Combined Small Interfering RNA Therapy and In Vivo Magnetic Resonance Imaging in Islet Transplantation

OBJECTIVE Recent advances in human islet transplantation are hampered by significant graft loss shortly after transplantation and inability to follow islet fate directly. Both issues were addressed by utilizing a dual-purpose therapy/imaging small interfering RNA (siRNA)-nanoparticle probe targeting apoptotic-related gene caspase-3. We expect that treatment with the probe would result in significantly better survival of transplanted islets, which could be monitored by in vivo magnetic resonance imaging (MRI). RESEARCH DESIGN AND METHODS We synthesized a probe consisting of therapeutic (siRNA to human caspase-3) and imaging (magnetic iron oxide nanoparticles, MN) moieties. In vitro testing of the probe included serum starvation of the islets followed by treatment with the probe. Caspase-3 gene silencing and protein expression were determined by RT-PCR and Western blot, respectively. In vivo studies included serial MRI of NOD-SCID mice transplanted with MN-small interfering (si)Caspase-3–labeled human islets under the left kidney capsule and MN-treated islets under the right kidney capsule. RESULTS Treatment with MN-siCaspase-3 probe resulted in decrease of mRNA and protein expression in serum-starved islets compared with controls. In vivo MRI showed that there were significant differences in the relative volume change between MN-siCaspase-3–treated grafts and MN-labeled grafts. Histology revealed decreased caspase-3 expression and cell apoptosis in MN-siCaspase-3–treated grafts compared with the control side. CONCLUSIONS Our data show the feasibility of combining siRNA therapy and in vivo monitoring of transplanted islets in mice. We observed a protective effect of MN-siCaspase-3 in treated islets both in vitro and in vivo. This study could potentially aid in increasing the success of clinical islet transplantation

Distinct gene expression pathways in islets from individuals with short‐ and long‐duration type 1 diabetes

Aims Our current understanding of the pathogenesis of type 1 diabetes (T1D) arose, in large part, from studies using the non‐obese diabetic (NOD) mouse model. In the present study, we chose a human‐focused method to investigate T1D disease mechanisms and potential targets for therapeutic intervention by directly analysing human donor pancreatic islets from individuals with T1D. Materials and Methods We obtained islets from a young individual with T1D for 3 years and from an older individual with T1D for 27 years and performed unbiased functional genomic analysis by high‐depth RNA sequencing; the T1D islets were compared with islets isolated from 3 non‐diabetic donors. Results The islets procured from these T1D donors represent a unique opportunity to identify gene expression changes in islets after significantly different disease duration. Data analysis identified several inflammatory pathways up‐regulated in short‐duration disease, which notably included many components of innate immunity. As proof of concept for translation, one of the pathways, governed by IL‐23(p19), was selected for further study in NOD mice because of ongoing human trials of biologics against this target for different indications. A mouse monoclonal antibody directed against IL‐23(p19) when administered to NOD mice resulted in a significant reduction in incidence of diabetes. Conclusion While the sample size for this study is small, our data demonstrate that the direct analysis of human islets provides a greater understanding of human disease. These data, together with the analysis of an expanded cohort to be obtained by future collaborative efforts, might result in the identification of promising novel targets for translation into effective therapeutic interventions for human T1D, with the added benefit of repurposing known biologicals for use in different indications

Treatment With Recombinant Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Alleviates the Severity of Streptozotocin-Induced Diabetes

