Association of Prehospital Neck Wound Survivability and Battlefield Medical Evacuation Time in Afghanistan.

INTRODUCTION: The U.S. Military's Golden Hour policy led to improved warfighter survivability during the Global War on Terror. The policy's success is well-documented, but a categorical evaluation and stratification of medical evacuation (MEDEVAC) times based on combat injury is lacking. METHODS: We queried the Department of Defense Joint Trauma System Prehospital Trauma Registry for casualties with documented penetrating neck trauma in Afghanistan requiring battlefield MEDEVAC from June 15, 2009, through February 1, 2021. Casualties were excluded if the time from the point of injury to reach higher level medical care was not documented, listed as zero, or exceeded 4 hours. They were also excluded if demographic data were incomplete or deemed unreliable or if their injuries occurred outside of Afghanistan.We designed a logistic regression model to test for associations in survivability, adjusting for composite injury severity score, patient age group, and type of next higher level of care reached. We then used our model to interpolate MEDEVAC times associated with 0.1%, 1%, and 10% increased risk of death for an incapacitated casualty with penetrating neck trauma. RESULTS: Of 1,147 encounters, 444 casualties met inclusion criteria. Of these casualties, 430 (96.9%) survived to discharge. Interpolative analysis of our multivariable logistic regression model showed that MEDEVAC times ≥8 minutes, ≥53 minutes, and ≥196 minutes are associated with a 0.1%, 1%, and 10% increased risk of mortality from baseline, respectively. CONCLUSIONS: Our data characterize the maximum MEDEVAC times associated with 0.1%, 1%, and 10% increased risk of death from baseline survivability for penetrating battlefield neck trauma in Afghanistan.

Market Analysis of Video Laryngoscopy Equipment for the Role 1 Setting.

INTRODUCTION: Airway compromise is the second leading cause of potentially preventable prehospital combat death. Endotracheal intubation (ETI) remains the most common role 1 airway intervention. Video laryngoscopy (VL) is superior to direct laryngoscopy (DL) for first-attempt intubation, especially in less-experienced providers and for trauma patients. The cost has been a major challenge in pushing VL technology far-forward; however, the cost of equipment continues to become more affordable. We conducted a market analysis of VL devices under $10,000 for possible options for role 1. MATERIALS AND METHODS: We searched Google, PubMed, and the Food and Drug Administration database from August 2022 to January 2023 with a combination of several keywords to identify current VL market options under $10,000. After identifying relevant manufacturers, we then reviewed individual manufacturer or distributor websites for pricing data and system specifications. We noted several characteristics regarding VL device design for comparison. These include monitor features, size, modularity, system durability, battery life, and reusability. When necessary, we requested formal price quotes from respective companies. RESULTS: We identified 17 VL options under $10,000 available for purchase, 14 of which were priced below $5,000 for individual units. Infium (n = 3) and Vimed Medical (n = 4) provided the largest number of unique models. VL options under $10,000 exist in both reusable and disposable modalities. These modalities included separate monitors as well as monitors attached to the VL handle. Disposable options, on a per-unit basis, cost less than reusable options. CONCLUSIONS: Several VL options exist within our goal price point in both reusable and disposable options. Clinical studies assessing the technology performance of ETI and deliberate downselection are needed to identify the most cost-effective solution for role 1 dispersion.

Lessons from the Fallen: An After-Action Review of Prehospital Casualty Data during the Global War on Terror.

BACKGROUND: The US military's recent involvement in long standing conflict has caused the pioneering of many lifesaving medical advances, often made possible by data-driven research. However, future advances in battlefield medicine will likely require greater data fidelity than is currently attainable. Continuing to improve survival rates will require data which establishes the relative contributions to preventable mortality and guides future interventions. Prehospital data, particularly that from Tactical Combat Casualty Care (TCCC) Cards and TCCC After Action Reports (TCCC AARs), are notoriously inconsistent in reaching searchable databases for formal evaluation. While the military has begun incorporating more modern technology in advanced data capture over the past few years like the Air Force's Battlefield Assisted Trauma Distributed Observation Kit (BATDOK) and the Army's Medical Hands-free Unified Broadcast system (MEDHUB), more analysis weighing the advantages and disadvantages of substituting analog solutions is needed. DISCUSSION: We propose 3 changes which may aid prehospital data capture and facilitate analysis: reexamine the current format of TCCC Cards and consider reducing the number of available datapoints to streamline completion, implement a military-wide mandate for all Role 1 providers to complete a TCCC AAR within 24 hours of a casualty event, and formalize the process of requesting de-identified data from the Armed Forces Medical Examiner System (AFMES) database. CONCLUSION: Reflecting on the state of US military medicine after 20 years of war, an important focus is improving the way prehospital data is gathered and analyzed by the military. There are steps we can take now to enhance our capabilities.

