Self-Adherent Biodegradable Gelatin-Based Hydrogel Electrodes for Electrocardiography Monitoring.

Patch-type hydrogel electrodes have received increasing attention in biomedical applications due to their high biocompatibility and conformal adherence. However, their poor mechanical properties and non-uniform electrical performance in a large area of the hydrogel electrode should be improved for use in wearable devices for biosignal monitoring. Here, we developed self-adherent, biocompatible hydrogel electrodes composed of biodegradable gelatin and conductive polymers for electrocardiography (ECG) measurement. After incorporating conductive poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS) into gelatin hydrogels crosslinked by natural crosslinkers (genipin), the mechanical properties and electrical conductivity of the hydrogel electrodes were improved and additionally optimized by adjusting the amounts of crosslinker and PEDOT:PSS, respectively. Furthermore, the effect of dimethyl sulfoxide, as a dopant, on the conductivity of hydrogels was investigated. The gelatin-based, conductive hydrogel patch displayed self-adherence to human skin with an adhesive strength of 0.85 N and achieved conformal contact with less skin irritation compared to conventional electrodes with a chemical adhesive layer. Eyelet-type hydrogel electrodes, which were compatible with conventional ECG measurement instruments, exhibited a comparable performance in 12-lead human ECG measurement with commercial ECG clinical electrodes (3M Red Dot). These self-adherent, biocompatible, gelatin-based hydrogel electrodes could be used for monitoring various biosignals, such as in electromyography and electroencephalography.

Metal Ion-Mediated Interliposomal Aggregation of Polydiacetylene Liposomes Incorporating a Phenolic Lipid.

Intercolloidal behaviors mediated by metal-ligand coordination have rarely been studied. In this work, such intercolloidal behaviors were demonstrated visibly using blue-colored polydiacetylene liposomes containing a phenolic lipid that acts as a binding ligand toward metal ions. The optimized liposomes were 150-200 nm in diameter and stable in aqueous solution. In incubation tests with various neocortical metal ions, iron(III) ions produced the most obvious colloidal aggregation of the liposomes. As the pH of the incubation medium was increased from acid to basic, stronger aggregation and increased precipitation behavior were observed. The phenolic lipid is believed to contribute to the interliposomal bridging interaction, and the pH dependence of the complexation between iron(III) and the phenolic lipid inserted in the liposomes were verified.

Tumor-specific delivery of therapeutic siRNAs by anti-EGFR immunonanoparticles.

BACKGROUND: Efficient target-specific siRNA delivery has always been a primary concern in the field of siRNA clinical application. PURPOSE: In this study, four different types of anti-epidermal growth factor receptor (EGFR) antibody-conjugated immunonanoparticles were prepared and tested for cancer cell-targeted therapeutic siRNA delivery. MATERIALS AND METHODS: The prepared nanoparticles encapsulating siRNAs were character-ized by gel retardation and particle analysis using a Zetasizer. In vitro transfection and reduction of target genes, vimentin and JAK3, were determined using quantitative reverse transcription polymerase chain reaction. In vivo tumor targeting and antitumoral efficacies of the nanoparticles were evaluated in mice carrying tumors. RESULTS: Among these immunonanoparticles, anti-EGFR immunolipoplexes and immunoviroplexes exhibited remarkable cell binding and siRNA delivery to EGFR-expressing tumor cells compared to immunoliposomes and immunovirosomes. Especially, the anti-EGFR immunoviroplexes exhibited the most efficient siRNA transfection to target tumor cells. Therefore, antitumoral vimentin and Janus kinase-3 siRNAs were loaded in the anti-EGFR immunolipoplexes and immunoviroplexes, which were tested in mice carrying SK-OV-3 tumor xenografts. In fact, the therapeutic siRNAs were efficiently delivered to the tumor tissues by both delivery vehicles, resulting in significant inhibition of tumor growth. Moreover, administration of doxorubicin in combination with anti-EGFR immunoviroplexes resulted in remarkable and synergistic tumor growth inhibition. CONCLUSION: This study provides experimental proof that cancer cell-targeted immunoviroplexes are an efficient siRNA delivery system for cancer therapy. Moreover, this study also suggests that a combination of conventional chemotherapy and tumor-directed anticancer siRNA therapy would be a better modality for cancer treatment.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy.

