Early over expression of messenger RNA for multiple genes, including insulin, in the Pancreatic Lymph Nodes of NOD mice is associated with Islet Autoimmunity

<p>Abstract</p> <p>Background</p> <p>Autoimmune diabetes (T1D) onset is preceded by a long inflammatory process directed against the insulin-secreting β cells of the pancreas. Deciphering the early autoimmune mechanisms represents a challenge due to the absence of clinical signs at early disease stages. The aim of this study was to identify genes implicated in the early steps of the autoimmune process, prior to inflammation, in T1D. We have previously established that insulin autoantibodies (E-IAA) predict early diabetes onset delineating an early phenotypic check point (window 1) in disease pathogenesis. We used this sub-phenotype and applied differential gene expression analysis in the pancreatic lymph nodes (PLN) of 5 weeks old Non Obese Diabetic (NOD) mice differing solely upon the presence or absence of E-IAA. Analysis of gene expression profiles has the potential to provide a global understanding of the disease and to generate novel hypothesis concerning the initiation of the autoimmune process.</p> <p>Methods</p> <p>Animals have been screened weekly for the presence of E-IAA between 3 and 5 weeks of age. E-IAA positive or negative NOD mice at least twice were selected and RNAs isolated from the PLN were used for microarray analysis. Comparison of transcriptional profiles between positive and negative animals and functional annotations of the resulting differentially expressed genes, using software together with manual literature data mining, have been performed.</p> <p>Results</p> <p>The expression of 165 genes was modulated between E-IAA positive and negative PLN. In particular, genes coding for insulin and for proteins known to be implicated in tissue remodelling and Th1 immunity have been found to be highly differentially expressed. Forty one genes showed over 5 fold differences between the two sets of samples and 30 code for extracellular proteins. This class of proteins represents potential diagnostic markers and drug targets for T1D.</p> <p>Conclusion</p> <p>Our data strongly suggest that the immune related mechanisms taking place at this early age in the PLN, correlate with homeostatic changes influencing tissue integrity of the adjacent pancreatic tissue. Functional analysis of the identified genes suggested that similar mechanisms might be operating during pre-inflammatory processes deployed in tissues i) hosting parasitic microorganisms and ii) experiencing unrestricted invasion by tumour cells.</p

Investigation of type 1 diabetes in NOD mice knockout for the osteopontin gene

International audienceObjective: Type 1 diabetes onset is preceded by a pre-inflammatory stage leading to insulitis and followed by targeted destruction of the insulin-producing beta cells of the pancreas. Osteopontin (OPN) is a secreted phos-phoprotein with cytokine properties, implicated in many physiological and pathological processes, including infection and autoimmunity. We have previously identified up-regulated osteopontin transcripts in the pan-creatic lymph nodes of the NOD (Non-Obese Diabetic) mouse at the pre-diabetic stages. Investigating the underlined disease initiating mechanisms may well contribute to the development of novel preventive therapies. Our aim was to construct opn null mice in a NOD autoimmune-prone genetic background and address the pathogenic or protective role of the osteopontin molecule in the early stages of type 1 diabetes. Methods: We generated opn null mutant mice in a NOD genetic background by serial backcrossing to the existing C57BL/6 opn knockout strain. The presence of opn wild type or null alleles in the congenic lines was evaluated by PCR amplification. We used NOD opn-null mice to assess the phenotypic evolution of type 1 diabetes. The presence of OPN in the serum was evaluated by ELISA and by immunostaining on the mouse tissues. The primary gene structure of the NOD opn encoding gene and protein sequences were compared to the known alleles of other mouse strains. Evaluation of Single Nucleotide Polymorphisms (SNPs) variation between opn alleles of the opn gene is reported. Results: In the absence of OPN, type 1 diabetes is accelerated, suggesting a protective role of this cytokine on the insulin-producing cells of the pancreatic islets. Conversely, in the presence of the opn gene, an increase of the OPN protein in the serum of young NOD mice indicates that this molecule might be involved in the immune regulatory events taking place at early stages, prior to disease onset. Our data support that OPN acts as a positive regulator of the early islet autoimmune damage, possibly by a shift of the steady-state of T1D pathogenesis. We report that the OPN protein structure of the NOD/ShiLtJ strain corresponds to the a-type allele of the osteopontin gene. Comparative analysis of the single nucleotide polymorphisms between the a-type and b-type alleles indicates that the majority of variations are within the non-coding regions of the gene. Conclusions: The construction of opn null mice in an autoimmune genetic background (NOD.B6.Cg-spp1-/-) provides important tools for the study of the implication of the OPN in type 1 diabetes, offering the possibility to address the significance of this molecule as an early marker of the disease and as a therapeutic agent in pre-clinical studies