Prehospital Pharmacotherapy in Moderate and Severe Traumatic Brain Injury: A Systematic Review.

BACKGROUND: Traumatic brain injury (TBI) affects civilian and military populations with high morbidity and mortality rates and devastating sequelae. As the US military shifts its operational paradigm to prepare for future large-scale combat operations, the need for prolonged casualty care is expected to intensify. Identifying efficacious prehospital TBI management strategies is therefore vital. Numerous pharmacotherapies are beneficial in the inpatient management of TBI, including beta blockers, calcium channel blockers, statins, and other agents. However, their utility in prehospital management of moderate or severe TBI is not well understood. We performed a systematic review to elucidate agents of potential prehospital benefit in moderate and severe TBI. METHODS: We searched 6 databases from January 2000 through December 2021 without limitations in outcome metrics using a variety of search terms designed to encapsulate all studies pertaining to prehospital TBI management. We identified 2,142 unique articles, which netted 114 studies for full review. Seven studies met stringent inclusion criteria for our aims. RESULTS: Studies meeting inclusion criteria assessed tranexamic acid (TXA) (n=6) and ethanol (n=1). Of the TXA studies, 3 were randomized controlled trials, 2 were retrospective cohort studies, 1 was a prospective cohort study, and 1 was a meta-analysis. Notably absent were papers investigating therapeutics shown to be beneficial in inpatient hospital treatment of TBI. Overall, data suggest TXA administration is potentially beneficial in moderate or severe TBI with or without intracranial hemorrhage. Severe TBI with or without penetrating trauma was associated with worse overall outcomes, regardless of TXA use. CONCLUSION: Effective interventions for treating moderate or severe TBI are lacking. TXA is the most widely studied pharmacologic intervention and appears to offer some benefit without adverse effects in moderate TBI (with or without intracranial hemorrhage) in the pre-hospital setting despite heterogeneous results. Limitations of these studies include heterogeneity in outcome metrics, patient populations, and circumstances of TXA use. We identified a gap in the literature in translating agents with demonstrated inpatient benefit to the prehospital setting. Further investigation into these and other novel therapeutic options in the prehospital arena is crucial to improving clinical outcomes in TBI.

Blood Product Administration During the Role 1 Phase of Care: The Prehospital Trauma Registry Experience.

BACKGROUND: The majority of combat deaths occur in the prehospital setting. Efforts to increase survival including blood transfusions are made in the prehospital setting. The blood products available in the Role 1 setting include whole blood (WB), red blood cells (RBCs), fresh frozen plasma (FFP), and lyophilized (freeze-dried) plasma (FDP). METHODS: This is a secondary analysis of a previously published dataset within the Prehospital Trauma Registry (PHTR) from 2003 through May 2019. Deterministic linking was used when possible with the DoD Trauma Registry for outcome data. Descriptive statistics were used to analyze the data. RESULTS: We identified 1,357 patient encounters in the PHTR. Within that group, 28 patients received a prehospital blood product, with 41 total administrations: WB (18), RBCs (12), FFP (6), FDP (3), and blood not otherwise specified (2). Outcome data were available for 17 of the 28 patients. The median injury severity score was 20, with the thorax being the most frequent seriously injured body region. Most (94%) patients survived to discharge. The median ICU days was 11 (Interquartile Range [IQR] 3-19), and the median hospital days was 19 (IQR 8-29). The average volume (units) of RBCs was 6.0 (95% CI 1.9-10.1), WB 2.8 (95% CI 0.0-5.6), platelets 0.7 (95% CI 0.0-1.4), and FFP 5.0 (95% CI 1.2-8.8). CONCLUSIONS: The use of prehospital blood products is uncommon in U.S. combat settings. Patients who received blood products sustained severe injuries but had a high survival rate. Given the infrequent but critical use and potentially increased need for adequate prolonged casualty care in future near-peer conflicts, optimizing logistical chain circulation is required.

Characterizing pediatric supermassive transfusion and the contributing injury patterns in the combat environment.

