Project description:In this study, an α2(VI) deficient mouse (Col6α2-KO) model was used to examine the role of Type VI collagen in oral tissues. To examine bone properties, µCT was employed, and bone volume and bone mineral density (BMD) was measured in oral tissues. To further investigate its molecular basis, proteome analysis was performed using protein extracted from alveolar bone. In addition, alveolar bone loss progression was evaluated with a periodontitis induced model. µCT analysis showed the Col6α2- KO mice had less volume of alveolar bone, dentin and dental pulp, while the width of periodontal ligament (PDL) was greater than WT. The BMD in alveolar bone and dentin were elevated in Col6α2-KO mice compared with WT. Our proteome analysis showed significant changes in proteins related to ECM organization and elevation of proteins associated with biomineralization in the Col6α2-KO mice. In induced periodontitis, Col6α2-KO mice had greater alveolar bone loss compared to WT. In conclusion, Type VI collagen has role in controlling biomineralization in alveolar bone and that changes in the ECM of alveolar bone could be associated with greater bone loss from periodontitis.
Project description:Pandemic influenza H1N1 (pdmH1N1) virus causes mild disease in humans but occasionally leads to severe complications and even death, especially in those who are pregnant or have underlying disease. Cytokine responses induced by pdmH1N1 viruses in vitro are comparable to other seasonal influenza viruses, suggesting the cytokine dysregulation as seen in H5N1 infection is not a feature of the pdmH1N1 virus. However, a comprehensive gene expression profile of pdmH1N1 in relevant primary human cells in vitro has not been reported. Type I alveolar epithelial cells are a key target cell in pdmH1N1 pneumonia. We carried out a comprehensive gene expression profiling using the Affymetrix microarray platform to compare the transcriptomes of primary human alveolar type I-like alveolar epithelial cells infected with pdmH1N1 or seasonal H1N1 virus. Primary type II alveolar epithelial cells were isolated from human non-malignant lung tissue of three patients who underwent lung resection, and cells were differentiated to type I-like before use. Type I-like alveolar epithelial cells were mock infected, or infected with pdmH1N1 or seasonal H1N1 viruses at a multiplicity of infection (MOI) of two. Total RNA was extracted from cells after 8h post-infection, and gene expression profiling was performed using an Affymetrix Human Gene 1.0 ST microarray platform.
Project description:GM-CSF receptor-β deficient (Csf2rbâ/â or KO) mice develop a lung disease identical to hereditary pulmonary alveolar proteinosis (hPAP) in humans with recessive CSF2RA or CSF2RB mutations that impair GM-CSF receptor function. We performed pulmonary macrophage transplantation (PMT) of bone marrow derived macrophages (BMDMs) without myeloablation in Csf2rbâ/âmice. BMDMs were administered by endotracheal instillation into 2 month-old Csf2rbâ/â mice. Results demonstrated that PMT therapeutic of hPAP in Csf2rbâ/â mice was highly efficacious and durable. Alveolar macrophages were isolated by bronchoalveolar lavage one year after administration subjected to microarray analysis to determine the effects of PMT therapy on the global gene expression profile. Total mRNA was isolated from alveolar macrophages PMT-treated Csf2rbâ/âmice (PMT) and from age-matched, untreated KO mice (KO) and wild-type (C57Bl/6) mice (WT). Total mRNA was evaluated using Affymetrix microarrays (Mouse Gene 1.0 ST Array) to compare the gene expression profiles among the three groups (3 mice/group).
Project description:Transcriptional profiling of TNF-M-NM-1 treated HUVECs comparing cells transfected microRNA negative control with cells transfected with miR-181b. Two-condition experiment, miR negative control VS. miR-181b transfected HUVECs. Biological replicates: 4 replicates of comparison of miR negative control VS. miR-181b
Project description:The many modifications involved in transcription are extensively coupled, and levels of histone modifications, insulators, and RNA polymerase II could be measured using genomic tiling microarrays. After 0, 30, 60 min of stimulation of HUVEC with TNF alpha, we crosslinked cells and fixed, then immunoprecipitated to prepare DNA using antibodies for modified histone, insulators and RNA polymerase. We apply samples collected every 7.5 min to arrays and monitor the growth of several long transcripts. Keywords: time course Using tumor necrosis factor a (TNF-alpha) to switch on transcription of selected human genes rapidly and synchronously, we apply samples collected every 30 min to arrays
Project description:A series of conditional mouse models of embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma and spindle cell sarcoma were generated and validated for relavence to corresponding human cancers. Conditional mouse models of embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma and spindle cell sarcoma were created by activation or deletion of Pax3:Fkhr, p53, Ptch1 or Rb1 genes.
