Heme oxygenase-1 regulates the immune response to influenza virus infection and vaccination in aged mice.

Underlying mechanisms of individual variation in severity of influenza infection and response to vaccination are poorly understood. We investigated the effect of reduced heme oxygenase-1 (HO-1) expression on vaccine response and outcome of influenza infection. HO-1-deficient and wild-type (WT) mice (kingdom, Animalia; phylum, Chordata; genus/species, Mus musculus) were infected with influenza virus A/PR/8/34 with or without prior vaccination with an adenoviral-based influenza vaccine. A genome-wide association study evaluated the expression of single-nucleotide polymorphisms (SNPs) in the HO-1 gene and the response to influenza vaccination in healthy humans. HO-1-deficient mice had decreased survival after influenza infection compared to WT mice (median survival 5.5 vs. 6.5 d, P=0.016). HO-1-deficient mice had impaired production of antibody following influenza vaccination compared to WT mice (mean antibody titer 869 vs. 1698, P=0.02). One SNP in HO-1 and one SNP in the constitutively expressed isoform HO-2 were independently associated with decreased antibody production after influenza vaccination in healthy human volunteers (P=0.017 and 0.014, respectively). HO-1 deficient mice were paired with sex- and age-matched WT controls. HO-1 affects the immune response to both influenza infection and vaccination, suggesting that therapeutic induction of HO-1 expression may represent a novel adjuvant to enhance influenza vaccine effectiveness.

Generation of a Kupffer cell-evading adenovirus for systemic and liver-directed gene transfer.

As much as 90% of an intravenously (i.v.) injected dose of adenovirus serotype 5 (Ad5) is absorbed and destroyed by liver Kupffer cells. Viruses that escape these cells can then transduce hepatocytes after binding factor X (FX). Given that interactions with FX and Kupffer cells are thought to occur on the Ad5 hexon protein, we replaced its exposed hypervariable regions (HVR) with those from Ad6. When tested in vivo in BALB/c mice and in hamsters, the Ad5/6 chimera mediated >10 times higher transduction in the liver. This effect was not due to changes in FX binding. Rather, Ad5/6 appeared to escape Kupffer cell uptake as evidenced by producing no Kupffer cell death in vivo, not requiring predosing in vivo, and being phagocytosed less efficiently by macrophages in vitro compared to Ad5. When tested as a helper-dependent adenovirus (Ad) vector, Ad5/6 mediated higher luciferase and factor IX transgene expression than either helper-dependent adenoviral 5 (HD-Ad5) or HD-Ad6 vectors. These data suggest that the Ad5/6 hexon-chimera evades Kupffer cells and may have utility for systemic and liver-directed therapies.

Prescription opioid misuse among university students: A systematic review.

Misuse of prescription opioids has substantially increased in the past decade among the general population, including among university students. Relative to the literature concerning opioid misuse among the general population, little information is available regarding the college student population. Objective: The purpose of the present study was to conduct a systematic review of the literature concerning the prevalence of prescription opioid misuse among the university student population. Results: The lifetime estimate for prescription opioid misuse among general populations of students ranged from 4% to 19.7%, with higher estimates for special student populations. Students most at risk for misuse of prescription opioids are those who report higher rates of psychological distress, depression, and suicidal thoughts and behaviors, and white, male students who use alcohol and illicit drugs. Conclusions: Findings from this study underscore the need for opioid prevention and intervention programs on university campuses.

Expanded anticancer therapeutic window of hexon-modified oncolytic adenovirus.

One of the significant hurdles toward safe and efficacious systemic treatment of cancer with oncolytic adenoviruses (Ads) is dose-limiting hepatotoxicity that prevents the increase of a therapeutic dose. In this study, we expanded the therapeutic window of oncolytic serotype 5 Ad (Ad5) by a genetic modification of hypervariable loop 5 (HVR5) in the capsid protein hexon that prevented infection of hepatocytes due to ablation of binding to blood factors. This oncolytic virus, Ad-GL-HB, had significantly reduced levels of hepatocyte transduction in immunocompetent and immunodeficient mice as compared to parental virus Ad-GL. The hepatocyte detargeting decreased liver damage and increased the maximum tolerated dose of Ad-GL-HB tenfold relative to that of Ad-GL. Intravenous (i.v.) injection of Ad-GL or Ad-GL-HB into tumor-bearing mice produced equally increased survival rates demonstrating that while Ad-GL-HB detargeted hepatocytes, it sustained tumor cell infection after systemic administration. The significantly improved safety of the virus allowed it to be used at increased doses for improved systemic antitumor efficacy. Our results suggest that hexon modifications provide valuable strategies for systemic oncolytic Ad therapy.

