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2.  C-Abl Inhibitor Imatinib Enhances Insulin Production by β Cells: C-Abl Negatively Regulates Insulin Production via Interfering with the Expression of NKx2.2 and GLUT-2 
PLoS ONE  2014;9(5):e97694.
Chronic myelogenous leukemia patients treated with tyrosine kinase inhibitor, Imatinib, were shown to have increased serum levels of C-peptide. Imatinib specifically inhibits the tyrosine kinase, c-Abl. However, the mechanism of how Imatinib treatment can lead to increased insulin level is unclear. Specifically, there is little investigation into whether Imatinib directly affects β cells to promote insulin production. In this study, we showed that Imatinib significantly induced insulin expression in both glucose-stimulated and resting β cells. In line with this finding, c-Abl knockdown by siRNA and overexpression of c-Abl markedly enhanced and inhibited insulin expression in β cells, respectively. Unexpectedly, high concentrations of glucose significantly induced c-Abl expression, suggesting c-Abl may play a role in balancing insulin production during glucose stimulation. Further studies demonstrated that c-Abl inhibition did not affect the major insulin gene transcription factor, pancreatic and duodenal homeobox-1 (PDX-1) expression. Of interest, inhibition of c-Abl enhanced NKx2.2 and overexpression of c-Abl in β cells markedly down-regulated NKx2.2, which is a positive regulator for insulin gene expression. Additionally, we found that c-Abl inhibition significantly enhanced the expression of glucose transporter GLUT2 on β cells. Our study demonstrates a previously unrecognized mechanism that controls insulin expression through c-Abl-regulated NKx2.2 and GLUT2. Therapeutic targeting β cell c-Abl could be employed in the treatment of diabetes or β cell tumor, insulinoma.
PMCID: PMC4023982  PMID: 24835010
3.  University of Florida and Shands Hospital Personalized Medicine Program: clinical implementation of pharmacogenetics 
Pharmacogenomics  2013;14(7):723-726.
The University of Florida and Shands Hospital recently launched a genomic medicine program focused on the clinical implementation of pharmacogenetics called the Personalized Medicine Program. We focus on a preemptive, chip-based genotyping approach that is cost effective, while providing experience that will be useful as genomic medicine moves towards genome sequence data for patients becoming available. The Personalized Medicine Program includes a regulatory body that is responsible for ensuring that evidence-based examples are moved to clinical implementation, and relies on clinical decision support tools to provide healthcare providers with guidance on use of the genetic information. The pilot implementation was with CYP2C19-clopidogrel and future plans include expansion to additional pharmacogenetic examples, along with aiding in implementation in other health systems across Florida.
PMCID: PMC3684075  PMID: 23651020
4.  Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes 
Journal of Immunology Research  2014;2014:326708.
Anti-CD3 antibody has been employed for various immune-mediated disorders. However, whether anti-CD3 administration leads to rapid metabolic alternation has not been well investigated. In the current study, we studied how anti-CD3 treatment affected blood glucose levels in mice. We found that anti-CD3 treatment induced immediate reduction of blood glucose after administration. Furthermore, a single dose of anti-CD3 treatment corrected hyperglycemia in all nonobese diabetic mice with recently diagnosed diabetes. This glucose-lowering effect was not attributable to major T cell produced cytokines. Of interest, when tested in a normal strain of mice (C57BL/6), the serum levels of C-peptide in anti-CD3 treated animals were significantly lower than control mice. Paradoxically, anti-CD3 treated animals were highly tolerant to exogenous glucose challenge. Additionally, we found that anti-CD3 treatment significantly induced activation of T and B cells in vitro and in vivo. Further studies demonstrated that anti-CD3 treatment lowered the glucose levels in T cell culture media and increased the intracellular transportation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2 deoxyglucose (2-NBDG) particularly in activated T and B cells. In addition, injection of anti-CD3 antibodies induced enhanced levels of Glut1 expression in spleen cells. This study suggests that anti-CD3 therapy-induced hypoglycemia likely results from increased glucose transportation and consumption by the activated lymphocytes.
