Genetic control of renal tumorigenesis by the mouse Rtm1 locus

BACKGROUND: The genetic basis of susceptibility to renal tumorigenesis has not yet been established in mouse strains. Mouse lines derived by bidirectional phenotypic selection on the basis of their maximal (AIRmax) or minimal (AIRmin) acute inflammatory responsiveness differ widely in susceptibility to spontaneous and urethane-induced renal tumorigenesis. To map the functional loci modulating renal tumor susceptibility in these mice, we carried out a genome-wide genetic linkage study, using SNP arrays, in an (AIRmax x AIRmin)F2 intercross population treated with a single urethane dose at 1 week of age and phenotyped for renal tumors at 35 weeks of age. RESULTS: AIRmax mice did not develop renal tumors spontaneously nor in response to urethane, whereas in AIRmin mice renal tumors formed spontaneously (in 52% of animals) and after urethane induction (89%). The tumors had a papillary morphology and were positive for alpha-methylacyl-CoA racemase and negative for CD10. By analysis of 879 informative SNPs in 662 mice, we mapped a single quantitative trait locus modulating the incidence of renal tumors in the (AIRmax x AIRmin)F2 intercross population. This locus, which we named Renal tumor modifier QTL 1 (Rtm1), mapped to chromosome 17 at 23.4 Mb (LOD score = 15.8), with SNPs rs3696835 and rs3719497 flanking the LOD score peak. The A allele of rs3719497 from AIRmin mice was associated with a 2.5-fold increased odds ratio for renal tumor development. The LOD score peak included the Tuberous sclerosis 2 (Tsc2) gene which has already been implicated in kidney disease: loss of function by germline retroviral insertion is associated with spontaneous renal tumorigenesis in the Eker rat, and heterozygous-null Tsc2((+/-)) mice develop renal cystadenomas. CONCLUSIONS: We mapped Rtm1 as a single major locus modulating renal tumorigenesis in a murine intercross population. Thus, the AIR mouse lines can be considered a new genetic model for studying the role of germline and somatic molecular alterations in kidney neoplastic disease

Controle genetico inespecifico da sintese de anticorpos : estudo dos genes envolvidos na resposta a eritrocitos heterologos em camundongos bons e maus respondedores a antigeno somatico de Salmonellae

Orientadora : Maria Siqueira PinheiroDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: 1- As linhagens H/s e L/s, obtidas usando como caráter selecionador a resposta elevada ou baixa contra antígeno s de Salmonellae (Seleção IV), não se diferenciaram na resposta contra eritrócitos de carneiro e humanos. Tomando como população inicial segregantes F2 entre as linhagens H/s e L/s, foi possível selecionar camundongos bons (H/hte) e maus (L/hte) respondedores contra eritrócitos de carneiro e humanos. Essas novas linhagens, por sua vez, apresentaram respostas semelhantes contra antígenos de Salmonella. 2 ? A análise genética dos dados obtidos nas cinco gerações estudadas da Seleção contra Eritrócitos Heterólogos (SE e HuE), demonstrou que a separação entre as linhagens vem se desenvolvendo progressivamente. A "herdabilidade realizada" foi cerca de 0,2; o ganho por geração 0,4; e a pressão de seleção 2,2, valores muito semelhantes aos obtidos nas outras seleções de camundongos já descritas (Biozzi et a1, 1979). O número de loci tornado homozigotos até o momento, foi cerca de 2, calculado a partir da herdabilidade e da variância da população inicial. Os genes selecionados atuam também, na resposta contra eritrócitos de pombo e de galinha, e possivelmente na resposta a RGG, porém, não tem nenhuma influência sobre a resposta contra os antígenos f e s de Salmonella e contra BGG. A intensidade do efeito inespecífico, portanto, irá depender, em cada seleção, não só do número de loci envolvidos, como também da importância destes, na resposta a cada um dos antígeno testados... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: Not informed.MestradoMestre em Ciências Biológica

miRNA Expression and Interaction with Genes Involved in Susceptibility to Pristane-Induced Arthritis

Pristane-induced arthritis (PIA) in mice is an experimental model that resembles human rheumatoid arthritis, a chronic autoimmune disease that affects joints and is characterized by synovial inflammation and articular cartilage and bone destruction. AIRmax and AIRmin mouse lines differ in their susceptibility to PIA, and linkage analysis in this model mapped arthritis severity QTLs in chromosomes 5 and 8. miRNAs are a class of small RNA molecules that have been extensively studied in the development of arthritis. We analyzed miRNA and gene expression profiles in peritoneal cells of AIRmax and AIRmin lines, in order to evaluate the genetic architecture in this model. Susceptible AIRmax mice showed higher gene (2025 vs 1043) and miRNA (240 vs 59) modulation than resistant AIRmin mice at the onset of disease symptoms. miR-132-3p/212-3p, miR-106-5p, miR-27b-3p, and miR-25-3p were among the miRNAs with the highest expression in susceptible animals, showing a negative correlation with the expression of predicted target genes (Il10, Cd69, and Sp1r1). Our study showed that global gene and miRNA expression profiles in peritoneal cells of susceptible AIRmax and resistant AIRmin lines during pristane-induced arthritis are distinct, evidencing interesting targets for further validation

Early Peritoneal CC Chemokine Production Correlates with Divergent Inflammatory Phenotypes and Susceptibility to Experimental Arthritis in Mice

