RSV Treatment Research Articles

THE ANTI-TUMOR EFFECTS OF RESVERATROL ON NON-SMALL CELL LUNG CANCER WITH RESISTANCE TO EGFR TYROSINE KINASE INHIBITORS THROUGH VEGF SUPPRESSION

SESSION TITLE: Lung Cancer Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: The efficacy of Epidermal growth factor receptor (EGFR)-tyosine kinase inhibitors is significantly limited by various resistance mechanisms to those drugs. Vascular endothelial growth factor (VEGF) and EGFR inhibitors have become key therapies in lung cancer. Resveratrol (RSV, 3, 5, 4-trihydroxystilbene), a naturally polyphenolic phytoalexin has anti-inflammatory and anti-carcinogenic and angiogenesis regulating properties. The purpose of this study was to investigate the effect of RSV in gefitinib resistant PC9 (PC9/G) cell lines and establish if VEGF signaling may be a potential target. METHODS: Two cell lines, PC9 and PC9/G, were selected for this study. Cells were exposed to 0.1, 1.0, 10, 20 uM gefitinib (Gef) and/or 40 uM RSV. Cell viability was measured by MTT assay. The expression levels of target genes were examined with ELISA and western blot assay according to gefitinib and RSV treatment. RESULTS: To assess whether RSV could sensitize PC9/G cells to Gef. We evaluated the effects combination treatment of Gef and RSV on the proliferation of PC9/G cells. Compared with Gef teatment alone, Gef + RSV treatment was more potent growth inhibitory effects. Cell migration was more inhibited by Gef+RSV treatment than Gef alone. Western blot analysis showed that RSV repressed the expressions of VEGFR and multiple downstream protein kinases such as Akt, mTOR, and p70S6K. Survivin and cleaved PARP, apoptotic proteins, were enhanced after RSV + Gef treatment in PC9/G cells. CONCLUSIONS: Our findings suggested that RSV combined with Gef had additional antitumor effects in PC-9/GR cells. CLINICAL IMPLICATIONS: Apoptotic effects was mediated by multiple mechanism involving suppression of VEGF expression via inhibition of PI3K/Akt/mTOR pathway. DISCLOSURES: No relevant relationships by Hye Seon Kang, source=Web Response No relevant relationships by Yong hyun Kim, source=Web Response No relevant relationships by SOON SEOG KWON, source=Web Response no disclosure on file for Eung Gu Lee; No relevant relationships by JIWON RYOO, source=Web Response

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing.

Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨm), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P<0.05), and RSV did not significantly increase the sperm motility (0.44 vs. 0.40, P>0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨm. RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 μmol/L vs. 0.25 μmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.

Immunogenic potency of engineered exosomes for prevention of respiratory syncytial virus

Abstract Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in infants and young children. Bovine RSV is a common cause of respiratory disease in young cattle and is a useful model to study RSV infection. There is currently no vaccine for RSV treatment in humans, and the vaccines available for BRSV have limited efficacy in the field. Exosomes (30–150nm) are cell-derived extracellular vesicles that play critical roles in cellular communication by carrying various molecules. Since exosomes secreted by virus or tumor infected cells can deliver cell-specific antigens and proteins to immune cells, immunogenic exosomes have emerged as a new vaccine platform for prevention of viral infections or therapy for cancer treatment. Here, dendritic-cell derived exosomes were engineered to present peptides from the RSV M, NS and L proteins, purified from PDMS microfluidic systems and were evaluated as an efficacious surface engineered antigenic exosome vaccine against RSV and BRSV in mice. The immunogenicity of the exosomes was confirmed in vitro by measuring their capacity to induce antigen-specific T cell clonal proliferation and IFNγ production. The in vivo immunogenicity and efficacy of the engineered exosomes was investigated by immunizing animals subcutaneously with escalating doses of engineered exosomes and measuring antigen-specific T cell responses in the spleen and lungs, and assessing morbidity and mortality. Collectively, dendritic-cell derived surface engineered antigenic exosomes have the capacity to elicit antigen-specific CD8 T cell activation in vitro and in vivo, and the potential to be developed as a vaccine platform to prevent RSV and other viral infections in humans and animals.