Importing a change in diet: the proposed food safety law of 2010 and the possible impact on importers and international trade.

The current combination of widespread consumer alarm about foodborne illness outbreaks and industry concern about profitability has encouraged Congress, for the first time in many years, to consider major food safety reform. The House of Representatives has already passed its version of reform, the Food Safety Enhancement Act of 2009. The Senate appears ready to pass its bill, the FDA Food Safety Modernization Act. Both bills will subject firms in the food industry to a number of new requirements and will considerably increase Food and Drug Administration's (FDA's) enforcement authority. This article addresses how the passage of major food safety reform in 2010 will potentially affect food importation into the United States, by using the Food Safety Enhancement Act of 2009, the bill passed in the House, as a model for what food safety reform will entail. Under the bill, food facilities and importers will have to register yearly with FDA and pay a fee. Customs brokers will also have to register with FDA. FDA will have the authority to subject certain foods to a certification requirement for obtaining entry into the United States. Food facilities will be required to evaluate hazards and implement preventive controls and food safety plans. FDA will establish mandatory performance standards and produce standards. Specific foods identified by FDA will be subject to traceability requirements. FDA will follow a mandatory risk-based inspection schedule, will have far greater access to records, and will have the authority to enforce mandatory recalls. U.S. trading partners may take issue with the substantial burdens placed on those importing food into the United States and may consider bringing a challenge against the United States claiming that the new food safety legislation violates World Trade Organization obligations.

The Protein Chaperone ClpX Targets Native and Non-native Aggregated Substrates for Remodeling, Disassembly, and Degradation with ClpP.

ClpX is a member of the Clp/Hsp100 family of ATP-dependent chaperones and partners with ClpP, a compartmentalized protease, to degrade protein substrates bearing specific recognition signals. ClpX targets specific proteins for degradation directly or with substrate-specific adaptor proteins. Native substrates of ClpXP include proteins that form large oligomeric assemblies, such as MuA, FtsZ, and Dps in Escherichia coli. To remodel large oligomeric substrates, ClpX utilizes multivalent targeting strategies and discriminates between assembled and unassembled substrate conformations. Although ClpX and ClpP are known to associate with protein aggregates in E. coli, a potential role for ClpXP in disaggregation remains poorly characterized. Here, we discuss strategies utilized by ClpX to recognize native and non-native protein aggregates and the mechanisms by which ClpX alone, and with ClpP, remodels the conformations of various aggregates. We show that ClpX promotes the disassembly and reactivation of aggregated Gfp-ssrA through specific substrate remodeling. In the presence of ClpP, ClpX promotes disassembly and degradation of aggregated substrates bearing specific ClpX recognition signals, including heat-aggregated Gfp-ssrA, as well as polymeric and heat-aggregated FtsZ, which is a native ClpXP substrate in E. coli. Finally, we show that ClpX is present in insoluble aggregates and prevents the accumulation of thermal FtsZ aggregates in vivo, suggesting that ClpXP participates in the management of aggregates bearing ClpX recognition signals.

Imaging luciferase-expressing viruses.

Optical imaging of luciferage gene expression has become a powerful tool to track cells and viruses in vivo in small animal models. Luciferase imaging has been used to study the location of infection by replication-defective and replication-competent viruses and to track changes in the distribution of viruses in mouse models. This approach has also been used in oncolytic studies as a noninvasive means to monitor the growth and killing of tumor cells modified with luciferase genes. In this chapter, we describe the techniques used for luciferase imaging as have been applied to track replication-defective and replication-competent adenoviruses in mouse and hamster models of oncolysis and virus pharmacology. Although these methods are simple, the process of obtaining accurate luciferase imaging data has many caveats that are discussed.

The effects of body mass on cremation weight.

