Shinya Ohkouchi

Multipotent Stromal Cells Are Activated to Reduce Apoptosis in Part by Upregulation and Secretion of Stanniocalcin‐1

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 coculture of MSCs with previously UV‐irradiated fibroblasts reduced apoptosis of the irradiated cells, but fresh MSC conditioned medium 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 antiapoptotic effects of MSCs in two distinct models of apoptosis in vitro. STEM CELLS 2009;27:670–681

Paracrine Factors of Multipotent Stromal Cells Ameliorate Lung Injury in an Elastase-induced Emphysema Model

Multipotent stromal cells (MSCs) ameliorate several types of lung injury. The differentiation of MSCs into specific cells at the injury site has been considered as the important process in the MSC effect. However, although MSCs reduce destruction in an elastase-induced lung emphysema model, MSC differentiation is relatively rare, suggesting that MSC differentiation into specific cells does not adequately explain the recuperation observed. Humoral factors secreted by MSCs may also play an important role in ameliorating emphysema. To confirm this hypothesis, emphysema was induced in the lungs of C57BL/6 mice by intratracheal elastase injection 14 days before intratracheal MSC or phosphate-buffered saline (PBS) administration. Thereafter, lungs were collected at several time points and evaluated. Our results showed that MSCs reduced the destruction in elastase-induced emphysema. Furthermore, double immunofluorescence staining revealed infrequent MSC engraftment and differentiation into epithelial cells. Real-time PCR showed increased levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Real-time PCR and western blotting showed enhanced production of secretory leukocyte protease inhibitor (SLPI) in the lung. In-vitro coculture studies confirmed the in vivo observations. Our findings suggest that paracrine factors derived from MSCs is the main mechanism for the protection of lung tissues from elastase injury.

Adenovirus Vector-Mediated in Vivo Gene Transfer of OX40 Ligand to Tumor Cells Enhances Antitumor Immunity of Tumor-Bearing Hosts

OX40 ligand (OX40L), the ligand for OX40 on activated CD4+ T cells, has adjuvant properties for establishing effective T-cell immunity, a potent effector arm of the immune system against cancer. The hypothesis of this study is that in vivo genetic engineering of tumor cells to express OX40L will stimulate tumor-specific T cells by the OX40L-OX40 engagement, leading to an induction of systemic antitumor immunity. To investigate this hypothesis, s.c. established tumors of three different mouse cancer cells (B16 melanoma, H-2b; Lewis lung carcinoma, H-2b; and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of a recombinant adenovirus vector expressing mouse OX40L (AdOX40L). In all tumor models tested, treatment of tumor-bearing mice with AdOX40L induced a significant suppression of tumor growth along with survival advantages in the treated mice. The in vivo AdOX40L modification of tumors evoked tumor-specific cytotoxic T lymphocytes in the treated host correlated with in vivo priming of T helper 1 immune responses in a tumor-specific manner. Consistent with the finding, the antitumor effect provided by intratumoral injection of AdOX40L was completely abrogated in a CD4+ T cell-deficient or CD8+ T cell-deficient condition. In addition, ex vivo AdOX40L-transduced B16 cells also elicited B16-specific cytotoxic T lymphocyte responses, and significantly suppressed the B16 tumor growth in the immunization-challenge experiment. All of these results support the concept that genetic modification of tumor cells with a recombinant OX40L adenovirus vector may be of benefit in cancer immunotherapy protocols.

Antitumor immune response by CX3CL1 fractalkine gene transfer depends on both NK and T cells

The CX3C chemokine fractalkine (CX3CL1) exists as both a membrane‐bound form promoting firm cell‐cell adhesion and a soluble form chemoattracting leukocytes expressing its receptor CX3CR1. When adenoviral vector expressing mouse fractalkine (AdFKN) was transduced to the tumor cells, fractalkine was expressed as both membrane‐bound form on the tumor cells and soluble form in the supernatant in vitro. Intratumoral injection of AdFKN (1×109PFU/tumor) into C26 and B16F10 tumors resulted in marked reduction of tumor growth compared to control (C26: 86.5%, p<0.001; B16F10: 85.5%, p<0.001). Histological examination of tumor tissues revealed abundant infiltration of NK cells, dendritic cells, and CD8+ T lymphocytes 3 and/or 6 days after treatment with AdFKN. Splenocytes from mice treated by AdFKN developed tumor‐specific cytotoxic T cells, and thereby protected from rechallenging with parental tumor cells. Antitumor effects by AdFKN were completely abrogated in both NK cell‐depleted mice and CD8–/– mice, and partially blocked in CD4–/– mice. These data indicated that fractalkine mediates antitumor effects by both NK cell‐dependent and T cell‐dependent mechanisms. This study suggests that fractalkine can be a suitable candidate for immunogene therapy of cancer because fractalkine induces both innate and adaptive immunity.

