IntroductionMultipotent stromal cells (MSCs) are currently in clinical trials for a number of inflammatory diseases. Recent studies have demonstrated the ability of MSCs to attenuate inflammation in rodent models of acute lung injury (ALI) suggesting that MSCs may also be beneficial in treating ALI.MethodsTo better understand how human MSCs (hMSCs) may act in ALI, the lungs of immunocompetent mice were exposed to lipopolysaccharide (LPS) and four hours later bone marrow derived hMSCs were delivered by oropharyngeal aspiration (OA). The effect of hMSCs on lung injury was assessed by measuring the lung wet/dry weight ratio and total protein in bronchoalveolar lavage (BAL) fluid 24 or 48 h after LPS. BAL fluid was also analyzed for the presence of inflammatory cells and cytokine expression by multiplex immunoassay. Microarray analysis of total RNA isolated from treated and untreated lungs was performed to elucidate the mechanism(s) involved in hMSC modulation of lung inflammation.ResultsAdministration of hMSCs significantly reduced the expression of pro-inflammatory cytokines, neutrophil counts and total protein in bronchoalveolar lavage. There was a concomitant reduction in pulmonary edema. The anti-inflammatory effects of hMSCs were not dependent on localization to the lung, as intraperitoneal administration of hMSCs also attenuated LPS-induced inflammation in the lung. Microarray analysis revealed significant induction of tumor necrosis factor (TNF)-α-induced protein 6 (TNFAIP6/TSG-6) expression by hMSCs 12 h after OA delivery to LPS-exposed lungs. Knockdown of TSG-6 expression in hMSCs by RNA interference abrogated most of their anti-inflammatory effects. In addition, intra-pulmonary delivery of recombinant human TSG-6 reduced LPS-induced inflammation in the lung.ConclusionsThese results show that hMSCs recapitulate the observed beneficial effects of rodent MSCs in animal models of ALI and suggest that the anti-inflammatory properties of hMSCs in the lung are explained, at least in part, by activation of hMSCs to secrete TSG-6.

Multipotent stromal cells (MSCs) have been shown to reduce apoptosis in injured cells by secretion of paracrine factors, but these factors were not fully defined. We observed that co-culture of MSCs with previously UV irradiated fibroblasts reduced apoptosis of the irradiated cells, but fresh MSC conditioned media was unable reproduce the effect. Comparative Microarray analysis of MSCs grown in the presence or absence of UV irradiated fibroblasts demonstrated that the MSCs were activated by the apoptotic cells to increase synthesis and secretion of stanniocalcin-1 (STC-1), a peptide hormone that modulates mineral metabolism and has pleiotrophic effects that have not been fully characterized. We showed that STC-1 was required but not sufficient for reduction of apoptosis of UV-irradiated fibroblasts. In contrast, we demonstrated that MSC-derived STC-1 was both required and sufficient for reduction of apoptosis of lung cancer epithelial cells made apoptotic by incubation at low pH in hypoxia. Our data demonstrate that STC-1 mediates the anti-apoptotic effects of MSCs in two distinct models of apoptosis in vitro.

Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (*92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrowand adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.

Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC – high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

Increased expression of transforming growth factor (TGF)-beta(1) and tumor necrosis factor (TNF)-alpha are thought to play important roles in the development of pulmonary fibrosis. We recently reported that TNF-alpha upregulates TGF-beta(1) expression in primary mouse lung fibroblasts (MLFs), a key cell population in fibrogenesis. In the present study, we have investigated signal transduction pathways involved in TNF-alpha upregulation of TGF-beta(1) in both primary MLFs and the Swiss 3T3 fibroblast cell line. Treatment of fibroblasts with TNF-alpha resulted in a significant increase in TGF-beta(1) protein as measured by ELISA. The increase in protein was preceded by a 200-400% increase in TGF-beta(1) mRNA detected by quantitative, real-time, reverse transcriptase-polymerase chain reaction. Western blot analysis showed that TNF-alpha activated the extracellular signal-regulated kinase (ERK), and inhibitors of the ERK-specific mitogen-activated protein kinase pathway (PD98059 or U0126) blocked TNF-alpha induction of TGF-beta(1) mRNA and protein. mRNA stability experiments showed that TNF-alpha increased the half-life of TGF-beta(1) mRNA to more than 24 h compared with approximately 15 h in unstimulated cells. Expression of constitutively active MEK1 that selectively phosphorylates ERK was sufficient for TGF-beta(1) mRNA stabilization in Swiss 3T3 fibroblasts. These results indicate that TNF-alpha activates the ERK-specific mitogen-activated protein kinase pathway leading to increased TGF-beta(1) production in fibroblasts, primarily via a post-transcriptional mechanism that involves stabilization of the TGF-beta(1) transcript.