Effect of the natural compound trans‑resveratrol on human MCM4 gene transcription.

trans‑Resveratrol (Rsv) is a natural compound contained in red wine and grape skins that has various beneficial effects for organisms such as lengthening of their life span. Rsv induces expression of the human TP53 and HELB genes, which are involved in the regulation of DNA maintenance. In the present study, a luciferase expression vector containing 309bp of the 5' upstream end of the human MCM4 gene was transfected into HeLa S3 cells. A luciferase assay revealed that Rsv treatment increased the minichromosome maintenance4 (MCM4) gene promoter activity by GC‑box and GGAA (TTCC) motifs. Electro phoretic mobility shift assay revealed that the specific binding factor (complex) contains PU.1 (SPI1). Quantitative reverse transcription‑polymerase chain reaction analysis indicated that MCM4 gene expression was transiently induced by Rsv. Moreover, western blotting revealed that the SP1/PU.1 ratio markedly increased after Rsv treatment, indicating that a balance or profile of these transcription factors may control Rsv‑inducible initiation of transcription. These observations indicated that the beneficial effects of Rsv can be attributed to induction of the chromosomal DNA maintenance factor encoding gene expression.

Capsaicinoid nonivamide improves nonalcoholic fatty liver disease in rats fed a high-fat diet

Nonalcoholic fatty liver disease (NAFLD) is a chronic disease that causes morbidity associated with metabolic syndrome. NAFLD is a worldwide problem and represents a major cause of liver injury, which can lead to liver cell death. We investigated the effects of nonivamide (pelargonic acid vanillylamide, PAVA; 1 mg/kg) and rosuvastatin (RSV; 10 mg/kg) on hepatic steatosis induced by a high-fat diet (HFD). Male Sprague–Dawley rats were fed a HFD for 16 weeks then received PAVA or RSV for 4 additional weeks. We examined the metabolic parameters, function, fat content, histological alterations, reactive oxygen species production, and apoptotic cell death of the liver, in addition to the expression of the following important molecules: transient receptor potential cation channel subfamily V member 1 (TRPV1) phosphorylation of sterol regulatory element binding protein (pSREBP-1c/SREBP-1c), total and membrane glucose transporter 2 (GLUT2), 4-hydroxynonenal (4-HNE), and cleaved caspase-3. HFD-induced hepatic steatosis was associated with significantly increased morphological disorganization, injury markers, oxidative stress, lipid peroxidation, and apoptosis. However, metabolic dysfunction and hepatic injury were reduced by RSV and PAVA treatment. PAVA regulated lipid deposition, improved insulin resistance, and decreased oxidative stress and apoptotic cell death. Therefore, PAVA represents a promising therapeutic approach for treating metabolic disorders in patients with NAFLD.

Cold Weather Viruses.

1. Asif Noor, MD* 2. Theresa Fiorito, MD* 3. Leonard R. Krilov, MD*,† 1. *Department of Pediatrics, Children's Medical Center, NYU Winthrop Hospital, Mineola, NY 2. †Department of Pediatrics, State University of New York, Stony Brook School of Medicine, Stony Brook, NY * Abbreviations: AAP: : American Academy of Pediatrics Adv: : adenovirus CDC: : Centers for Disease Control and Prevention FDA: : Food and Drug Administration hMPV: : human metapneumovirus PCR: : polymerase chain reaction PIV: : parainfluenza virus RSV: : respiratory syncytial virus RV: : rhinovirus Clinicians must learn to identify viral infections in children during the winter months and must practice caution with the use of unnecessary medications in such cases. Recognition of the clinical pattern of viral infection (eg, bronchiolitis) in conjunction with judicious use of viral tests (either office-based immunoassays or newer molecular tests) may assist in epidemiological monitoring, cohorting patients in the hospital, withholding unnecessary therapies, and providing a definitive diagnosis. After completing this article, readers should be able to: 1. Review the epidemiological aspects and clinical signs and symptoms of common cold weather viruses. 2. Recognize situations in which viral testing is indicated. 3. Recognize situations in which treatment is indicated. In early November you are evaluating a 9-month-old boy born at 33 weeks of gestation. The infant presents with 2 days of fevers (101°F–102°F [38.3°C–38.8°C]), copious rhinorrhea, and 1 day of coughing with difficulty breathing. He is otherwise feeding well and has had adequate urination. His 4-year-older sister has an upper respiratory tract infection. On physical examination, the infant has a respiratory rate of 45 breaths/min without chest wall retractions. On auscultation there is good air entry with scattered rhonchi bilaterally. What is the most appropriate next step in management? 1. Obtain respiratory syncytial virus (RSV) and influenza antigen testing. 2. Obtain a chest radiograph to look for focal infiltrate. 3. Provide supportive care with nasal …