PMCID: PMC3987876  PMID: 24741590
5.  Autologous Umbilical Cord Blood Infusion followed by Oral Docosahexaenoic Acid and Vitamin D Supplementation for C-Peptide Preservation in Children with Type 1 Diabetes 
We sought to determine if autologous umbilical cord blood (UCB) infusion followed by 1 year of supplementation with vitamin D and docosahexaenoic acid (DHA) can preserve C-peptide in children with type 1 diabetes. We conducted an open-label, 2:1 randomized study in which 15 type 1 diabetes subjects with stimulated C-peptide > .2 pmol/mL received either (1) autologous UCB infusion, 1 year of daily oral vitamin D (2000 IU), and DHA (38 mg/kg) and intensive diabetes management or (2) intensive diabetes management alone. Primary analyses were performed 1 year after UCB infusion. Treated (N = 10) and control (N = 5) subjects had median ages of 7.2 and 6.6 years, respectively. No severe adverse events were observed. Although the absolute rate of C-peptide decline was slower in treated versus control subjects, intergroup comparisons failed to reach significance (P = .29). Area under the curve C-peptide declined and insulin use increased in both groups (P <.01). Vitamin D levels remained stable in treated subjects but declined in control subjects (P = .01). DHA levels rose in treated subjects versus control subjects (P = .003). CD4/CD8 ratio remained stable in treated subjects but declined in control subjects (P = .03). No changes were seen in regulatory T cell frequency, total CD4 counts, or autoantibody titers. Autologous UCB infusion followed by daily supplementation with vitamin D and DHA was safe but failed to preserve C-peptide. Lack of significance may reflect small sample size. Future efforts will require expansion of specific immunoregulatory cell subsets, optimization of combined immunoregulatory and anti-inflammatory agents, and larger study cohorts.
PMCID: PMC3852705  PMID: 23611977
Autologous cord blood; Type 1 diabetes; Vitamin D; Docosahexaenoic acid; C-Peptide
6.  Persistent STAT5 Phosphorylation and Epigenetic Dysregulation of GM-CSF and PGS2/COX2 Expression in Type 1 Diabetic Human Monocytes 
PLoS ONE  2013;8(10):e76919.
STAT5 proteins are adaptor proteins for histone acetylation enzymes. Histone acetylation at promoter and enhancer chromosomal regions opens the chromatin and allows access of transcription enzymes to specific genes in rapid response cell signals, such as in inflammation. Histone acetylation-mediated gene regulation is involved in expression of 2 key inflammatory response genes: CSF2, encoding granulocyte-macrophage colony stimulating factor (GM-CSF), and PTGS2, encoding prostaglandin synthase 2/cyclooxygenase 2 (PGS2/COX2). Prolonged CSF2 expression, high GM-CSF production, and GM-CSF activation of PTGS2 gene expression all are seen in type 1 diabetes (T1D) monocytes. Persistent phosphorylation activation of monocyte STAT5 (STAT5Ptyr) is also found in individuals with or at-risk for T1D. To examine whether elevated T1D monocyte STAT5Ptyr may be associated with aberrant inflammatory gene expression in T1D, blood monocytes from non-autoimmune controls and T1D patients were analyzed by flow cytometry for STAT5Ptyr activation, and by chromatin immuno-precipitation (ChIP) analyses for STAT5Ptyr’s ability to bind at CSF2 and PTGS2 regulatory sites in association with histone acetylation. In unstimulated monocytes, STAT5Ptyr was elevated in 59.65% of T1D, but only 2.44% of control subjects (p<0.0001). Increased STAT5Ptyr correlated with T1D disease duration (p = 0.0030, r2 = 0.0784). Unstimulated (p = 0.140) and GM-CSF-stimulated (p = 0.0485) T1D monocytes, had greater STAT5Ptyr binding to epigenetic regulatory sites upstream of CSF2 than control monocytes. Increased STAT5Ptyr binding in T1D monocytes was concurrent with binding at these sites of STAT6Ptyr (p = 0.0283), CBP/P300 histone acetylase, acetylated histones H3, SMRT/NCoR histone deacetylase (p = 0.0040), and RNA Polymerase II (p = 0.0040). Our study indicates that in T1D monocytes, STAT5Ptyr activation is significantly higher and that STAT5Ptyr is found bound to CSF2 promoter and PTGS2 enhancer regions coincident with histone acetylation and RNA polymerase II. These findings suggest that the persistent activation of STAT5 by GM-CSF may be involved in altering the epigenetic regulation of these inflammatory response genes in T1D monocytes.
