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
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....
Radiation therapy methods have evolved remarkably in recent years which have resulted in more effective local tumor control with negligible toxicity of surrounding normal tissues. However, local recurrence and distant metastasis often occur following radiation therapy mostly due to the development of radioresistance through the deregulation of the cell cycle, apoptosis, and inhibition of DNA damage repair mechanisms. Over the last decade, extensive progress in radiotherapy and gene therapy combinatorial approaches has been achieved to overcome resistance of tumor cells to radiation. In this review, we summarize the results from experimental cancer therapy studies on the combination of radiation therapy and gene therapy.
Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter were characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.
Adenoviral (Ad) vector vaccines represent one of the most promising modern vaccine platforms, and Ad vector vaccines are currently being investigated in human clinical trials for infectious disease and cancer. Our studies have shown that specific targeting of adenovirus to dendritic cells dramatically enhanced vaccine efficacy. However, this was achieve using a molecular adapter, thereby necessitating a two component vector approach. To address the mandates of clinical translation of our strategy, we here sought to accomplish the goal of DC targeting with a single component adenovirus vector approach. To redirect the specificity of Ad vector vaccines, we replaced the Ad fiber knob with fiber-fibritin chimeras fused to DC1.8, a single domain antibody (sdAb) specific for murine immature DC. We engineered a fiber-fibritin-sdAb chimeric molecule using the coding sequence for DC1.8, and then replaced the native Ad5 fiber knob sequence by homologous recombination. The resulting Ad5 virus, Ad5FF1.8, expresses the chimeric fiber-fibritin sdAb chimera. Infection with Ad5FF1.8 dramatically enhances transgene expression in DC 2.4 dendritic cells compared to infection with native Ad5. Ad5FF1.8 infection of bone marrow derived DC demonstrates that Ad5FF1.8 selectively infects immature DC consistent with the known specificity of DC1.8. Thus, sdAb can be used to selectively redirect the tropism of Ad5 vector vaccines, providing the opportunity to engineer Ad vector vaccines that are specifically targeted to DC, or specific DC subsets.
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