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

Effect of vitamin D3 on production of progesterone in porcine granulosa cells by regulation of steroidogenic enzymes.

1,25-dihydroxyvitamin D3 (VD3), an active form of Vitamin D, is photosynthesized in the skin of vertebrates in response to solar ultraviolet B radiation (UV-B). VD3 deficiency can cause health problems such as immune disease, metabolic disease, and bone disorders. It has also been demonstrated that VD3 is involved in reproductive functions. Female sex hormones such as estrogen and progesterone are biosynthesized mainly in ovarian granulosa cells as the ovarian follicle develops. The functions of sex hormones include regulation of the estrus cycle and puberty as well as maintenance of pregnancy in females. In this study, we isolated granulosa cells from porcine ovaries and cultured them for experiments. To examine the effects of VD3 on ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by Real-time PCR and Western blotting assay. Production of progesterone from granulosa cells was also measured by ELISA assay. As a result, transcriptional and translational regulation of progesterone biosynthesis-related genes in granulosa cells was significantly altered by VD3. Furthermore, progesterone concentrations in porcine granulosa cell-cultured media decreased in response to VD3. These results show that VD3 was a strong regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

Sendai viroplexes for epidermal growth factor receptor-directed delivery of interleukin-12 and salmosin genes to cancer cells.

BACKGROUND: The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor-directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off-target effects of gene transfection. METHODS: The system consists of a well-verified cationic O,O'-dimyristyl-N-lysyl glutamate (DMKE), Sendai virus fusion (F) protein and hemagglutinin-neuraminidase (HN) protein, referred to as cationic Sendai F/HN virosomes. To achieve tumor-specific recognition, anti-epidermal growth factor (EGF) receptor antibody was coupled to the surface of the virosomes containing interleukin-12 (IL-12) and/or salmosin genes that have potent anti-angiogenetic functions. RESULTS: Among the virosomal formulations, the anti-EGF receptor (EGFR) viroplexes, prepared via complexation of plasmid DNA (pDNA) with cationic DMKE lipid, exhibited more efficient gene transfection to tumor cells over-expressing EGF receptors compared to the neutrally-charged anti-EGFR virosomes encapsulating pDNA. In addition, the anti-EGFR viroplexes with IL-12 and salmosin genes exhibited the most effective therapeutic efficacy in a mouse tumor model. Especially when combined with doxorubicin, transfection of the two genes via the anti-EGFR viroplexes exhibited an enhanced inhibitory effect on tumor growth and metastasis in lungs. CONCLUSIONS: The results of the present study suggest that anti-EGFR viroplexes can be utilized as an effective strategy for tumor-directed gene delivery. Copyright © 2016 John Wiley & Sons, Ltd.

Chitin-fibroin-hydroxyapatite membrane for guided bone regeneration: micro-computed tomography evaluation in a rat model.

BACKGROUND: In guided bone regeneration (GBR) technique, many materials have been used for improving biological effectiveness by adding on membranes. The new membrane which was constructed with chitin-fibroin-hydroxyapatite (CNF/HAP) was compared with a collagen membrane (Bio-Gide®) by means of micro-computed tomography. METHODS: Fifty-four rats were used in this study. A critical-sized (8 mm) bony defect was created in the calvaria with a trephine bur. The CNF/HAP membrane was prepared by thermally induced phase separation. In the experimental group (n = 18), the CNF/HAP membrane was used to cover the bony defect, and in the control group (n = 18), a resorbable collagen membrane (Bio-Gide®) was used. In the negative control group (n = 18), no membrane was used. In each group, six animals were euthanized at 2, 4, and 8 weeks after surgery. The specimens were analyzed using micro-CT. RESULTS: Bone volume (BV) and bone mineral density (BMD) of the new bone showed significant difference between the negative control group and membrane groups (P < 0.05). However, between two membranes, the difference was not significant. CONCLUSIONS: The CNF/HAP membrane has significant effect on the new bone formation and has the potential to be applied for guided bone regeneration.

Hepatotoxicity and nephrotoxicity of gallotannin-enriched extract isolated from Galla Rhois in ICR mice.

