BackgroundRhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the defects present in these cells that block myogenesis.MethodsProtein and RNA analysis identified the loss of MEF2D in RMS cells. MEF2D was expressed in RD and RH30 cells by transient transfection and selection of stable cell lines, respectively, to demonstrate the rescue of muscle differentiation observed. A combination of techniques such as proliferation assays, scratch assays and soft agar assays were used with RH30 cells expressing MEF2D to demonstrate the loss of oncogenic growth in vitro and xenograft assays were used to confirm the loss of tumor growth in vivo.ResultsHere, we show that one member of the MEF2 family of proteins required for normal myogenesis, MEF2D, is largely absent in RMS cell lines representing both major subtypes of RMS as well as primary cells derived from an embryonal RMS model. We show that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find that MyoD and myogenin are bound with their dimerization partner, the E proteins, to the promoters of muscle specific genes in RMS cells. However, we cannot detect MEF2D binding at any promoter tested. We find that exogenous MEF2D expression can activate muscle specific luciferase constructs, up regulate p21 expression and increase muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility and anchorage independent growth in vitro. We have confirmed the inhibition of tumorigenicity by MEF2D in a tumor xenograft model, with a complete regression of tumor growth.ConclusionsOur data indicate that the oncogenic properties of RMS cells can be partially attributed to the loss of MEF2D expression and that restoration of MEF2D may represent a useful therapeutic strategy to decrease tumorigenicity.

Helminths stimulate the secretion of T helper 2 (Th2) cytokines, like interleukin-4 (IL4) and suppress lethal graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). This suppression depends on the production of immune-modulatory TGFβ and is associated with TGFβ-dependent in vivo expansion of Foxp3+ regulatory T cells (Treg). In vivo expansion of Tregs is under investigation for its potential as a therapy for GVHD. Nonetheless, the mechanism of induced and TGFβ-dependent, in vivo expansion of Tregs - in a Th2 polarized environment after helminth infection - is unknown. Here we show that helminth-induced IL4 production by host cells is critical to the induction and maintenance of TGFβ secretion, TGFβ-dependent expansion of Foxp3+ Tregs, and the suppression of GVHD. In mice with GVHD, the expanding donor Tregs express the Th2-driving transcription factor, GATA3, which is required for helminth-induced production IL4 and TGFβ. On the other hand, TGFβ is not necessary for GATA3 expression by Foxp3+ Tregs or by Foxp3− CD4 T cells. Various cell types of innate or adaptive immune compartments produce high quantities of IL4 after helminth infection. As a result, IL4-mediated suppression of GVHD does not require invariant NKT (iNKT) cells of the host - a cell type known to produce IL4 and suppress GVHD in other models. Thus, TGFβ generation – in a manner dependent on IL4 secretion by host cells and GATA3 expression - constitutes a critical effector arm of helminthic immune modulation that promotes the in vivo expansion of Tregs and suppresses GVHD.

Production of transforming growth factor-β (TGFβ) by T cells is key to various aspects of immune homeostasis, with defects in this process causing or aggravating immune-mediated disorders. The molecular mechanisms that lead to TGFβ generation by T cells remain largely unknown. To address this issue, we take advantage of the fact that intestinal helminths stimulate T helper 2 (Th2) cells besides triggering TGFβ generation by T lymphocytes and regulate immune-mediated disorders. We show that the Th2 cell-inducing transcription factor STAT6 is necessary and sufficient for the expression of TGFβ pro-peptide (pro-TGFβ) in T cells. STAT6 is also necessary for several helminth-triggered events, such as TGFβ-dependent suppression of alloreactive inflammation in graft-versus-host disease (GVHD), in mice. Besides STAT6, helminth-induced secretion of active TGFβ requires cleavage of pro-TGFβ by the endopeptidase furin. Thus - for the immune regulatory pathway necessary for TGFβ production by T cells - our results support a two-step model, comprised by STAT6 and furin.

BackgroundPatients with inflammatory bowel disease have higher incidence of airway hyperresponsiveness compared to the general population. Lung inflammation leading to airway hyperresponsiveness causes illnesses for more than ten percent of the population in USA.AimsWe investigated the lung response to bacterial endotoxin in colitic mice.MethodsRag-1 mice were transplanted with negatively selected splenic T cells. Some mice groups were treated with NSAID to develop colitis. All mice were treated with bacterial endotoxin and necropsied 3 weeks later.ResultsColitic mice developed intensified lung inflammation on day 21 of treatment with bacterial endotoxin. Pulmonary lymphocytes from colitic mice displayed a proinflammatory cytokine profile, expressed high ICAM1 and low FoxP3. CD11c+, CD8+ cells bound and responded to non-systemic antigens from gut-localized microbiota and had higher expression of TLR4.ConclusionsColitic mice developed exacerbated lung inflammation in response to bacterial endotoxin compared to non-colitic mice. Proinflammatory cytokines from pulmonary lymphocytes induced high expression of ICAM1 and suppressed FoxP3 on CD4+ cells. CD11c+, CD8+ cells binding and responding to gut-localized antigens as well as high expression of TLR4 indicate innate and adaptive lung response to bacterial endotoxin. Inflammatory cells from colons of colitic mice homed in the lungs as well as the intestine suggesting recirculation of sensitized immunocompetent cells. These data support our hypothesis that colitis intensifies lung inflammation.

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