OBJECTIVE: To evaluate the potential therapeutic effect of recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) treatment in a model of type 1 diabetes. RESEARCH DESIGN AND METHODS: Recombinant TRAIL was added in vitro to primary human and mouse peripheral blood mononuclear cells (PBMCs) and isolated human islets to evaluate the expression of the immunoregulatory gene SOCS1. Diabetes was induced by five consecutive daily injections of low-concentration (50 mg/kg) streptozotocin (STZ) in C57 black mice (n = 24). A group of these mice (n = 12) was co-injected with recombinant TRAIL (20 microg/day) for 5 days, and the diabetic status (glycemia and body weight) was followed over time. After 6 weeks, circulating levels of insulin, TNF-alpha, and osteoprotegerin (OPG) were measured, and animals were killed to perform the histological analysis of the pancreas. RESULTS: The in vitro exposure of both PBMCs and human islets to recombinant TRAIL significantly upregulated the expression of SOCS1. With respect to STZ-treated animals, mice co-injected with STZ+TRAIL were characterized by 1) lower levels of hyperglycemia, 2) higher levels of body weight and insulinemia, 3) a partial preservation of pancreatic islets with normal morphology, and 4) a lower expression of both systemic (TNF-alpha and OPG) and pancreatic (vascular cell adhesion molecule [VCAM]-1) inflammatory markers. CONCLUSIONS: Overall, these data demonstrate that the administration of recombinant TRAIL ameliorates the severity of STZ-induced type 1 diabetes, and this effect was accompanied by the upregulation of SOCS1 expressio

Inhibition of Th17 Cells Regulates Autoimmune Diabetes in NOD Mice

OBJECTIVE: The T helper 17 (Th17) population, a subset of CD4-positive T-cells that secrete interleukin (IL)-17, has been implicated in autoimmune diseases, including multiple sclerosis and lupus. Therapeutic agents that target the Th17 effector molecule IL-17 or directly inhibit the Th17 population (IL-25) have shown promise in animal models of autoimmunity. The role of Th17 cells in type 1 diabetes has been less clear. The effect of neutralizing anti-IL-17 and recombinant IL-25 on the development of diabetes in NOD mice, a model of spontaneous autoimmune diabetes, was investigated in this study. RESEARCH DESIGN AND METHODS AND RESULTS: Although treatment with either anti-IL-17 or IL-25 had no effect on diabetes development in young (<5 weeks) NOD mice, either intervention prevented diabetes when treatment was started at 10 weeks of age (P < 0.001). Insulitis scoring and immunofluorescence staining revealed that both anti-IL-17 and IL-25 significantly reduced peri-islet T-cell infiltrates. Both treatments also decreased GAD65 autoantibody levels. Analysis of pancreatic lymph nodes revealed that both treatments increased the frequency of regulatory T-cells. Further investigation demonstrated that IL-25 therapy was superior to anti-IL-17 during mature diabetes because it promoted a period of remission from new-onset diabetes in 90% of treated animals. Similarly, IL-25 delayed recurrent autoimmunity after syngeneic islet transplantation, whereas anti-IL-17 was of no benefit. GAD65-specific ELISpot and CD4-positive adoptive transfer studies showed that IL-25 treatment resulted in a T-cell-mediated dominant protective effect against autoimmunity. CONCLUSIONS: These studies suggest that Th17 cells are involved in the pathogenesis of autoimmune diabetes. Further development of Th17-targeted therapeutic agents may be of benefit in this disease

Immune Cell and Cytokine Patterns in Children with Type 1 Diabetes Mellitus Undergoing a Remission Phase: A Longitudinal Study

Objective: Type 1 diabetes (T1D) develops in distinct stages, before and after disease onset. Whether the natural course translates into different immunologic patterns is still uncertain. This study aimed at identifying peripheral immune patterns at key time-points, in T1D children undergoing remission phase. Methods: Children with new-onset T1D and healthy age and gender-matched controls were recruited at a pediatric hospital. Peripheral blood samples were evaluated by flow cytometry at 3 longitudinal time-points: onset (T1), remission phase (T2) and established disease (T3). Cytokine levels were quantified by multiplex assay. Fasting C-peptide, HbA1c, and 25OHD were also measured. Results: T1D children (n = 28; 10.0 ± 2.6 years) showed significant differences from controls in circulating neutrophils, T helper (Th)17 and natural killer (NK) cells, with relevant variations during disease progression. At onset, neutrophils, NK, Th17 and T cytotoxic (Tc)17 cells were decreased. As disease progressed, neutrophil counts recovered whereas NK counts remained low. Th17 and Tc17 cells behavior followed the neutrophil variation pattern. B-cells were lowest in the remission phase and regulatory T-cells significantly declined after remission. Two cytokine response profiles were identified. Low cytokine-responders showed higher circulating fasting C-peptide levels at onset and longer remission periods. C-peptide inversely correlated with pro-inflammatory and cytotoxic cells. Conclusions: Our data suggest an association between immune cells, cytokine patterns and metabolic counterparts. The dynamic changes of circulating immune cells during disease progression involve key innate and acquired immune cell types. This longitudinal picture of T1D progression may enable disease staging and patient stratification, essential for individualized treatment.info:eu-repo/semantics/publishedVersio

