Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

L-alanyl-L-glutamine modified perfusate improves human lung cell functions and extend porcine ex vivo lung perfusion.

BACKGROUND: The clinical application of normothermic ex vivo lung perfusion (EVLP) has increased donor lung utilization for transplantation through functional assessment. To develop it as a platform for donor lung repair, reconditioning and regeneration, the perfusate should be modified to support the lung during extended EVLP. METHODS: Human lung epithelial cells and pulmonary microvascular endothelial cells were cultured, and the effects of Steen solution (commonly used EVLP perfusate) on basic cellular function were tested. Steen solution was modified based on screening tests in cell culture, and further tested with an EVLP cell culture model, on apoptosis, GSH, HSP70, and IL-8 expression. Finally, a modified formula was tested on porcine EVLP. Physiological parameters of lung function, histology of lung tissue, and amino acid concentrations in EVLP perfusate were measured. RESULTS: Steen solution reduced cell confluence, induced apoptosis, and inhibited cell migration, compared to regular cell culture media. Adding L-alanyl-L-glutamine to Steen solution improved cell migration and decreased apoptosis. It also reduced cold preservation and warm perfusion-induced apoptosis, enhanced GSH and HSP70 production, and inhibited IL-8 expression on an EVLP cell culture model. L-alanyl-L-glutamine modified Steen solution supported porcine lungs on EVLP with significantly improved lung function, well-preserved histological structure, and significantly higher levels of multiple amino acids in EVLP perfusate. CONCLUSIONS: Adding L-alanyl-L-glutamine to perfusate may provide additional energy support, antioxidant, and cytoprotective effects to lung tissue. The pipeline developed herein, with cell culture, cell EVLP, and porcine EVLP models, can be used to further optimize perfusates to improve EVLP outcomes.

Inhibitory effect of soluble platelet-derived growth factor receptor ß on intraosseous growth of breast cancer cells in nude mice.

Bone metastasis is a frequent complication of advanced breast cancer. On the basis of functional and molecular evidence, signaling mediated by the binding of platelet-derived growth factor (PDGF)-BB and -DD to PDGF receptor ß (PDGFRß) is critical for the survival and growth of metastatic breast cancer cells within the bone microenvironment. In this study, we propose a new approach to blocking PDGFRß signaling using soluble PDGFRß (sPDGFRß) as a decoy receptor for PDGF-BB and -DD secreted from tumor cells and bone marrow stromal cells. A bone-seeking TNBCT/Bo cell line was established by in vivo selection from TNBCT human breast cancer cells, which are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 protein expression. The TNBCT/Bo cells were transfected with a mammalian expression vector encoding the extracellular domain of PDGFRß. A stable transfectant (TNBCT/Bo-sPDGFRß) grew at a similar rate to that of control cells under normal culture conditions, although growth stimulation of human fibroblasts with PDGF-BB was neutralized by the culture medium from TNBCT/Bo-sPDGFRß cells. Intratibial injection of TNBCT/Bo-sPDGFRß cells into athymic nude mice resulted in a significant decrease in tumor incidence compared with control mice (P < 0.01). This attenuated growth correlated with decreased cancer cell proliferation, angiogenesis, and recruitment of stromal cells, and with an increase in the number of apoptotic cells. These findings suggest that sPDGFRß is useful for the treatment of breast cancer bone metastasis.

Engineered mesenchymal stromal cell therapy during human lung ex vivo lung perfusion is compromised by acidic lung microenvironment.

Ex vivo lung perfusion (EVLP) is an excellent platform to apply novel therapeutics, such as gene and cell therapies, before lung transplantation. We investigated the concept of human donor lung engineering during EVLP by combining gene and cell therapies. Premodified cryopreserved mesenchymal stromal cells with augmented anti-inflammatory interleukin-10 production (MSCIL-10) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSCIL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSCIL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSCIL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that "off-the-shelf" MSCIL-10 therapy of human lungs during EVLP is safe and feasible, and results in rapid IL-10 elevation, and that the acidic target-tissue microenvironment may compromise the efficacy of cell-based therapies.

TAZ induces lung cancer stem cell properties and tumorigenesis by up-regulating ALDH1A1.

Recent studies suggest that lung cancer stem cells (CSCs) may play major roles in lung cancer. Therefore, identification of lung CSC drivers may provide promising targets for lung cancer. TAZ is a transcriptional co-activator and key downstream effector of the Hippo pathway, which plays critical roles in various biological processes. TAZ has been shown to be overexpressed in lung cancer and involved in tumorigenicity of lung epithelial cells. However, whether TAZ is a driver for lung CSCs and tumor formation in vivo is unknown. In addition, the molecular mechanism underlying TAZ-induced lung tumorigenesis remains to be determined. In this study, we provided evidence that constitutively active TAZ (TAZ-S89A) is a driver for lung tumorigenesis in vivo in mice and formation of lung CSC. Further RNA-seq and qRT-PCR analysis identified Aldh1a1, a well-established CSC marker, as critical TAZ downstream target and showed that TAZ induces Aldh1a1 transcription by activating its promoter activity through interaction with the transcription factor TEAD. Most significantly, inhibition of ALDH1A1 with its inhibitor A37 or CRISPR gene knockout in lung cancer cells suppressed lung tumorigenic and CSC phenotypes in vitro, and tumor formation in mice in vivo. In conclusion, this study identified TAZ as a novel inducer of lung CSCs and the first transcriptional activator of the stem cell marker ALDH1A1. Most significantly, we identified ALDH1A1 as a critical meditator of TAZ-induced tumorigenic and CSC phenotypes in lung cancer. Our studies provided preclinical data for targeting of TAZ-TEAD-ALDH1A1 signaling to inhibit CSC-induced lung tumorigenesis in the future.

Recurrent acute pancreatitis and its relative factors.

AIM: To evaluate the causes and the relative factors of recurrent acute pancreatitis. METHODS: From 1997 to 2000, acute pancreatitis relapsed in 77 of 245 acute pancreatitis patients. By reviewing the clinical treatment results and the follow-up data, we analyzed the recurrent factors of acute pancreatitis using univariate analysis and multivariate analysis. RESULTS: Of the 245 acute pancreatitis patients, 77 were patients with recurrent acute pancreatitis. Of them, 56 patients relapsed two times, 19 relapsed three times, each patient relapsed three and four times. Forty-seven patients relapsed in hospital and the other 30 patients relapsed after discharge. Eighteen patients relapsed in 1 year, eight relapsed in 1-3 years, and four relapsed after 3 years. There were 48 cases of biliary pancreatitis, 3 of alcohol pancreatitis, 5 of hyperlipidemia pancreatitis, 21 of idiopathic pancreatitis. Univariate analysis showed that the patients with local complications of pancreas, obstructive jaundice and hepatic function injury were easy to recur during the treatment period of acute pancreatitis (P=0.022<0.05, P=0.012<0.05 and P=0.002<0.05, respectively). Multivariate analysis showed that there was no single factor related to recurrence. Of the 47 patients who had recurrence in hospital, 16 had recurrence in a fast period, 31 after refeeding. CONCLUSION: Acute pancreatitis is easy to recur even during treatment. The factors such as changes of pancreas structure and uncontrolled systemic inflammatory reaction are responsible for the recurrence of acute pancreatitis. Early refeeding increases the recurrence of acute pancreatitis. Defining the etiology is essential for reducing the recurrence of acute pancreatitis.