The use of replication-defective adenoviruses (RDAd) for human gene therapy has been limited by host immune responses that result in transient recombinant gene expression in vivo. It remained unclear whether these immune responses were directed predominantly against viral proteins or, alternatively, against foreign transgene-encoded proteins. In this report, we have compared the stability of recombinant gene expression in adult immunocompetent mice following intramuscular (i.m.) injection with identical RDAd encoding self (murine) or foreign (human) erythropoietin. Our results demonstrate that immune responses direct against foreign transgene-encoded proteins are the major determinants of the stability of gene expression following i.m. injection of RDAd. Moreover, we demonstrate long-term recombinant gene expression in immunocompetent animals following a single i.m. injection of RDAd encoding a self protein. These findings are important for the design of future preclinical and clinical gene therapy trials.

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Erythropoietin (Epo)-responsive anemia is a common and debilitating complication ofchronic renal failure and human immunodeficiency virus infection. Current therapy for this condition involves repeated intravenous or subcutaneous injections of recombinant Epo. In this report, we describe the development of a novel muscle-based gene transfer approach that produces long-term expression of physiologically significant levels of Epo in the systemic circulation of mice. We have constructed a plasmid expression vector, pVRmEpo, that contains the murine Epo cDNA under the transcriptional control of the cytomegalovirus immediate early (CMV-IE) promoter, the CMV-IE 5' untranslated region, and intron A. A single intramuscular (i.m.) injection of as little as 10 ,ug of this plasmid into immunocompetent adult mice produced physiologically significant elevations in serum Epo levels and increased hematocrits from preinjection levels of 48 ± 0.4% to levels of 64 ± 3.3% 45 days after injection. Hematocrits in these animals remained elevated at greater than 60% for at least 90 days after a single i.m. injection of 10 ,ug of pVRmEpo. We observed a dose-response relationship between the amount of plasmid DNA injected and subsequent 10876The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Objective: The current national opioid epidemic is a public health emergency. We have identified an outbreak of exaggerated opioid toxicity caused by fentanyl adulterated tablets purchased on the street as hydrocodone/ acetaminophen.Methods: Over an 8-day period in late March 2016, a total of 18 patients presented to our institution with exaggerated opioid toxicity. The patients provided a similar history: ingesting their "normal dose" of hydrocodone/ acetaminophen tablets but with more pronounced symptoms. Toxicology testing and analysis was performed on serum, urine, and surrendered pills.Results: One of the 18 patients died in hospital. Five patients underwent cardiopulmonary resuscitation, one required extracorporeal life support, three required intubation, and two received bag-valve-mask ventilation. One patient had recurrence of toxicity after 8 hours after naloxone discontinuation. Seventeen of 18 patients required boluses of naloxone, and four required prolonged naloxone infusions (26-39 hours). All 18 patients tested positive for fentanyl in the serum. Quantitative assays conducted in 13 of the sera revealed fentanyl concentrations of 7.9 to 162 ng/mL (mean = 52.9 ng/mL). Pill analysis revealed fentanyl amounts of 600-6,900 lg/pill. The pills are virtually indistinguishable from authentic hydrocodone/acetaminophen tablets and are similar in weight. To date, our county has reported 56 cases of fentanyl opioid toxicity, with 15 fatalities. In our institution, the outbreak has stressed the capabilities and resources of the emergency department and intensive care units. Conclusions:A serious outbreak of exaggerated opioid toxicity caused by fentanyl-adulterated tablets purchased on the street as hydrocodone/acetaminophen is under way in California. These patients required higher dosing and prolonged infusions of naloxone. Additionally, observation periods off naloxone were extended due to delayed, recurrent toxicity. The outbreak has serious ramifications for public health and safety, law enforcement, and healthcare facilities and resources.