The tumor suppressor gene, SMARCA4 (or BRG1), which encodes the ATPase component of the chromatin remodeling complex SWI/SNF, is commonly inactivated by mutations and deletions in lung cancer cell lines. However, SMARCA4 alterations appear to be rare in lung primary tumors. Ultra‐deep sequencing technologies provide a promising alternative to achieve a sensitivity superior to that of current sequencing strategies. Here we used ultra‐deep pyrosequencing to screen for mutations over the entire SMARCA4 coding region in 12 lung tumors without detectable BRG1 protein. While automatic‐fluorescence‐based sequencing detected one somatic mutation (p.K586X), the pyrosequencing revealed additional variants, thus increasing the sensitivity. One of the variants, which affected a consensus splice site, was confirmed by individual cloning of PCR products, ruling out the possibility of PCR or pyrosequencing artifacts. This mutation, confirmed to be somatic, was present at a frequency of ten percent, suggesting normal cell contamination in the tumor. Our analysis also allowed us to determine the sensitivity and to identify some limitations of the technology. In conclusion, in addition to cell lines, SMARCA4 is biallelically inactivated in a significant proportion of lung primary tumors, thereby constituting one of the most important genes contributing to the development of this type of cancer.

Increasing evidence suggests that the infectiousness of patients for the sand fly vector of visceral leishmaniasis is linked to parasites found in the skin. Using a murine model that supports extensive skin infection withLeishmania donovani, spatial analyses at macro-(quantitative PCR) and micro-(confocal microscopy) scales indicate that parasite distribution is markedly skewed. Mathematical models accounting for this heterogeneity demonstrate that while a patchy distribution reduces the expected number of sand flies acquiring parasites, it increases the infection load for sand flies feeding on a patch, increasing their potential for onward transmission. Models representing patchiness at both macro- and micro-scales provide the best fit with experimental sand fly feeding data, pointing to the importance of the skin parasite landscape as a predictor of host infectiousness. Our analysis highlights the skin as a critical site to consider when assessing treatment efficacy, transmission competence and the impact of visceral leishmaniasis elimination campaigns.

Type 1 diabetes results from a co-ordinated autoimmune attack of insulin producing beta cells in the pancreas by the innate and adaptive immune systems, beta cell death being predominantly T cell-mediated. In addition to T cells, peripheral B cells are important in type 1 diabetes progression. The thymus of mice and man also contain B cells, and lately they have been linked to central tolerance of T cells. The role of thymic B cells in type 1 diabetes is undefined. Here we show there are abnormalities in the thymic B cell compartment prior to beta cell destruction and type 1 diabetes manifestation. Using non-obese diabetic (NOD) mice, we document that preceding type 1 diabetes development, there is significant accumulation of thymic B cells-partly through in situ development- and the putative formation of ectopic germinal centres. In addition, in NOD mice we quantify thymic plasma cells and observe in situ binding of immunoglobulins to undefined antigens on a significant proportion of medullary thymic epithelial cells. In contrast, no ectopic germinal centres, or pronounced intrathymic autoantibodies are detectable in animals not genetically predisposed to developing type 1 diabetes. Binding of autoantibodies to thymic stroma correlates with apoptosis of medullary thymic epithelial cells, including insulin-expressing cells. In contrast, apoptosis of medullary thymic epithelial cells was decreased by 50% in B cell deficient NOD mice suggesting intrathymic autoantibodies may selectively target certain medullary thymic epithelial cells for destruction. Futhermore, we observe that these thymic B cell-associated events correlated with an increased prevalence of premature thymic emigration of T cells. Together our data suggests that the thymus may be a principal autoimmune target in type 1 diabetes and contributes to disease progression.

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150⁺ CD34^- CD48^- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4⁺ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4⁺ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ⁺CD4⁺ T cells that promote the irreversible loss of BM function. These findings provide new insights into the pathogenic potential of CD4⁺ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously. Visceral leishmaniasis (VL) is a chronic often fatal disease caused by the protozoan parasites Leishmania donovani and L. infantum. Progressive disease in humans and in animal models is associated with parasite replication at systemic sites, including the bone marrow (BM) and results in significant changes in hematological profile. The mechanisms underlying hematologic dysregulation during infection are largely unknown. Using a panel of stem cell markers, we characterized murine haematopoietic stem and progenitor cells in the BM over the course of L. donovani-infection in C57BL/6 (B6) mice. Most long-term hematopoietic stem cells (LT-HSCs) in naïve mice are found in a quiescent state, representing cells with the highest degree of reconstitution potential. In contrast, during L. donovani infection, most LT-HSCs had entered cell-cycle and this correlated with a reduced potential to engraft into syngeneic recipients. HSC exhaustion and other alterations in the hematopoietic compartment did not occur in infected immunodeficient mice, but adoptive transfer of IFNγ-sufficient CD4⁺ T cells restored this phenotype. Using mixed BM chimeras, we established that IFNγ signalling and TNF signalling pathways converge at the level of BM CD4⁺ T cells, with intrinsic TNF signalling being critical for the expansion / differentiation of CD4⁺ T cells that are responsible for HSC exhaustion. Contrary to commonly held views, in the setting of experimental visceral leishmaniasis neither IFNγ nor TNF signalling in HSCs was required for their functional exhaustion. Hence, pro-inflammatory cytokines commonly associated with host protection in leishmaniasis and many other infectious diseases can also drive the development of pathogenic CD4⁺ T cells that cause long term irreversible alterations in HSC function.

Human visceral leishmaniasis, a parasitic disease of major public health importance in developing countries, is characterized by variable degrees of severity of anemia, but the mechanisms underlying this change in peripheral blood have not been thoroughly explored. Here, we used an experimental model of visceral leishmaniasis in C57BL/6 mice to explore the basis of anemia following infection with Leishmania donovani. 28 days post-infection, mice showed bone marrow dyserythropoiesis by myelogram, with a reduction of TER119⁺ CD71^−/+ erythroblasts. Reduction of medullary erythropoiesis coincided with loss of CD169^high bone marrow stromal macrophages and a reduction of CXCL12-expressing stromal cells. Although the spleen is a site of extramedullary erythropoiesis and erythrophagocytosis, splenectomy did not impact the extent of anemia or affect the repression of medullary hematopoiesis that was observed in infected mice. In contrast, these changes in bone marrow erythropoiesis were not evident in B6.Rag2^−/− mice, but could be fully reconstituted by adoptive transfer of IFNγ-producing but not IFNγ-deficient CD4⁺ T cells, mimicking the expansion of IFNγ-producing CD4⁺ T cells that occurs during infection in wild type mice. Collectively, these data indicate that anemia during experimental murine visceral leishmaniasis can be driven by defects associated with the bone marrow erythropoietic niche, and that this represents a further example of CD4⁺ T cell-mediated immunopathology affecting hematopoietic competence.