PMCID: PMC3799903  PMID: 24204704
8.  Anti-thymocyte globulin (ATG) differentially depletes naïve and memory T cells and permits memory-type regulatory T cells in nonobese diabetic mice 
BMC Immunology  2012;13:70.
ATG has been employed to deplete T cells in several immune-mediated conditions. However, whether ATG administration affects naïve and memory T cell differently is largely unknown.
The context and purpose of the study
In this study, we assessed how murine ATG therapy affected T cell subsets in NOD mice, based on their regulatory and naïve or memory phenotype, as well as its influence on antigen-specific immune responses.
Peripheral blood CD4+ and CD8+ T cells post-ATG therapy declined to their lowest levels at day 3, while CD4+ T cells returned to normal levels more rapidly than CD8+ T cells. ATG therapy failed to eliminate antigen-primed T cells. CD4+ T cell responses post-ATG therapy skewed to T helper type 2 (Th2) and possibly IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) were less sensitive to ATG depletion and remained at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory T cell phenotype was significantly increased in ATG-treated animals.
ATG therapy may modulate antigen-specific immune responses through inducing memory-like regulatory T cells as well as other protective T cells such as Th2 and IL-10-producing Tr1 cells.
PMCID: PMC3547787  PMID: 23237483
Anti-thymocyte globulin; Naïve and memory T cells; Regulatory T cells; T helper cell; Autoimmune diabetes; Nonobese diabetic mouse
9.  Erythrocyte membrane omega-3 fatty acid levels and omega-3 fatty acid intake are not associated with conversion to type 1 diabetes in children with islet autoimmunity: The Diabetes Autoimmunity Study in the Young (DAISY) 
Pediatric diabetes  2011;12(8):669-675.
We investigated whether omega-3 fatty acid intake and erythrocyte membrane omega-3 fatty acid levels are associated with conversion to type 1 diabetes in children with islet autoimmunity (IA).
The Diabetes Autoimmunity Study in the Young is following children at increased genetic risk for type 1 diabetes for the development of persistent IA, as defined as being positive for glutamic acid decarboxylase 65, i, or insulin autoantibodies on two consecutive visits, and then for the development of type 1 diabetes, as diagnosed by a physician. One hundred and sixty-seven children with persistent IA were followed for a mean of 4.8 yr, and 45 of these developed type 1 diabetes at a mean age of 8.7 yr. Erythrocyte membrane fatty acids (as a percent of total lipid) and dietary fatty acid intake (estimated via food frequency questionnaire) were analyzed as time-varying covariates in proportional hazards survival analysis, with follow-up time starting at detection of the first autoantibody.
Neither dietary intake of omega-3 fatty acids nor omega-6 fatty acids were associated with conversion to type 1 diabetes, adjusting for human leukocyte antigen (HLA)-DR, family history of type 1 diabetes, age at first IA positivity, maternal age, maternal education, and maternal ethnicity. Adjusting for HLA-DR, family history of type 1 diabetes and age at first IA positivity, omega-3 and omega-6 fatty acid levels of erythrocyte membranes were not associated with conversion to type 1 diabetes.
In this observational study, omega-3 fatty acid intake and status are not associated with conversion to type 1 diabetes in children with IA.
PMCID: PMC3475955  PMID: 21435137
dietary intake; IA; omega-3 fatty acids; type 1 diabetes mellitus
10.  Reduced Serum Vitamin D–Binding Protein Levels Are Associated With Type 1 Diabetes 
Diabetes  2011;60(10):2566-2570.
