David Y. Huang

Integrin αvβ3 is a coreceptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that causes birth defects in newborns and severe disease in immunocompromised individuals. The broad tropism of HCMV infection suggests that it uses multiple receptors. We recently showed that the epidermal growth factor receptor (EGFR) serves as a receptor for HCMV. Here we show that HCMV also uses integrin αvβ3 as a coreceptor. Upon infection, HCMV glycoproteins gB and gH independently bind to EGFR and αvβ3, respectively, to initiate viral entry and signaling. αvβ3 then translocates to lipid rafts where it interacts with EGFR to induce coordinated signaling. The coordination between EGFR and αvβ3 is essential for the early events of HCMV infection, including viral entry, RhoA downregulation, stress-fiber disassembly and viral nuclear trafficking. Our findings support a model in which EGFR and αvβ3 work together as coreceptors for HCMV entry and signaling. This discovery is fundamental to understanding HCMV pathogenesis and developing treatment strategies targeted to viral receptors.

Integrin alphavbeta3 is a coreceptor for human cytomegalovirus.

Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that causes birth defects in newborns and severe disease in immunocompromised individuals. The broad tropism of HCMV infection suggests that it uses multiple receptors. We recently showed that the epidermal growth factor receptor (EGFR) serves as a receptor for HCMV. Here we show that HCMV also uses integrin αvβ3 as a coreceptor. Upon infection, HCMV glycoproteins gB and gH independently bind to EGFR and αvβ3, respectively, to initiate viral entry and signaling. αvβ3 then translocates to lipid rafts where it interacts with EGFR to induce coordinated signaling. The coordination between EGFR and αvβ3 is essential for the early events of HCMV infection, including viral entry, RhoA downregulation, stress-fiber disassembly and viral nuclear trafficking. Our findings support a model in which EGFR and αvβ3 work together as coreceptors for HCMV entry and signaling. This discovery is fundamental to understanding HCMV pathogenesis and developing treatment strategies targeted to viral receptors.

Stem Cells as an Emerging Paradigm in Stroke 3 Enhancing the Development of Clinical Trials

Cell-based therapy continues to grow as a new field to explore investigational treatments for stroke. Leaders from academia and industry convened an inaugural meeting in 2007 with members of the National Institutes of Health and Food and Drug Administration (FDA) to generate consensus-based guidelines on the development of cell therapies for stroke, entitled “Stem Cells as an Emerging Paradigm in Stroke” (STEPS).1 These guidelines focused on preclinical studies that are considered important as part of a development program to support clinical testing of cell therapies. The STEPS meeting also provided recommendations on the conduct of early-stage clinical trials. Given the rapid advances in the field, a second meeting was held in 2009 to update and expand these guidelines, which were published as STEPS 2.2 In December 2011, investigators in academia, industry leaders, and members of the National Institutes of Health and FDA gathered at a third meeting, STEPS 3, to discuss emerging data on the mechanisms of action of cell therapy, the barriers to successful translation from animal models to patients, and the design of current clinical trials for acute and chronic stroke. Since the prior STEPS meeting, there are now several active cell therapy platforms for stroke and other neurological disorders, in stages that range from preclinical to clinical trials, and with sponsors that include industry, the National Institutes of Health, and the California Institute of Regenerative Medicine. As the field continues to progress and as pilot clinical studies are starting to show safety for some cell types, it has become necessary to formulate a new set of guidelines that address topics not covered in prior STEPS publications. Specifically, the current document reflects a compilation of recommendations that focus on more advanced stages of clinical testing, as well as the testing of cell therapies in a broader …

Transcranial Laser Therapy in Acute Stroke Treatment Results of Neurothera Effectiveness and Safety Trial 3, a Phase III Clinical End Point Device Trial

