Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis.

L-selectin ligands might be relevant for inflammatory cell trafficking into the small intestine in a spontaneous model of chronic ileitis (i.e., SAMP1/YitFc mice). Immunoblockade of peripheral node addressin or mucosal addressin cell adhesion molecule 1 failed to ameliorate ileitis, whereas P-selectin glycoprotein ligand 1 (PSGL-1) neutralization attenuated both the adoptively transferred and spontaneous disease. PSGL-1 was detected in venules of mesenteric lymph node and small intestine by immunohistochemistry and confirmed by real-time reverse transcription polymerase chain reaction and flow cytometry. In addition, reconstitution of wild-type mice with PSGL-1(-/-) bone marrow demonstrated that PSGL-1 messenger RNA and PSGL-1 protein expression remained on endothelium, localized within mesenteric lymph node and small intestine. Endothelial PSGL-1 bound P-selectin-IgG and its blockade or genetic deletion altered the recruitment of lymphocytes to the small intestine, as revealed by intravital microscopy and homing studies. Endothelial expression of PSGL-1 adds a new dimension to the various cellular interactions involved in small intestinal recruitment. Thus, the multiple roles of PSGL-1 may explain why targeting this single adhesion molecule results in attenuation of chronic murine ileitis, a disease previously resistant to antiadhesion molecule strategies.

Beta7 integrin deficiency suppresses B cell homing and attenuates chronic ileitis in SAMP1/YitFc mice.

Lymphocyte recruitment to intestinal tissues depends on ß(7) integrins. In this study, we studied disease severity and lymphocyte recruitment into the small intestine in SAMP1/YitFc mice, which develop chronic ileitis with similarity to human Crohn's disease. To assess the role of ß(7) integrins in chronic ileitis, we generated SAMP1/YitFc lacking ß(7) integrins (SAMP1/YitFc Itgb7(-/-)) using a congenic strain developed via marker-assisted selection. We analyzed ileal inflammation in SAMP1/YitFc and SAMP1/YitFc Itgb7(-/-) mice by histopathology and the distribution of T and B lymphocytes in the mesenteric lymph nodes (MLNs) by flow cytometry. Short-term (18 h) adoptive transfer experiments were used to study the in vivo homing capacity of T and B lymphocytes. In both young (<20 wk) and old (20-50 wk) SAMP1/YitFc Itgb7(-/-) mice, ileitis was reduced by 30-50% compared with SAMP1/YitFc mice. SAMP1/YitFc Itgb7(-/-) mice showed a dramatic 67% reduction in the size of their MLNs, which was caused by a 85% reduction in lymphocyte numbers and reduced short-term B cell homing. Flow cytometric analysis revealed a highly significant decrease in the percentage of B cells in MLNs of SAMP1/YitFc Itgb7(-/-) mice. Cotransfer of SAMP1/YitFc MLN B cells but not SAMP1/YitFc Itgb7(-/-) MLN B cells along with CD4(+) T cells resulted in exacerbated ileitis severity in SCID mice. Our findings suggest that ß(7) integrins play an essential role in spontaneous chronic ileitis in vivo by promoting homing of disease-exacerbating B cells to MLNs and other intestinal tissues.

Proinflammatory signaling in islet ß cells propagates invasion of pathogenic immune cells in autoimmune diabetes.

Type 1 diabetes is a disorder of immune tolerance that leads to death of insulin-producing islet ß cells. We hypothesize that inflammatory signaling within ß cells promotes progression of autoimmunity within the islet microenvironment. To test this hypothesis, we deleted the proinflammatory gene encoding 12/15-lipoxygenase (Alox15) in ß cells of non-obese diabetic mice at a pre-diabetic time point when islet inflammation is a feature. Deletion of Alox15 leads to preservation of ß cell mass, reduces populations of infiltrating T cells, and protects against spontaneous autoimmune diabetes in both sexes. Mice lacking Alox15 in ß cells exhibit an increase in a population of ß cells expressing the gene encoding the protein programmed death ligand 1 (PD-L1), which engages receptors on immune cells to suppress autoimmunity. Delivery of a monoclonal antibody against PD-L1 recovers the diabetes phenotype in knockout animals. Our results support the contention that inflammatory signaling in ß cells promotes autoimmunity during type 1 diabetes progression.

Myeloid lineage cell-restricted insulin resistance protects apolipoproteinE-deficient mice against atherosclerosis.