Previous studies have noted a specific association between type 1 diabetes and insufficient levels of vitamin D, as well as polymorphisms within genes related to vitamin D pathways. Here, we examined whether serum levels or genotypes of the vitamin D–binding protein (VDBP), a molecule key to the biologic actions of vitamin D, specifically associate with the disorder.
A retrospective, cross-sectional analysis of VDBP levels used samples from 472 individuals of similar age and sex distribution, including 153 control subjects, 203 patients with type 1 diabetes, and 116 first-degree relatives of type 1 diabetic patients. Single nucleotide polymorphism (SNP) typing for VDBP polymorphisms (SNP rs4588 and rs7041) was performed on this cohort to determine potential genetic correlations. In addition, SNP analysis of a second sample set of banked DNA samples from 1,502 type 1 diabetic patients and 1,880 control subjects also was used to determine genotype frequencies.
Serum VDBP levels were highest in healthy control subjects (median 423.5 µg/mL [range 193.5–4,345.0; interquartile range 354.1–]586), intermediate in first-degree relatives (402.9 µg/mL [204.7–4,850.0; 329.6–492.4]), and lowest in type 1 diabetic patients (385.3 µg/mL [99.3–1,305.0; 328.3–473.0]; P = 0.003 vs. control subjects). VDBP levels did not associate with serum vitamin D levels, age, or disease duration. However, VDBP levels were, overall, lower in male subjects (374.7 µg/mL [188.9–1,602.0; 326.9–449.9]) than female subjects (433.4 µg/mL [99.3–4,850.0; 359.4–567.8]; P < 0.0001). It is noteworthy that no differences in genotype frequencies of the VDBP polymorphisms were associated with serum VDBP levels or between type 1 diabetic patients and control subjects.
Serum VDBP levels are decreased in those with type 1 diabetes. These studies suggest that multiple components in the metabolic pathway of vitamin D may be altered in type 1 diabetes and, collectively, have the potential to influence disease pathogenesis.
PMCID: PMC3178281  PMID: 21844098
11.  The Tyrphostin Agent AG490 Prevents and Reverses Type 1 Diabetes in NOD Mice 
PLoS ONE  2012;7(5):e36079.
Recent studies in the NOD (non-obese diabetic) mouse model of type 1 diabetes (T1D) support the notion that tyrosine kinase inhibitors have the potential for modulating disease development. However, the therapeutic effects of AG490 on the development of T1D are unknown.
Materials and Methods
Female NOD mice were treated with AG490 (i.p, 1 mg/mouse) or DMSO starting at either 4 or 8 week of age, for five consecutive week, then once per week for 5 additional week. Analyses for the development and/or reversal of diabetes, insulitis, adoptive transfer, and other mechanistic studies were performed.
AG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points). Monotherapy of newly diagnosed diabetic NOD mice with AG490 markedly resulted in disease remission in treated animals (n = 23) in comparision to the absolute inability (0%; 0/10, p = 0.003, Log-rank test) of DMSO and sustained eugluycemia was maintained for several months following drug withdrawal. Interestingly, adoptive transfer of splenocytes from AG490 treated NOD mice failed to transfer diabetes to recipient NOD.Scid mice. CD4 T-cells as well as bone marrow derived dendritic cells (BMDCs) from AG490 treated mice, showed higher expression of Foxp3 (p<0.004) and lower expression of co-stimulatory molecules, respectively. Screening of the mouse immune response gene arrary indicates that expression of costimulaotry molecule Ctla4 was upregulated in CD4+ T-cell in NOD mice treated with AG490, suggesting that AG490 is not a negative regulator of the immune system.
The use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease.
PMCID: PMC3351395  PMID: 22615750
12.  Multifunctional dendritic cell-targeting polymeric microparticles 
Human Vaccines  2011;7(1):37-44.