The Presence of IL-17A and T Helper 17 Cells in Experimental Mouse Brain Tumors and Human Glioma

Background: Recently, CD4 + IL-17A + T helper 17 (Th17) cells were identified and reported in several diseased states, including autoimmunity, infection and various peripheral nervous system tumors. However, the presence of Th17 in gliaderived tumors of the central nervous system has not been studied. Methodology/Principal Findings: In this report, we demonstrate that mRNA expression for the Th17 cell cytokine IL-17A, as well as Th17 cells, are present in human glioma. The mRNA expression for IL-17A in glioma was recapitulated in an immunocompetent mouse model of malignant glioma. Furthermore, the presence of Th17 cells was confirmed in both human and mouse glioma. Interestingly, some Th17 cells present in mouse glioma co-expressed the Th1 and Th2 lineage markers, IFN-c and IL-4, respectively, but predominantly co-expressed the Treg lineage marker FoxP3. Conclusions: These data confirm the presence of Th17 cells in glia-derived CNS tumors and provide the rationale for further investigation into the role of Th17 cells in malignant glioma

Influence of microenvironment on engraftment of transplanted β-cells

Pancreatic islet transplantation into the liver provides a possibility to treat selected patients with brittle type 1 diabetes mellitus. However, massive early β-cell death increases the number of islets needed to restore glucose homeostasis. Moreover, late dysfunction and death contribute to the poor long-term results of islet transplantation on insulin independence. Studies in recent years have identified early and late challenges for transplanted pancreatic islets, including an instant blood-mediated inflammatory reaction when exposing human islets to the blood microenvironment in the portal vein and the low oxygenated milieu of islets transplanted into the liver. Poor revascularization of remaining intact islets combined with severe changes in the gene expression of islets transplanted into the liver contributes to late dysfunction. Strategies to overcome these hurdles have been developed, and some of these interventions are now even tested in clinical trials providing a hope to improve results in clinical islet transplantation. In parallel, experimental and clinical studies have, based on the identified problems with the liver site, evaluated the possibility of change of implantation organ in order to improve the results. Site-specific differences clearly exist in the engraftment of transplanted islets, and a more thorough characterization of alternative locations is needed. New strategies with modifications of islet microenvironment with cells and growth factors adhered to the islet surface or in a surrounding matrix could be designed to intervene with site-specific hurdles and provide possibilities to improve future results of islet transplantation

β-Cells with Relative Low HIMP1 Overexpression Levels in a Transgenic Mouse Line Enhance Basal Insulin Production and Hypoxia/Hypoglycemia Tolerance

Rodent pancreatic β-cells that naturally lack hypoglycemia/hypoxia inducible mitochondrial protein 1 (HIMP1) are susceptible to hypoglycemia and hypoxia influences. A linkage between the hypoglycemia/hypoxia susceptibility and the lack of HIMP1 is suggested in a recent study using transformed β-cells lines. To further illuminate this linkage, we applied mouse insulin 1 gene promoter (MIP) to control HIMP1-a isoform cDNA and have generated three lines (L1 to L3) of heterozygous HIMP1 transgenic (Tg) mice by breeding of three founders with C57BL/6J mice. In HIMP1-Tg mice/islets, we performed quantitative polymerase chain reaction (PCR), immunoblot, histology, and physiology studies to investigate HIMP1 overexpression and its link to β-cell function/survival and body glucose homeostasis. We found that the HIMP1 level increased steadily in β-cells of L1 to L3 heterozygous HIMP1-Tg mice. HIMP1 overexpression at relatively lower levels in L1 heterozygotes results in a negligible decline in blood glucose concentrations and an insignificant elevation in blood insulin levels, while HIMP1 overexpression at higher levels are toxic, causing hyperglycemia in L2/3 heterozygotes. Follow-up studies in 5–30-week-old L1 heterozygous mice/islets found that HIMP1 overexpression at relatively lower levels in β-cells has enhanced basal insulin biosynthesis, basal insulin secretion, and tolerances to low oxygen/glucose influences. The findings enforced the linkage between the hypoglycemia/hypoxia susceptibility and the lack of HIMP1 in β-cells, and show a potential value of HIMP1 overexpression at relatively lower levels in modulating β-cell function and survival