The autoimmune contrivance: Genetics in the mouse model

International audienceAutoimmunity and inheritance of complex characters behold an explosive interest in biology over the last 15 years. Research in the genetics of autoimmunity has been impelled by the isolation of genetic markers allowing tracing of heredity. The annotation and sequencing of the human and mouse genomes provide with the potential for further advancements, through the development of new technologies. This review aims to summarize advances made in the autoimmunity field, centered in type 1 diabetes in the NOD mouse model. It also aims to demonstrate that animal models, albeit some phenotypic and genetic dissimilarities with the human diseases, still remain the best way to move towards an understanding of the molecular mechanisms involved in autoimmunity. Assessing the current state of research in this field together with the increasing potential of novel biotechnology advancements, new insights to disease pathogenesis and discovery of molecular targets for intervention strategies are anticipated in the coming years.

Mecanisme d'action des hormones steroides: Regulation de l'expression genique par les androgenes

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RNA Extraction from Ears and Draining Lymph Nodes of Mice Infected with Leishmania amazonensis

International audienceParasites of the genus Leishmania infect the mammalian hosts, including mice and humans and cause cutaneous or visceral leishm aniasis depending upon the parasite species transmitted by the vector sandfly. Leishmania amazonensis is one of the Leishmania species responsible for th e cutaneous form of the disease. We have inoculated with these parasites the ear dermis of mice. RNA preparations were performed from fragmented tissues using a buffer containing guanidin isothiocynate (RLT buffer, RNeasy Mini Kit, Qiagen, SAS, France) and β mercaptoethanol. Both reagents facilitate the isolation of intact RNA from tissues and the use of the RNeasy Kits present with several advantages that facilitate the isolation of pure non degraded total RNA: i) T his method allows to avoid the presence o f phenol in the RNA extraction buffer, commonly used in alternative protocols; ii) Moreover Diethylpyrocarbonate (DEPC) treatment of glassware, to avoid RNAses contamination of the samples, is not required with this protocol; iii) F inally, it is a fast pro cedure and the isolated total RNA may be concentrated in a small volume thus facilitating its use for downstream experimental procedures

Functional Genomics in Early Autoimmunity

International audienceThe molecular mechanisms initiating the autoimmune process in type 1 diabetes mellitus (T1DM) remain unknown, and studies aiming to address this question have been compromised by the difficulty of predicting the disease at an early age both in humans and in animal models. An additional hindrance in selecting individuals at an early age has been the complex genetic inheritance of autoimmune diabetes, implicating not only several genes but also environmental factors. We have previously demonstrated the predictive value of insulin autoantibodies (IAAs) at an early age, between three to five weeks in the NOD mouse. Animals positive for early appearance of IAAs (E-IAAs) develop auto-immune diabetes earlier. We showed a correlation between the presence of IAAs in the mothers during pregnancy, E-IAAs in the litters, and the early appearance of T1DM. NOD mice, E-IAA–positive, within litters from IAA-positive mothers during pregnancy, develop diabetes earlier and at a much greater rate than animals that are IAA-negative and from IAA-negative mothers. The molecular mechanisms responsible for this early autoimmune subphenotype were addressed by a global approach to differential gene expression analysis in the pancreatic lymph nodes (PaLNs). Although the data analysis is currently in progress, gene expression signatures were observed that are characteristic for PaLNs with regard to the presence or absence of IAAs. Overall, these data are consistent with the hypothesis of an early environmental influence from the autoimmune maternal environment on the genetic predisposition of the offspring, characterized by specific gene signatures leading to autoimmune disease

Genetics of Type 1A (immune mediated) diabetes

International audienceType 1A (immune mediated) diabetes is genetically heterogeneous with important examples for man and animal models with major mutations (autosomal recessive and X-linked recessive) identified as well as oligogenic/polygenic inheritance. For the most common forms of type 1A diabetes alleles of DQ and DR within the major histocompatibility complex are important determinants of disease and allow identification of high risk individuals at birth. Further understanding of both common and rare genetic determinants of type 1A diabetes will contribute to understanding the pathogenesis of diabetes and of autoimmunity