RESUMO
BACKGROUND: Environmental factors are critical in the development of age-related cognitive decline and dementia. A western diet (WD) can cause nutrient deficiency and inflammation that could impact cognition directly. It is increasingly recognized that innate immune responses by brain myeloid cells, such as resident microglia, and infiltrating peripheral monocytes/macrophages may represent an essential link between a WD, cognitive decline, and dementia. Our previous data demonstrated that chronic consumption of a WD induced inflammation through brain myeloid cells in aging mice and a mouse model of Alzheimer's disease (AD). However, the subtypes of myeloid cells that contribute to the WD-induced inflammation remain unclear. METHODS: C57BL/6J (B6), myeloid cell reporter mice (B6.Ccr2RFP/+Cx3cr1GFP/+), and Ccr2-deficient mice (B6.Ccr2RFP/RFP) were fed a WD or a control chow diet (CD) from 2 to 6 or 12 months of age. CD11b+CD45lo and CD11b+CD45hi cells from WD- and CD-fed B6 or Ccr2-deficient mice were characterized using flow cytometry, RNA-sequencing, and immunofluorescence. RESULTS: Ccr2::RFP expressing myeloid cells were significantly increased in brains of WD- compared to CD-fed mice, but were not elevated in Ccr2-deficient WD-fed mice. The percent of CD11b+CD45hi cells was significantly increased in WD- compared to CD-fed mice. Comparison of RNA-sequencing data with immune cell data in ImmGen supports that CD11b+CD45hi cells from WD-fed mice are enriched for peripheral monocytes and neutrophils. Ingenuity pathway analysis predicted these cells elicit proinflammatory responses that may be damaging to the brain. Using stringent criteria for gene expression levels between CD11b+CD45hi and CD11b+CD45lo cells, we identified approximately 70 genes that we predict are uniquely expressed in infiltrating cells, including Itgal, Trem1, and Spp1 (osteopontin, OPN). Finally, we show a significantly greater number of OPN+IBA1- cells in WD- compared to CD-fed mice that we propose are activated neutrophils based on ImmGen data. OPN+IBA1- cells are not significantly increased in Ccr2-deficient WD-fed mice. CONCLUSIONS: These data further support the model that peripheral myeloid cells enter the brain in response to diet-induced obesity. Elucidating their contribution to age-related cognitive decline and age-related neurodegenerative diseases should offer new avenues for therapeutic intervention in Alzheimer's disease and related dementias, where diet/obesity are major risk factors.
Assuntos
Antígeno CD11a/metabolismo , Dieta Ocidental/efeitos adversos , Perfilação da Expressão Gênica/métodos , Obesidade/metabolismo , Osteopontina/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/metabolismo , Obesidade/induzido quimicamente , Obesidade/genética , Osteopontina/genética , Receptor Gatilho 1 Expresso em Células Mieloides/genéticaRESUMO
B lymphocytes play a key role in type 1 diabetes (T1D) development by serving as a subset of APCs preferentially supporting the expansion of autoreactive pathogenic T cells. As a result of their pathogenic importance, B lymphocyte-targeted therapies have received considerable interest as potential T1D interventions. Unfortunately, the B lymphocyte-directed T1D interventions tested to date failed to halt ß cell demise. IgG autoantibodies marking humans at future risk for T1D indicate that B lymphocytes producing them have undergone the affinity-maturation processes of class switch recombination and, possibly, somatic hypermutation. This study found that CRISPR/Cas9-mediated ablation of the activation-induced cytidine deaminase gene required for class switch recombination/somatic hypermutation induction inhibits T1D development in the NOD mouse model. The activation-induced cytidine deaminase protein induces genome-wide DNA breaks that, if not repaired through RAD51-mediated homologous recombination, result in B lymphocyte death. Treatment with the RAD51 inhibitor 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid also strongly inhibited T1D development in NOD mice. The genetic and small molecule-targeting approaches expanded CD73+ B lymphocytes that exert regulatory activity suppressing diabetogenic T cell responses. Hence, an initial CRISPR/Cas9-mediated genetic modification approach has identified the AID/RAD51 axis as a target for a potentially clinically translatable pharmacological approach that can block T1D development by converting B lymphocytes to a disease-inhibitory CD73+ regulatory state.
