Determination of gastric emptying in nonobese diabetic mice

Animal studies on diabetic gastroparesis are limited by inability to follow gastric emptying changes in the same mouse. The study aim was to validate a nonlethal gastric emptying method in nonobese diabetic (NOD) LtJ mice, a model of type 1 diabetes, and study sequential changes with age and early diabetic status. The reliability and responsiveness of a [(13)C]octanoic acid breath test in NOD LtJ mice was tested, and the test was used to measure solid gastric emptying in NOD LtJ mice and nonobese diabetes resistant (NOR) LtJ mice. The (13)C breath test produced results similar to postmortem recovery of a meal. Bethanechol accelerated gastric emptying [control: 92 +/- 9 min; bethanechol: 53 +/- 3 min, mean half emptying time (T(1/2)) +/- SE], and atropine slowed gastric emptying (control: 92 +/- 9 min; atropine: 184 +/- 31 min, mean T(1/2) +/- SE). Normal gastric emptying (T(1/2)) in nondiabetic NOD LtJ mice (8-12 wk) was 91 +/- 2 min. Aging had differing effects on gastric emptying in NOD LtJ and NOR LtJ mice. Onset of diabetes was accompanied by accelerated gastric emptying during weeks 1-2 of diabetes. Gastric emptying returned to normal by weeks 3-5 with no delay. The [(13)C]octanoic acid breath test accurately measures gastric emptying in NOD LtJ mice, is useful to study the time course of changes in gastric emptying in diabetic NOD LtJ mice, and is able to detect acceleration in gastric emptying early in diabetes. Opposing changes in gastric emptying between NOD LtJ and NOR LtJ mice suggest that NOR LtJ mice are not good controls for the study of gastric emptying in NOD LtJ mice.     

Islet allograft rejection in nonobese diabetic mice involves the common gamma-chain and CD28/CD154-dependent and -independent mechanisms

Once nonobese diabetic (NOD) mice become diabetic, they are highly resistant to islet transplantation. The precise mechanism of such resistance remains largely unknown. In the present study we tested the hypothesis that islet allograft survival in the diabetic NOD mouse is determined by the interplay of diverse islet-specific T cell subsets whose activation is regulated by CD28/CD154 costimulatory signals and the common gamma-chain (gammac; a shared signaling element by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). We found that common gammac blockade is remarkably effective in blocking the onset and the ongoing autoimmune diabetes, whereas CD28/CD154 blockade has no effect in suppressing the ongoing diabetes. However, CD28/CD154 blockade completely blocks the alloimmune-mediated islet rejection. Also, a subset of memory-like T cells in the NOD mice is resistant to CD28/CD154 blockade, but is sensitive to the common gammac blockade. Nonetheless, neither common gammac blockade nor CD28/CD154 blockade can prevent islet allograft rejection in diabetic NOD mice. Treatment of diabetic NOD recipients with CD28/CD154 blockade plus gammac blockade markedly prolongs islet allograft survival compared with the controls. However, allograft tolerance is not achieved, and all CTLA-4Ig-, anti-CD154-, and anti-gammac-treated diabetic NOD mice eventually rejected the islet allografts. We concluded that the effector mechanisms in diabetic NOD hosts are inherently complex, and rejection in this model involves CD28/CD154/gammac-dependent and -independent mechanisms.     

Insulin binding to circulating erythrocytes in nonobese diabetic mice

Insulin binding to circulating erythrocytes was studied in nonobese diabetic (NOD) mice which develop insulinopenic diabetes mellitus spontaneously. NOD mice with a short duration of diabetes mellitus and mild insulinopenia did not show any change in insulin binding, while those with a long duration of diabetes mellitus and severe insulinopenia showed an increase in insulin binding compared with nondiabetic NOD mice (6.85 +/- 0.38% bound vs. 4.19 +/- 0.24% bound, p less than 0.01). This increase in insulin binding was due to an increase in the number of receptors. Insulin treatment of diabetic NOD mice significantly reduced the insulin binding by 64%, which resulted from a decrease in the number of the receptors. These results indicate that insulin binding to erythrocytes in NOD mice is controlled mostly by up-and-down regulation.     

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonobese diabetic (NOD) mice, recombinant congenic nonobese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [(3)H]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-generative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.     