BACKGROUND: Trauma is the leading cause of pediatric mortality in the United States. Often, these patients require supermassive transfusion (SMT), which we define as receipt of >80 mL/kg blood products, double the proposed volume for standard pediatric massive transfusion (MT). Evaluating the blood volumes, injury patterns, clinical findings, and prehospital interventions predictive for SMT are critical to reducing pediatric mortality. We describe the pediatric casualties, injury patterns, and clinical findings that comprise SMT. METHODS: We retrospectively analyzed pediatric trauma data from the Department of Defense Trauma Registry from January 2007-2016. We stratified patients into two cohorts based on blood products received in the first 24 h after injury: 1) those who received 40-80 mL/kg (MT), or 2) those who received >80 mL/kg (SMT). We evaluated demographics, injury patterns, prehospital interventions, and clinical findings. RESULTS: Our original dataset included 3439 pediatric casualties. We identified 536 patients who met inclusion parameters (receipt of ≥40 mL/kg of blood products [whole blood, packed red blood cells, fresh frozen plasma, platelets, or cryoprecipitate]). The MT cohort included 271 patients (50.6%), and the SMT cohort comprised 265 patients (49.4%). Survival to discharge was significantly lower (78% for SMT, 86% for MT; p < 0.011) in the SMT cohort. Multivariable analysis of injury patterns revealed serious injuries (Abbreviated Injury Scale 3-6) to the extremities (OR 2.13, 95% CI 1.45-3.12) and abdomen (OR 1.65, 1.08-2.53) were associated with SMT. Wound dressings (41% versus 29%; p = 0.003), tourniquets (23% vs 12%; p = 0.001), and IO access (17% vs 10%; p = 0.013) were more common in the SMT group. Age-adjusted hypotension was significantly higher in the SMT group (41%, n = 100 vs 23%, n = 59; p < 0.001) with no statistical difference detected in tachycardia (87%, n = 223 vs 87%, n = 228; p = 0.932). CONCLUSIONS: Our research demonstrates that pediatric SMT patients are at increased risk of mortality. Our study highlights the seriousness of extremity injuries in pediatric trauma patients, identifying associations between severe injuries to the extremities and abdomen with the receipt of SMT. Prehospital interventions of wound dressing, tourniquets, and IO access were more frequent in the SMT cohort. Our research determined that hypotension was associated with SMT, but tachycardia was not a reliable predictor of SMT over MT.

A Descriptive Analysis of Battlefield First Responder and Combat Lifesaver Interventions during the Role 1 Phase of Care.

BACKGROUND: Battlefield first responders (BFR) are the first non-medical personnel to render critical lifesaving interventions for combat casualties, especially for massive hemorrhage where rapid control will improve survival. Soldiers receive medical instruction during initial entry training (IET) and unit-dependent medical training, and by attending the Combat Lifesaver (CLS) course. We seek to describe the interventions performed by BFRs on casualties with only BFRs listed in their chain of care within the Prehospital Trauma Registry (PHTR). METHODS: This is a secondary analysis of a dataset from the PHTR from 2003-2019. We excluded encounters with a documented medical officer, medic, or unknown prehospital provider at any time in their chain of care during the Role 1 phase to isolate only casualties with BFR medical care. RESULTS: Of the 1,357 encounters in our initial dataset, we identified 29 casualties that met inclusion criteria. Pressure dressing was the most common intervention (n=12), followed by limb tourniquets (n=4), IV fluids (n=3), hemostatic gauze (n=2), and wound packing (n=2). Bag-valve-masks, chest seals, extremity splints, and nasopharyngeal airways (NPA) were also used (n=1 each). Notably absent were backboards, blizzard blankets, cervical collars, eye shields, pelvic splints, hypothermia kits, chest tubes, supraglottic airways (SGA), intraosseous (I/O) lines, and needle decompression (NDC). CONCLUSIONS: Despite limited training, BFRs employ vital medical skills in the prehospital setting. Our data show that BFRs largely perform medical interventions within the scope of their medical knowledge and training. Better datasets with efficacy and complication data are needed.

Modeling the functional state of the reverse transcriptase of hepatitis B virus and its application to probing drug-protein interaction.