Background and Purpose— On the basis of phase II trials, we considered that transcranial laser therapy could have neuroprotective effects in patients with acute ischemic stroke. Methods— We studied transcranial laser therapy in a double-blind, sham-controlled randomized clinical trial intended to enroll 1000 patients with acute ischemic stroke treated ⩽24 hours after stroke onset and who did not undergo thrombolytic therapy. The primary efficacy measure was the 90-day functional outcome as assessed by the modified Rankin Scale, with hierarchical Bayesian analysis incorporating relevant previous data. Interim analyses were planned after 300 and 600 patients included. Results— The study was terminated on recommendation by the Data Monitoring Committee after a futility analysis of 566 completed patients found no difference in the primary end point (transcranial laser therapy 140/282 [49.6%] versus sham 140/284 [49.3%] for good functional outcome; modified Rankin Scale, 0–2). The results remained stable after inclusion of all 630 randomized patients (adjusted odds ratio, 1.024; 95% confidence interval, 0.705–1.488). Conclusions— Once the results of the interim futility analysis became available, all study support was immediately withdrawn by the capital firms behind PhotoThera, and the company was dissolved. Proper termination of the trial was difficult but was finally achieved through special efforts by former employees of PhotoThera, the CRO Parexel and members of the steering and the safety committees. We conclude that transcranial laser therapy does not have a measurable neuroprotective effect in patients with acute ischemic stroke when applied within 24 hours after stroke onset. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01120301.

Human Cytomegalovirus IE1-72 Protein Interacts with p53 and Inhibits p53-Dependent Transactivation by a Mechanism Different from That of IE2-86 Protein

ABSTRACT Infection of host cells with human cytomegalovirus (HCMV) induces cell cycle dysregulation. Two HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, are promiscuous transactivators that have been implicated in the dysregulatory events. Cellular p53 protein is accumulated to high levels in HCMV-infected cells, but the indicative marker of p53 transcriptional activity, p21, is markedly decreased. Both IE1-72 and IE2-86 were able to transactivate the p53 promoter and interact with p53 protein in DNA-transfected or HCMV-infected cells. HCMV UL84, a multiregulatory protein expressed in early periods of HCMV infection, also interacted with p53. HCMV IE1-72 prevented or disrupted p53 binding to p53-specific DNA sequences, while IE2-86 and/or UL84 enhanced p53 binding and induced supershift of this DNA-protein complex. Both HCMV IE1-72 and IE2-86 were able to inhibit p53-dependent transcriptional activation in plasmid-transfected cells. IE1-72, rather than IE2-86, was found to be responsible for p21 downregulation in HCMV-infected HEL cells. DNA transfection analysis using IE1-72 mutants revealed that exon 2/3 and the zinc finger region of IE1-72 are essential for IE1-72s effect on the repression of p53-dependent transcriptional activation. These data suggest that HCMV IE1-72 and/or IE2-86 transactivates the p53 promoter and induces p53 accumulation, but HCMV IE1-72 represses the p53 transactivation activity by a unique binding hindrance mechanism different from that of IE2-86. Thus, various modes of viral IE proteins and p53 interactions might result in multiple outcomes, such as stimulation of cellular DNA synthesis, cell cycle progression and cell cycle arrest, and prevention of program cell death.

Interactions between Human Cytomegalovirus IE1-72 and Cellular p107: Functional Domains and Mechanisms of Up-Regulation of Cyclin E/cdk2 Kinase Activity

ABSTRACT Previous work has demonstrated that the human cytomegalovirus IE1-72 protein is able to bind to the N terminus of p107, and IE1-72 alone is sufficient for alleviation of p107-mediated cell growth suppression. However, the mechanism of this alleviation is unclear. Here, we show that IE1-72 can alleviate p107 inhibition of cyclin E/cdk2 kinase activity. We cotransfected various IE1-72 and p107 constructs into C33A cells and demonstrated that IE1-72 could activate the kinase activity of cyclin E/cdk2. Conversely, IE2-86 did not activate this activity, suggesting that the interaction between p107 and IE1-72 and the subsequent kinase activation are specific. By the use of a series of deletion and point mutants of IE1-72 and p107, we observed that a mutation of the loop region of helix-loop-helix-turn-helix in exon 3 of IE1-72 as well as a mutation of the leucine zipper-2 region in exon 4 of IE1-72 abolished binding to p107. In addition, these two IE1-72 mutants did not alleviate p107 inhibition of cyclin E/cdk2 kinase activity and also failed to alleviate p107 inhibition of the E2F-responsive promoter. Meanwhile, deletion of the N-terminal aa 1 to 175 of p107 abolished both p107 binding with IE1-72 and p107 inhibition of cyclin E/cdk2 kinase activity. This result confirms that the N-terminus aa 1 to 175 region of p107 is a common region where both IE1-72 protein and cyclin E/cdk2 bind. We propose a mechanism in which binding of IE1-72 to p107 displaces cyclin E/cdk2 from p107. Once released from p107, cyclin E/cdk2 is able to function as an active kinase.