Assessing the Incidence of Symptomatic Respiratory Syncytial Virus Illness Within a Prospective Birth Cohort in Managua, Nicaragua.

BackgroundRespiratory syncytial virus (RSV) causes substantial morbidity and mortality among children worldwide, commonly through acute lower respiratory tract infections (ALRI). To assess the incidence rate of symptomatic RSV illness among young children, we conducted a prospective birth cohort study following children from 0–2 years of age in Managua, Nicaragua.MethodsChildren meeting the testing criteria (fever, history of fever, or severe respiratory symptoms [apnea, stridor, nasal flaring, wheezing, chest indrawing, and/or central cyanosis]) were tested for RSV infections using real-time reverse transcriptase-polymerase chain reaction. An acute lower respiratory infection was defined as a diagnosis of pneumonia, bronchiolitis, bronchitis, or bronchial hyperreactivity. The incidence rate was calculated, and 95% confidence intervals were estimated using a Poisson distribution.ResultsA total of 833 children participated in the cohort: 289 (34.7%) had at least 1 episode of laboratory-confirmed RSV, and 156 (18.7%) of had an episode of RSV-associated ALRI (RSV-ALRI). The incidence rate of symptomatic RSV was 248.1 cases per 1000 person-years (95% confidence interval [CI] 223.2–275.7). While infants aged 6–11 months had the highest incidence of symptomatic RSV (361.3/1000 person-years, 95% CI 304.4–428.8), infants <3 months had the highest incidence of severe RSV (RSV-associated hospitalizations and/or severe ALRI). RSV was also associated with 25.0–37.5% of deaths from medical causes (n = 8).ConclusionsA substantial burden of RSV exists among children aged <2 years in Nicaraguan communities. RSV was also a leading cause of infant mortality among study participants. The development and implementation of effective RSV prevention and treatment measures represent an opportunity to substantially reduce severe illness and death among children worldwide.

Divergent Effects of Resveratrol on Rat Cardiac Fibroblasts and Cardiomyocytes.

In this study, we tested the potential cardioprotective effects of the phytoalexin resveratrol (Rsv) on primary adult rat cardiac fibroblasts (CF), myofibroblasts (MF) and cardiomyocytes. Adult rat CF and cardiomyocytes were isolated from male 10-week old Sprague–Dawley rats, cultured for either 24 h (cardiomyocytes) or 48 h (CF) before treatments. To isolate MF, CF were trypsinized after 48 h in culture, seeded in fresh plates and cultured for 24 h prior to treatment. All three cells were then treated for a further 24 h with a range of Rsv doses. In CF and MF, cell proliferation, viability, apoptosis assays were performed with or without Rsv treatment for 24 h. In cardiomyocytes, cell viability and apoptosis assay were performed 24 h after treatment. In separate experiments, CF was pre-incubated with estrogen, tamoxifen and fulvestrant for 30 min prior to Rsv treatment. Rsv treatment decreased proliferation of both fibroblasts and myofibroblasts. Rsv treatment also increased the proportion of dead CF and MF in a dose dependent manner. However, treatment with Rsv did not induce cell death in adult cardiomyocytes. There was an increase in the percentage of cells with condensed nuclei with Rsv treatment in both CF and MF, but not in cardiomyocytes. Treatment with estrogen, tamoxifen and fulvestrant alone or in combination with Rsv did not have any additional effects on CF survival. Our results demonstrate that treatment with Rsv can inhibit cell proliferation and induce cell death in rat CF and MF, while not affecting cardiomyocyte survival. We also demonstrated that the induction of cell death in CF with Rsv treatment was independent of estrogen receptor alpha (ERα) signaling.

