic phenotype was characterised. Mice, 9 weeks old, were treated with PLAD.Fc protein at 5 mg/kg of body weight twice per week for 16 weeks, and presence of colitis was monitored by the appearance of diarrhoea, weight loss, and by histological colonic scoring. Activation status, cytokine profiles, and transcription factors in T cells were further analysed. Results The colitogenic phenotype in BLIMP-1 knockout mice was alleviated when an interleukin (IL)-23 knockdown transgene was introduced, indicating a therapeutic potential by downregulating IL-23-Th17 axis in these knockout mice. In PLAD.Fc-treated group, the mouse body weight remained stable and only mild disease scores were revealed. The percentage of naive CD4 T cells was increased and that of effector/memory CD4 T cells was decreased after PLAD.Fc-treatment. Moreover, the levels of IFN-γ, IL-17, IL-21, IL-22, IL-23R, granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α were diminished. Strikingly, Th2-associated cytokines (IL-4, IL-13 and IL-10) in sera, as well as percentages of Th2 cells, were increased in PLAD.Fc-treated mice. However, PLAD.Fc-mediated suppression of effector phenotypes in Th1/Th17 was abrogated after neutralising IL-10. Conclusions The Th2 cytokine milieu induced by PLAD.Fc rebalanced T-helper cell subsets and conferred a protection against colitis in BLIMP-1 knockout mice.

Recent reports indicate that B lymphocyte-induced maturation protein 1 (BLIMP-1), encoded by the Prdm1 gene, expands its control over T cells and is associated with susceptibility to colitis in mice with T cell-specific BLIMP-1 deficiency. In this study, we aimed to investigate the potential role of BLIMP-1 in regulating autoimmune diabetes and T helper type 17 (Th17) cells.We generated T cell-specific Blimp1 (also known as Prdm1) transgenic (Tg) or conditional knockout (CKO) NOD mice, in which Blimp1 is overexpressed or deleted in T cells, respectively. By side-by-side analysing these Tg or CKO mice, we further dissected the potential mechanisms of BLIMP-1-mediated modulation on autoimmune diabetes.Overproduction of BLIMP-1 in T cells significantly attenuated insulitis and the incidence of diabetes in NOD mice. Consistent with these results, the diabetogenic effect of splenocytes was remarkably impaired in Blimp1 Tg mice. Moreover, overproduction of BLIMP-1 repressed the proliferation and activation of lymphocytes and enhanced the function of regulatory T cells (Tregs) in NOD mice. In contrast, mice lacking BLIMP-1 in T cells markedly increased Th1 and Th17 cells, and developed highly proliferative and activated lymphocytes. Strikingly, overexpansion of Th1 and Th17 cells in CKO mice was significantly reduced by introducing a Blimp1 transgene, reinforcing the emerging role of BLIMP-1 in autoimmunity.We conclude that BLIMP-1 orchestrates a T cell-specific modulation of autoimmunity by affecting lymphocyte proliferation and activation, Th1 and Th17 cell differentiation, and Treg function. Our results provide a theoretical basis for developing BLIMP-1-manipulated therapies for autoimmune diabetes.

The complementary role of hyperglycemia and suppression on islet beta cell regeneration was investigated in a syngeneic mouse model. gene silencing was performed by infecting islets of C57BL/6 with shRNA lentiviral particles. At 54 hours after viral infection, protein content in cultured targeting islets was 22% of that in freshly isolated islets. Six days after transplantation to diabetic mice, targeting islet graft had considerably more cells with Ki67-staining nuclei than nontargeting islets. The mice in the targeting-islet group had a significantly shorter duration of temporary hyperglycaemia than mice in the non-targeting-islet group. The long-termex vivobeneficial effect of silencing on graft function was also indicated by the significantly higher cumulative cure rate for diabetes in mice receiving 200 targeting islets than that in mice receiving 200 non-targeting islets. Our data suggest that hyperglycemia and persistent suppression have a synergistic effect on islet beta cell replication in adult mice.