BACKGROUND: Herein, the predicted atomic structures of five representative sequence variants of the reverse transcriptase protein (RT) of hepatitis B virus (HBV), sampled from patients with rapid or slow response to tenofovir disoproxil fumarate (TDF) treatment, have been examined to identify structural variations between them in order to assess structural and functional properties of HBV-RT variants associated with the differential responses to TDF treatment. RESULTS: We utilized a hybrid computational approach to model the atomistic structures of HBV-RT/DNA-RNA/dATP and HBV-RT/DNA-RNA/TFV-DP (tenofovir diphosphate) complexes with the native hybrid DNA-RNA substrate in place. Multi-nanosecond molecular dynamics (MD) simulations of HBV-RT/DNA-RNA/dATP complexes revealed strong coupling of the natural nucleotide substrate, dATP, to the active site of the RT, and the differential involvement of the two putative magnesium cations (Mg(2+)) at the active site, whereby one Mg(2+) directly bridges the interaction between dATP and HBV-RT and the other serves as a coordinator to maintain an optimal configuration of the active site. Solvated interaction energy (SIE) calculated in MD simulations of HBV-RT/DNA-RNA/TFV-DP complexes indicate no differential binding affinity between TFV-DP and HBV-RT variants identified in patients with slow or rapid response to TDF treatment. CONCLUSION: The predicted atomic structures accurately represent functional states of HBV-RT. The equivalent interaction between TFV-DP and each examined HBV-RT variants suggests that binding affinity of TFV-DP to HBV-RT is not associated with delayed viral clearance.

Dysregulation of distal cholesterol biosynthesis in association with relapse and advanced disease in CHC genotype 2 and 3 treated with sofosbuvir and ribavirin.

BACKGROUND & AIMS: Hepatitis C virus (HCV) modulates host lipid metabolism for its replication and lifecycle. Our aims were to assess changes in the serum lipid and distal (post-squalene) cholesterol biosynthesis metabolite profile of HCV genotypes (GT) 2 and 3 patients treated with sofosbuvir+ribavirin. METHODS: Serum samples [baseline, treatment week 12, 4weeks post-treatment] were analyzed for apolipoproteins B and E (apoB/E), total cholesterol, HDL, LDL, and 11 post-squalene sterol metabolites using a GC/MS platform. RESULTS: We selected 127 patients (GT2 n=50, GT3 n=77), 50% cirrhotic patients, and 42% who experienced a virological relapse. At baseline, GT3 patients had lower level of serum lipids, apoB/E, 7-dehydrocholesterol, desmosterol, lathosterol, compared to GT2 (p<0.006). Baseline lathosterol was lower in relapsers with cirrhosis compared to cirrhotic patients with SVR (p=0.003). From baseline to treatment week 12, serum lipids, apoB/E, and key sterol pathway metabolites (7-dehydrocholesterol, desmosterol, lathosterol, lanosterol) increased in GT3. In contrast, in GT2 patients, apoB/E and dihydrolanosterol decreased with viral suppression (p<0.025). At follow-up week 4, cirrhotic SVR patients showed substantially greater increases in apoB and total sterols compared to cirrhotic relapsers regardless of HCV genotype. After adjustment for genotype and gender, baseline lathosterol was independently associated with virologic response (p=0.04). CONCLUSION: HCV GT3 is associated with reduced circulation of lipids involved in the distal cholesterol biosynthesis pathway, resulting in relative hypocholesterolemia. HCV suppression during sofosbuvir+ribavirin restores distal sterol metabolites indicating viral interference with de novo lipogenesis or selective retention by hepatocytes.

Temporal dynamics of inflammatory cytokines/chemokines during sofosbuvir and ribavirin therapy for genotype 2 and 3 hepatitis C infection.

UNLABELLED: The analysis of inflammatory cytokines and chemokines produced during hepatitis C virus (HCV) infection has advanced our understanding of viral-host interactions and identified predictors of treatment response. Administration of interferons (IFNs) made it difficult to interpret biomarkers of immune activation during treatment. Direct-acting antiviral (DAA) regimens without IFN are now being used to treat HCV with excellent efficacy. To gain insight into HCV-host interactions occurring before, during, and after HCV treatment, we performed a case-control study that measured serial plasma levels of IFN-γ-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 beta (MIP-1ß), and interleukin-18 (IL-18) in 131 patients with chronic HCV treated with sofosbuvir (SOF) plus ribavirin (RBV). A linear regression analysis using baseline factors identified strong positive associations between elevated alanine aminotransferase and pretreatment IP-10 and between the presence of cirrhosis and elevated pretreatment IL-18. Mean IP-10, MCP-1, MIP-1ß, and IL-18 levels all decline on therapy, but display different dynamics late in treatment and after cessation of therapy. On treatment, IP-10 and MIP-1ß levels were significantly higher in individuals who achieved sustained virological response (SVR). Logistic regression analyses examining treatment response in all patients demonstrated significant associations between higher baseline MIP-1ß levels and smaller decreases in MIP-1ß early in treatment and SVR. Higher early MIP-1ß levels were also significantly associated with SVR in subsets of patients with cirrhosis and individuals with genotype 3 (GT3) infection, two factors associated with decreased responsiveness to treatment. CONCLUSION: Changes in IP-10 levels mirror HCV RNA, suggesting that IP-10 is an indicator of innate immune viral recognition. MIP-1ß levels remain elevated in GT2/3 patients who achieved SVR, suggesting differential immune activation in those who respond to SOF/RBV therapy and a potential role in predicting treatment responses.