Inflammatory processes play an important role in the pathogenesis of vascular diseases, and insulin-resistant diabetes mellitus type 2 represents an important risk factor for the development of atherosclerosis. To directly address the role of insulin resistance in myeloid lineage cells in the development of atherosclerosis, we have created mice with myeloid lineage-specific inactivation of the insulin receptor gene. On an ApoE-deficient background, MphIRKO mice developed smaller atherosclerotic lesions. There was a dramatic decrease in LPS-stimulated IL-6 and IL-1beta expression in the presence of macrophage autonomous insulin resistance. Consistently, while insulin-resistant IRS-2-deficient mice on an ApoE-deficient background display aggravated atherosclerosis, fetal liver cell transplantation of IRS-2(-/-) ApoE(-/-) cells ameliorated atherosclerosis in Apo-E-deficient mice. Thus, systemic versus myeloid cell-restricted insulin resistance has opposing effects on the development of atherosclerosis, providing direct evidence that myeloid lineage autonomous insulin signaling provides proinflammatory signals predisposing to the development of atherosclerosis.

Cutting edge: genetic characterization of IFN-producing killer dendritic cells.

The combined phenotypic expression of CD11c(low)B220(+)CD122(+)DX5(+) has been used to define a novel cell type termed IFN-producing killer dendritic cells (IKDC). IKDC readily produce IFN-gamma and demonstrate spontaneous cytotoxic activity toward tumors, suggesting that a modulation of IKDC number may be beneficial in cancer treatment. We examined various mouse strains and found that IKDC number was highly variable between the different strains. A linkage analysis associated the distal arm of chromosome 7 with variations in IKDC number. The genetic contribution of chromosome 7 to the regulation of IKDC number was confirmed through the use of congenic mice. We further demonstrate that IKDC proportion is regulated by intrinsic hematopoietic factors. We discuss the role of various candidate genes in the regulation of this newly described cell type and its implication in therapy.

Breaking tolerance to double stranded DNA, nucleosome, and other nuclear antigens is not required for the pathogenesis of lupus glomerulonephritis.

In lupus-prone NZM2328 mice, a locus Cgnz1 on chromosome 1 was linked to chronic glomerulonephritis, severe proteinuria, and early mortality in females. A locus Adnz1 on chromosome 4 was linked to antinuclear antibody (ANA) and anti-double stranded DNA (dsDNA) antibody (Ab) production. In this investigation, two congenic strains, NZM2328.C57L/Jc1 (NZM.C57Lc1) and NZM2328.C57L/Jc4 (NZM.C57Lc4), were generated by replacing the respective genetic intervals containing either Cgnz1 or Adnz1 with those from C57L/J, a nonlupus-prone strain. The NZM.C57Lc1 females had markedly reduced incidence of chronic glomerulonephritis and severe proteinuria. NZM.C57Lc4 females had chronic glomerulonephritis and severe proteinuria without circulating ANA, anti-dsDNA, and antinucleosome Ab. These data confirm the linkage analysis. Unexpectedly, NZM.C57Lc1 females had little anti-dsDNA and related Ab, suggesting the presence of a second locus Adnz2 on chromosome 1. The diseased NZM.C57Lc4 kidneys had immune complexes by immunofluorescence and electron microscopy. The eluates from these kidneys did not contain ANA, anti-dsDNA, and antinucleosome Ab, indicative of the presence of non-anti-dsDNA nephritogenic Ab. Thus, breaking tolerance to dsDNA and chromatin is not required for the pathogenesis of lupus nephritis. These results reaffirm that anti-dsDNA and related Ab production and chronic glomerulonephritis are under independent genetic control. These findings have significant implications in the pathogenesis of systemic lupus erythematosus.

Impaired Crkl expression contributes to the defective DNA binding of Stat5b in nonobese diabetic mice.

A point mutation in the Stat5b DNA binding domain in the nonobese diabetic (NOD) mouse was shown to have weaker DNA binding compared with the B6 Stat5b. Here, we assessed the binding ability of the mutant Stat5b in the B6 genetic background (B6.NOD-c11) and the wild-type Stat5b in the NOD background (NOD.Lc11). To our surprise, the binding ability of Stat5b is inconsistent with the presence or absence of the Stat5b mutation in these congenic mice but is correlated with the expression levels of the Crkl protein, which was coprecipitated by an anti-Stat5b antibody. Both the expression of Crkl and the Stat5b binding ability are the highest in B6.NOD-c11 and the lowest in NOD while intermediate in B6 and NOD.Lc11 mice. We demonstrated that the adapter molecule Crkl can bind Stat5b and that the Crkl protein is a Stat5b binding cofactor. More importantly, profection of Crkl recombinant protein significantly increased Stat5b binding ability and rescued the binding defect of the NOD mutant Stat5b, suggesting that Crkl is a key regulatory molecule for Stat5b binding. Therefore, the defective Crkl expression may contribute to the development of diabetes in the NOD mice by exacerbating the defective Stat5b binding ability.

Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow-derived cells.

Activation of A2A adenosine receptors (A2ARs) protects kidneys from ischemia-reperfusion injury (IRI). A2ARs are expressed on bone marrow-derived (BM-derived) cells and renal smooth muscle, epithelial, and endothelial cells. To measure the contribution of A2ARs on BM-derived cells in suppressing renal IRI, we examined the effects of a selective agonist of A2ARs, ATL146e, in chimeric mice in which BM was ablated by lethal radiation and reconstituted with donor BM cells derived from GFP, A2AR-KO, or WT mice to produce GFP-->WT, A2A-KO-->WT, or WT-->WT mouse chimera. We found little or no repopulation of renal vascular endothelial cells by donor BM with or without renal IRI. ATL146e had no effect on IRI in A2A-KO mice or A2A-KO-->WT chimera, but reduced the rise in plasma creatinine from IRI by 75% in WT mice and by 60% in WT-->WT chimera. ATL146e reduced the induction of IL-6, IL-1beta, IL-1ra, and TGF-alpha mRNA in WT-->WT mice but not in A2A-KO-->WT mice. Plasma creatinine was significantly greater in A2A-KO than in WT mice after IRI, suggesting some renal protection by endogenous adenosine. We conclude that protection from renal IRI by A2AR agonists or endogenous adenosine requires activation of receptors expressed on BM-derived cells.

The Runx3 distal transcript encodes an additional transcriptional activation domain.

The runt family transcriptional regulator, Runx3, is upregulated during the differentiation of CD8 single-positive thymocytes and is expressed in peripheral CD8(+) T cells. Mice carrying targeted deletions in Runx3 have severe defects in the development and activation of CD8(+) T cells, resulting in decreased CD8(+) T-cell numbers, aberrant coexpression of CD4, and failure to expand CD8(+) effector cells after activation in vivo or in vitro. Expression of each of the three vertebrate runt family members, including Runx3, is controlled by two promoters that generate proteins with alternative N-terminal sequences. The longer N-terminal region of Runx3, expressed from the distal promoter, is highly conserved among family members and across species. We show that transcripts from the distal Runx3 promoter are selectively expressed in mature CD8(+) T cells and are upregulated upon activation. We show that the N-terminal region encoded by these transcripts carries an independent transcriptional activation domain. This domain can activate transcription in isolation, and contributes to the increased transcriptional activity observed with this isoform as compared to those expressed from the ancestral, proximal promoter. Together, these data suggest an important role for the additional N-terminal Runx3 activation domain in CD8(+) T-cell function.

Quantitative trait locus analysis of atherosclerosis in an intercross between C57BL/6 and C3H mice carrying the mutant apolipoprotein E gene.

Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) differ significantly in atherosclerosis susceptibility and plasma lipid levels on the apolipoprotein E-deficient (apoE-/-) background when fed a Western diet. To determine genetic factors contributing to the variations in these phenotypes, we performed quantitative trait locus (QTL) analysis using an intercross between the two strains carrying the apoE-/- gene. Atherosclerotic lesions at the aortic root and plasma lipid levels of 234 female F2 mice were analyzed after being fed a Western diet for 12 weeks. QTL analysis revealed one significant QTL, named Ath22 (42 cM, LOD 4.1), on chromosome 9 and a suggestive QTL near D11mit236 (20 cM, LOD 2.4) on chromosome 11 that influenced atherosclerotic lesion size. One significant QTL on distal chromosome 1, which accounted for major variations in plasma LDL/VLDL cholesterol and triglyceride levels, coincided with a QTL having strong effects on body weight. Plasma LDL/VLDL cholesterol or triglyceride levels of F2 mice were significantly correlated with body weight, but they were not correlated with atherosclerotic lesion sizes. These data indicate that atherosclerosis susceptibility and plasma cholesterol levels are controlled by separate genetic factors in the B6 and C3H mouse model and that genetic linkages exist between body weight and lipoprotein metabolism.

Alterations in redox homeostasis and prostaglandins impair endothelial-dependent vasodilation in euglycemic autoimmune nonobese diabetic mice.

