In this study, we explored the regulation and the role of up-regulated microRNAs in idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly upregulated microRNAs. Twenty-four of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299-5p, miR-410, miR-382, miR-409-3p, miR-487b, miR-31, and miR-127 by quantitative RT-PCR and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region, but analysis of transcription factor binding sites identified multiple SMAD3-binding elements in the 14q32 microRNA cluster. TGF-b1 stimulation of normal human lung fibroblasts (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts, and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-b was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/b-catenin pathway, reduced the proliferative effect of miR-154. The potential role of miR-154, one of multiple chr14q32 micro-RNA cluster members up-regulated in IPF and a regulator of fibroblast migration and proliferation, should be further explored in IPF.
There are considerable differences in tumour biology between adult and paediatric cancers. The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear. Here, we show the successful propagation of primary human Wilms' tumour (WT), a common paediatric renal malignancy, in immunodeficient mice, demonstrating the presence of a population of highly proliferative CIC/CSCs capable of serial xenograft initiation. Cell sorting and limiting dilution transplantation analysis of xenograft cells identified WT CSCs that harbour a primitive undifferentiated – NCAM1 expressing – “blastema” phenotype, including a capacity to expand and differentiate into the mature renal-like cell types observed in the primary tumour. WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family. Complete eradication of WT in multiple xenograft models was achieved with a human NCAM antibody drug conjugate. The existence of CIC/CSCs in WT provides new therapeutic targets.
Our results suggest that smaller TNs (smaller than about 2.0 cm) are associated with increased probabilities of malignant disease irrespective of Bethesda class. Routine diagnostic thyroid lobectomy solely owing to TN size of 3.0 cm or greater need not be performed.
BackgroundExperimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is induced by immunization of mice with myelin oligodendrocytic glycoprotein (MOG35-55) injections, and after 9 days, mice develop behavioral signs of chronic progressive EAE. Proliferation of T and B cells located in peripheral lymph tissues such as spleen and inguinal lymph nodes of C57BL/6J mice are stimulated. The opioid growth factor-opioid growth factor receptor (OGF-OGFr) axis has been shown to effectively limit progression of chronic EAE when mice are treated at the time of induction or at time of established disease. In addition to repressed behavioral profiles, spinal cord neuropathology is diminished in mice treated with OGF or low dosages of naltrexone (LDN). However, there is little or no information on peripheral lymphocyte dynamics following immunization of mice with MOG antigen and treatment with OGF or LDN.MethodsSix-week old female mice were immunized with MOG35-55 and were injected intraperitoneally with OGF or a low dosage of naltrexone (LDN) beginning at the time of immunization; saline-injected immunized mice served as controls. Normal mice received saline for all injections. Periodically over a 2 week period, spleens and inguinal lymph nodes were removed, total lymphocytes counted, and subpopulations of CD4+ and CD8+ specific T-cells, as well as B lymphocytes, were determined by flow cytometry. On day 15 of treatment, lumbar spinal cord tissue was removed; CNS lymphocytes isolated, and assayed for Th1, Th2, and Th17 markers by flow cytometry. ResultsExogenous OGF or endogenous OGF following LDN suppressed T and B lymphocyte proliferation in the spleen and inguinal lymph nodes of MOG-immunized mice. Suppression of peripheral immune cell CD4+ and CD8+ T cell proliferation at 5 and 12 days correlated with reductions in clinical behavior. EAE mice treated with OGF for 15 days displayed elevated Th1 and Th17 cells; no subpopulations of Th2-specific T cells were noted.ConclusionsOGF or LDN repress proliferation of CD4+ and CD8+T cells and B220+ B lymphocytes in the spleen and lymph nodes of immunized mice within a week of immunization. These data provide novel mechanistic pathways underlying the efficacy of OGF and LDN therapy for MS.
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