Desiderata for Major Eligibility Criteria in Breast Cancer Clinical Trials.

Use of major eligibility criteria is a popular but unstudied folk practice for improving patient screening efficiency for clinical studies. This mixed-methods research study derived the desiderata for major eligibility criteria in breast cancer clinical trials. We randomly selected thirty interventional breast cancer clinical trials conducted at The New York-Presbyterian Hospital on the Columbia University Medical Center campus to create training (N=20) and testing (N=10) datasets. We utilized the Think-aloud protocol to gauge how clinical researchers identify and use major eligibility criteria to prescreen patients for clinical trials during an audio-recorded interview. A focus group session was held to understand the current prescreening process and investigate how it could be optimized to maximize recruitment rates. Using the grounded theory method, we annotated transcriptions to discover user rationale and desiderata behind major eligibility criteria in breast cancer clinical trials, which were later evaluated in a follow-up survey.

Interferon γ-induced protein 10 kinetics in treatment-naive versus treatment-experienced patients receiving interferon-free therapy for hepatitis C virus infection: implications for the innate immune response.

We measured interferon γ-induced protein 10 (IP-10) levels in 428 patients at baseline, week 1, and week 2 of all-oral treatment for hepatitis C virus (HCV) infection. An increased baseline IP-10 level was associated with a T allele in the IL28B gene, an increased alanine aminotransferase level in treatment-naive but not experienced patients, and an increased body mass index. At week 1, the mean decline in plasma IP-10 levels was the same in treatment-naive and treatment-experienced patients (-49%), whereas during week 2 the mean decline in IP-10 levels in treatment-naive patients (-14%) was significantly larger than in treatment-experienced patients (-2%; P = .0176). IP-10 thus may be a surrogate marker of the rate of intracellular viral replication complex decay.

Genome-wide association study to characterize serum bilirubin elevations in patients with HCV treated with GS-9256, an HCV NS3 serine protease inhibitor.

BACKGROUND: Protease inhibitors for the treatment of HCV can cause mild and reversible elevations of unconjugated bilirubin. We sought to characterize genetic determinants of bilirubin elevations using a genome-wide approach among patients with genotype 1 HCV who received combination therapy that included GS-9256, a novel potent inhibitor of HCV NS3 serine protease, as part of a Phase IIb trial. METHODS: Of the 200 patients sampled, 176 had confirmed European ancestry and were included in the analysis. Infinium HumanOmni5BeadChip (Illumina, Inc., San Diego, CA, USA) was used for genotyping. A categorical analysis of low (grade 0-1) versus high (grade 2-4) bilirubin toxicity grade and a quantitative trait locus mapping of peak bilirubin concentrations was performed. RESULTS: A total of 4,466,809 genetic markers were analysed. No single variant showed a statistically significant association with observed bilirubin elevations in this patient population. In a targeted analysis of single nucleotide polymorphisms in genes known to be involved in bilirubin transport, no significant differences in allele frequency between high and low bilirubin toxicity grade were observed. CONCLUSIONS: These results indicate that risk for bilirubin elevation in patients receiving GS-9256 is unlikely to be strongly influenced by common genetic variants with large effects. The current study cannot rule out a role for common variants of weak effect, or a more complex model, including multiple contributing factors, such as rare variants and as yet unidentified environmental influences.

A small-molecule inhibitor of hepatitis C virus infectivity.

One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.

Mechanistic characterization of GS-9190 (Tegobuvir), a novel nonnucleoside inhibitor of hepatitis C virus NS5B polymerase.

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the ß-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the ß-hairpin in the thumb subdomain.

Selection of clinically relevant protease inhibitor-resistant viruses using the genotype 2a hepatitis C virus infection system.