PMCID: PMC3679212  PMID: 21157186
vaccine; diabetes; dendritic cell; microparticle; microsphere; immunotherapy
13.  Erythrocyte Membrane Fatty Acid Content in Infants Consuming Formulas Supplemented with Docosahexaenoic Acid (DHA) and Arachidonic Acid (ARA): an Observational Study 
Maternal & child nutrition  2010;6(4):338-346.
In this observational study, we compared erythrocyte membrane fatty acids in infants consuming formula supplemented with docosahexaenoic acid (DHA) and arachidonic acid (ARA) with those consuming other types of milks. In 110 infants who were participants in a cohort study of otherwise healthy children at risk for developing type 1 diabetes, erythrocytes were collected at approximately 9 months of age, and fatty acid content was measured as a percent of total lipids. Parents reported the type of milk the infants consumed in the month of and prior to erythrocyte collection – infant formula supplemented with ARA and DHA (supplemented formula), formula with no ARA and DHA supplements (non-supplemented formula), breast-milk, or non-supplemented formula plus breast-milk. Membrane DHA (4.42 versus 1.79, p < 0.001) and omega-3 fatty acid (5.81 versus 3.43, p < 0.001) levels were higher in infants consuming supplemented versus non-supplemented formula. Omega-6 fatty acids were lower in infants consuming supplemented versus non-supplemented formula (26.32 versus 29.68, p = 0.023); ARA did not differ between groups. Infants given supplemented formula had higher DHA (4.42 versus 2.81, p < 0.001) and omega-3 fatty acids (5.81 versus 4.45, p = 0.008) than infants drinking breast-milk. In infants whose mothers did not receive any dietary advice, use of supplemented formula is associated with higher omega-3 and lower omega-6 fatty acid status.
PMCID: PMC2992442  PMID: 21050388
Arachidonic Acid; Docosahexaenoic Acid; Breastfeeding; Infant Feeding; Infant Formula; Infant Feeding Behavior
14.  Extracorporeal photopheresis-induced immune tolerance: a focus on modulation of antigen-presenting cells and induction of regulatory T cells by apoptotic cells 
Purpose of review
This review is intended to introduce recent advances in the research surrounding extracorporeal photopheresis (ECP) with a focus on how apoptotic cells modulate antigen-presenting cells and induce regulatory T cells, given that ECP therapy induces apoptosis of leukocytes collected through leukapheresis.
Recent findings
It has been suggested that ECP therapy, unlike other immunosuppressive regimens, does not cause global immunosuppression, but induces immune tolerance. Recent clinical and animal studies demonstrate that ECP therapy induces antigen-specific regulatory T cells, including CD4+CD25+FoxP3+ T cells and IL-10-producing Tr1 cells, that may arise secondarily to the induction of tolerogenic antigen-presenting cells (APCs) by infusion of apoptotic cells. It has also been suggested that ECP therapy may induce IL-10-producing regulatory B cells and regulatory CD8+ T cells. Finally, several recent studies, which examined the cellular elements involved in the uptake of apoptotic cells, demonstrated that apoptotic cells modulate APCs through binding to specific receptors, particularly TAM receptors that provide inhibitory signals that block APC activation.
ECP therapy induces immune tolerance through modulation of antigen-presenting cells as well as induction of regulatory T cells. ECP therapy has great potential in the management of allogeneic transplantation and autoimmune diseases.
PMCID: PMC2785117  PMID: 19444106
apoptosis; extracorporeal photopheresis; immune tolerance; regulatory T cell
15.  Comparison between omega-3 and omega-6 polyunsaturated fatty acid intakes as assessed by a food frequency questionnaire and erythrocyte membrane fatty acid composition in young children 
We conducted a dietary validation study in youth aged 1 to 11 years by comparing dietary intake of omega-3 and omega-6 polyunsaturated fatty acids (PUFA) as assessed by a parent-completed semi-quantitative food frequency questionnaire (FFQ) over time to erythrocyte membrane composition of the same fatty acids.