Assuntos
Linfócitos B Reguladores/imunologia , Proteínas de Transporte/antagonistas & inibidores , Citidina Desaminase/antagonistas & inibidores , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Ativação Linfocitária , Proteínas Nucleares/antagonistas & inibidores , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , 5'-Nucleotidase/imunologia , Animais , Autoanticorpos/imunologia , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Proteínas de Ligação a DNA , Diabetes Mellitus Experimental , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos NOD , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA , Hipermutação Somática de ImunoglobulinaRESUMO
The pleiotropic cytokine IL-21 is implicated in the pathogenesis of human systemic lupus erythematosus by polymorphisms in the molecule and its receptor (IL-21R). The systemic lupus erythematosus-like autoimmune disease of BXSB.Yaa mice is critically dependent on IL-21 signaling, providing a model for understanding IL-21/IL-21R signaling in lupus pathogenesis. In this study, we generated BXSB.Yaa mice selectively deficient in IL-21R on B cells, on all T cells, or on CD8(+) T cells alone and examined the effects on disease. We found that IL-21 signaling to B cells is essential for the development of all classical disease manifestations, but that IL-21 signaling also supports the expansion of central memory, CD8(+) suppressor cells and broadly represses the cytokine activity of CD4(+) T cells. These results indicate that IL-21 has both disease-promoting and disease-suppressive effects in the autoimmune disease of BXSB.Yaa mice.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Interleucinas/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Interleucina-21/metabolismo , Animais , Linfócitos B , Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Interleucinas/genética , Masculino , Camundongos , Camundongos Transgênicos , Receptores de Interleucina-21/genética , Transdução de Sinais/imunologiaRESUMO
Dysregulation of the T cell-dependent Ab response can lead to numerous immunological disorders, ranging from systemic lupus erythematosus to B cell lymphomas. Cellular processes governed by MHC class II proteins play a major role in this response and its dysregulation. The extent to which processes controlled by the diverse family of MHC class I proteins impact such autoimmune and neoplastic disorders, however, is less clear. In this study, we genetically dissect the contributions of individual MHC class I family members and the pathological processes under their control in the systemic lupus erythematosus-like disease of BXSB.Yaa mice and B cell lymphomagenesis of SJL mice. This study reveals a powerful repressive regulatory axis comprised of MHC class I-dependent CD8(+) T cells and NK cells. These results indicate that the predominant role of the MHC class I protein family in such immunological disorders is to protect from more aggressive diseases.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/prevenção & controle , Linfoma de Células B/imunologia , Linfoma de Células B/prevenção & controle , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Antígenos H-2/genética , Antígeno de Histocompatibilidade H-2D , Antígenos de Histocompatibilidade Classe I/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Lúpus Eritematoso Sistêmico/mortalidade , Linfoma de Células B/mortalidade , Camundongos , Camundongos Endogâmicos , Vírus da Leucemia Murina de Moloney/imunologia , Microglobulina beta-2/deficiência , Microglobulina beta-2/genéticaRESUMO
The lack of genetically diverse preclinical animal models in basic biology and efficacy testing has been cited as a potential cause of failure in clinical trials. We developed and characterized five diverse RAG1 null mouse strains as models that allow xenografts to grow. In these strains, we characterized the growth of breast cancer, leukemia and glioma cell lines. We found a wide range of growth characteristics that were far more dependent on strain than tumor type. For the breast cancer cell line, we characterized the spectrum of xenograft/tumor growth at structural, histological, cellular and molecular levels across each strain, and found that each strain captures unique structural components of the stroma. Furthermore, we showed that the increase in tumor-infiltrating myeloid CD45+ cells and the amount of circulating cytokine IL-6 and chemokine KC (also known as CXCL1) is associated with a higher tumor size in different strains. This resource is available to study established human xenografts, as well as difficult-to-xenograft tumors and growth of hematopoietic stems cells, and to decipher the role of myeloid cells in the development of spontaneous cancers.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Knockout , Transplante Heterólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inbred mouse strains are routinely used as genetically defined animal models for studying a wide assortment of biological and pathological processes, including immune system function. However, no studies have presented large-scale data on the immune cell populations among the inbred strains in physiological conditions. Here we present a systematic, quantitative analysis of peripheral blood cell phenotypes of 30 mouse strains assessed by flow cytometry. This cohort of mice represents a wide range of genetic origins and includes most of the strains used in genetic, physiological, and immunological studies. We evaluated the relative percentages of peripheral blood leukocyte subtypes (lymphocytes, granulocytes, and monocytes) and lymphocyte subpopulations (CD4+ T, CD8+ T, B220+ B, and natural killer cells) of mature (6-mo-old) mice. Our comprehensive study demonstrated: 1) marked differences in the relative proportions of blood cell populations among the strains at this age, 2) considerable variation of each immune trait with more than twofold difference between strains with the highest and the lowest trait values, and 3) haplotype analysis revealed a strong correlation between eosinophil percentage and a single region on chromosome 14 containing two candidate genes. The strain differences described here provide important information for researchers applying immunophenotyping of peripheral blood in immunological and genetic studies. The data from this study are available as part of the Mouse Phenome Database at http://www.jax.org/phenome.