Cremains have become increasingly frequent in forensic contexts, while higher body mass in the general population has simultaneously made cremation a more cost-effective mortuary practice. This study analyzed the relationship between body mass and bone mass, as reflected through cremation weight. Antemortem data were recorded for samples used in the multi-regional data set. Each was rendered through commercial crematoriums and reweighed postincineration. Pearson's correlation demonstrates clear association between body mass and cremation weight (r=0.56; p<0.0001). However, multiple linear regression revealed sex and age variables also have a significant relationship (t=7.198; t=-2.5, respectively). Regressed in conjunction, body mass, sex, and age contribute approximately 67% of all variation observed in cremation weight (r=0.668). Analysis of covariance indicates significant regional variation in body and cremation weight. Explanations include bone modification resulting from increased loading stress, as well as glucose intolerance and altered metabolic pathways related to obesity.

Real-time dynamic imaging of virus distribution in vivo.

The distribution of viruses and gene therapy vectors is difficult to assess in a living organism. For instance, trafficking in murine models can usually only be assessed after sacrificing the animal for tissue sectioning or extraction. These assays are laborious requiring whole animal sectioning to ascertain tissue localization. They also obviate the ability to perform longitudinal or kinetic studies in one animal. To track viruses after systemic infection, we have labeled adenoviruses with a near-infrared (NIR) fluorophore and imaged these after intravenous injection in mice. Imaging was able to track and quantitate virus particles entering the jugular vein simultaneous with injection, appearing in the heart within 500 milliseconds, distributing in the bloodstream and throughout the animal within 7 seconds, and that the bulk of virus distribution was essentially complete within 3 minutes. These data provide the first in vivo real-time tracking of the rapid initial events of systemic virus infection.

Comparison of adenoviruses as oncolytics and cancer vaccines in an immunocompetent B cell lymphoma model.

We have screened human adenoviruses (Ads) for oncolytic activity against a variety of mouse and hamster cell lines and have found a number that are susceptible to a variety of Ad serotypes. A20 lymphoma is derived from BALB/c mice and is susceptible to infection and killing by a variety of human Ads. A20 is also a suitable cancer vaccine model, because these cells express a unique immunoglobulin variable region that can be targeted by vaccination. To compare Ads as cancer vaccines versus Ads as oncolytics, A20 tumors were initiated in immunocompetent BALB/c mice. Mice immunized with first-generation Ad5 expressing the A20 immunoglobulin ScFv immunogen (Ad-A20) were protected against A20 lymphomas only when the vaccine was delivered before tumor. In contrast, vaccination after tumor initiation failed to increase survival or delay tumor growth. When Ad serotypes from species B, C, D, and E were tested as oncolytics in vitro, A20 cells were most efficiently killed by species D Ads, with intermediate activity by species B Ads. When tested in vivo in immunocompetent BALB/c mice bearing A20 tumors, single intratumoral injection of species D Ad26 and Ad48 were effective at controlling tumor growth. These data demonstrate that in this immunocompetent mouse cancer model, the oncolytic activity of adenoviruses is more potent than their use as a cancer vaccine. These data in immunocompetent mice lend further support to species D Ads as promising oncolytic viruses against B cell cancers.

Species D adenoviruses as oncolytics against B-cell cancers.

PURPOSE: Oncolytic viruses are self-amplifying anticancer agents that make use of the natural ability of viruses to kill cells. Adenovirus serotype 5 (Ad5) has been extensively tested against solid cancers, but less so against B-cell cancers because these cells do not generally express the coxsackie and adenoviral receptor (CAR). To determine whether other adenoviruses might have better potency, we "mined" the adenovirus virome of 55 serotypes for viruses that could kill B-cell cancers. EXPERIMENTAL DESIGN: Fifteen adenoviruses selected to represent Ad species B, C, D, E, and F were tested in vitro against cell lines and primary patient B-cell cancers for their ability to infect, replicate in, and kill these cells. Select viruses were also tested against B-cell cancer xenografts in immunodeficient mice. RESULTS: Species D adenoviruses mediated most robust killing against a range of B-cell cancer cell lines, against primary patient marginal zone lymphoma cells, and against primary patient CD138+ myeloma cells in vitro. When injected into xenografts in vivo, single treatment with select species D viruses Ad26 and Ad45 delayed lymphoma growth. CONCLUSIONS: Relatively unstudied species D adenoviruses have a unique ability to infect and replicate in B-cell cancers as compared with other adenovirus species. These data suggest these viruses have unique biology in B cells and support translation of novel species D adenoviruses as oncolytics against B-cell cancers.