To study effects of glucose on growth-related proteins of adult islets, we cultured mice islets in medium containing either 5.5 mmol/L (LG) or 20 mmol/L (HG) glucose. Total islet proteins were processed for sodium dodecyl sulfate polyacrylamide gel and Western blotting using antibodies against β-actin (housekeeping), p27kip1 (G1/G0 checkpoint), cyclin D1 (G1/S), cyclin B1 (G2/M), and FoxM1. At day 1, protein levels p27, B1, D1, and FoxM1 of islets on LG and HG were 0.48- and 0.63-fold; 7.09- and 11.58-fold; 1.25- and 1.38-fold; and 1.75- and 1.75-folds, the value of day 0, determinations respectively. At day 3, the proteins of p27, B1, D1, and FoxM1 of islets in LG and HG were 0.84- and 0.84-fold; 3.08- and 17.17-fold; 1.41- and 1.54-fold; and 0.83- and 1.17-fold of those on day 0, respectively. On day 7 the values were 1.19- and 1.09-fold; 3.15- and 14.81-fold; 0.86- and 1.44-fold; and 2.75- and 3.42-fold that of day 0, respectively. At day 1, the ratios of protein in islets after HG verse LG were 1.25, 2.38, 0.94, and 1.00 for p27, B1, D1, and FoxM1, respectively. At days 3 and 7, the protein ratios of HG/LG were 0.81 and 0.82, 5.47 and 2.64, 0.81 and 1.51, and 1.11 and 1.24 for p27, B1, D1, and FoxM1, respectively. In conclusion, adult mouse islets rapidly respond to cultivation by reducing p27 and increasing B1; HG attenuates p27 elevation but enhances B1 and D1 elevations, which favor islet entry into the cell cycle.

We co-transplanted silica gel-entrapping 4H-1,3-Thiazin-2-amine,5,6-dihydro-6-methyl monohydrochloride (AMT) with islets to evaluate the effects of AMT on early graft dysfunction in a syngeneic mouse model. The mean diameter of AMT-embedding silica gel particles was 595 +/- 275 nm. The cumulative release of AMT was 29% at 1 hour and 45% at 72 hours. Sixteen streptozotocin-induced diabetic mice were separated into 3 groups. Group A received 50 islets (n = 4). Group B received 50 islets and blank silica gel (n = 6). Group C received 50 islets plus silica-gel containing 6.4 microg AMT (n = 6). Mice in group C required significantly less time for temporary posttransplantation hyperglycemia than those in groups A and B (A, 39 +/- 7 vs B, 40 +/- 5 vs C, 24 +/- 2 days; P.05). The insulin contents of grafts retrieved at 13 weeks were 1.17 +/- 0.11 (n = 4), 1.01 +/- 0.16 (n = 6), and 1.68 +/- 0.30 microg (n = 6) for mice in groups A, B, and C, respectively. Pancreatic remnant insulin did not differ significantly between the 3 groups (A, 0.32 +/- 0.04 [n = 4] vs B, 0.29 +/- 0.06 [n = 6] vs C, 0.40 +/- 0.05 microg [n = 6]; P.05). In vitro study revealed that 4 and 20 nmol/L of sol-gel-embedded AMT protected 87% and 96% RIN-m5F cells from 1 ng/mL interleukin-1beta-mediated destruction, respectively. Silica-gel-entrapped AMT protects islet graft from a nonspecific inflammatory destruction, which is partly mediated via interleukin-1beta.