To evaluate the hepatotoxicity and nephrotoxicity of Galla Rhois (GR) toward the liver and kidney of ICR mice, alterations in related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed after oral administration of 250, 500 and 1,000 mg/kg body weight/day of gallotannin-enriched extract of GR (GEGR) for 14 days. GEGR contained 68.7±2.5% of gallotannin, 25.3±0.9% of gallic acid and 4.4±0.1% of methyl gallate. Also, the level of malondialdehyde (MDA), a marker of lipid peroxidation, was decreased with 19% in the serum of high dose GEGR (HGEGR)-treated mice. The body and organ weight, clinical phenotypes, urine parameters and mice mortality did not differ among GEGR-treated groups and the vehicle-treated group. Furthermore, no significant increase was observed in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) and the serum creatinine (Cr) in the GEGR-treated group relative to the vehicle-treated group. Moreover, the specific pathological features induced by most toxic compounds such as CCl4 were not observed upon liver and kidney histological analysis. Overall, the results of the present study suggest that GEGR does not induce any specific toxicity in liver and kidney organs of ICR at doses of 1,000 mg/kg body weight/day, indicating that this is no observed adverse effect level (NOAEL).

Insulin enhances RANKL-induced osteoclastogenesis via ERK1/2 activation and induction of NFATc1 and Atp6v0d2.

Insulin is one of the main factors affecting bone and energy metabolism, however, the direct effect of insulin on osteoclast differentiation remains unclear. Thus, in order to help elucidate that puzzle, the authors investigated the roles and regulatory mechanisms of insulin on osteoclasts differentiation. Co-stimulation with insulin and RANKL significantly enhanced the number of larger (>100 µm) osteoclastic cells and of TRAP-positive multinucleated cells compared with treatment by RANKL alone. Conversely, the insulin receptor shRNA markedly decreased osteoclast differentiation induced by insulin and RANKL. Insulin treatment significantly activated ERK1/2 MAP kinase as well as markedly induced the expression of NFATc1, an osteoclast marker gene, and Atp6v0d2, an osteoclast fusion-related gene. The pretreatment of PD98059, an ERK1/2 inhibitor, or insulin receptor shRNA effectively suppressed osteoclast differentiation and, in addition, blocked the expression of NFATc1 and Atp6vod2 induced by insulin stimulation. These data reveal insights into the regulation of osteoclast differentiation and fusion through ERK1/2 activation and the induction of NFATc1 and Atp6v0d2 by insulin.

Gene expression profiling in osteoclast precursors by insulin using microarray analysis.

The balance between bone formation by osteoblasts and destruction of mineralized bone matrix by osteoclasts is important for bone homeostasis. The increase of osteoclast differentiation by RANKL induces bone diseases such as osteoporosis. Recent studies have shown that insulin is one of main factors mediating the cross-talk between bone remodeling and energy metabolism. However, the systemic examination of insulin-induced differential gene expression profiles in osteoclasts has not been extensively studied. Here, we investigated the global effects of insulin on osteoclast precursors at the level of gene transcription by microarray analysis. The number of genes that were up-regulated by ≥ 1.5 fold after insulin treatment for 6 h, 12 h, or 24 h was 76, 73, and 39; and 96, 83, and 54 genes were down-regulated, respectively. The genes were classified by 20 biological processes or 24 molecular functions and the number of genes involved in 'development processes' and 'cell proliferation and differentiation' was 25 and 18, respectively, including Inhba, Socs, Plk3, Tnfsf4, and Plk1. The microarray results of these genes were verified by real-time RT-PCR analysis. We also compared the effects of insulin and RANKL on the expression of these genes. Most genes had a very similar pattern of expressions in insulin- and RANKL-treated cells. Interestingly, Tnfsf4 and Inhba genes were affected by insulin but not by RANKL. Taken together, these results suggest a potential role for insulin in osteoclast biology, thus contributing to the understanding of the pathogenesis and development of therapeutics for numerous bone and metabolic diseases.

Comparative study of chitosan/fibroin-hydroxyapatite and collagen membranes for guided bone regeneration in rat calvarial defects: micro-computed tomography analysis.