Impact of Dietary Gluten on Regulatory T Cells and Th17 Cells in BALB/c Mice

Dietary gluten influences the development of type 1 diabetes (T1D) and a gluten-free (GF) diet has a protective effect on the development of T1D. Gluten may influence T1D due to its direct effect on intestinal immunity; however, these mechanisms have not been adequately studied. We studied the effect of a GF diet compared to a gluten-containing standard (STD) diet on selected T cell subsets, associated with regulatory functions as well as proinflammatory Th17 cells, in BALB/c mice. Furthermore, we assessed diet-induced changes in the expression of various T cell markers, and determined if changes were confined to intestinal or non-intestinal lymphoid compartments. The gluten-containing STD diet led to a significantly decreased proportion of γδ T cells in all lymphoid compartments studied, although an increase was detected in some γδ T cell subsets (CD8+, CD103+). Further, it decreased the proportion of CD4+CD62L+ T cells in Peyer's patches. Interestingly, no diet-induced changes were found among CD4+Foxp3+ T cells or CD3+CD49b+cells (NKT cells) and CD3−CD49b+ (NK) cells. Mice fed the STD diet showed increased proportions of CD4+CD45RBhigh+ and CD103+ T cells and a lower proportion of CD4+CD45RBlow+ T cells in both mucosal and non-mucosal compartments. The Th17 cell population, associated with the development of autoimmunity, was substantially increased in pancreatic lymph nodes of mice fed the STD diet. Collectively, our data indicate that dietary gluten influences multiple regulatory T cell subsets as well as Th17 cells in mucosal lymphoid tissue while fewer differences were observed in non-mucosal lymphoid compartments

Decreased Numbers of Blood Dendritic Cells and Defective Function of Regulatory T Cells in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

BACKGROUND: Dendritic cells (DC) and regulatory cells (Treg) play pivotal roles in controlling both normal and autoimmune adaptive immune responses. DC are the main antigen-presenting cells to T cells, and they also control Treg functions. In this study, we examined the frequency and phenotype of DC subsets, and the frequency and function of Treg from patients with ANCA-associated vasculitis (AAV). METHODOLOGY/PRINCIPAL FINDINGS: Blood samples from 19 untreated patients with AAV during flares and before any immunosuppressive treatment were analyzed, along with 15 AAV patients in remission and 18 age-matched healthy controls. DC and Treg numbers, and phenotypes were assessed by flow cytometry, and in vitro suppressive function of Treg was determined by co-culture assay. When compared to healthy volunteers, absolute numbers of conventional and plasmacytoid DC were decreased in AAV patients. During the acute phase this decrease was significantly more pronounced and was associated with an increased DC expression of CD62L. Absolute numbers of Treg (CD4(+)CD25(high)CD127(low/-) Tcells) were moderately decreased in patients. FOXP3 and CD39 were expressed at similar levels on Treg from patients as compared to controls. The suppressive function of Treg from AAV patients was dramatically decreased as compared to controls, and this defect was more pronounced during flares than remission. This Treg functional deficiency occurred in the absence of obvious Th17 deviation. CONCLUSION: In conclusion, these data show that AAV flares are associated with both a decrease number and altered phenotype of circulating DC and point to a role for Treg functional deficiency in the pathogenesis of AAV