Modulation of ionizing radiation-induced damage in human blood lymphocytes by in vivo treatment with resveratrol

Purpose: Radiotherapy is an effective tool for cancer control, but side effects on normal tissue limit its therapeutic effectiveness. Thus, the search for agents that may allow the use of high doses of radiation but exerting a differential protection to healthy tissue is of current concern. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) (RSV) is a polyphenol with pleiotropic benefits for health due to its antioxidant and anti-inflammatory properties. Recent findings suggest that RSV could be promising in the fight against cancer since it inhibits the growth of tumor cells and optimizes radiotherapy. However, evidence in rodents and human beings is inconsistent. The aim of this study was to evaluate the radiomodulatory capacity of RSV on human lymphocytes.Materials and methods: To study these properties of RSV, human peripheral blood lymphocytes from 20 healthy women undergoing in vivo RSV treatment with 50 mg/day doses were irradiated. The genotoxic damage was assessed by the comet assay, also called single cell gel electrophoresis (it makes it possible to measure the extent of the DNA migration from individual cells, detecting the genomic damage present in each cell).Results: No differences were observed in basal clastogenic damage among samples without irradiation. There was only a slight radiation-induced clastogenic damage. The damage index (DI) value had a statistically significant increase in the exposed groups in comparison with the control groups (p < .0001), but a statistically significant decrease of the DI value was observed in samples irradiated after treatment with RSV compared to pretreatment samples (p < .0001).Conclusion: The RSV used as a dietary supplement had radioprotective properties, without exerting a cytotoxic effect. The potential utility of RSV to optimize the radiotherapeutic ratio in cancer treatments using radiotherapy should be considered.

Resveratrol exerts dose-dependent anti-fibrotic or pro-fibrotic effects in kidneys: A potential risk to individuals with impaired kidney function

BackgroundRenal fibrosis is the pathological feature of chronic kidney disease (CKD) which leads to end-stage renal disease (ESRD) and renal failure. Resveratrol [3,5,4′-trihydroxy-trans-stilbene (RSV)] has shown benefits for metabolic diseases and anti-cancer therapy, but its potential risk on renal health has not been fully evaluated. PurposeTo investigate the global effects of RSV on renal fibrosis in human tubular epithelial cell (TEC) line HK-2, and in mice with unilateral ureteral obstruction (UUO). MethodsA TGF-β-induced in vitro model of epithelial-mesenchymal transition (EMT) in TEC was established. The effects of RSV on cell viability, pro-fibrotic factors, oxidative stress, mitochondria function, and underlying pathway proteins were analyzed. In vivo, the effects of RSV on renal function and fibrosis were assayed in UUO mice. ResultsOur results showed that low concentrations of RSV (5–20 μM) decreased TGF-β-induced EMT via Sirt1-dependent deacetylation of Smad3/Smad4 mechanism. By contrast, long-term (72 h) exposure to high concentrations of RSV (≥ 40 μM) promoted EMT in HK-2 cells via mitochondrial oxidative stress and ROCK1-mediated disordered cytoskeleton remodeling. In vivo, low-dose treatment of RSV (≤ 25 mg/kg) partly improved renal function, whereas high-dose treatment of RSV (≥ 50 mg/kg) lost its anti-fibrotic role and even aggravated renal fibrosis. However, mice with UUO were more susceptible to high RSV-induced renal injury than normal mice. ConclusionDependent on dose, RSV activated either anti-fibrotic or pro-fibrotic effects in kidneys. The risk of RSV consumption in individuals with impaired kidney function should be carefully considered.