We report herein the novel observation that alterations in oxidant/antioxidant balance are evident and cause vascular dysfunction in aortae of prediabetic nonobese-diabetic mice (NOD). We found that nitrotyrosine, a biochemical marker of oxidant stress, was higher in the NOD aortae when compared to age-matched non-autoimmune BALB/c controls or the diabetes-resistant NOD congenic strain, NOD.Lc7. The oxidant stress was localized to the intimal and medial layers, and endothelium-dependent relaxation to acetylcholine was decreased in isolated aortic rings from NOD mice. Inhibition of nitric oxide synthesis caused an endothelium-dependent contraction, and treatment with either a selective thromboxane A2/prostaglandin H2 receptor antagonist or a non-isozyme-specific cyclooxygenase inhibitor reversed this effect. Aortic rings from NOD.Lc7 did not display the paradoxical vasoconstriction. Furthermore, the vascular dysfunction was caused by oxidative stress, as treatment with a superoxide dismutase mimetic in vivo or with native antioxidant enzymes ex vivo inhibited the tissue oxidant stress and restored endothelium-dependent relaxation. Endothelial function was also restored by the inhibitors of NAD(P)H oxidase, diphenylene iodonium or apocynin. Our studies indicate that an oxidant stress that occurs prior to the onset of diabetes in this mouse model contributes to endothelial dysfunction independently of overt diabetes.

Csf2 and Ptgs2 Epigenetic Dysregulation in Diabetes-prone Bicongenic B6.NODC11bxC1tb Mice.

In Type 1 diabetic (T1D) human monocytes, STAT5 aberrantly binds to epigenetic regulatory sites of two proinflammatory genes, CSF2 (encoding granulocyte-macrophage colony-stimulating factor) and PTGS2 (encoding prostaglandin synthase 2/cyclooxygenase 2). Bicongenic B6.NOD C11bxC1tb mice re-create this phenotype of T1D monocytes with only two nonobese diabetic (NOD) Idd subloci (130.8 Mb-149.7 Mb, of Idd5 on Chr 1 and 32.08-53.85 Mb of Idd4.3 on Chr11) on C57BL/6 genetic background. These two Idd loci interact through STAT5 binding at upstream regulatory regions affecting Csf2 (Chr 11) and Ptgs2 (Chr 1) expression. B6.NODC11bxC1tb mice exhibited hyperglycemia and immune destruction of pancreatic islets between 8 and 30 weeks of age, with 12%-22% penetrance. Thus, B6.NODC11bxC1tb mice embody NOD epigenetic dysregulation of gene expression in myeloid cells, and this defect appears to be sufficient to impart genetic susceptibility to diabetes in an otherwise genetically nonautoimmune mouse.

Role for oxidative stress in the regeneration of islet beta cells?

In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have found that there are increased markers of oxidative stress in islet beta cells in prediabetic animals when compared with control strains. Treatment of these mice with a superoxide dismutase (SOD) mimetic can markedly reduce the level of nitrotyrosine found in islets. In a diabetes-resistant NOD congenic mouse, the NOD.Lc7 mouse, we found increased beta cell proliferation and decreased apoptosis in islets. There are also lower levels of nitrotyrosine in islets of NOD.Lc7 mice than in NOD mice, suggesting that NOD.Lc7 islets are less susceptible to oxidative damage. We hypothesize that there may be a link between the ability of islet cells to regenerate and their resistance to oxidative stress.

Manganese-enhanced magnetic resonance imaging detects declining pancreatic ß-cell mass in a cyclophosphamide-accelerated mouse model of type 1 diabetes.

Currently, there is no ideal noninvasive method to quantify the progressive loss of pancreatic ß-cell mass (BCM) that occurs in type 1 diabetes. Magnetic resonance imaging has detected gross differences in BCM between healthy and diabetic mice using the contrast agent manganese, which labels functional ß-cells and increases the water proton relaxation rate (R1), but its ability to measure gradations in BCM during disease progression is unknown. Our objective was to test the hypothesis that measurements of the manganese-enhanced pancreatic R1 could detect decreasing BCM in a mouse model of type 1 diabetes. We used cyclophosphamide-accelerated BDC2.5 T-cell receptor transgenic nonobese diabetic mice, which experience development of type 1 diabetes during a 7-day time period after cyclophosphamide injection, whereas transgene-negative mice do not. We measured the manganese-enhanced pancreatic R1 before cyclophosphamide injection (day 0) and on days 3, 4, 5, and 7 afterward. Pancreatic R1 remained constant in transgene-negative mice and decreased stepwise day-to-day in transgene-positive mice, mirroring their loss of BCM, confirmed by pancreatic insulin measurements and histology. Changes in R1 in transgene-positive mice occurred before elevations in blood glucose, a clinical indicator of diabetes, suggesting potential for early noninvasive detection of changes in functional BCM.