Assuntos
Células Sanguíneas/citologia , Linhagem da Célula , Imunidade/genética , Animais , Citometria de Fluxo , Haplótipos , Leucócitos/citologia , Camundongos , Fenótipo , Caracteres Sexuais , Especificidade da EspécieRESUMO
The major histocompatibility complex (MHC) of the type 1 diabetes-prone NOD mouse lacks a functional class II H2-Ea gene such that antigen presenting cells (APCs) are I-E null. Transgenic expression of Ea in NOD mice both restores I-E expression and confers complete protection from diabetes progression. Non-myeloablative neonatal transplantation of bone marrow cells from such I-E+ transgenic donors into NOD recipients resulted in low-level but long-term haematopoietic stem cell (HSC) engraftment. Despite low levels of I-E antigen expression in blood (averaging 0.4-3.8% of total MHC class II-positive population), chimeric recipients were protected from overt diabetes, although not insulitis development. Adoptive transfer of diabetes into immunodeficient NOD-Rag recipients that received chimeric splenocytes from primary recipients confirmed the presence of an autoreactive T cell repertoire. The demonstration that purified T cells from these weak chimeras were not tolerant to irradiated transgenic I-E+ splenocytes indicated that I-E+ donor cells provide a constant, low-level immune stimulation capable of up-regulating nominally deficient immunoregulatory networks. This study raises the possibility that cord blood HSCs from infants with high risk HLA haplotypes and a family history of type 1 diabetes might be re-introduced without myoablative treatments following transfection with a single HLA class II allele associated with diabetes resistance.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Alelos , Animais , Transplante de Medula Óssea , Diabetes Mellitus Tipo 1/genética , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fatores SexuaisRESUMO
Interleukin (IL)-1 beta and IL-18 are two cytokines associated with the immunopathogenesis of diabetes in NOD mice. Both of these cytokines are cleaved by caspase-1 to their biologically active forms. IL-1 is a proinflammatory cytokine linked to beta-cell damage, and IL-18 stimulates production of interferon (IFN)gamma in synergy with IL-12. To examine the effects produced by caspase-1 deficiency on diabetes development in NOD/Lt mice, a disrupted Casp1 gene was introduced by a speed congenic technique. Casp1(-/-) bone marrow-derived macrophages stimulated with lipopolysaccharide produced no detectable IL-18, fourfold lower IL-1 beta, and 20-30% less IL-1 alpha than macrophages from wild-type Casp1(+/+) or Casp1(+/-) controls. Unexpectedly, despite reduced IL-1 and IL-18, there was no change in the rate of diabetes or in total incidence as compared with that in wild-type NOD mice. IL-1 reportedly makes an important pathological contribution in the multidose streptozotocin model of diabetes; however, there was no difference in sensitivity to streptozotocin between NOD mice and NOD.Casp1(-/-) mice at 40 mg/kg body wt or at 25 mg/kg body wt dosage levels. These findings show that caspase-1 processing of IL-1 beta and IL-18 is not absolutely required for mediation of spontaneous or chemically induced diabetes pathogenesis in the NOD mouse.
