Farhaan Vahidy

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 …

Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System

BACKGROUND The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohns disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times. METHODS We evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials. RESULTS After only two passages, we were able to expand a mean of 6.6 × 10(8) MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10(8) cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. CONCLUSIONS The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.

Systemic thrombolysis in patients with acute ischemic stroke and Internal Carotid ARtery Occlusion: the ICARO study

Background and Purpose— The beneficial effect of intravenous thrombolytic therapy in patients with acute ischemic stroke attributable to internal carotid artery (ICA) occlusion remains unclear. The aim of this study was to evaluate the efficacy and safety of intravenous recombinant tissue-type plasminogen activator in these patients. Methods— ICARO was a case-control multicenter study on prospectively collected data. Patients with acute ischemic stroke and ICA occlusion treated with intravenous recombinant tissue-type plasminogen activator within 4.5 hours from symptom onset (cases) were compared to matched patients with acute stroke and ICA occlusion not treated with recombinant tissue-type plasminogen activator (controls). Cases and controls were matched for age, gender, and stroke severity. The efficacy outcome was disability at 90 days assessed by the modified Rankin Scale, dichotomized as favorable (score of 0–2) or unfavorable (score of 3–6). Safety outcomes were death and any intracranial bleeding. Results— Included in the analysis were 253 cases and 253 controls. Seventy-three cases (28.9%) had a favorable outcome as compared with 52 controls (20.6%; adjusted odds ratio (OR), 1.80; 95% confidence interval [CI], 1.03–3.15; P=0.037). A total of 104 patients died, 65 cases (25.7%) and 39 controls (15.4%; adjusted OR, 2.28; 95% CI, 1.36–3.22; P=0.001). There were more fatal bleedings (2.8% versus 0.4%; OR, 7.17; 95% CI, 0.87–58.71; P=0.068) in the cases than in the controls. Conclusions— In patients with stroke attributable to ICA occlusion, thrombolytic therapy results in a significant reduction in the proportion of patients dependent in activities of daily living. Increases in death and any intracranial bleeding were the trade-offs for this clinical benefit.

Changes in spleen size in patients with acute ischemic stroke: a pilot observational study.

Background In animal models, the spleen contracts after acute ischemic stroke, followed by release of inflammatory cells leading to secondary brain injury. Aims We aim to characterize splenic responses in patients with acute ischemic stroke. Methods In this prospective observational study, we measured daily spleen sizes with abdominal ultrasound in 30 patients with suspected acute ischemic stroke. Splenic ultrasounds were also performed in 20 healthy individuals. Results A generalized estimating equation, longitudinal regression model for adjusted spleen measurements showed the difference between baseline spleen volume (within six-hours of stroke onset) and the volume at the last measured time point (up to seven-days) to be statistically significant (volume difference of 51·9 cm3, P = 0·04). Healthy controls had significantly smaller day-to-day variations; the maximum observed difference in mean spleen volume between any two time points was 9·5 cm3, with the average change over the period of observation being 1·24 cm3. A statistically significant negative association was also observed between the pattern of change of total white blood cell count and spleen volume (P = 0·01). An analysis of individual cases demonstrated possible associations between daily spleen volume changes and clinical course. Conclusions We hypothesize that the spleen may initially contract after ischemic stroke followed by a re-expansion and that it contributes to ischemic brain injury mediated via cellular components. Characterization of the splenic response after stroke and its contribution to cerebral ischemic injury has the potential to provide new opportunities for the development of novel stroke therapies.

Prehospital Utility of Rapid Stroke Evaluation Using In-Ambulance Telemedicine: A Pilot Feasibility Study