Importance of proper patient selection and endpoint selection in evaluation of new therapies in acute stroke: further analysis of the SENTIS trial.

Background The magnitude of treatment effect in acute stroke depends on several factors, including time from symptom onset (TFSO) to treatment and severity of the initial insult. Objective To report further evaluation of NeuroFlo therapy, focusing on the effect of time and stroke severity. Methods SENTIS was a prospective randomized trial (N=515) comparing standard medical therapy with/without NeuroFlo therapy. For this analysis, we evaluated outcomes in groups of patients based on TFSO and stroke severity: patients randomized <6 h, 6–10 h, and >10 h with mild (NIHSS<8), moderate (8–14), and severe (>14) symptoms at randomization. 90-Day mRS (modified Rankin Scale) scores and stroke-related death rates were compared between treatment groups. Results For patients randomized <6 h TFSO (n=128), the OR for mRS 0–2 was 3.11 (CI 1.30 to 7.46, p=0.011) for treated versus non-treated patients. In patients with disease of moderate severity (NIHSS 8–14, n=214), NeuroFlo-treated patients were more likely to have a good outcome (mRS 0–2; OR=1.84, CI 1.02 to 3.33, p=0.043). The stroke-related death rate was better in the treated group with TFSO >10 h and NIHSS >14 (n=42) (OR=7.10, CI 1.13 to 44.55, p=0.036). Conclusions The results of our analysis support the importance of careful selection of outcome measures and the impact that rapid treatment and initial stroke severity have on outcome. Clinical trial registration URL://http.clinicaltrials.gov.Unique identifier: NCT00119717.

Reduced Mortality and Severe Disability Rates in the SENTIS Trial

BACKGROUND AND PURPOSE: The Safety and Efficacy of NeuroFlo Technology in Ischemic Stroke trial showed a trend for reduced all-cause mortality and positive secondary safety end point outcomes. We present further analyses of the mortality and severe disability data from the Safety and Efficacy of NeuroFlo Technology in Ischemic Stroke trial. MATERIALS AND METHODS: The Safety and Efficacy of NeuroFlo Technology in Ischemic Stroke trial was a multicenter, randomized, controlled trial that evaluated the safety and effectiveness of the NeuroFlo catheter in patients with stroke. The current analysis was performed on the as-treated population. All-cause and stroke-related mortality rates at 90 days were compared between groups, and logistic regression models were fit to obtain ORs and 95% CIs for the treated versus not-treated groups. We categorized death-associated serious adverse events as neurologic versus non-neurologic events and performed multiple logistic regression analyses. We analyzed severe disability and mortality by outcomes of the mRS. Patient allocation was gathered by use of a poststudy survey. RESULTS: All-cause mortality trended in favor of treated patients (11.5% versus 16.1%; P = .079) and stroke-related mortality was significantly reduced in treated patients (7.5% versus 14.2%; P = .009). Logistic regression analysis for freedom from stroke-related mortality favored treatment (OR, 2.41; 95% CI, 1.22, 4.77; P = .012). Treated patients had numerically fewer neurologic causes of stroke-related deaths (52.9% versus 73.0%; P = .214). Among the 90-day survivors, nominally fewer treated patients were severely disabled (mRS 5) (5.6% versus 7.5%; OR, 1.72; 95% CI, 0.72, 4.14; P = .223). Differences in allocation of care did not account for the reduced mortality rates. CONCLUSIONS: There were consistent reductions in all-cause and stroke-related mortality in the NeuroFlo-treated patients. This reduction in mortality did not result in an increase in severe disability.