The inflammatory and autoimmune events preceding clinical symptoms in rheumatoid arthritis (RA) and other autoimmune diseases are difficult to study in human patients. Therefore, animal models that share immunologic and clinical features with human RA, such as pristane-induced arthritis (PIA), are valuable tools for assessing the primordial events related to arthritis susceptibility. PIA-resistant HIII and susceptible LIII mice were injected i.p. with pristane, and peritoneal lavage fluid was harvested in the early (7 days) and late (35 days) preclinical phases of PIA. Chemokine and cytokine levels were measured in lavage supernatant with ELISA, peritoneal inflammatory leukocytes were immunophenotyped by flow cytometry, and gene expression was determined by qRT-PCR. Leukocyte recruitment was quantitatively and qualitatively divergent in the peritoneum of HIII and LIII mice, with an early increase of CC chemokines (CCL2/CCL3/CCL5/CCL12/CCL22) in the susceptible LIII strain. Also, cytokines such as IL-12p40, IL-23, and IL-18 were elevated in LIII mice while IL-6 was increased in HIII animals. The results show that an early peritoneal CC chemokine response is an important feature of arthritis susceptibility and defines potential biomarkers in this model

Genetic Predisposition to Hepatocarcinogenesis in Inbred and Outbred Mouse Lines Selected for High or Low Inflammatory Response

AIRmax and AIRmin mouse strains phenotypically selected for high and low acute inflammatory responsiveness (AIR) are, respectively, susceptible or resistant to developing hepatocellular carcinoma (HCC) induced by the chemical carcinogens urethane and diethylnitrosamine (DEN). Early production of TNF-α, IL-1β, and IL-6 in the liver after DEN treatment correlated with tumor development in AIRmax mice. Transcriptome analysis of livers from untreated AIRmax and AIRmin mice showed specific gene expression profiles in each line, which might play a role in their differential susceptibility to HCC. Linkage analysis with SNP markers in F2 (AIRmax×AIRmin) intercross mice revealed two quantitative trait loci (QTL) in chromosomes 2 and 9, which are significantly associated with the number and progression of urethane-induced liver tumors. An independent linkage analysis with an intercross population from A/J and C57BL/6J inbred mice mapped regions in chromosomes 1 and 7 associated with the progression of urethane-induced liver tumors, evidencing the heterogeneity of HCC genetic control

Ovariectomized OVA-sensitized mice display increased frequency of CD4+Foxp3+ T regulatory cells in the periphery

It is well established that female sex hormones have a pivotal role in inflammation. For instance, our group has previously reported that estradiol has proinflammatory actions during allergic lung response in animal models. Based on these findings, we have decided to further investigate whether T regulatory cells are affected by female sex hormones absence after ovariectomy. We evaluated by flow cytometry the frequencies of CD4+Foxp3+ T regulatory cells (Tregs) in central and peripheral lymphoid organs, such as the thymus, spleen and lymph nodes. Moreover, we have also used the murine model of allergic lung inflammation a to evaluate how female sex hormones would affect the immune response in vivo. To address that, ovariectomized or sham operated female Balb/c mice were sensitized or not with ovalbumin 7 and 14 days later and subsequently challenged twice by aerosolized ovalbumin on day 21. Besides the frequency of CD4+Foxp3+ T regulatory cells, we also measured the cytokines IL-4, IL-5, IL-10, IL-13 and IL-17 in the bronchoalveolar lavage from lungs of ovalbumine challenged groups. Our results demonstrate that the absence of female sex hormones after ovariectomy is able to increase the frequency of Tregs in the periphery. As we did not observe differences in the thymus-derived natural occurring Tregs, our data may indicate expansion or conversion of peripheral adaptive Tregs. In accordance with Treg suppressive activity, ovariectomized and ovalbumine-sensitized and challenged animals had significantly reduced lung inflammation. This was observed after cytokine analysis of lung explants showing significant reduction of pro-inflammatory cytokines, such as IL-4, IL-5, IL-13 and IL-17, associated to increased amount of IL-10. In summary, our data clearly demonstrates that OVA sensitization 7 days after ovariectomy culminates in reduced lung inflammation, which may be directly correlated with the expansion of Tregs in the periphery and further higher IL-10 secretion in the lungs

Flow cytometric analysis of Tregs in spleen, lymph nodes of ovariectomized and sensitized mice.

<p>7 days after sensitization, OVx -OVA and Sham -OVA mice were sacrificed and spleen and lymph node cells were submitted to flow cytometry protocol and stained for CD4– FITC, CD8–PeCy5 and Foxp3– PE. In A) zebra plots demonstrate the gates used and the percentage of positive cells. In B, relative and C, absolute numbers of Tregs. Graph representative of three independent experiments. One way-ANOVA p<0.01. n = 5 animals per group.</p

Flow cytometric analysis of Tregs in spleen, lymph nodes and thymus of ovariectomized mice.

<p>7 days after ovariectomy, OVx and Sham mice were sacrificed and spleen, lymph nodes and thymus cells were submitted to flow cytometry protocol and stained for CD4– FITC, CD8–PeCy5 and Foxp3– PE. In A) Zebra plots demonstrate the gates used and the percentage of positive cells. In B relative and C absolute numbers of Tregs. Graphepresentative of three independent experiments. One way-ANOVA p<0.01. n = 5 animals per group.</p