Background and Purpose— Prehospital evaluation using telemedicine may accelerate acute stroke treatment with tissue-type plasminogen activator. We explored the feasibility and reliability of using telemedicine in the field and ambulance to help evaluate acute stroke patients. Methods— Ten unique, scripted stroke scenarios, each conducted 4 times, were portrayed by trained actors retrieved and transported by Houston Fire Department emergency medical technicians to our stroke center. The vascular neurologists performed remote assessments in real time, obtaining clinical data points and National Institutes of Health (NIH) Stroke Scale, using the In-Touch RP-Xpress telemedicine device. Each scripted scenario was recorded for a subsequent evaluation by a second blinded vascular neurologist. Study feasibility was defined by the ability to conduct 80% of the sessions without major technological limitations. Reliability of video interpretation was defined by a 90% concordance between the data derived during the real-time sessions and those from the scripted scenarios. Results— In 34 of 40 (85%) scenarios, the teleconsultation was conducted without major technical complication. The absolute agreement for intraclass correlation was 0.997 (95% confidence interval, 0.992–0.999) for the NIH Stroke Scale obtained during the real-time sessions and 0.993 (95% confidence interval, 0.975–0.999) for the recorded sessions. Inter-rater agreement using &kgr;-statistics showed that for live-raters, 10 of 15 items on the NIH Stroke Scale showed excellent agreement and 5 of 15 showed moderate agreement. Matching of real-time assessments occurred for 88% (30/34) of NIH Stroke Scale scores by ±2 points and 96% of the clinical information. Conclusions— Mobile telemedicine is reliable and feasible in assessing actors simulating acute stroke in the prehospital setting.

Optimizing Prediction Scores for Poor Outcome After Intra-Arterial Therapy in Anterior Circulation Acute Ischemic Stroke

Background and Purpose— Intra-arterial therapy (IAT) promotes recanalization of large artery occlusions in acute ischemic stroke. Despite high recanalization rates, poor clinical outcomes are common. We attempted to optimize a score that combines clinical and imaging variables to more accurately predict poor outcome after IAT in anterior circulation occlusions. Methods— Patients with acute ischemic stroke undergoing IAT at University of Texas (UT) Houston for large artery occlusions (middle cerebral artery or internal carotid artery) were reviewed. Independent predictors of poor outcome (modified Rankin Scale, 4–6) were studied. External validation was performed on IAT-treated patients at Emory University. Results— A total of 163 patients were identified at UT Houston. Independent predictors of poor outcome (P⩽0.2) were identified as score variables using sensitivity analysis and logistic regression. Houston Intra-Arterial Therapy 2 (HIAT2) score ranges 0 to 10: age (⩽59=0, 60–79=2, ≥80 years=4), glucose (<150=0, ≥150=1), National Institute Health Stroke Scale (⩽10=0, 11–20=1, ≥21=2), the Alberta Stroke Program Early CT Score (8–10=0, ⩽7=3). Patients with HIAT2≥5 were more likely to have poor outcomes at discharge (odds ratio, 6.43; 95% confidence interval, 2.75–15.02; P<0.001). After adjusting for reperfusion (Thrombolysis in Cerebral Infarction score ≥2b) and time from symptom onset to recanalization, HIAT2≥5 remained an independent predictor of poor outcome (odds ratio, 5.88; 95% confidence interval, 1.96–17.64; P=0.02). Results from the cohort of Emory (198 patients) were consistent; patients with HIAT2 score ≥5 had 6× greater odds of poor outcome at discharge and at 90 days. HIAT2 outperformed other previously published predictive scores. Conclusions— The HIAT2 score, which combines clinical and imaging variables, performed better than all previous scores in predicting poor outcome after IAT for anterior circulation large artery occlusions.

Posterior circulation stroke is associated with prolonged door-to-needle time

Background Lack of recognition of early symptoms of acute posterior circulation ischaemic stroke might delay timely diagnosis and treatment with tissue plasminogen activator. Aims and hypothesis We hypothesized that patients with posterior circulation stroke receive delayed thrombolytic treatment in comparison to anterior circulation stroke. We investigated the differences in times to evaluation or treatment between patients with anterior circulation ischaemic stroke and posterior circulation stroke in our aim to understand the barriers that might have caused these delays. Methods A cross-sectional study was conducted using consecutive patients presenting to our tertiary academic centre with acute ischaemic stroke who were treated with intravenous tissue plasminogen activator within 4·5 h from symptom onset. We compared demographics, stroke severity, symptoms and signs, and time intervals among onset, emergency department arrival, emergency department physician evaluation, neurologist evaluation, brain imaging, and tissue plasminogen activator treatment in patients with anterior circulation stroke and posterior circulation stroke. Results Among 252 patients treated with intravenous tissue plasminogen activator, 12% had posterior circulation stroke. Patients with posterior circulation stroke had significantly lower median baseline the National Institutes of Health and Stroke Scale (NIHSS) score (P = 0·01), higher frequency of nausea (P < 0·01), vomiting (P < 0·01), dizziness (P < 0·01), and lower frequency of aphasia (P = 0·002) or neglect (P = 0·048). The emergency department physician evaluation-to-neurologist evaluation and door-to-needle intervals were significantly longer for posterior circulation stroke patients compared with anterior circulation stroke patients. The neurologist-to-needle time, however, was similar in the two groups. The presence of nausea and vomiting was associated with a longer time from emergency department evaluation to neurology evaluation and had a significant association with delayed treatment. Conclusions Posterior circulation stroke patients had a delay in neurology evaluation after initial emergency department evaluation and a delay in intravenous tissue plasminogen activator administration compared with anterior circulation stroke patients. There may be difficulties in rapidly recognizing the symptoms of posterior circulation stroke, in contrast to anterior circulation stroke, in the emergency department.