The study population included youth aged 1 to 11 years who were participants in the Diabetes Autoimmunity Study in the Young (DAISY), a longitudinal study in Denver, Colorado that is following a cohort of youth at risk for developing Type I diabetes. Four hundred four children who had erythrocyte membrane fatty acid data matched to an FFQ corresponding to the same time frame for a total of 917 visits (matches) were included. PUFA intake was expressed as both g/day (adjusted for total energy) and as percent of total fat intake. We used mixed models to test the association and calculate the correlation between the erythrocyte membrane estimates and PUFA intake using all records of data for each youth.
Intakes of total omega-3 fatty acids (β=0.52, p<0.0001, ρ=0.23) and marine PUFAs (β=1.62, p<0.0001, ρ=0.42), as a percent of total fat in the diet, were associated with percent of omega-3 and marine PUFAs in the erythrocyte membrane. Intakes of omega-6 PUFAs (β=0.04, p=0.418, ρ=0.05) and arachidonic acid (β=0.31, p=0.774, ρ=0.01) were not associated.
In these young children, a FFQ using parental report provided estimates of average long-term intakes of marine PUFAs that correlated well with their erythrocyte cell membrane fatty acid status.
PMCID: PMC2896066  PMID: 17440518
16.  Rabbit Polyclonal Anti-Mouse Thymocyte Globulin Administration Alters Dendritic Cell Profile and Function in NOD Mice to Suppress Diabetogenic Responses 
Anti-mouse thymocyte globulin (mATG) prevents, as well as reverses, type-1 diabetes (T1D) in NOD mice, through mechanisms involving modulation of the immunoregulatory activities of T lymphocytes. Dendritic cells (DC) play a pivotal role in the generation of T cell responses, including those relevant to the autoreactive T cells enabling T1D. As antibodies against DC are likely generated during production of mATG, we examined the impact of this preparation on the phenotype and function of DC in order to elucidate novel mechanism(s) underlying its beneficial activities. In vivo, mATG treatment transiently induced the trafficking of mature CD8− predominant DC into the pancreatic lymph node of NOD mice. Splenic DC from mATG treated mice also exhibited a more mature phenotype characterized by reduced CD8 expression and increased IL-10 production. The resultant DC possessed a potent capacity to induce Th2 responses when cultured ex vivo with diabetogenic CD4+ T cells obtained from BDC2.5 TCR transgenic mice. Co-transfer of these Th2 deviated CD4+ T cells with splenic cells from newly diabetic NOD mice into NOD.RAG−/− mice significantly delayed the onset of diabetes. These studies suggest the alteration of DC profile and function by mATG may skew the Th1/Th2 balance in vivo and through such actions, represent an additional novel mechanism by which this agent provides its beneficial activities.
PMCID: PMC2770841  PMID: 19342635
Dendritic cells; Antibodies; Diabetes; Rodent; Th1/Th2 Cells
17.  Apoptotic Non–β Cells Suppress β Cell Antigen–Reactive T Cells and Induce β Cell Antigen–Specific Regulatory T Cells: Transfusion of Apoptotic Splenic Stromal Cells Prevents Type 1 Diabetes 
Steady-state cell apoptosis plays an important role in maintenance of self-tolerance. Based on this notion, the use of apoptotic cells to restore self-tolerance to β cell antigens is a rational approach to type 1 diabetes (T1D) prevention. Our previous study demonstrated that transfusion of apoptotic β cells induced immune tolerance to β cell antigens in NOD mice. However, concerned about the limited β cell source for future clinical applications, we attempted in the present study to develop a more practical approach for T1D prevention using apoptotic non–β cells. We found that UVB-irradiation-induced apoptotic NOD splenic stromal cells significantly suppressed β cell antigen-specific T cell proliferation in vitro and in vivo. Furthermore, TCR-transgenic CD4+ T cells primed by the antigens to which they were specific in the presence of UVB-irradiated stromal cells were rendered unresponsive to the antigen restimulation, a result that was partially attributed to the induced IL-10-producing regulatory T cells. Of more interest, transfusion of UVB-irradiated stromal cells appeared to induce β cell antigen–responding IL-10-producing regulatory T cells in vivo. Most importantly, transfusion of UVB-irradiated stromal cells effectively prevented T1D in NOD mice, which is consistent with these findings. This study suggests that it is possible to use apoptotic non–β cells such as peripheral blood mononuclear cells to induce β cell antigen–specific tolerance, thereby preventing T1D in humans.