Targeting adenoviruses with factor x-single-chain antibody fusion proteins.

Abstract It has been shown that blood clotting factors, including factor X (FX), bind to the adenovirus serotype 5 (Ad5) hexon protein and target the virus to liver hepatocytes after intravenous injection. These factors bind to hexon via their conserved vitamin K-dependent gamma-carboxyglutamic acid (GLA) domains with subnanomolar affinity. In this work, we have used this strong interaction to retarget Ad to new receptors, using the GLA domain of FX fused to single-chain antibody variable fragment (ScFv). We demonstrate that fusion of the GLA domain of human FX to receptor-specific ScFvs will target Ad5 vectors to cells expressing these receptors. Fusion of an alphaHer2 ScFv to GLA increased in vitro transduction of Her2-positive versus Her2-negative cells when compared with untargeted virus. Similar results were obtained with ScFvs against the epidermal growth factor receptor (EGFR) and against the stem cell marker ATP-binding cassette protein G2 (ABCG2). Direct expression of GLA fusion protein from replication-defective or replication-competent Ad increased infection and killing of cancer cells in vitro and in vivo. These data demonstrate the potential of using GLA domains to bridge secreted ligands with intracellularly produced Ad5 vectors for vector targeting.

Characterization of human adenovirus serotypes 5, 6, 11, and 35 as anticancer agents.

Human adenovirus type 5 (Ad5) has been the most popular platform for the development of oncolytic Ads. Alternative Ad serotypes with low seroprevalence might allow for improved anticancer efficacy in Ad5-immune patients. We studied the safety and efficacy of rare serotypes Ad6, Ad11 and Ad35. In vitro cytotoxicity of the Ads correlated with expression of CAR and CD46 in most but not all cell lines. Among CAR-binding viruses, Ad5 was often more active than Ad6, among CD46-binding viruses Ad35 was generally more cytotoxic than Ad11 in cell culture studies. Ad5, Ad6, and Ad11 demonstrated similar anticancer activity in vivo, whereas Ad35 was not efficacious. Hepatotoxicity developed only in Ad5-injected mice. Predosing with Ad11 and Ad35 did not increase infection of hepatocytes with Ad5-based vector demonstrating different interaction of these Ads with Kupffer cells. Data obtained in this study suggest developing Ad6 and Ad11 as alternative Ads for anticancer treatment.

Chemical modification with high molecular weight polyethylene glycol reduces transduction of hepatocytes and increases efficacy of intravenously delivered oncolytic adenovirus.

Oncolytic adenoviruses are anticancer agents that replicate within tumors and spread to uninfected tumor cells, amplifying the anticancer effect of initial transduction. We tested whether coating the viral particle with polyethylene glycol (PEG) could reduce transduction of hepatocytes and hepatotoxicity after systemic (intravenous) administration of oncolytic adenovirus serotype 5 (Ad5). Conjugating Ad5 with high molecular weight 20-kDa PEG but not with 5-kDa PEG reduced hepatocyte transduction and hepatotoxicity after intravenous injection. PEGylation with 20-kDa PEG was as efficient at detargeting adenovirus from Kupffer cells and hepatocytes as virus predosing and warfarin. Bioluminescence imaging of virus distribution in two xenograft tumor models in nude mice demonstrated that PEGylation with 20-kDa PEG reduced liver infection 19- to 90-fold. Tumor transduction levels were similar for vectors PEGylated with 20-kDa PEG and unPEGylated vectors. Anticancer efficacy after a single intravenous injection was retained at the level of unmodified vector in large established prostate carcinoma xenografts, resulting in complete elimination of tumors in all animals and long-term tumor-free survival. Anticancer efficacy after a single intravenous injection was increased in large established hepatocellular carcinoma xenografts, resulting in significant prolongation of survival as compared with unmodified vector. The increase in efficacy was comparable to that obtained with predosing and warfarin pretreatment, significantly extending the median of survival. Shielding adenovirus with 20-kDa PEG may be a useful approach to improve the therapeutic window of oncolytic adenovirus after systemic delivery to primary and metastatic tumor sites.