This study examined the combinatory effect on graft survival of neonatal pig pancreatic cell clusters (NPCC) with nordihydroguaiaretic acid (NDGA), a 5-lipoxygenase inhibitor, with systemic CTLA4Ig expression, with local CTLA4Ig and with interleukin-1 (IL-1) receptor antagonist (IL-1ra) expression using a pig to mouse model. About 2000 NPCCs, which were infected with both adenoviruses carrying CTLA4Ig and IL1-1ra genes (each 500 pfu/NPCC), were transplanted beneath the kidney capsule of diabetic BALB/c mice. Two days before transplantation, the recipient mice were either injected with (group C, n = 4; group D, n = 6) or without (group A, n = 7; group B, n = 9) 1 x 10(13) pfu/kg body weight of adenovirus carrying the CTLA4Ig gene. Mice in groups B and D received daily injections of NDGA (20 mg/kg body weight) subcutaneously for 4 weeks. Blood glucose levels less than 200 mg/dL were defined to be normoglycemic and the transplant termed as a functioning graft for the purpose of calculating mean graft function time (MFT). Four weeks posttransplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed to calculate the area under the curve (AUC). Blood glucose levels in groups C and D were significantly lower than groups A and B at 1, 2, and 3 weeks after transplantation. Graft MFT and AUC of IPGTT in group D were significantly different from those in groups A and B. Our data suggested that a high dosage of systemic expression of CTLA4Ig was effective to enhance xenograft survival and that in it was reinforced by a combination with the macrophage inhibitor NDGA.

Sodium 4-phenylbutyrate (4-SPB), an aromatic derivative of butyric acid, was examined to elucidate its effect on islet engraftment in a syngeneic transplantation model using C57BL/6 mice. Diabetic mice that received subrenal implantation of 150 islets on day 0 and oral administration of twice daily 4-SPB (500 mg/kg body weight) on days -2 through 28 displayed a significantly shorter duration of posttransplantation temporary hyperglycemia than diabetic mice that received islets in isotonic sodium chloride solution (NaCl), namely 16 +/- 2 (n = 12) vs 23 +/- 2 days (n = 7; P.05). Four weeks after transplantation, the insulin content (IC) of grafts from mice treated with islets and 4-SPB was substantially higher than that of grafts from mice treated with islets and NaCl, namely 2.59 +/- 0.37 (n = 8) vs 1.36 +/- 0.36 mug (n = 13; P.01). The IC of pancreatic remnants showed no significant difference between groups after 2 and 4 weeks of incubation. In vitro studies demonstrated that the net glucose-stimulated insulin secretion (GSIS) and the ratio of net GSIS to the IC of islets cultured with 4-SPB (1 mM) did not differ significantly from those cultured with NaCl. The lipopolysaccharide-stimulated secretions of IL-1beta, IL-10, and IFNgamma from peritoneal exudate monocytes were significantly reduced by co-incubation with 4-SPB (1 mM). In conclusion, our data suggest that daily administration of 4-SPB reduces primary nonfunction and enhances islet engraftment in a syngeneic mouse transplantation model.

Successful human islet transplantation has led to insulin independence in type 1 diabetes. Dogs constitute an animal model for preclinical studies. We present our recent experience in canine islet isolation, cryopreservation and transplantation.Twenty-seven pancreases from mongrel dogs, weighing 9-31 kg, were removed. Each pancreas was digested with collagenase, and then purified by density gradients. Islet number and purity were counted, and the viability of isolated islets was assessed in vitro by static incubation, perifusion study and in vivo transplantation into nondiabetic or diabetic nude mice. Additionally. freshly isolated islets were cryopreserved for 1 week, and then studied in vitro.The islet yield and purity were 121,000 +/- 135,000 IEQ per pancreas and 81.4 +/- 1.2%, respectively. The stimulation index (insulin release in 300 mg/dl glucose/insulin release in 100 mg/dl glucose) of the isolated islets was 6.6 +/- 1.9 (N = 7), and first and second phases of insulin secretion were demonstrated during perifusion study. After 1-week cryopreservation, the islet number decreased from 1,000 to 540 (N = 1) and insulin content decreased from 50.95 to 39.23 microg/150 islets (N = 1). These islets maintained their insulin response to high glucose. Four weeks after transplantation, the grafts showed abundant beta-cells and significant insulin content. Normoglycemia was achieved in 14 of 23 diabetic recipients after transplantation with 2,000 freshly isolated islets.Canine islets isolated at our laboratory were viable and maintained their physiological function both in vitro and in vivo.