Project description:The proteasome is a central regulatory hub for intracellular signaling by degrading numerous signaling mediators. Immunoproteasomes are specialized types of proteasomes known to be involved in shaping adaptive immune responses, but their role for innate immune signaling is elusive. Here, we analyzed immunoproteasome function for polarization of alveolar macrophages which are highly specialized tissue macrophages of the alveolar surface of the lung. Classical activation (M1 polarization) of primary alveolar macrophages by LPS/IFNγ transcriptionally induced all three immunoproteasome subunits LMP2, LMP7, and MECL-1. In contrast, IL-4 triggered alternative (M2) activation was accompanied by posttranscriptional upregulation of LMP2 and LMP7. Accordingly, immunoproteasome activity increased in M1 cells, and to some extent under M2 conditions. Analyzing the polarization capability from LMP7 deficient mice revealed no effect on the LPS/IFNγ triggered M1 profile, but uncovered a distorted M2 profile for IL-4 stimulated LMP7-/- alveolar macrophages as characterized by increased M2 marker gene expression and CCL17 cytokine release. This shift in immunoproteasome-dependent M2 polarization was accompanied by amplified AKT/STAT6 activation and IRF4 expression in LMP7-/- alveolar macrophages. IL-13 stimulation of LMP7 deficient cells induced a similar M2 skewed profile and IL4Rα protein expression was generally elevated in LMP7-/- alveolar macrophages, indicating that amplified IL4R signaling in immunoproteasome defective cells may contribute to augmented M2 polarization. Importantly, treatment with an LMP7-specific proteasome inhibitor recapitulated the findings of genetic LMP7 inactivation. Our results thus suggest a novel role of immunoproteasome function for regulating innate immune function of macrophages by limiting IL4R expression and signaling. Expression data of M0 and M2 macrophages derived from Lmp7 k.o. and control animals
Project description:Purpose: The goal of this study is to investigate the alteration of gene expression pattern of alveolar macrophages by allergen challenge in human asthmatics. Method: By using subsegmental bronchial provocation with allergen (SBP-AG) protocol, we obtained BAL fluids, before and 48 hours after allergen challenge in the subjects enrolled in the protocol. Alveolar macrophages were purified from the BAL fluids and total RNA was isolated. Next-generation sequencing data were generated by using the Illumina system. Results: Using an optimized data analysis workflow, we mapped about 75 million sequence reads per sample to the human genome and identified 29,691 transcripts in the macrophage mRNAs. Among them, the change in the expression profiles of 37 transcripts were statistically significant. Conclusions: It has been well accepted that Th2 cytokine enriched environment transforms the phenotype of macrophages into alternatively activated form. However, the details of a genome-wide gene expression profiles of macrophages were not well investigated. Using RNA-seq technology, we provided comprehensive data of macrophage gene expression profiles in allergic lung inflammation. Our data could offer a framework to study biologic functions of alternatively activated macrophage in chronic inflammatory diseases. mRNA profiles of alveolar macrophages obtained from asthmatics, before and after allergen challenge.
Project description:CD8 SP Thymocytes were sorted from F5 Rag1KO TCR transgenic mice and stimulated for 4 hr with 30ng/ml TNF (samples 8-11). Controls were samples either purified thymocytes left untreated (samples 1-3), or cultured for 4 hr with the addition of PBS vehicle alone (Samples 4-7).
Project description:This project is based upon the fundamental observation that alveolar macrophage-derived extracellular vesicles (AM-EVs), when internalized by neighboring epithelial cells, inhibit their infection by influenza virus. This inhibitory activity of AM-EVs is abolished when AMs are treated with cigarette smoke extract (CSE). We chose to survey the AM-EV proteome in an effort to identify candidate proteins whose abundance within EVs was downregulated by CSE treatment of AMs, thus explaining the ability of CSE to abrogate the inhibitory activity against influenza.