Mesenchymal stromal cells protect cancer cells from ROS-induced apoptosis and enhance the Warburg effect by secreting STC1.

Previous studies have demonstrated that mesenchymal stromal cells (MSCs) enhance cell survival through upregulation and secretion of stanniocalcin-1 (STC1). This study shows that MSC-derived STC1 promotes survival of lung cancer cells by uncoupling oxidative phosphorylation, reducing intracellular reactive oxygen species (ROS), and shifting metabolism towards a more glycolytic metabolic profile. MSC-derived STC1 upregulated uncoupling protein 2 (UCP2) in injured A549 cells in an STC1-dependent manner. Knockdown of UCP2 reduced the ability of MSCs and recombinant STC1 (rSTC1) to reduce cell death in the A549 population. rSTC1-treated A549 cells displayed decreased levels of ROS, mitochondrial membrane potential (MMP), and increased lactate production, all of which were dependent on the upregulation of UCP2. Our data suggest that MSCs can promote cell survival by regulating mitochondrial respiration via STC1.

Gradual Increase of High Mobility Group Protein B1 in the Lungs after the Onset of Acute Exacerbation of Idiopathic Pulmonary Fibrosis

The pathogenesis of acute exacerbation of idiopathic pulmonary fibrosis (IPF) remains to be elucidated. To evaluate the roles of inflammatory mediators in acute exacerbation, the concentrations of high mobility group protein B1 (HMGB1), a chief mediator of acute lung injury, and 18 inflammatory cytokines were measured in the bronchoalveolar lavage fluid, serially sampled from seven IPF patients after the onset of acute exacerbation. HMGB1 gradually increased in the alveolar fluid after the onset of acute exacerbation, in positive correlation with monocytes chemotactic protein-1 (MCP-1), a potent fibrogenic mediator. In the lung tissues of eight IPF patients autopsied after acute exacerbation, intense cytoplasmic staining for HMGB1 was observed in the alveolar epithelial cells in alveolar capillary augmented lesions, where the capillary endothelial cells remarkably reduced the expression of thrombomodulin, an intrinsic antagonist of HMGB1. These results suggest pathogenic roles for HMGB1 and MCP-1 in the late phase of acute exacerbation of IPF.

Phase 1 clinical study of cyclophilin B peptide vaccine for patients with lung cancer.

Cyclophilin B (CypB) possesses two antigenic epitopes (CypB84–92 and CypB91–99) recognized by HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). To determine the safety of CypB-derived peptides and its ability to generate antitumor immune responses, patients with advanced lung cancer received subcutaneous vaccinations of these peptides or their modified peptides. All 16 patients were vaccinated with CypB91–99 or its modified peptide, whereas only two patients were vaccinated with the modified CypB84–92, as immediate-type hypersensitivity to CypB84–92 or its modified peptide was observed in the remaining patients. No severe adverse events were associated with the vaccination. No significant increase in cellular responses to either peptides or tumor cells was observed in the postvaccination PBMCs by the conventional CTL assays in any patients tested. These results suggest that the vaccination of CypB91–99 peptide was safe, but failed to induce objective immune responses at this regimen.