To study the effectiveness of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), in the reduction of primary nonfunction, an insufficient number of syngeneic islets were transplanted underneath the renal capsule with NDGA administered daily for 4 weeks. After transplantation of the 150 islets, the decrement of blood glucose levels was significantly faster in the mice that had received NDGA than in the mice that had received no drug at all or dimethyl sulfoxide (DMSO) (p < 0.005, p < 0.05). The mean duration of temporary posttransplant hyperglycemia was 22.3 ± 3.2 (n = 10), 35.9 ± 2.3 (n = 14), and 33.7 ± 4.1 (n = 6) days for the respective groups. The diabetic mice that received 300 islets had their blood glucose levels decrease faster than those that received 150 islets (19.7 ± 1.6 vs. 35.9 ± 2.3 days, n = 14, p < 0.0001). There was no significant difference in the blood glucose reducing effect between the mice that received 150 islets with NDGA and the mice that received 300 islets [22.3 ± 3.2 (n = 10) vs. 19.7 ± 1.6 (n = 14) days, p > 0.05]. The insulin content of the graft from the mice treated with 150 islets and NDGA (3.02 ± 0.24 μg, n = 4) was higher than that from the mice that received 150 islets but no treatment (1.10 ± 0.26 μg, n = 15, p < 0.005) or that had been treated with DMSO (1.21 ± 0.30 μg, n = 4, p < 0.05). The insulin content of the pancreas remnant had no significant differences among the three groups. The net glucose-stimulated insulin secretion was 0.82 ± 0.14 vs. 0.20 ± 0.10 μIU/islet × 60 min (n = 8, p < 0.005) and 0.59 ± 0.08 vs. 0.04 ± 0.02 μIU/islet × 60 min (n = 8, p < 0.0001) for islets cultured without NDGA vs. with NDGA at 1 and 2 weeks, respectively. However, the insulin content of the cultured islets was similar between the two groups for up to 2 weeks of incubation (at 1 week: 0.71 ± 0.01 vs. 0.67 ± 0.04 ng/islet, n = 8, p > 0.05; at 2 weeks: 0.71 ± 0.02 vs. 0.80 ± 0.07 ng/islet, n = 8, p > 0.05). Serum leukotriene B4 (LTB4) concentrations before and between the fifth and seventh days after transplantation were determined. For diabetic mice that received 150 islets, serum LTB4 levels were 25,835 ± 3,335 and 27,631 ± 3,136 pg/ml (n = 4, p > 0.05). For diabetic mice that received 150 islets and NDGA, the corresponding figures were 22,401 ± 2,706 pg/ml and 27,530 ± 2,190 pg/ml (n = 8, p > 0.05). The graft histology revealed viable islet cells and networks of close vascular structures around the islets and did not reveal microscopic differences among the samples of all four groups. In conclusion, our data revealed that daily administration of NDGA for 4 weeks enhanced isoislet engraftment and preserved three times more mass of the islet beta cells in the isografts. This result indicates that NDGA reduces primary nonfunction of islet syngeneic grafts in diabetic mice.

SEVERAL bioartificial liver researchers adopt C3A/ HepG2 hepatoma cell line in their designs. Advantages of using this cell line in contrast to rat or porcine hepatocyte primary cells include stability, ease of proliferation, and possibility of long-term maintenance. Although some researchers have carried out comparative studies of C3A/HepG2 with other types of hepatocytes, there are no comprehensive surveys of the characteristics of C3A/ HepG2. Besides, to get a quantitative picture of threedimensional growth of hepatocyte, it is important to first realize the cell growth, toxin production and proteins synthesis kinetics in two-dimensional growth condition. In this study, we analyze the kinetics of concentration changes of ammonia (a key toxin of metabolism and a clinical index of liver detoxification function), albumin, and alpha-fetoprotein (a survival evidence of the hepatocyte) in the culture media, followed by finding out the relationship between cell density and those anabolites by using a linear type expression.