Effects of Resveratrol on IL‐1α and Nitric Oxide Levels in GTM‐3 Cells

ObjectivesResveratrol (RSV), an antioxidant polyphenol found in many fruit and plant products, could potentially have a role in reducing the trabecular meshwork (TM) tissue abnormalities observed in primary open‐angle glaucoma (POAG). The mechanisms by which this occurs are not completely understood. This study was designed to evaluate the effects of RSV on the expression and levels of selected nitric oxide synthase (NOS) enzymes, and nitric oxide (NO), respectively, and the inflammatory biomarker interleukin‐1 alpha (IL‐1 α) in human glaucomatous TM (GTM‐3) cells.MethodsGTM‐3 cells were grown to approximately 90% confluence and analyzed by Western blot analysis for the expression of endothelial and inducible nitric oxide synthases (eNOS; iNOS). GAPDH loading control was used to confirm accurate protein loading. The concentration‐related effects of RSV (0.1 – 100 mM) on IL‐1 α and NO levels were assessed. Values were normalized to protein. Cells were pre‐treated with RSV for 24 hours before analysis. Each experiment was performed at least three times.ResultsDensitometry data showed concentration‐related increases in eNOS protein expression and reduction in expression of iNOS at high RSV concentrations in GTM‐3 cells. Significant increases in eNOS expression were observed to be dependent on RSV concentrations. At 10 μM concentration, an average protein value of 1.33 ± 0.23 was obtained, while at 100 μM an average protein value of 1.47 ± 0.09 was obtained as compared to control (0.76 ± 0.09). Our results indicate a significant decrease in iNOS protein expression at RSV concentration of 100 μM. Our results indicated that NO levels increased with increasing concentrations of RSV. NO levels obtained following RSV treatment (0.1, 1, 10 and 100 μM), were 6 ± 0.7, 7 ± 0.8, 7.3 ± 0.7 and 9.5 ± 1 nM/mg protein, respectively. The average value obtained for the control group was 4.8 ± 0.6 nM/mg protein. Significant increases in IL‐1α levels were observed with increasing concentrations of RSV up to a concentration of 10 μM. There was a decrease in IL‐1 levels from 10 μM to100 μM RSV. The values obtained for concentrations of 0.1 – 100 μM were significantly different when compared to the control.ConclusionsOur results confirmed that resveratrol increases NO levels in glaucomatous TM cells. This increase is possibly due to increased eNOS expression in these cells. Decreased NO production in TM cells have been associated as a risk factor for primary open‐angle glaucoma, and increasing NO production as a result of RSV supports the beneficial effects of this antioxidant in these cells. Studies suggest that iNOS activation is a potential contributor to oxidative stress. Therefore, our results showing a reduction in iNOS expression further support RSV's antioxidant capabilities.Support or Funding InformationThis research was supported by NIH RCMI G12 Grant: 8G12MD007602 and Renovation and Research Core Consolidation Grant number 1G20RR031196This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Addressing the selectivity and toxicity of antiviral nucleosides

Nucleoside and nucleotide analogs have played significant roles in antiviral therapies and are valued for their impressive potency and high barrier to resistance. They have been approved for treatment of herpes simplex virus-1, HIV, HBV, HCV, and influenza, and new drugs are being developed for the treatment of RSV, Ebola, coronavirus MERS, and other emerging viruses. However, this class of compounds has also experienced a high attrition rate in clinical trials due to toxicity. In this review, we discuss the utility of different biochemical and cell-based assays and provide recommendations for assessing toxicity liability before entering animal toxicity studies.

Antitumor activity of resveratrol is independent of Cu(II) complex formation in MCF-7 cell line

Resveratrol (Rsv) is widely reported to possess anticarcinogenic properties in a plethora of cellular and animal models having limited toxicity toward normal cells. In the molecular level, Rsv can act as a suppressive agent for several impaired signaling pathways on cancer cells. However, Fukuhara and Miyata have shown a non-proteic reaction of Rsv, which can act as a prooxidant agent in the presence of copper (Cu), causing cellular oxidative stress accompanied of DNA damage. After this discovery, the complex Rsv-Cu was broadly explored as an antitumor mechanism in multiples tumor cell lines. The aim of the study is to explore the anticarcinogenic behavior of resveratrol–Cu(II) complex in MCF-7 cell line.Selectivity of Rsv binding to Cu ions was analyzed by HPLC and UV–VIS. The cells were enriched with concentrations of 10 and 50µM CuSO4 solution and treated with 25µM of Rsv. Copper uptake after enrichment of cells, as its intracellular distribution in MCF-7 line, was scanned by ICP-MS and TEM-EDS. Cell death and intracellular ROS production were determined by flow cytometry.Different from the extracellular model, no relationship of synergy between Rsv–Cu(II) and reactive oxidative species (ROS) production was detected in vitro. ICP-MS revealed intracellular copper accumulation to both chosen concentrations (0.33±0.09 and 1.18±0.13ppb) but there is no promotion of cell death by Rsv–Cu(II) complex. In addition, significant attenuation of ROS production was detected when cells were exposed to CuSO4 after Rsv treatment, falling from 7.54% of ROS production when treated only with Rsv to 3.07 and 2.72% with CuSO4.Based on these findings antitumor activity of resveratrol when in copper ions presence, is not mediated by Rsv-Cu complex formation in MCF-7 human cell line, suggesting that the antitumoral reaction is dependent of a cancer cellular model.