Systematic Review and Meta-Analysis of Bone Marrow–Derived Mononuclear Cells in Animal Models of Ischemic Stroke

Background and Purpose— Bone marrow–derived mononuclear cells (BMMNCs) offer the promise of augmenting poststroke recovery. There is mounting evidence of safety and efficacy of BMMNCs from preclinical studies of ischemic stroke; however, their pooled effects have not been described. Methods— Using Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines, we conducted a systematic review of preclinical literature for intravenous use of BMMNCs followed by meta-analyses of histological and behavioral outcomes. Studies were selected based on predefined criteria. Data were abstracted by 2 independent investigators. After quality assessment, the pooled effects were generated using mixed-effect models. Impact of possible biases on estimated effect size was evaluated. Results— Standardized mean difference and 95% confidence interval for reduction in lesion volume was significantly beneficial for BMMNC treatment (standardized mean difference: −3.3; 95% confidence interval, −4.3 to −2.3). n=113 each for BMMNC and controls. BMMNC-treated animals (n=161) also had improved function measured by cylinder test (standardized mean difference: −2.4; 95% confidence interval, −3.1 to −1.6), as compared with controls (n=205). A trend for benefit was observed for adhesive removal test and neurological deficit score. Study quality score (median: 6; Q1–Q3: 5–7) was correlated with year of publication. There was funnel plot asymmetry; however, the pooled effects were robust to the correction of this bias and remained significant in favor of BMMNC treatment. Conclusions— BMMNCs demonstrate beneficial effects across histological and behavioral outcomes in animal ischemic stroke models. Although study quality has improved over time, considerable degree of heterogeneity calls for standardization in the conduct and reporting of experimentation.

Acute splenic responses in patients with ischemic stroke and intracerebral hemorrhage

Animal models provide evidence of spleen mediated post-stroke activation of the peripheral immune system. Translation of these findings to stroke patients requires estimation of pre-stroke spleen volume along with quantification of its day-to-day variation. We enrolled a cohort of 158 healthy volunteers and measured their spleen volume over the course of five consecutive days. We also enrolled a concurrent cohort of 158 stroke patients, measured initial spleen volume within 24 h of stroke symptom onset followed by daily assessments. Blood samples for cytokine analysis were collected from a subset of patients. Using data from healthy volunteers, we fit longitudinal quantile regression models to construct gender and body surface area based normograms of spleen volume. We quantified day-to-day variation and defined splenic contraction. Based on our criteria, approximately 40% of stroke patients experienced substantial post-stroke reduction in splenic volume. African Americans, older patients, and patients with past history of stroke have significantly higher odds of post-stroke splenic contraction. All measured cytokine levels were elevated in patients with splenic contraction, with significant differences for interferon gamma, interleukin 6, 10, 12, and 13. Our work provides reference standards for further work, validation of pre-clinical findings, and characterization of patients with post-stroke splenic contraction.

Reporting Standards for Preclinical Studies of Stroke Therapy

The unmet need for development of new stroke therapies is enormous. Evidence generated from positive, null, or negative preclinical studies for various therapeutic agents is crucial to enhancing scientific progress. The scientific community shares a societal responsibility to practice and promote meticulous conduct and reporting of all experimental studies. A systematic survey conducted by the UK government–sponsored National Center for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) reported that only 59% of biomedical animal studies stated the hypotheses and objectives, and ≤87% did not use randomization.1 This, in part, led to the development of the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines,2 modeled after the CONSORT (Consolidated Standards for Reporting …