The impact of genetic background on neurodegeneration and behavior in seizured mice

We used pilocarpine-induced seizures in mice to determine the impact of genetic background on the vulnerability of hippocampal neurons and associated changes of behavioral performance. The susceptibility of hippocampal neurons to seizure-induced cell death paralleled the severity of the seizures and depended on genetic background. Hippocampal neurons in C57BL/6 mice were most resistant to cell death, whereas they were highly vulnerable in FVB/N mice. The degree of neuronal degeneration in F1 hybrid mice obtained by crossing the two strains was at an intermediate level between the parent strains. Two weeks after the severe seizures, performance in a water-maze place navigation task showed a bimodal distribution. Seventeen of 19 (90%) F1 mice were completely unable to learn while the other two learned reasonably well. Of 28 C57BL/6 mice with similarly severe seizures, six were as strongly impaired as their F1 counterparts (22%). The remaining 22 performed normally, indicating a much lower probability of C57BL/6 mice to be affected. Treated mice showed a deficit of open-field exploration which was strongly correlated with the impairment in the place navigation task and was again more severe in F1 mice. Our results show that the vulnerability of hippocampal neurons to pilocarpine-induced seizures, as well as the associated behavioral changes, depended on genetic background. Furthermore, they confirm and extend our earlier finding that a relatively modest reduction of hippocampal cell death can be associated with dramatic changes of behavioral performance and emphasize the importance of tightly-controlled genetic backgrounds in biological studies.     

Characterization of the anti-tissue transglutaminase antibody response in nonobese diabetic mice

Type 1 diabetes mellitus is an autoimmune disorder characterized by destruction of insulin-producing pancreatic beta cells by T lymphocytes. In nonobese diabetic (NOD) mice, a role has been hypothesized for dietary gluten proteins in the onset of diabetes, and because gluten dependence is the major feature of celiac disease, together with production of Abs to the autoantigen tissue transglutaminase (tTG), we looked for the presence of anti-tTG Abs in the serum of NOD mice and, to establish their origin, analyzed the Ab repertoire of NOD mice using phage display Ab libraries. We found significant levels of serum anti-tTG Abs and were able to isolate single-chain Ab fragments to mouse tTG mainly from the Ab libraries made from intestinal lymphocytes and to a lesser extent from splenocytes. Data from NOD mice on a gluten-free diet suggest that the anti-tTG response is not gluten-dependent. The intestinal Ab response to tTG is a feature of NOD mice, but the underlying mechanisms remain obscure.     

A new autoantigen reactive with prediabetic nonobese diabetic mice sera.

The identification and characterization of new autoantigens would widen the knowledge of the pathogenic mechanism of insulin dependent diabetes mellitus. Screening of lambda gt11 mouse insulinoma (MIN6N8a) cell cDNA library with prediabetic nonobese diabetic (NOD) mice sera resulted in the isolation of a strong positive clone, named the clone 3-5, of 1579 nucleotides without a poly A region. After 5'-rapid amplification of the cDNA end (RACE), complete nucleotide sequence of the clone 3-5 gene consisting of 2231 nucleotides showed that the 3-5 gene had the theoretical open reading frame of 634 amino acids. However, the real antigenic protein of the clone 3-5 was only 21 amino acids long encoded by only 63 nucleotides. The 21 amino acids were expressed as a fusion protein in E. coli and purified by affinity chromatography. The purified 3-5 recombinant protein was examined for its reactivity with prediabetic NOD mice sera by immunoblotting. The only non-denatured form of the 3-5 protein showed a binding reactivity with NOD mice sera, demonstrating that the conformational epitope of 3-5 protein was important for antibody recognition. The prevalence of autoantibody reactive to the 3-5 protein was about 78% (14/18) and 46% (11/24) in prediabetic and acute diabetic NOD mice sera, respectively. However, the sera from other mouse strains such as BALB/c, ICR, C57BL/6, SJL/J, and NOD/SCID did not show a positive reactivity to the 3-5 protein, which indicated that immune reactivity against the 3-5 protein was autoimmune diabetic mouse-specific.     

IL-17 silencing does not protect nonobese diabetic mice from autoimmune diabetes

The long-held view that many autoimmune disorders are primarily driven by a Th1 response has been challenged by the discovery of Th17 cells. Since the identification of this distinct T cell subset, Th17 cells have been implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. Type 1 diabetes has also long been considered a Th1-dependent disease. In light of the emerging role for Th17 cells in autoimmunity, several recent studies investigated the potential of this subset to initiate autoimmune diabetes. However, direct evidence supporting the involvement of Th17 cells in actual pathogenesis, particularly during spontaneous onset, is lacking. In this study, we sought to directly address the role of IL-17, the cytokine by which Th17 cells are primarily characterized, in the pathogenesis of autoimmune diabetes. We used lentiviral transgenesis to generate NOD mice in which IL-17 is silenced by RNA interference. The loss of IL-17 had no effect on the frequency of spontaneous or cyclophosphamide-induced diabetes. In contrast, IL-17 silencing in transgenic NOD mice was sufficient to reduce the severity of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, consistent with reports that IL-17 deficiency is protective in this experimental model of multiple sclerosis. We concluded that IL-17 is dispensable, at least in large part, in the pathogenesis of autoimmune diabetes.     