Use of Oral Ribavirin for the Treatment of RSV Infections in Hematopoietic Cell Transplant (HCT) Recipients

BackgroundThe benefit of aerosolized ribavirin (AR) in reducing the risk of progression of RSV infections and RSV-associated mortality in HCT recipients has been recognized, yet there is a paucity of data assessing the use of oral ribavirin (OR) in this patient population. We evaluated outcomes associated with the use of OR compared with AR in HCT recipients.MethodsRetrospective review of all HCT recipients with RSV infection treated with OR or AR during three RSV seasons (September 2014 – February 2017). An established immunodeficiency Scoring Index (ISI) was applied to identify patients at high risk for progression and death based on host risk factors. Mortality, progression to lower respiratory infection (LRI), and need for ICU admission was compared among recipients of AR and OR.ResultsA total of 107 patients were treated with OR (n = 42, 39%) or AR (n = 65, 61%). Recipients of AR and OR were equally likely to be high-risk by ISI scoring (11% vs. 10%, P = 1.00). Fifty-three patients (50%) presented with upper respiratory infection (URI) of whom 13 (25%) progressed to LRI. There was no difference in the rate of progression to LRI between patients who received AR and OR (28% vs. 19%, P = 0.53). No difference was found in 30-day mortality rates based on treatment strategy (8% AR vs. 5% OR, P = 0.70). Interestingly, 90-day mortality was found to be significantly lower among patients who received OR vs. AR (20% vs. 5%, P = 0.04). No differences in rates of ICU admission and requirement for mechanical ventilation were found between the two groups. For the 99 inpatients at time of diagnosis, median (interquartile range) length of stay was 7 (5 – 19) days, and was similar for patients on either treatment modality. Eight patients were treated for RSV on an outpatient basis and all received OR.ConclusionHCT patients with RSV had similar outcomes when treated with AR and OR. OR may be a safe and effective alternative to AR for prevention and treatment of RSV in HCT patients with significantly reduced cost.Disclosures R. Chemaly, Gilead: Consultant and Investigator, Consulting fee and Research grant. Ansun: Investigator, Research grant. GSK: Investigator, Research grant.

SIRT1 activator ameliorates the renal tubular injury induced by hyperglycemia in vivo and in vitro via inhibiting apoptosis.