Duration of Benefit in Patients With Autoimmune Pulmonary Alveolar Proteinosis After Inhaled Granulocyte-Macrophage Colony-Stimulating Factor Therapy

BACKGROUND Treatment of autoimmune pulmonary alveolar proteinosis (aPAP) by subcutaneous injection or inhaled therapy of granulocyte-macrophage colony-stimulating factor (GM-CSF) has been demonstrated to be safe and efficacious in several reports. However, some reports of subcutaneous injection described transient benefit in most instances. The durability of response to inhaled GM-CSF therapy is not well characterized. METHODS To elucidate the risk factors for recurrence of aPAP after GM-CSF inhalation, 35 patients were followed up, monitoring for the use of any additional PAP therapies and disease severity score every 6 months. Physiologic, serologic, and radiologic features of the patients were analyzed for the findings of 30-month observation after the end of inhalation therapy. RESULTS During the observation, 23 patients remained free from additional treatments, and twelve patients required additional treatments. There were no significant differences in age, sex, symptoms, oxygenation indexes, or anti-GM-CSF antibody levels at the beginning of treatment between the two groups. Baseline vital capacity (% predicted, %VC) were higher among those who required additional treatment (P<.01). Those patients not requiring additional treatment maintained the improved disease severity score initially achieved. A significant difference in the time to additional treatment between the high %VC group (%VC≥80.5) and the low %VC group was seen by a Kaplan-Meier analysis and a log-rank test (P<.0005). CONCLUSIONS These results demonstrate that inhaled GM-CSF therapy sustained remission of aPAP in more than one-half of cases, and baseline %VC might be a prognostic factor for disease recurrence. TRIAL REGISTRY ISRCTN Register and JMACCT Clinical Trial Registry; No.: ISRCTN18931678 and JMAIIA00013; URL: http://www.isrctn.org and http://www.jmacct.med.or.jp.

Mesenchymal Stem Cells Correct Inappropriate Epithelial–mesenchyme Relation in Pulmonary Fibrosis Using Stanniocalcin-1

Current hypotheses suggest that aberrant wound healing has a critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). In these hypotheses, continuous TGF-β1 secretion by alveolar epithelial cells (AECs) in abnormal wound healing has a critical role in promoting fibroblast differentiation into myofibroblasts. Mesenchymal stem cells (MSCs) home to the injury site and reduce fibrosis by secreting multifunctional antifibrotic humoral factors in IPF. In this study, we show that MSCs can correct the inadequate-communication between epithelial and mesenchymal cells through STC1 (Stanniocalcin-1) secretion in a bleomycin-induced IPF model. Inhalation of recombinant STC1 shows the same effects as the injection of MSCs. Using STC1 plasmid, it was possible to enhance the ability of MSCs to ameliorate the fibrosis. MSCs secrete large amounts of STC1 in response to TGF-β1 in comparison to AECs and fibroblasts. The antifibrotic effects of STC1 include reducing oxidative stress, endoplasmic reticulum (ER) stress, and TGF-β1 production in AECs. The STC1 effects can be controlled by blocking uncoupling protein 2 (UCP2) and the secretion is affected by the PI3/AKT/mTORC1 inhibitors. Our findings suggest that STC1 tends to correct the inappropriate epithelial-mesenchymal relationships and that STC1 plasmid transfected to MSCs or STC1 inhalation could become promising treatments for IPF.

Deterioration in regional health status after the acute phase of a great disaster: respiratory physicians' experiences of the Great East Japan Earthquake.

BACKGROUND The Great East Japan Earthquake occurred on March 11, 2011. The source of the ensuing devastation was not the tremors, but the subsequent tsunami. Responding emergency medical teams could not provide sufficient assistance, which led to many people dying before the rescue teams arrived. Thus, the main objective of healthcare professionals became to prevent deterioration in peoples health statuses in the disaster area. METHODS One month after the earthquake, the Health-Promoting Association of Respiratory Medicine of Tohoku conducted a survey regarding changing disease prevalence among inpatients in respiratory medicine departments of regional core hospitals in Miyagi Prefecture, the area that suffered the most damage. RESULTS The number of patients from March 11 to April 10, 2011 was 2.7 times greater than that during the same period in 2010 (1223 vs. 443, respectively). The prevalence of asthma, exacerbations of chronic obstructive pulmonary disease, and community-acquired pneumonia were also 2-3 times greater in 2011 than in 2010 (98 vs. 32, 117 vs. 46, and 443 vs. 202, respectively) among all ages. Half of the community-acquired pneumonia cases originated in evacuation shelters. The number of inpatients with other diseases, including those who drowned, was relatively small, and mortality did not increase significantly at these hospitals. CONCLUSIONS The findings may result from poor shelter or dwelling conditions, as well as overpopulation and lack of basic resources. Adequate shelters, supply systems, and protection from infection, including vaccinations, are needed to prevent deteriorations in health status after the acute phase of a natural disaster.