Buildup from birth onward of short telomeres in human hematopoietic cells

Telomere length (TL) limits somatic cell replication. However, the shortest among the telomeres in each nucleus, not mean TL, is thought to induce replicative senescence. Researchers have relied on Southern blotting (SB), and techniques calibrated by SB, for precise measurements of TL in epidemiological studies. However, SB provides little information on the shortest telomeres among the 92 telomeres in the nucleus of human somatic cells. Therefore, little is known about the accumulation of short telomeres with age, or whether it limits the human lifespan. To fill this knowledge void, we used the Telomere-Shortest-Length-Assay (TeSLA), a method that tallies and measures single telomeres of all chromosomes. We charted the age-dependent buildup of short telomeres (<3 kb) in human hematopoietic cells from 334 individuals (birth-89 years) from the general population, and 18 patients with dyskeratosis congenita-telomere biology disorders (DC/TBDs), whose hematopoietic cells have presumably reached or are close to their replicative limit. For comparison, we also measured TL with SB. We found that in hematopoietic cells, the buildup of short telomeres occurs in parallel with the shortening with age of mean TL. However, the proportion of short telomeres was lower in octogenarians from the general population than in patients with DC/TBDs. At any age, mean TL was longer and the proportion of short telomeres lower in females than in males. We conclude that though converging to the TL-mediated replicative limit, hematopoietic cell telomeres are unlikely to reach this limit during the lifespan of most contemporary humans.     

Plasmid vaccination with insulin B chain prevents autoimmune diabetes in nonobese diabetic mice

The insulin B (InsB) chain bears major type 1 diabetes-associated epitopes of significance for disease in humans and nonobese diabetic (NOD) mice. Somatic expression of InsB chain initiated early in life by plasmid inoculation resulted in substantial protection of female NOD mice against disease. This was associated with a T2 shift in spleen, expansion of IL-4-producing and, to a lesser extent, of IFN-gamma-secreting T cells in pancreatic lymph nodes, as well as intermolecular Th2 epitope spreading to glutamic acid decarboxylase determinants. A critical role of IL-4 for the Ag-specific protective effect triggered by plasmid administration was revealed in female IL-4(-/-) NOD mice that developed diabetes and higher Th1 responses. Coadministration of IL-4-expressing plasmid or extension of the vaccination schedule corrected the unfavorable response of male NOD mice to DNA vaccination with InsB chain. Thus, plasmid-mediated expression of the InsB chain early in diabetes-prone mice has the potential to prevent transition to full-blown disease depending on the presence of IL-4.     

ICOS mediates the development of insulin-dependent diabetes mellitus in nonobese diabetic mice

Initiation of diabetes in NOD mice can be mediated by the costimulatory signals received by T cells. The ICOS is found on Ag-experienced T cells where it acts as a potent regulator of T cell responses. To determine the function of ICOS in diabetes, we followed the course of autoimmune disease and examined T cells in ICOS-deficient NOD mice. The presence of ICOS was indispensable for the development of insulitis and hyperglycemia in NOD mice. In T cells, the deletion of ICOS resulted in a decreased production of the Th1 cytokine IFN-gamma, whereas the numbers of regulatory T cells remained unchanged. We conclude that ICOS is critically important for the induction of the autoimmune process that leads to diabetes.     

Molecular genetic analysis of the Idd4 locus implicates the IFN response in type 1 diabetes susceptibility in nonobese diabetic mice