This study aimed to utilize micro-computed tomography (micro-CT) analysis to compare new bone formation in rat calvarial defects using chitosan/fibroin-hydroxyapatite (CFB-HAP) or collagen (Bio-Gide) membranes. Fifty-four (54) rats were studied. A circular bony defect (8 mm diameter) was formed in the centre of the calvaria using a trephine bur. The CFB-HAP membrane was prepared by thermally induced phase separation. In the experimental group (n=18), the CFB-HAP membrane was used to cover the bony defect, and in the control group (n=18), a resorbable collagen membrane (Bio-Gide) was used. In the negative control group (n=18), no membrane was used. In each group, six animals were euthanized at 2, 4 and 8 weeks after surgery. The specimens were then analysed using micro-CT. There were significant differences in bone volume (BV) and bone mineral density (BMD) (P<0.05) between the negative control group and the membrane groups. However, there were no significant differences between the CFB-HAP group and the collagen group. We concluded that the CFB-HAP membrane has significant potential as a guided bone regeneration (GBR) membrane.

Sendai F/HN viroplexes for efficient transfection of leukemic T cells.

PURPOSE: Most chemical transfection reagents are ineffective for the transfection of cells in suspension, such as leukemic cell and stem cell lineages. We developed two different types of viroplexes, cationic Sendai F/HN viroplexes (CSVs) and protamine sulfate-condensed cationic Sendai F/HN viroplexes (PCSVs) for the efficient transfection of T-leukemic cells. MATERIALS AND METHODS: The viroplex systems were prepared by reconstitution of fusogenic Sendai F/HN proteins in DMKE (O,O'-dimyristyl-N-lysyl glutamate) cationic liposomes. The viroplexes were further optimized for plasmid DNA and siRNA delivery to suspension cells. The particle size and surface charge of the viroplexes were analyzed with a ζ-sizer. Transfection of plasmid DNA (pDNA) and small interfering RNA (siRNA) by CSVs or PCSV was evaluated by measurement of transgene expression, confocal microscopy, FACS, and RT-PCR. RESULTS: The optimized CSVs and PCSVs exhibited enhanced gene and siRNA delivery in the tested suspension cell lines (Jurkat cells and CEM cells), compared with conventional cationic liposomes. In the case of pDNA transfection, the CSVs and PCSVs show at least 10-fold and 100-fold higher transgene expression compared with DMKE lipoplexes (or lipofectamine 2000), respectively. The CSVs showed more effective siRNA delivery to the suspension cells than cationic liposomes, as assessed by confocal microscopy, FACS, and RT-PCR. The effective transfection by the CSVs and PCSVs is presumably due to fusogenic activity of F/HN proteins resulting in facilitated internalization of pDNA and siRNA. CONCLUSION: This study suggests that Sendai F/HN viroplexes can be widely applicable for the transfection of pDNA and siRNA to suspension cell lines.

Caffeine enhances osteoclast differentiation and maturation through p38 MAP kinase/Mitf and DC-STAMP/CtsK and TRAP pathway.

The consumption of caffeine from some common beverages has been associated with low bone mass by inducing urinary calcium loss and deceasing bone mineral density. However, the effect of caffeine on osteoclast differentiation is still unclear. Here, we demonstrate that caffeine directly enhances osteoclast differentiation and maturation. TRAP staining showed that the number of larger (>100 µm) osteoclastic cells as well as of TRAP-positive multinucleated cells was increased by caffeine treatment. Among the MAP kinases, caffeine specifically activated p38 MAP kinase, which in turn, controlled osteoclast differentiation and maturation. This is evidenced by the abolishment of activated p38 MAP kinase by pretreatment with SB203580, a p38-specific inhibitor, resulting in suppressed osteoclast differentiation and maturation that should be increased by caffeine. Caffeine significantly induced the expression of Mitf and pretreatment with SB203580 markedly suppressed the expression of Mitf induced by caffeine. Whereas it failed to regulate the expression of NFATc1 and Oscar, the expressions of Cathepsin K and TRAP were induced by caffeine treatment in primary preosteoclasts. Real-time PCR and luciferase assays showed that the increase of osteoclastic cell-cell fusion by caffeine was through the transcriptional up-regulation of DC-STAMP expression but not of Atp6v0d2. These results strongly suggest that caffeine directly enhances osteoclast differentiation and maturation through p38 MAP kinase activation, thus inducing Mitf expression and transcriptional activation of DC-STAMP, and finally CtsK and TRAP.

Synergistic antitumoral effect of IL-12 gene cotransfected with antiangiogenic genes for angiostatin, endostatin, and saxatilin.