PMCID: PMC2785112  PMID: 19120288
type 1 diabetes; apoptosis; immune tolerance; regulatory T cells
18.  Infusion of UVB-treated splenic stromal cells induces suppression of β cell antigen-specific T cell responses in NOD mice 
Journal of autoimmunity  2008;30(4):283-292.
Our previous study has demonstrated that transfusion of UVB-irradiation-induced apoptotic β cells effectively prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the limitation of β cell source would preclude the clinical application of this approach. Therefore, in the present study, we have attempted to establish a more practical approach by utilizing apoptotic non-β cells to prevent T1D. We find that apoptotic splenic stromal cells significantly suppress β cell antigen-reactive T cell proliferation in vitro and in vivo. Moreover, β cell antigen-specific T cells primed by β cell antigens in the presence of apoptotic stromal cells have markedly reduced responsiveness to the re-stimulation of the same β cell antigen. We also find that β cell antigen-specific IL-10-producing CD4+ T cells are induced in the presence of apoptotic splenic stromal cells. As expected, transfusion of apoptotic stromal cells effectively protected NOD mice from developing T1D. Furthermore, the proliferation of adoptively transferred β cell antigen-specific TCR-transgenic T cells in pancreatic draining lymph nodes is markedly suppressed in UVB-stroma-treated mice, indicating that UVB-stroma treatment induces immune tolerance to multiple β cell antigens. This study provides an effective and convenient approach for managing T1D by utilizing apoptotic non-β cells.
PMCID: PMC2785115  PMID: 18226498
Apoptosis; Immune tolerance; Type 1 diabetes; Regulatory T cell; Cell therapy
19.  Nonobese Diabetic Mouse Congenic Analysis Reveals Chromosome 11 Locus Contributing to Diabetes Susceptibility, Macrophage STAT5 Dysfunction, and Granulocyte-Macrophage Colony-Stimulating Factor Overproduction1 
Unstimulated monocytes of at-risk/type 1 diabetic humans and macrophages of the NOD mouse have markedly elevated autocrine GM-CSF production and persistent STAT5 phosphorylation. We analyzed the relationship between GM-CSF production and persistent STAT5 phosphorylation in NOD macrophages using reciprocal congenic mouse strains containing either diabetes-susceptible NOD (B6.NODC11), or diabetes-resistant C57L (NOD.LC11) loci on chromosome 11. These intervals contain the gene for GM-CSF (Csf2; 53.8 Mb) and those for STAT3, STAT5A, and STAT5B (Stat3, Stat5a, and Stat5b; 100.4–100.6 Mb). High GM-CSF production and persistent STAT5 phosphorylation in unactivated NOD macrophages can be linked to a region (44.9–55.7 Mb) containing the Csf2 gene, but not the Stat3/5a/5b genes. This locus, provisionally called Idd4.3, is upstream of the previously described Idd4.1 and Idd4.2 loci. Idd4.3 encodes an abundance of cytokine genes that use STAT5 in their macrophage activation signaling and contributes ~50% of the NOD.LC11 resistance to diabetes.
PMCID: PMC2605964  PMID: 16177100
20.  Dendritic Cells post-Maturation Are Reprogrammed with Heightened IFN-γ and IL-10 
Mature Dendritic cells (mDCs) undergo “exhaustion” in producing cytokines. Nevertheless, whether this “exhaustion” of mDCs is selective to certain cytokines, or whether mDC have specific cytokine-producing profiles has yet to be defined. Herein, we investigated the cytokine production in vitro by immature DCs (iDCs) and LPS-induced mDCs. Compared to iDCs, mDCs produced comparable levels of IL-6 and TNF-α. Strikingly, mDCs produced significantly higher IFN-γ and IL-10. IL-12 production of mDCs was suppressed. Kinetic studies of the responses of iDCs and mDCs to LPS or CD40L showed that mDCs acquired progressively heightened activity in producing IFN-γ and IL-10. TNF-α-, IL-6-producing capability of mDCs was maintained. Nevertheless, IL-12 production by mDCs was not recovered at any time point. Mature DCs were potent in priming both Th1 and Th2 cells. In conclusion, upon maturation, DCs are reprogrammed with a distinct cytokine-secreting profile, which may play an important role in regulating T cell functions.