High-resolution mapping and identification of the genes responsible for type 1 diabetes (T1D) has proved difficult because of the multigenic etiology and low penetrance of the disease phenotype in linkage studies. Mouse congenic strains have been useful in refining Idd susceptibility loci in the NOD mouse model and providing a framework for identification of genes underlying complex autoimmune syndromes. Previously, we used NOD and a nonobese diabetes-resistant strain to map the susceptibility to T1D to the Idd4 locus on chromosome 11. Here, we report high-resolution mapping of this locus to 1.4 megabases. The NOD Idd4 locus was fully sequenced, permitting a detailed comparison with C57BL/6 and DBA/2J strains, the progenitors of T1D resistance alleles found in the nonobese diabetes-resistant strain. Gene expression arrays and quantitative real-time PCR were used to prioritize Idd4 candidate genes by comparing macrophages/dendritic cells from congenic strains where allelic variation was confined to the Idd4 interval. The differentially expressed genes either were mapped to Idd4 or were components of the IFN response pathway regulated in trans by Idd4. Reflecting central roles of Idd4 genes in Ag presentation, arachidonic acid metabolism and inflammation, phagocytosis, and lymphocyte trafficking, our combined analyses identified Alox15, Alox12e, Psmb6, Pld2, and Cxcl16 as excellent candidate genes for the effects of the Idd4 locus.     

Abnormal anxiety-related behavior in serotonin transporter null mutant mice: the influence of genetic background

Serotonin transporter (5-HTT) null mutant mice provide a model system to study the role genetic variation in the 5-HTT plays in the regulation of emotion. Anxiety-like behaviors were assessed in 5-HTT null mutants with the mutation placed on either a B6 congenic or a 129S6 congenic background. Replicating previous findings, B6 congenic 5-HTT null mutants exhibited increased anxiety-like behavior and reduced exploratory locomotion on the light ↔ dark exploration and elevated plus-maze tests. In contrast, 129S6 congenic 5-HTT null mutant mice showed no phenotypic abnormalities on either test. 5-HTT null mutants on the 129S6 background showed reduced 5-HT_1A receptor binding (as measured by quantitative autoradiography) and reduced 5-HT_1A receptor function (as measured by 8-OH-DPAT-indcued hypothermia). These data confirm that the 5-HTT null mutation produced alterations in brain 5-HT function in mice on the 129S6 background, thereby discounting the possibility that the absence of an abnormal anxiety-like phenotype in these mice was due to a suppression of the mutation by 129 modifier genes. Anxiety-like behaviors in the light ↔ dark exploration and elevated plus-maze tests were significantly higher in 129S6 congenic +/+ mice as compared to B6 congenic +/+ mice. This suggests that high baseline anxiety-like behavior in the 129S6 strain might have precluded detection of the anxiety-like effects of the 5-HTT null mutation on this background. Present findings provide further evidence linking genetic variation in the 5-HTT to abnormalities in mood and anxiety. Furthermore, these data highlight the utility of conducting behavioral phenotyping of mutant mice on multiple genetic backgrounds.     

Absence of SH2B3 mutation in nonobese diabetic mice

Type 1 diabetes is a chronic progressive autoimmune disease characterized by mononuclear cell infiltration, with subsequent destruction of insulin-producing β-cells. Studies have identified strong associations between type 1 diabetes and several chromosome regions, including 12q24. Association between type 1 diabetes and 12q24 arises from SNP rs3184504; rs3184504 is a nonsynonymous SNP in exon 3 of SH2B3 (also known as LNK). Nonobese diabetic (NOD) mice recapitulate many aspects of the pathogenesis of type 1 diabetes in humans and are therefore frequently used in studies addressing the cellular and molecular mechanisms of this disease. It is of interest to know whether there is a similar mutation of SH2B3 in NOD mice. We found that the SH2B3 mutation is absent in NOD mice. To our knowledge, this is the first report of the sequence and the protein levels of SH2B3 in NOD mice.     

Elimination of maternally transmitted autoantibodies prevents diabetes in nonobese diabetic mice

The influence of maternally transmitted immunoglobulins on the development of autoimmune diabetes mellitus in genetically susceptible human progeny remains unknown. Given the presence of islet beta cell-reactive autoantibodies in prediabetic nonobese diabetic (NOD) mice, we abrogated the maternal transmission of such antibodies in order to assess their influence on the susceptibility of progeny to diabetes. First, we used B cell-deficient NOD mothers to eliminate the transmission of maternal immunoglobulins. In a complementary approach, we used immunoglobulin transgenic NOD mothers to exclude autoreactive specificities from the maternal B-cell repertoire. Finally, we implanted NOD embryos in pseudopregnant mothers of a non-autoimmune strain. The NOD progeny in all three groups were protected from spontaneous diabetes. These findings demonstrate that the maternal transmission of antibodies is a critical environmental parameter influencing the ontogeny of T cell-mediated destruction of islet beta cells in NOD mice. It will be important to definitively determine whether the transmission of maternal autoantibodies in humans affects diabetes progression in susceptible offspring.     