We aimed to explore the role of SIRT1 in apoptosis in human kidney proximal tubule epithelial (HK-2) cells, and to determine whether resveratrol (RSV, a SIRT1 activator) could ameliorate apoptosis in rats with streptozotocin-induced diabetes mellitus (DM) and/or in high glucose (HG, 30mM) - stimulated HK-2 cells. Rats were distributed randomly into three groups: 1) control group, 2) DM group, and 3) DM with RSV group (DM+RSV; rats treated with 30mg/kg/d of RSV for 16 weeks). The physical, biochemical, and morphological parameters were then examined. Additionally, the deacetylase activity of SIRT1, and the expression levels of SIRT1 and of representative apoptosis markers, such as p53, acetylated p53, cleaved caspase-3, caspase-9, and cleaved PARP, were measured. HK-2 cells were stimulated by HG for different lengths of time to study the effect of HG on apoptosis. HK-2 cells were treated with or without RSV (25μM) to investigate if RSV has a protective effect on HG-induced apoptosis. A gene-specific small interfering RNA against SIRT1 was used to study the role of SIRT1 in apoptosis. More apoptosis was found in the DM rats than in the control rats. Similarly, the expression levels of cleaved caspase-3, cleaved PARP, and acetylated p53 were significantly higher, and the level of SIRT1 was significantly lower, in the HK-2 cells that were cultured under HG conditions than those in the HK-2 cells that were cultured under low glucose (5.5mM) conditions. Notably, treatment with RSV lessened the HG-induced changes in the levels of apoptosis indicators, and this inhibition of HG-induced apoptosis in HK-2 cells by RSV treatment was abolished by SIRT1 silencing. Our study showed that hyperglycemia contributes to apoptosis in rat kidney and HK-2 cells. SIRT1 activation by RSV can reduce urinary albumin excretion and proximal tubule epithelial apoptosis both in vitro and in vivo. Based on our study, SIRT1/p53 axis played an important role in the hyperglycemia induced apoptosis. These findings indicated that the increased expression of SIRT1, mediated by RSV, is a possible mechanism by which RSV prevents renal tubular injury in diabetic nephropathy (DN). So RSV has great clinical significance and could provide the basis for the new way to effective treatment to contain the morbidity and mortality associated with DN.

Identification of Respiratory Syncytial Virus Nonstructural Protein 2 Residues Essential for Exploitation of the Host Ubiquitin System and Inhibition of Innate Immune Responses.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development. RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly, and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV overattenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance between antigenicity and safety required for an effective vaccine. We coupled bioinformatics analysis with reverse genetics to introduce mutations into RSV's negative-sense genome. In this way we constructed rRSV NS2 ubiquitin mutants that limited NS2's ability to antagonize the innate immune system, thereby attenuating rRSV growth and increasing innate immune responses.

Characterization of the 5'-flanking region of the human DNA helicase B (HELB) gene and its response to trans-Resveratrol.

Human DNA helicase B (HELB/HDHB) regulates DNA replication through association with human DNA polymerase α-primase. In the present study, an 866-base pair (bp) of the 5′-flanking region of the human HELB gene-containing Luciferase (Luc) reporter plasmid, pHDHB-Luc was transfected into various cell lines and Luc activity was analyzed. Deletion analyses revealed that a 121-bp containing the major transcription start site (TSS) was essential for the basal promoter activity in all tested cells. TF-SEARCH analysis indicated that GC-box/Sp1 and duplicated GGAA-motifs containing putative STAT-x and c-ETS binding sites are located close to the TSS. Furthermore, chromatin immunoprecipitation (ChIP) analysis showed that PU.1 and Sp1 bind to the 121-bp region. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analyses showed the HELB gene and protein expression was up-regulated by trans-Resveratrol (Rsv) treatment in HeLa S3 cells. Moreover, transfection experiment indicated that mutations on the GC-boxes and the duplicated GGAA-motif greatly reduced promoter activity and the response to Rsv in HeLa S3 cells. These results suggest that Rsv, which is a natural compound that has been found to elongate the lifespan of various organisms, regulates HELB promoter activity through co-operation of the GC-boxes and the duplicated GGAA-motif in the 121-bp.

A Randomized Placebo Controlled Trial of Ibuprofen for Respiratory Syncytial Virus Infection in a Bovine Model.