CHANCE Trial: Early Short-Term Dual Antiplatelet Treatment for Stroke Prevention

The Clopidogrel in High-Risk Patients with Acute Nondisabling Cerebrovascular Events (CHANCE) trial tested the effect of early dual antiplatelet treatment for the prevention of secondary stroke within the first 90 days following a qualifying transient ischemic attack (TIA) or minor stroke in 5170 Chinese patients. The trial showed a significant reduction in secondary stroke in patients treated with dual antiplatelet therapy (hazard ratio, 0.68; 95% confidence interval, 0.57–0.81; P <0.001). There was no difference in the rate of moderate or severe hemorrhages (0.3% in both groups; P =0.73).1 CHANCE is critical support for early-onset and short-term intervention of antiplatelet therapy in patients with TIAs and mild strokes. The trial builds on knowledge gained during the past decade, beginning with observations that the risks of stroke early after a TIA or minor stroke are substantial,2–4 with a 2-day risk of stroke following TIA of ≈5%.2,4 Greater attention has since been paid to early evaluation, identification, and treatment of modifiable risk factors and the initiation of antiplatelet treatment for noncardioembolic events within 48 hours of the initial event.5 Previous lysis and recanalization studies have shown the importance of immediate medical management following ischemic events. More recently, studies have addressed the use of antiplatelet agents in an earlier time window, seeking to address the period of highest stroke recurrence risk. The Fast Assessment of Stroke and Transient Ischaemic Attack to Prevent Early Recurrence (FASTER) pilot trial randomized 392 patients with TIA/stroke presenting within 24 hours to aspirin plus clopidogrel or aspirin plus placebo. There was a reduction of stroke within 90 days seen with dual therapy (absolute risk reduction, 3.8%; 95% confidence interval, −9.4 to 1.9; P =0.19) and no significant difference in bleeding events between treatment arms.5 …

Strategies for improving outcomes in the acute management of ischemic stroke in rural emergency departments: a quality improvement initiative in the Stroke Belt

Background The timely evaluation and initiation of treatment for acute ischemic stroke (AIS) is critical to optimal patient outcomes. However, clinical practice often falls short of guideline-established goals. Hospitals in rural regions of the USA, and notably those in the Stroke Belt, are particularly challenged to meet timing goals since the vast majority of primary stroke centers (PSCs) are concentrated in urban academic institutions. Methods Between May 2015 and May 2017, emergency department (ED) teams from 5 non-PSC hospitals in the Stroke Belt participated in a quality improvement (QI) initiative. The intervention included a baseline practice assessment survey, repeat audit-and-feedback cycles with patient data on AIS treatment timing, personalized Continuing Medical Education/Continuing Education-certified grand rounds sessions at each participating site with expert study faculty, targeted reinforcement of best practices, and follow-up to evaluate the benefits and limitations of the intervention. Results At the start of the initiative, clinical staff from participating EDs overestimated the proportion of patients with AIS who received alteplase within the guideline-recommended 60-minute door-to-needle window at their facility. At the end of the 6-month intervention period, significantly more patients were treated with alteplase within 60 minutes of ED arrival compared to baseline across the entire sample (1.9% of patients at baseline vs. 5.2% at 6 months; P < 0.01). Similarly, there was a trend toward a decrease in the percentage of patients whose alteplase treatment was initiated more than 60 minutes after their arrival at the ED (67.3% at baseline vs. 22.2% at 6 months). Conclusion Structured QI interventions that engage ED care teams to reflect on processes related to AIS diagnosis and treatment and deploy repeat audit-and-feedback cycles with real-time patient data have the potential to support an increase in the number of patients who receive alteplase within the guideline-recommended timeframe of 60 minutes from hospital arrival.