Dysregulated TLR3-dependent signaling and innate immune activation in superoxide-deficient macrophages from nonobese diabetic mice

In type 1 diabetes (T1D), reactive oxygen species (ROS) and proinflammatory cytokines produced by macrophages and other innate immune cells destroy pancreatic β cells while promoting autoreactive T cell maturation. Superoxide-deficient nonobese diabetic mice (NOD.Ncf1^m1J) are resistant to spontaneous diabetes, revealing the integral role of ROS signaling in T1D. Here, we evaluate the innate immune activation state of bone marrow-derived macrophages (BM-M?) from NOD and NOD.Ncf1^m1J mice after poly(I:C)-induced Toll-like receptor 3 (TLR3) signaling. We show that ROS synthesis is required for efficient activation of the NF-κB signaling pathway and concomitant expression of TLR3 and the cognate adaptor molecule, TRIF. Poly(I:C)-stimulated NOD.Ncf1^m1J BM-M? exhibited a 2- and 10-fold decrease in TNF-α and IFN-β proinflammatory cytokine synthesis, respectively, in contrast to NOD BM-M?. Optimal expression of IFN-α/β is not solely dependent on superoxide synthesis, but requires p47^phox to function in a NOX-independent manner to mediate type I interferon synthesis. Interestingly, MHC-II I-A^g7 expression necessary for CD4 T cell activation is increased 2-fold relative to NOD, implicating a role for superoxide in I-A^g7 downregulation. These findings suggest that defective innate immune-pattern-recognition receptor activation and subsequent decrease in TNF-α and IFN-β proinflammatory cytokine synthesis necessary for autoreactive T cell maturation may contribute to the T1D protection observed in NOD.Ncf1^m1J mice.     

Synchronized gastric electrical stimulation improves delayed gastric emptying in nonobese mice with diabetic gastroparesis.

The aim of this study was to investigate the effect and mechanism of synchronized gastric electrical stimulation (SGES) on gastric emptying in nonobese mice with diabetic gastroparesis (DB-GP). Eight control mice and 48 nonobese diabetic (NOD) mice with two pairs of gastric electrodes were used in this study. The study included seven groups in a randomized order [control, diabetes (DB), DB-GP, DB + SGES, DB-GP + SGES, DB-GP + Atropine, and DB-GP + SGES + Atropine groups]. In the control, DB, DB-GP, and DB-GP + Atropine groups, gastric emptying was measured in BLAB/cJ mice (control group) or NOD mice with a duration of diabetes of 0-7 days (DB group) or 28-35 days (DB-GP or DB-GP + Atropine group). In the DB + SGES, DB-GP + SGES, and DB-GP + SGES + Atropine groups, the experiment was the same as the corresponding DB, DB-GP, and DB-GP + Atropine groups except that SGES was applied during the experiment. SGES was applied via the proximal pair of electrodes and synchronized with the intrinsic gastric slow waves. The following results were obtained: 1) gastric emptying was delayed in NOD mice with a duration of diabetes of 28-35 days; 2) SGES was able to significantly increase gastric emptying in both diabetic mice and diabetic gastroparetic mice; and 3) the excitatory effect of SGES was completely blocked by atropine. SGES accelerates gastric emptying in NOD mice with diabetic gastroparesis. The effect of SGES on gastric emptying is mediated via the cholinergic pathway. These findings suggest that SGES may have a therapeutic potential for treating patients with diabetic gastroparesis.     

Manipulation of CD98 resolves type 1 diabetes in nonobese diabetic mice

The interplay of CD4(+) and CD8(+) T cells targeting autoantigens is responsible for the progression of a number of autoimmune diseases, including type 1 diabetes mellitus (T1D). Understanding the molecular mechanisms that regulate T cell activation is crucial for designing effective therapies for autoimmune diseases. We probed a panel of Abs with T cell-modulating activity and identified a mAb specific for the H chain of CD98 (CD98hc) that was able to suppress T cell proliferation. The anti-CD98hc mAb also inhibited Ag-specific proliferation and the acquisition of effector function by CD4(+) and CD8(+) T cells in vitro and in vivo. Injection of the anti-CD98hc mAb completely prevented the onset of cyclophosphamide-induced diabetes in NOD mice. Treatment of diabetic NOD mice with anti-CD98hc reversed the diabetic state to normal levels, coincident with decreased proliferation of CD4(+) T cells. Furthermore, treatment of diabetic NOD mice with CD98hc small interfering RNA resolved T1D. These data indicate that strategies targeting CD98hc might have clinical application for treating T1D and other T cell-mediated autoimmune diseases.