PMCID: PMC1850984  PMID: 17157804
Dendritic cells; immature/mature; Th1/Th2 cells; cytokines
21.  MyD88-dependent expansion of an immature GR-1+CD11b+ population induces T cell suppression and Th2 polarization in sepsis 
The Journal of Experimental Medicine  2007;204(6):1463-1474.
Polymicrobial sepsis alters the adaptive immune response and induces T cell suppression and Th2 immune polarization. We identify a GR-1+CD11b+ population whose numbers dramatically increase and remain elevated in the spleen, lymph nodes, and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly myeloid progenitors that express interleukin 10 and several other cytokines and chemokines. Splenic GR-1+ cells effectively suppress antigen-specific CD8+ T cell interferon (IFN) γ production but only modestly suppress antigen-specific and nonspecific CD4+ T cell proliferation. GR-1+ cell depletion in vivo prevents both the sepsis-induced augmentation of Th2 cell–dependent and depression of Th1 cell–dependent antibody production. Signaling through MyD88, but not Toll-like receptor 4, TIR domain–containing adaptor-inducing IFN-β, or the IFN-α/β receptor, is required for complete GR-1+CD11b+ expansion. GR-1+CD11b+ cells contribute to sepsis-induced T cell suppression and preferential Th2 polarization.
PMCID: PMC2118626  PMID: 17548519
22.  CapG−/− Mice Have Specific Host Defense Defects That Render Them More Susceptible than CapG+/+ Mice to Listeria monocytogenes Infection but Not to Salmonella enterica Serovar Typhimurium Infection  
Infection and Immunity  2003;71(11):6582-6590.
Loss of the actin filament capping protein CapG has no apparent effect on the phenotype of mice maintained under sterile conditions; however, bone marrow-derived macrophages from CapG−/− mice exhibited distinct motility defects. We examined the ability of CapG−/− mice to clear two intracellular bacteria, Listeria monocytogenes and Salmonella enterica serovar Typhimurium. The 50% lethal dose of Listeria was 10-fold lower for CapG−/− mice than for CapG+/+ mice (6 × 103 CFU for CapG−/− mice and 6 × 104 CFU for CapG+/+ mice), while no difference was observed for Salmonella. The numbers of Listeria cells in the spleens and livers were significantly higher in CapG−/− mice than in CapG+/+ mice at days 5 to 9, while the bacterial counts were identical on day 5 for Salmonella-infected mice. Microscopic analysis revealed qualitatively similar inflammatory responses in the spleens and livers of the two types of mice. Specific immunofluorescence staining analyzed by fluorescence-activated cell sorting revealed similar numbers of macrophages and dendritic cells in infected CapG−/− and CapG+/+ spleens. However, analysis of bone marrow-derived macrophages revealed a 50% reduction in the rate of phagocytosis of Listeria in CapG−/− cells but a normal rate of phagocytosis of Salmonella. Stimulation of bone marrow-derived dendritic cells with granulocyte-macrophage colony-stimulating factor resulted in a reduction in the ruffling response of CapG−/− cells compared to the response of CapG+/+ cells, and CapG−/− bone-marrowed derived neutrophils migrated at a mean speed that was nearly 50% lower than the mean speed of CapG+/+ neutrophils. Our findings suggest that specific motility deficits in macrophages, dendritic cells, and neutrophils render CapG−/− mice more susceptible than CapG+/+ mice to Listeria infection.
PMCID: PMC219612  PMID: 14573680

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