The aromatic fatty acid phenylbutyrate (PB) induces cytostasis, differentiation, and apoptosis in primary myeloid leukemic cells at clinically achievable concentrations. In the present study, we have investigated the structural and cellular basis for PB-induced cytostasis, using the ML-1 human myeloid leukemia cell line as a model system. PB induced a dose-dependent increase in cells in G1 with a corresponding decrease in cells in S-phase of the cell cycle. At comparable doses, PB induced expression of CD11b, indicating myeloid differentiation. At higher doses, the drug induced apoptosis. The antitumor activity was independent of the aromatic ring, as butyric acid (BA) was of equal or greater potency at producing these biological changes. In contrast, shortening of the fatty acid carbon chain length, as demonstrated with phenylacetate (PA), significantly diminished drug potency. Consistent with their effects on cell cycle, PB and BA, but not PA, induced the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1), and led to the appearance of hypophosphorylated Rb, suggesting a role for p21(WAF1/CIP1) in PB-induced cytostasis. Therefore, it appears that the fatty acid moiety of PB, rather than its aromatic ring, is critical for its activity in myeloid leukemic cells. These data provide a potential mechanistic basis for the increased potency of PB over PA previously demonstrated in primary leukemic samples, and support the further clinical development of PB in the treatment of hematologic malignancies.

Inspired by the idea of “artificial cell” proposed by Dr T.M. Chang 30 years ago, Dr A.M. Sun's group developed alginate-poly- l -lysine-alginate (A-P-A) microcapsules in 1980 to immunoisolate rat islet transplant which reversed hyperglycemia of streptozotocin-induced diabetic mice for a couple of weeks. Although reversal of hyperglycemia in various diabetic animal models and human subjects by A-P-A microencapsulated islets have been reported since 1980, the graft function was found to be variable in degree and duration. In 1994, Dr Soon-Shiong and colleagues presented a case of insulin-dependent diabetes (IDDM) who received a total of 960,000 A-P-A microencapsulated human islets intraperitoneally had a period, although for only 2 months, without the heed of exogenous insulin. We intend to identify and correct adverse factors that reduce graft survival of A-P-A microencapsulated islets to maintain long-term normoglycemia of treated diabetic patients. The pathogenesis of graft rejection of A-P-A microencapsulated xenoislet is not clear. It was noted that pericapsular cellular infiltration of A-P-A microencapsulated xenoislet grafts occurred as early as 28 days after intraperitoneal implantation. Many researchers found that the majority of pericapsular cellular components were macrophages and fibroblasts. Pericapsular macrophages may secrete proinflammatory cytokines, such as interleukin-1β which may suppress islet beta-cells and result in graft dysfunction. The inflammatory reaction induced by A-P-A microencapsulated islets may suffocate the islets by exhausting oxygen. Many unidentified factors are possibly involved in degradating A-P-A microcapsules in vivo. It is interesting to note that macrophages secreted urokinase plasminogen activator in response to inflammation. One of the principal players of the fibrinolytic system is plasminogen which binds to the exposed lysyl residues in fibrin. Polylysine was found to mimic the cofactor (enhancing) function of fibrin in tissue activator-induced and low molecular weight urokinase-induced plasminogen activation. Since A-P-A microcapsules were formed by polycondensation of poly- l -lysine and alginate, it contained terminal and interchain lysine residues on their surface. The exposed lysine residues of A-P-A microcapsule confer binding sites for plasminogen and its activators. If the membrane-bound tissue activator is activated for plasminogen activation, the catalytic domain of plasmin will cleave poly- l -lysine and disintegrate capsular membrane. Therefore, we hypothesize that the plasminogen-plasmin fibrinolytic system is involved in degradation of A-P-A microcapsules. To verify this hypothesis, we studied the lytic activity of the plasminogen-plasmin fibrinolytic system on the physical durability of A-P-A microcapsules in vitro.