Treatment of patients infected with hepatitis C virus (HCV) with direct acting antivirals can lead to the emergence of drug-resistant variants that may pose a long-term threat to viral eradication. HCV replicons have been used to select resistance mutations; however, genotype 2a JFH-1-based viruses provide the opportunity to perform resistance selection in a bona fide infection system. In this study, we used a tissue culture-adapted J6/JFH-1 virus to select resistance to the NS3 protease inhibitors BILN-2061 and VX-950. Lunet-CD81 cells were infected with J6/JFH-1 virus and maintained in the presence of inhibitors until high-titer viral supernatant was produced. Viral supernatants were passaged over naive cells at escalating drug concentrations, and the resulting viruses were then characterized. Three NS3 resistance mutations were identified in BILN-2061-resistant viruses: A156G, D168A, and D168V. Interestingly, D168A, D168V, and A156T/V, but not A156G, were selected in parallel using a genotype 2a replicon. For VX-950, the T54A and A156S NS3 resistance mutations were identified in the virus selections, whereas only A156T/V emerged in genotype 2a replicon selections. Of note, VX-950 resistance mutations selected using the 2a virus (T54A and A156S) were also observed during VX-950 clinical studies in genotype 2 patients. We also performed viral fitness evaluations and determined that the mutations selected in the viral system did not confer marked reductions in virus production kinetics or peak titers. Overall, the HCV infection system is an efficient tool for drug resistance selections and has advantages for the rapid identification and characterization of clinically relevant resistance mutations.

Novel mutations in a tissue culture-adapted hepatitis C virus strain improve infectious-virus stability and markedly enhance infection kinetics.

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 × 10(8) 50% tissue culture infective doses (TCID(50))/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.

A feasibility study of bevacizumab plus dose-dense doxorubicin-cyclophosphamide (AC) followed by nanoparticle albumin-bound paclitaxel in early-stage breast cancer.

PURPOSE: Bevacizumab confers benefits in metastatic breast cancer but may be more effective as adjuvant therapy. We evaluated the cardiac safety of bevacizumab plus dose-dense doxorubicin-cyclophosphamide (ddAC) → nanoparticle albumin-bound (nab)-paclitaxel in human epidermal growth factor receptor 2 normal early-stage breast cancer. EXPERIMENTAL DESIGN: Eighty patients with normal left ventricular ejection fraction (LVEF) were enrolled. Bevacizumab was administered for 1 year, concurrently with ddAC → nab-paclitaxel then as a single agent. LVEF was evaluated at months 0, 2, 6, 9, and 18. This regimen was considered safe if fewer than three cardiac events or fewer than two deaths from left ventricular dysfunction occurred. Correlative studies of cardiac troponin (cTn) and plasma renin activity (PRA) were conducted. RESULTS: The median age was 48 years (range, 27-75 years), and baseline LVEF was 68% (53%-82%). After 39 months' median follow-up (5-45 months): median LVEF was 68% (53%-80%) at 2 months (n = 78), 64% (51%-77%) at 6 months (n = 66), 63% (48%-77%) at 9 months (n = 61), and 66% (42%-76%) at 18 months (n = 54). One patient developed symptomatic LV dysfunction at month 15. Common toxicities necessitating treatment discontinuation were hypertension (HTN, 4%), wound-healing complications (4%), and asymptomatic LVEF declines (4%). Neither cTn nor PRA predicted congestive heart failure (CHF) or HTN, respectively. CONCLUSIONS: Bevacizumab with ddAC → nab-paclitaxel had a low rate of cardiac events; cTn and PRA levels are not predictive of CHF or HTN, respectively. The efficacy of bevacizumab as adjuvant treatment will be established in several ongoing phase III trials.

Generation and quantitation of infectious hepatitis C virus derived from cell culture (HCVcc).

The development of robust genotype 1b and genotype 1a hepatitis C virus (HCV) replicon systems has enabled the convenient in vitro study of part of the virus life cycle. This unit describes detailed protocols for generating and measuring infectious HCV, or cell-culture-derived infectious HCV (HCVcc). The HCVcc infectious system has two essential components: (1) cells that are permissive to de novo infection and allow effective replication of the full virus life cycle; and (2) a virus genome that has robust and efficient replication in tissue culture. The assays in this unit are based on protocols designed for Huh-7-derived cell lines that allow robust replication of HCV and are permissive to infection. These protocols are important for the implementation of drug discovery efforts relative to the entire infectious virus life cycle.