Assuntos
Caspase 1/genética , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/genética , Animais , Caspase 1/deficiência , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/fisiopatologia , Feminino , Incidência , Interleucina-1/metabolismo , Interleucina-18/metabolismo , Cinética , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Caracteres Sexuais , Especificidade da Espécie , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Molecular cancer diagnostics are an important clinical advance in cancer management, but new methods are still needed. In this context, gene expression signatures obtained by microarray represent a useful molecular diagnostic. Here, we describe novel probe-level microarray analyses that reveal connections between mRNA processing and neoplasia in multiple tumor types, with diagnostic potential. We now show that characteristic differences in mRNA processing, primarily in the 3'-untranslated region, define molecular signatures that can distinguish similar tumor subtypes with different survival characteristics, with at least 74% accuracy. Using a mouse model of B-cell leukemia/lymphoma, we find that differences in transcript isoform abundance are likely due to both alternative polyadenylation (APA) and differential degradation. While truncation of the 3'-UTR is the most common observed pattern, genes with elongated transcripts were also observed, and distinct groups of affected genes are found in related but distinct tumor types. Genes with elongated transcripts are overrepresented in ontology categories related to cell-cell adhesion and morphology. Analysis of microarray data from human primary tumor samples revealed similar phenomena. Western blot analysis of selected proteins confirms that changes in the 3'-UTR can correlate with changes in protein expression. Our work suggests that alternative mRNA processing, particularly APA, can be a powerful molecular biomarker with prognostic potential. Finally, these findings provide insights into the molecular mechanisms of gene deregulation in tumorigenesis.
Assuntos
Linfoma de Células B/genética , Linfoma de Células B/patologia , RNA Mensageiro/genética , Regiões 3' não Traduzidas , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma de Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Isoformas de Proteínas , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Primary immunodeficiencies are rare but serious diseases with diverse genetic causes. Accumulating evidence suggests that defects in DNA double-strand break (DSB) repair can underlie many of these syndromes. In this context, the nonhomologous end joining pathway of DSB repair is absolutely required for lymphoid development, but possible roles for the homologous recombination (HR) pathway have remained more controversial. While recent evidence suggests that HR may indeed be important to suppress lymphoid transformation, the specific relationship of HR to normal lymphocyte development remains unclear. We have investigated roles of the X-ray cross-complementing 2 (Xrcc2) HR gene in lymphocyte development. We show that HR is critical for normal B-cell development, with Xrcc2 nullizygosity leading to p53-dependent early S-phase arrest. In the absence of p53 (encoded by Trp53), Xrcc2-null B cells can fully develop but show high rates of chromosome and chromatid fragmentation. We present a molecular model wherein Xrcc2 is important to preserve or restore replication forks during rapid clonal expansion of developing lymphocytes. Our findings demonstrate a key role for HR in lymphoid development and suggest that Xrcc2 defects could underlie some human primary immunodeficiencies.
Assuntos
Linfócitos B/citologia , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Linfopoese/fisiologia , Recombinação Genética , Animais , Células Cultivadas/citologia , Aberrações Cromossômicas , Quebra Cromossômica , Técnicas de Cocultura , Deleção de Genes , Genes p53 , Imunoglobulina M/biossíntese , Interleucina-7/metabolismo , Antígenos Comuns de Leucócito/biossíntese , Fígado/citologia , Fígado/embriologia , Linfopoese/genética , Camundongos , Camundongos Knockout , Células NIH 3T3/metabolismo , Fase S , Homologia de Sequência do Ácido Nucleico , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Ubiquitously expressed CD38 and T cell-expressed ADP-ribosyltransferase 2 (ART2) are ectoenzymes competing for NAD substrate. CD38 exerts pleiotropic actions in hemopoietic and nonhemopoietic compartments via effects on calcium mobilization. ART2 is an ADP-ribosyltransferase on naive CD4+ and CD8+ T cells. ART2-catalyzed ADP-ribosylation of the P2X7 purinoreceptor elicits apoptosis. Transfer of a genetically disrupted CD38 allele into the autoimmune diabetes-prone NOD/Lt background accelerated diabetes onset in both sexes, whereas transfer of a disrupted ART2 complex had no effect. However, the fact that the accelerated pathogenesis mediated by CD38 deficiency required ART2 activity was demonstrated by combining both ART2 and CD38 deficiencies. Reciprocal bone marrow reconstitution studies demonstrated accelerated diabetes only when CD38-deficient bone marrow was transferred into CD38-deficient recipients. Neither decreases in beta cell function nor viability were indicated. Rather, the balance between T-effectors and T-regulatory cells was disturbed in CD38-deficient but ART2-intact NOD mice. In these mice, significant reductions in total viable CD8+ T cells were observed. This was accompanied by an age-dependent increase in a diabetogenic CD8 clonotype. This in turn correlated with impaired T-regulatory development (10-fold reduction in Foxp3 mRNA expression). These changes were corrected when CD38 deficiency was combined with ART2 deficiency. Both ART2-deficient and CD38/ART2 combined deficient T cells were resistant to NAD-induced killing in vitro, whereas CD38-deficient but ART2-intact T cells showed increased sensitivity, particularly the CD4+ CD25+ subset. Unexpectedly, diabetes development in the combined CD38/ART2 stock was strongly suppressed, possibly through epistatic interactions between genes linked to the targeted CD38 on Chromosome 5 and the ART2 complex on Chromosome 7.