BackgroundRespiratory syncytial virus (RSV) is the most common cause of bronchiolitis and hospital admission in infants. An analogous disease occurs in cattle and costs US agriculture a billion dollars a year. RSV causes much of its morbidity indirectly via adverse effects of the host response to the virus. RSV is accompanied by elevated prostaglandin E2 (PGE2) which is followed by neutrophil led inflammation in the lung. Ibuprofen is a prototypical non-steroidal anti-inflammatory drug that decreases PGE2 levels by inhibiting cyclooxygenase.HypothesesWe hypothesized that treatment of RSV with ibuprofen would decrease PGE2 levels, modulate the immune response, decrease clinical illness, and decrease the histopathological lung changes in a bovine model of RSV. We further hypothesized that viral replication would be unaffected.MethodsWe performed a randomized placebo controlled trial of ibuprofen in 16 outbred Holstein calves that we infected with RSV. We measured clinical scores, cyclooxygenase, lipoxygenase and endocannabinoid products in plasma and mediastinal lymph nodes and interleukin (Il)-4, Il-13, Il-17 and interferon-γ in mediastinal lymph nodes. RSV shedding was measured daily and nasal Il-6, Il-8 and Il-17 every other day. The calves were necropsied on Day 10 post inoculation and histology performed.ResultsOne calf in the ibuprofen group required euthanasia on Day 8 of infection for respiratory distress. Clinical scores (p<0.01) and weight gain (p = 0.08) seemed better in the ibuprofen group. Ibuprofen decreased cyclooxygenase, lipoxygenase, and cytochrome P450 products, and increased monoacylglycerols in lung lymph nodes. Ibuprofen modulated the immune response as measured by narrowed range of observed Il-13, Il-17 and IFN-γ gene expression in mediastinal lymph nodes. Lung histology was not different between groups, and viral shedding was increased in calves randomized to ibuprofen.ConclusionsIbuprofen decreased PGE2, modulated the immune response, and improved clinical outcomes. However lung histopathology was not affected and viral shedding was increased.

Characterization of the 5'-flanking region of the human TP53 gene and its response to the natural compound, Resveratrol.

Tumour suppressor p53, which is encoded by theTP53gene, is widely known to play an important role in response to DNA damage and various stresses. It has recently been reported that p53 regulates glucose metabolism and that an increase in p53 protein level is induced after serum deprivation or treatments with a natural compound,trans-Resveratrol (Rsv). In this study, we constructed a Luciferase expression vector, pGL4-TP53-551, containing 551 bp of the 5'-upstream region of the humanTP53gene, which was then transfected into HeLa S3 cells. A Luciferase assay showed that Rsv treatment increased the promoter activity of theTP53gene in comparison to that ofPIF1 Detailed deletion and mutation analyses revealed that Nkx-2.5 and E2F-binding elements are required in addition to duplicated GGAA (TTCC), for the regulation ofTP53promoter activity. In this study, it is suggested that the transient induction ofTP53gene expression by Rsv treatment might be partly involved in its anti-aging effect through maintenance of chromosomal DNAs.

Dose-related effects of chronic resveratrol administration on neurogenesis, angiogenesis, and corticosterone secretion are associated with improved spatial memory retention following global cerebral ischemia

Objectives: The polyphenol resveratrol has shown regulatory effects on hippocampal neurogenesis, which according to the neurovascular niche hypothesis, is likely to involve stimulation of angiogenesis. In rodents, global cerebral ischemia leads to selective CA1 neuronal damage, spatial memory impairments, lasting changes in corticosterone (CORT) secretion, and increased neurogenesis. This study examined dose-related effects of 21-day RSV treatment on markers associated with neurogenesis (DCX, PSA-NCAM) and angiogenesis (CD31) in the hippocampus at short (7-day) and long-term (85-day) intervals following global ischemia. In parallel, post-ischemic and stress-induced CORT levels and spatial memory in the Morris water maze were determined.Methods: Five groups of male Wistar rats were included: sham/saline, ischemia/saline, ischemia/1 mg/kg RSV, ischemia/10 mg/kg RSV, and sham/10 mg/kg RSV. Changes in expression of plasticity markers were paralleled by assessment of basal- and stress-induced CORT secretions, and spatial memory performance.Results: Our findings revealed a significant attenuation of ischemia-induced DCX/PSA-NCAM expression in RSV-treated rats, whereas RSV treatment increased angiogenesis in the injured CA1 region. RSV attenuated CORT levels 3 days post-ischemia and a trend toward attenuating CORT secretion in response to 15 minutes restraint stress. Increased swimming latencies in the target quadrant during the MWM probe trial in RSV-treated ischemic rats support beneficial effects of RSV on spatial memory retention.Discussion: Our findings uncover time- and dose-related effects of RSV and global ischemia on the regulation of hippocampal plasticity. Changes in neuro- and angiogenesis are consistent with RSV neuroprotective effects, but appear independent of RSV regulatory effects on corticosterone secretion.