Previously, it was reported that the cotransfection of angiostatin K1-3, endostatin, and saxatilin genes using cationic liposomes significantly inhibited tumor progression. IL-12 is a well-known immune modulator that promotes Th1-type antitumor immune responses and also induces antiangiogenic effects. In this study, we have examined the antitumoral function of the IL-12 gene cotransfected with antiangiogenic genes for angiostatin K1-3, endostatin, and saxatilin by O,O'-dimyristyl-N-lysyl glutamate (DMKE) cationic liposomes in a mouse tumor model. According to our results, the administration of the IL-12 gene or the genes for angiostatin K1-3, endostatin, and saxatilin exhibited effective inhibition of B16BL6 melanoma growth in mice. In particular, intravenous administration of the IL-12 gene along with intratumoral administration of the three antiangiogenic genes synergistically inhibited the B16BL6 tumor growth. These results suggest that systemically expressed IL-12 enhances antitumoral efficacy of locally expressed antiangiogenic proteins.

Altered expression of γ-secretase components in animal model of major depressive disorder induced by reserpine administration.

Altered expression of neurotrophic factors as well as neuroinflammation is commonly associated with Major depressive disorder (MDD) and Alzheimer's disease (AD). To investigate whether or not reserpine-induced MDD affects the expression of AD-related proteins, the expression of γ-secretase components and substrate were measured in brains of ICR mice following reserpine treatment for 15 days. In active avoidance test, total response time and peak slightly increased in the 2 mg/kg reserpine (RSP2)-treated group compared to vehicle-treated group (P<0.05). Expression and phosphorylation of MKP-1, which is a key factor in MDD pathology, were both higher in the RSP2-treated group than the vehicle- and 1 mg/kg reserpine (RSP1)-treated groups (P<0.02). Furthermore, full-length expression of amyloid precursor protein (APP) was enhanced in the RSP1 and RSP2-treated groups compared to the vehicle-treated group, whereas expression of γ-secretase components decreased (P<0.03). Among the three components of the γ-secretase complex, nicastrin protein underwent the largest decrease in expression, as detected by Western blotting (P<0.03). Therefore, the data presented here provide additional evidence about the pathological correlation between MDD and AD.

Hepatic control elements promote long-term expression of human coagulation factor IX gene in hydrodynamically transfected mice.

BACKGROUND: Long-term expression of the delivered target gene is critical for successful gene therapy. Recently, hepatic control region I (HCR I) originating from the apolipoprotein (apo)C-I pseudogene was shown to be a critical element for long-term gene expression in the liver of mice. HCR II is another hepatic control region of apoC-I. METHODS: HCR I, HCR II and HCR I/II-containing plasmids encoding factor IX were prepared and hydrodynamically transferred into the liver of normal and hemophilia B mice. Factor IX expression, clotting activity and formation of antibodies against the expressed gene product were compared. RESULTS: HCR I-, HCR II- and HCR I/II-containing plasmids all induced long-term gene expression in both normal and hemophilia B mice. Post-transfection factor IX expression in the hemophilia B mice remained above 500 ng/ml for 210 days. Antibodies against human factor IX were detected at a low level in the serum, although they had no effect on the levels and clotting activity of the expressed factor IX. CONCLUSIONS: We have shown in mouse models that hydrodynamic transfection of pBS-HCRII-HP-FIXA and pBS-HCRI/II-HP-FIXA was able to induce and maintain the expression and clotting activity of human factor IX for a long period of time at a potentially therapeutic level. With an appropriate delivery system, this type of plasmid vector could be clinically useful for the hepatic expression of therapeutic genes including human factor IX.

Peroxiredoxin I regulates the component expression of γ-secretase complex causing the Alzheimer's disease.

Peroxiredoxin I (Prx I) is a member of the peroxiredoxins (Prxs) family, which are antioxidant enzymes that regulate various cellular process via intracellular oxidative signal pathways. In order to investigate the correlation between Prx I and the γ-secretase complex, which causes Alzheimer's disease (AD), the expression level of Prx I was firstly evaluated in an animal model for AD. NSE/hPen-2 transgenic (Tg) mice, which were used as animal model in this study, showed a high level of Pen-2 expression and accumulation of Aß-42 peptides in the hippocampus of brain. The expression level of Prx I was significantly higher on the mRNA and protein level in the brain of this model, while not change in Prx VI expression was observed. Furthermore, to verify the effect of Prx I on the γ-secretase components in vitro, the expression level of these components was analyzed in the Prx I transfectants. Of the components of the γ-secretase complex, the expression of PS-2 and Pen-2 was lower in the transfectants overexpressing Prx I compared to the vector transfectants. However, the expression of APP, NCT and APH-1 did not change in Prx I transfectants. Therefore, these results suggested that the expression of Prx I may be induced by the accumulation of Aß-42 peptides and the overexpression of Prx I in neuroblastoma cells may regulate the expression of γ-secretase components.

