In recent years, it has been established that programmed cell death protein ligand 1 (PD-L1)-mediated inhibition of activated PD-1 + T lymphocytes plays a major role in tumor escape from immune system during cancer progression. Lately, the anti-PD-L1 and -PD-1 immune therapies have become an important tool for treatment of advanced human cancers, including bladder cancer. However, the underlying mechanisms of PD-L1 expression in cancer are not fully understood. We found that coculture of murine bone marrow cells with bladder tumor cells promoted strong expression of PD-L1 in bone marrowderived myeloid cells. Tumor-induced expression of PD-L1 was limited to F4/80 + macrophages and Ly-6C + myeloid-derived suppressor cells. These PD-L1-expressing cells were immunosuppressive and were capable of eliminating CD8 T cells in vitro. Tumor-infiltrating PD-L1 + cells isolated from tumor-bearing mice also exerted morphology of tumorassociated macrophages and expressed high levels of prostaglandin E 2 (PGE 2 )-forming enzymes microsomal PGE 2 synthase 1 (mPGES1) and COX2. Inhibition of PGE 2 formation, using pharmacologic mPGES1 and COX2 inhibitors or genetic overexpression of PGE 2 -degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH), resulted in reduced PD-L1 expression. Together, our study demonstrates that the COX2/mPGES1/PGE 2 pathway involved in the regulation of PD-L1 expression in tumor-infiltrating myeloid cells and, therefore, reprogramming of PGE 2 metabolism in tumor microenvironment provides an opportunity to reduce immune suppression in tumor host.PD-L1 | tumor-associated macrophages | PGE 2 metabolism | myeloid cells | bone marrow

The promise of gene therapy for health care will not be realized until gene delivery systems are capable of achieving efficient, cell-specific gene delivery in vivo. Here we describe an adenoviral system for achieving cell-specific transgene expression in pulmonary endothelium. The combination of transductional targeting to a pulmonary endothelial marker (angiotensin-converting enzyme, ACE) and an endothelial-specific promoter (for vascular endothelial growth factor receptor type 1, flt-1) resulted in a synergistic, 300,000-fold improvement in the selectivity of transgene expression for lung versus the usual site of vector sequestration, the liver. This combined approach should be useful for the design of other gene delivery systems.

It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is expressed in human cancers but is undetectable in normal differentiated tissues. We employed a recombinant adenoviral vector (reAdGL3BSurvivin) in which a tumor-specific survivin promoter and a luciferase reporter gene were inserted into the E1-deleted region of adenovirus vector. Luciferase activity was measured in both multiple tumor cell lines and two primary melanoma cells infected with reAdGL3BSurvivin. Human fibroblast and mammary epithelial cell lines were used as negative controls. A reAdGL3CMV, containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity generated by the survivin promoter. Our data revealed that the survivin promoter showed high activity in both established tumor cell lines and the primary melanoma cells. In contrast, the in vivo studies indicated that the activities of survivin promoter were extremely low in the major mouse organs. The survivin promoter appears to be a promising tumor-specific promoter exhibiting a ''tumor on'' and ''liver off'' profile, and therefore, it may prove to be a good candidate for transcriptional targeting of cancer gene therapy in a wide variety of tumors.

Mutations in the ATP-binding cassette transporter A3 gene (ABCA3) result in severe neonatal respiratory distress syndrome and childhood interstitial lung disease. As most ABCA3 mutations are rare or private, determination of mutation pathogenicity is often based on results from in silico prediction tools, identification in unrelated diseased individuals, statistical association studies, or expert opinion. Functional biologic studies of ABCA3 mutations are needed to confirm mutation pathogenicity and inform clinical decision making. Our objective was to functionally characterize two ABCA3 mutations (p.R288K and p.R1474W) identified among term and latepreterm infants with respiratory distress syndrome with unclear pathogenicity in a genetically versatile model system. We performed transient transfection of HEK293T cells with wild-type or mutant ABCA3 alleles to assess protein processing with immunoblotting. We used transduction of A549 cells with adenoviral vectors, which concurrently silenced endogenous ABCA3 and expressed either wildtype or mutant ABCA3 alleles (p.R288K and p.R1474W) to assess immunofluorescent localization, ATPase activity, and organelle ultrastructure. Both ABCA3 mutations (p.R288K and p.R1474W) encoded proteins with reduced ATPase activity but with normal intracellular localization and protein processing. Ultrastructural phenotypes of lamellar body-like vesicles in A549 cells transduced with mutant alleles were similar to wild type. Mutant proteins encoded by ABCA3 mutations p.R288K and p.R1474W had reduced ATPase activity, a biologically plausible explanation for disruption of surfactant metabolism by impaired phospholipid transport into the lamellar body. These results also demonstrate the usefulness of a genetically versatile, human model system for functional characterization of ABCA3 mutations with unclear pathogenicity.Keywords: surfactant; childhood interstitial lung disease; neonatal respiratory distress; respiratory distress syndrome ATP-binding cassette transporter A3 (ABCA3), a member of a large family of proteins that hydrolyze ATP to transport substances across biologic membranes, is encoded by an 80-kb gene (ABCA3; NM_001089.2, Gene ID 21) and consists of 1,704 amino acids with two membranespanning domains and two nucleotide binding domains. ABCA3 is expressed in alveolar type II cells, localizes to the membranes of lamellar bodies, and transports phospholipids into the lamellar body, where surfactant, a complex protein-phospholipid mixture that reduces surface tension and prevents alveolar collapse at end expiration, is assembled and stored (1-3). ABCA3 is also required for lamellar body biogenesis (4).Biallelic loss-of-function (nonsense, frameshift) mutations in ABCA3 result in severe, progressive neonatal respiratory distress syndrome (RDS) that is lethal by 1 year of age without lung transplantation (5). However, for individuals with missense, T.C., F.V.W., A.H., B.P.H., and F.S.C.; drafting and revising the manuscript for important intellectual content-J.A.W., P.Y....

Many cancers are known to produce high amounts of PGE2, which is involved in both tumor progression and tumor-induced immune dysfunction. The key enzyme responsible for the biological inactivation of PGE2 in tissue is NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). It is well established that cancer cells frequently show down-regulated expression of 15-PGDH, which plays a major role in catabolism of the PGE2. Here we demonstrate that tumor-infiltrated CD11b cells are also deficient for the 15-PGDH gene. Targeted adenovirus-mediated delivery of 15-PGDH gene resulted in substantial inhibition of tumor growth in mice with implanted CT-26 colon carcinomas. PGDH-mediated antitumor effect was associated with attenuated tumor-induced immune suppression and substantially reduced secretion of immunosuppressive mediators and cytokines such as PGE2, IL-10, IL-13, and IL-6 by intratumoral CD11b cells. We show also that introduction of 15-PGDH gene in tumor tissue is sufficient to redirect the differentiation of intratumoral CD11b cells from immunosuppressive M2-oriented F4/80+ tumor-associated macrophages (TAM) into M1-oriented CD11c+ MHC class II-positive myeloid APCs. Notably, the administration of the 15-PGDH gene alone demonstrated a significant therapeutic effect promoting tumor eradication and long-term survival in 70% of mice with preestablished tumors. Surviving mice acquired antitumor T cell-mediated immune response. This study for the first time demonstrates an important role of the 15-PGDH in regulation of local antitumor immune response and highlights the potential to be implemented to enhance the efficacy of cancer therapy and immunotherapy.