Deletion of 12/15-lipoxygenase alters macrophage and islet function in NOD-Alox15(null) mice, leading to protection against type 1 diabetes development.

AIMS: Type 1 diabetes (T1D) is characterized by autoimmune depletion of insulin-producing pancreatic beta cells. We showed previously that deletion of the 12/15-lipoxygenase enzyme (12/15-LO, Alox15 gene) in NOD mice leads to nearly 100 percent protection from T1D. In this study, we test the hypothesis that cytokines involved in the IL-12/12/15-LO axis affect both macrophage and islet function, which contributes to the development of T1D. METHODS: 12/15-LO expression was clarified in immune cells by qRT-PCR, and timing of expression was tested in islets using qRT-PCR and Western blotting. Expression of key proinflammatory cytokines and pancreatic transcription factors was studied in NOD and NOD-Alox15(null) macrophages and islets using qRT-PCR. The two mouse strains were also assessed for the ability of splenocytes to transfer diabetes in an adoptive transfer model, and beta cell mass. RESULTS: 12/15-LO is expressed in macrophages, but not B and T cells of NOD mice. In macrophages, 12/15-LO deletion leads to decreased proinflammatory cytokine mRNA and protein levels. Furthermore, splenocytes from NOD-Alox15(null) mice are unable to transfer diabetes in an adoptive transfer model. In islets, expression of 12/15-LO in NOD mice peaks at a crucial time during insulitis development. The absence of 12/15-LO results in maintenance of islet health with respect to measurements of islet-specific transcription factors, markers of islet health, proinflammatory cytokines, and beta cell mass. CONCLUSIONS: These results suggest that 12/15-LO affects islet and macrophage function, causing inflammation, and leading to autoimmunity and reduced beta cell mass.

Phospholemman deficiency in postinfarct hearts: enhanced contractility but increased mortality.

Phospholemman (PLM) regulates [Na(+) ](i), [Ca(2+)](i) and contractility through its interactions with Na(+)-K(+)-ATPase (NKA) and Na(+) /Ca(2+) exchanger (NCX1) in the heart. Both expression and phosphorylation of PLM are altered after myocardial infarction (MI) and heart failure. We tested the hypothesis that absence of PLM regulation of NKA and NCX1 in PLM-knockout (KO) mice is detrimental. Three weeks after MI, wild-type (WT) and PLM-KO hearts were similarly hypertrophied. PLM expression was lower but fractional phosphorylation was higher in WT-MI compared to WT-sham hearts. Left ventricular ejection fraction was severely depressed in WT-MI but significantly less depressed in PLM-KO-MI hearts despite similar infarct sizes. Compared with WT-sham myocytes, the abnormal [Ca(2+) ], transient and contraction amplitudes observed in WT-MI myocytes were ameliorated by genetic absence of PLM. In addition, NCX1 current was depressed in WT-MI but not in PLM-KO-MI myocytes. Despite improved myocardial and myocyte performance, PLM-KO mice demonstrated reduced survival after MI. Our findings indicate that alterations in PLM expression and phosphorylation are important adaptations post-MI, and that complete absence of PLM regulation of NKA and NCX1 is detrimental in post-MI animals.

Prevention, but not cure, of autoimmune diabetes in a NOD.scid transfer model by FTY720 despite effective modulation of blood T cells.

FTY720 modulates lymphocyte trafficking through blood (peripheral blood lymphocyte, PBL) and peripheral lymph nodes (PLN). Treatment with FTY720 causes retention of most blood lymphocytes in PLN. Long-term treatment can slow and/or prevent Type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse model. B and T cells are both affected by FTY720 binding to sphingosine-1-phosphate receptor 1 (S1P1). However, little has been done to elucidate which T-cell subsets are differentially affected by FTY720 under healthy conditions, and how this affects disease pathogenesis in T1D. In healthy C57BL/6J (B6) mice, total CD4(+) and CD8(+) T-cell subsets were diminished by FTY720, but recently activated and memory subsets were spared and constituted significantly higher percentage of remaining T cells in blood. FTY720 also lowered PBL counts in NOD mice, but less severely than in B6 mice. This is consistent with a different ratio of naïve, activated, and memory cells in NOD mice compared to those in B6 mice, as well as alterations in S1P1 and sphingosine-1-phosphate (S1P) levels in PBLs and blood of NOD mice, respectively. To address the functional consequences of PBL T-cell depletion, we studied the effects of FTY720 on disease progression in a timed adoptive transfer model of T1D. Continuous treatment with FTY720 eliminated T1D, if treatment was started before splenocyte transfer. FTY20 treatment started after disease onset slowed disease progression. The inability to fully suppress memory and effector T-cell circulation may explain why FTY720 is only partially effective in the NOD adoptive transfer model of T1D.