Assuntos
ADP Ribose Transferases/metabolismo , ADP-Ribosil Ciclase 1/deficiência , Diabetes Mellitus Tipo 1/etiologia , Glicoproteínas de Membrana/deficiência , ADP Ribose Transferases/genética , ADP-Ribosil Ciclase 1/genética , Animais , Apoptose , Autoimunidade , Transplante de Medula Óssea , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Epistasia Genética , Feminino , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , NAD/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
Adding NAD to murine T lymphocytes inhibits their functions and induces annexin V binding. This report shows that NAD induces cell death in a subset of T cells within seconds whereas others do not die until many hours later. Low NAD concentrations (<10 microM) suffice to trigger rapid cell death, which is associated with annexin V binding and membrane pore formation, is not blocked by the caspase inhibitor Z-VADfmk, and requires functional P2X7 receptors. The slower induction of death requires higher NAD concentrations (>100 microM), is blocked by caspase inhibitor Z-VADfmk, is associated with DNA fragmentation, and does not require P2X7 receptors. T cells degrade NAD to ADP-ribose (ADPR), and adding ADPR to T cells leads to slow but not rapid cell death. NAD but not ADPR provides the substrate for ADP-ribosyltransferase (ART-2)-mediated attachment of ADP-ribosyl groups to cell surface proteins; expression of ART-2 is required for NAD to trigger rapid but not slow cell death. These results support the hypothesis that cell surface ART-2 uses NAD but not ADPR to attach ADP-ribosyl groups to the cell surface, and that these groups act as ligands for P2X7 receptors that then induce rapid cell death. Adding either NAD or ADPR also triggers a different set of mechanisms, not requiring ART-2 or P2X7 receptors that more slowly induce cell death.
Assuntos
Inibidores do Crescimento/farmacologia , NAD/análogos & derivados , NAD/farmacologia , Receptores Purinérgicos P2/fisiologia , Linfócitos T/citologia , Linfócitos T/metabolismo , ADP Ribose Transferases/fisiologia , Adenosina Difosfato Ribose/farmacologia , Animais , Apoptose/imunologia , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , NAD/metabolismo , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Especificidade por Substrato , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Fatores de TempoRESUMO
Penetrance of the complex of genes predisposing the nonobese diabetic (NOD) mouse to autoimmune diabetes is affected by the maternal environment. NOD.CBALs-Tyr(+)/Lt is an agouti-pigmented Chromosome 7 congenic stock of NOD/Lt mice produced as a resource for embryo transfer experiments to provide the necessary maternal factors and allow the easy identification of NOD (albino) embryo donor phenotype. CBcNO6/Lt, a recombinant congenic agouti stock already containing approximately 50% NOD genome, was used as the donor source of a wild-type CBA tyrosinase allele. When the incidence of diabetes was assessed after nine generations of backcrossing and one generation of sib-sib mating, significant reduction in diabetes development was observed. No difference in diabetes development was observed in Tyr/Tyr(c) heterozygotes, showing that protection was recessive. Analysis of diabetes progression in another NOD stock congenic for C57BL/6 alleles on Chromosome 7 linked to the glucose phosphate isomerase (Gpi1(b)) locus provided no protection, indicating that the diabetes resistance (Idd) gene was distal to 34 cM (D7Mit346). Approximately 5 cM of the distal congenic region overlaps a region from C57L previously associated with protection when homozygous. The delayed onset and reduced frequency of diabetes in the NOD.CBALs-Tyr(+)/Lt stock is an advantage when females of this stock are used as surrogate mothers in studies involving hysterectomy or embryo transfers. Indeed, a newly developed NOD embryonic stem (ES) cell line injected into NOD.CBALs- Tyr(+)/Lt blastocysts produced approximately 50% live-born mice, of which approximately 11% were chimeric. Presumably because of high genomic instability, no germline transmission was observed.