This study examined whether treating donor mice with a single-dose of cobalt protoporphyrin (CoPP) could induce heme oxygenase-1 (HO-1) and thus protect islet cells from suppression by high-dose glucocorticoid. Islets were isolated from mice receiving either a single dose of CoPP (20 mg/kg body weight) (CoPP-islets) or isotonic sodium chloride solution (control islets) at 24 hours before isolation. Following incubation in the absence or presence of methylprednisolone (100 and 1000 ng/mL) for 24 hours, glucose-stimulated insulin secretion and insulin content of cultured islets were determined. Data were expressed as the mean ± standard error. HO-1 protein level of CoPP-islets was significantly higher than that of normal islets at 12 hours (P

To study the effect of treatment with cobalt-protoporphyrin (CoPP) for the induction of the heme oxygenase-1 (HO-1) enzyme on islet engraftment donor mice received either a single intraperitoneal injection of CoPP (20 mg/kg body weight) 1 day prior to islet isolation or this injection plus a 9 day posttransplantation course of Copp. After a single injection of CoPP, the CoPP-induced islets contained higher HO-1 proteins than did the normal islets both at 12 (5.3 +/- 1.5 vs 0.1 +/- 0.1 ng/mg protein, P.01) and at 30 hours (6.8 +/- 2.1 vs 0.4 +/- 0.3 ng/mg protein, P.05), but not at 56 hours (1.9 +/- 0.8 vs 1.6 +/- 0.8 ng/mg protein, P.05). In contrast, diabetic mice that received 75 CoPP-induced islets and a 9-day CoPP injection course posttransplantation showed better improvement in blood glucose levels and body weights than did the mice that only received CoPP-induced islets. Mice of both CoPP-treated groups displayed better improvement in glycemic control than mice that received control islets. At 8 weeks after transplantation, the insulin content of grafts from both CoPP groups was significantly higher than that in the control group. In conclusion, CoPP treatment for the induction of HO-1 enhances engraftment of islets in a syngeneic murine transplantation model.

This study examined whether treating donor mice with a single dose of cobalt protoporphyrin (CoPP) induced heme oxygenase-1 (HO-1) and protected islet cells from interleukin-1 beta (IL-1 beta) suppression. Islets were isolated from mice receiving a single dose of either CoPP (20 mg/kg of body weight, CoPP islets) or isotonic NaCl solution vehicle (control islets), 24 hours before isolation. Glucose-stimulated insulin secretion (GSIS) and insulin content (IC) of the islets were determined following incubation in the presence versus absence of murine IL-1 beta for 21 or 65 hours. The HO-1 protein level of CoPP-induced islets, as determined by an enzyme immunoassay, was significantly higher than that of control islets at 12 hours (P.01) and 30 hours (P.05), and returned to basal levels at 56 hours (P = NS). Following a 21-hour incubation with IL-1 beta, CoPP islets secreted significantly more insulin upon glucose stimulation and preserved significantly more IC than control islets. After 65-hour incubation with IL-1 beta, CoPP islets secreted significantly less insulin upon glucose stimulation than control islets and preserved significantly less IC compared to islets incubated without IL-1 beta. In conclusion, treatment with cobalt-protoporphyrin to induce heme oxygenase-1 protects islets against the suppressive effects of IL-1 beta.

Background Clozapine, an atypical antipsychotic drug, induces derangements in glucose homeostasis in certain patients. This study investigated the mechanisms of clozapine-induced beta-cell toxicity. Methods Fifty-two healthy C57BL/6 male mice were randomized into 4 groups to study the effects of clozapine (group C, D) and a high-fat diet (group B, D). Three mice from each group were randomly selected to determine the amount of food intake on days 8-10, and their pancreases were removed for histological examination on day 11. The remaining 10 mice in each group were sacrificed at the 8th week to measure pancreatic insulin content (PIC). Results Mice given clozapine for 8 weeks demonstrated trends of lower PIC. The histological examination of the pancreases retrieved on day 11 already revealed apoptotic changes and suppression of cell proliferation. Although mice fed high-fat chow gained weight, mice given both clozapine and a high-fat diet showed less weight gain and more severe histological deterioration, and had the lowest PIC levels of the 4 groups. Conclusion Pancreatic beta-cell apoptosis, suppression of cell proliferation, and trends of reduction in pancreatic insulin content were observed in mice taking clozapine. The findings of clozapine induced beta-cell toxicity were further aggravated when mice were concomitantly fed a high-fat diet.