Production and characterization of cellulose by Acetobacter sp. V6 using a cost-effective molasses-corn steep liquor medium.

In order to reduce of the manufacturing cost of bacterial cellulose (BC), BC production by Acetobacter sp. V6 was investigated in shaking culture using molasses and corn steep liquor (CSL) as the sole carbon and nitrogen sources, respectively. The highest BC production was obtained with Ca3(PO4)2-treated molasses. Maximum BC yield (2.21+/-0.04 g/l) was obtained at 5% (w/v) total sugar in molasses. In improved medium containing molasses and CSL, BC production was observed in the medium after 1 day of incubation and increased rapidly thereafter with maximum yield (3.12+/-0.03 g/l) at 8 days. This value was approximately twofold higher than the yield in the complex medium. Physical properties of BC from the complex and molasses media were studied using Fourier-transform infrared (FT-IR) spectroscopy and X-ray diffractometer. By FT-IR, all the BC were found to be of cellulose type I, the same as typical native cellulose. The relative crystallinity of BC produced in the complex and molasses media were 83.02 and 67.27%, respectively. These results suggest that molasses and CSL can be useful low-cost substrates for BC production by Acetobacter sp. V6 without supplementation with expensive nitrogen complexes such as yeast extract and polypeptone, leading to the reduction in the production costs.

Host lymphodepletion enhances the therapeutic activity of an oncolytic vaccinia virus expressing 4-1BB ligand.

Oncolytic viral vectors have shown promise as antitumor therapeutic agents but their effectiveness is complicated by induction of antiviral antibody responses and rapid host clearance of recombinant vectors. We developed a recombinant oncolytic vaccinia virus expressing the 4-1BBL T-cell costimulatory molecule (rV-4-1BBL) and showed modest tumor regression in the poorly immunogenic B16 murine melanoma model. To improve the therapeutic potential of this vector, we tested the antitumor activity of local intratumoral injection in the setting of host lymphodepletion, which has been shown to augment vaccination and adoptive T-cell therapy. In this model, rV-4-1BBL injection in the setting of lymphodepletion promoted MHC class I expression, reduced antiviral antibody titers, promoted viral persistence, and rescued effector-memory CD8(+) T cells, significantly improving the therapeutic effectiveness of the oncolytic vector. These data suggest that vaccination with rV-4-1BBL in the setting of host nonmyeloablative lymphodepletion represents a logical strategy for improving oncolytic vaccination in melanoma, and perhaps other cancers as well.

Local IL-21 promotes the therapeutic activity of effector T cells by decreasing regulatory T cells within the tumor microenvironment.

The eradication of tumors by the immune system depends on the generation of antigen-specific T cells which can migrate to sites of tumor growth and maintain their effector functions despite local tumor-derived T-cell inhibitory factors. Interleukin-21 (IL-21) is an IL-2-related cytokine that has shown limited evidence of antitumor activity in murine models and early phase clinical trials. Effect of local IL-21 on T-cell responses within the tumor microenvironment, however, has not been extensively evaluated. Thus, we developed a stably transfected IL-21-secreting B16 melanoma cell line to test the effects of local IL-21 on endogenous and adoptively transferred T-cell responses. Tumors expressing IL-21 exhibited delayed growth in vivo, which was associated with an increase in activated systemic effector and memory CD8(+) T-cell responses. Local IL-21 also enhanced the therapeutic effects of adoptively transferred gp100-specific T cells and was synergistic with IL-2. The effect was also associated with an increased proliferation of local CD8(+) T cells and decreased accumulation of regulatory CD4(+)FOXP3(+) T cells within the tumor microenvironment. These data suggest that local IL-21 enhances endogenous and adoptively transferred T-cell immunity through increased effector CD8(+) T cells and decreased CD4(+) regulatory T cells in the tumor microenvironment.