Age-dependent tolerance to an endogenous tumor-associated antigen.

Immunologic tolerance to endogenous antigens reduces antitumor responses. Gp70 is an endogenous tumor-associated antigen (TAA) of the BALB/c-derived colon carcinoma CT26. We found that expression of gp70 mRNA is detectable in tissues of mice 8 months of age and older. We showed that expression of gp70 establishes immunologic tolerance and affects antitumor immunity in a similarly age-dependent manner using gp70-deficient mice. We found that tumors grew in all gp70-sufficient mice, while approximately half of gp70-deficient mice controlled tumor growth with endogenous T-cell responses. Protection in gp70-deficient mice correlated with more robust gp70-specific CTL responses, and increased numbers and avidity of responding antigen-specific T cells after vaccination. We conclude that immunosurveillance may decline with age due to increased or de novo peripheral expression of endogenous TAAs.

Nonobese diabetic (NOD) mice congenic for a targeted deletion of 12/15-lipoxygenase are protected from autoimmune diabetes.

OBJECTIVE: 12/15-lipoxygenase (12/15-LO), one of a family of fatty acid oxidoreductase enzymes, reacts with polyenoic fatty acids to produce proinflammatory lipids. 12/15-LO is expressed in macrophages and pancreatic beta-cells. It enhances interleukin 12 production by macrophages, and several of its products induce apoptosis of beta-cells at nanomolar concentrations in vitro. We had previously demonstrated a role for 12/15-LO in beta-cell damage in the streptozotocin model of diabetes. Since the gene encoding 12/15-LO (gene designation Alox15) lies within the Idd4 diabetes susceptibility interval in NOD mice, we hypothesized that 12/15-LO is also a key regulator of diabetes susceptibility in the NOD mouse. RESEARCH DESIGN AND METHODS: We developed NOD mice carrying an inactivated 12/15-LO locus (NOD-Alox15(null)) using a "speed congenic" protocol, and the mice were monitored for development of insulitis and diabetes. RESULTS: NOD mice deficient in 12/15-LO develop diabetes at a markedly reduced rate compared with NOD mice (2.5 vs. >60% in females by 30 weeks). Nondiabetic female NOD-Alox15(null) mice demonstrate improved glucose tolerance, as well as significantly reduced severity of insulitis and improved beta-cell mass, when compared with age-matched nondiabetic NOD females. Disease resistance is associated with decreased numbers of islet-infiltrating activated macrophages at 4 weeks of age in NOD-Alox15(null) mice, preceding the development of insulitis. Subsequently, islet-associated infiltrates are characterized by decreased numbers of CD4(+) T cells and increased Foxp3(+) cells. CONCLUSIONS: These results suggest an important role for 12/15-LO in conferring susceptibility to autoimmune diabetes in NOD mice through its effects on macrophage recruitment or activation.

Truncated pStat5B is associated with the Idd4 locus in NOD mice.

We investigate JAK-STAT5 activation and its relationship to full-length Stat5B (FL-Stat5) and constitutive phosphorylated carboxy-truncated Stat5B (ct-pStat5) in four different strains of mouse. Our electrophoresis mobility shift assays data indicate constitutive phosphorylation of full-length-Stat5 (p<0.001) and DNA binding in NOD but not in B6 mice. Our data suggest that the relative ratio of FL-Stat5: ct-Stat5 in NOD is 5- to 8-fold lower (p<0.0001) when compared with normal B6 mice. Additionally, EMSAs data from B6.NOD/c11 suggest contribution of Idd4 susceptibility locus on chromosome 11 in constitutive phosphorylation of Stat5 in NOD mice. The presence of ct-pStat5 in regulatory T cells of NOD mice suggests this form of Stat5 is associated with impaired function of Tregs in NOD mouse. In agreement with our previous report the JAK-Stat5B defective pathway in NOD mice along with other defective factors is associated with the pathogenesis of autoimmune type 1 diabetes in NOD mice.