Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling.

Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amenable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed "nanodysferlins," designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.

Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice.

Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.

Autoantibodies targeting TRIM72 compromise membrane repair and contribute to inflammatory myopathy.

Idiopathic inflammatory myopathies (IIM) involve chronic inflammation of skeletal muscle and subsequent muscle degeneration due to an uncontrolled autoimmune response; however, the mechanisms leading to pathogenesis are not well understood. A compromised sarcolemmal repair process could promote an aberrant exposure of intramuscular antigens with the subsequent initiation of an inflammatory response that contributes to IIM. Using an adoptive transfer mouse model of IIM, we show that sarcolemmal repair is significantly compromised in distal skeletal muscle in the absence of inflammation. We identified autoantibodies against TRIM72 (also known as MG53), a muscle-enriched membrane repair protein, in IIM patient sera and in our mouse model of IIM by ELISA. We found that patient sera with elevated levels of TRIM72 autoantibodies suppress sarcolemmal resealing in healthy skeletal muscle, and depletion of TRIM72 antibodies from these same serum samples rescues sarcolemmal repair capacity. Autoantibodies targeting TRIM72 lead to skeletal muscle fibers with compromised membrane barrier function, providing a continuous source of autoantigens to promote autoimmunity and further amplifying humoral responses. These findings reveal a potential pathogenic mechanism that acts as a feedback loop contributing to the progression of IIM.

Enhancing membrane repair increases regeneration in a sciatic injury model.

Various injuries to the neural tissues can cause irreversible damage to multiple functions of the nervous system ranging from motor control to cognitive function. The limited treatment options available for patients have led to extensive interest in studying the mechanisms of neuronal regeneration and recovery from injury. Since many neurons are terminally differentiated, by increasing cell survival following injury it may be possible to minimize the impact of these injuries and provide translational potential for treatment of neuronal diseases. While several cell types are known to survive injury through plasma membrane repair mechanisms, there has been little investigation of membrane repair in neurons and even fewer efforts to target membrane repair as a therapy in neurons. Studies from our laboratory group and others demonstrated that mitsugumin 53 (MG53), a muscle-enriched tripartite motif (TRIM) family protein also known as TRIM72, is an essential component of the cell membrane repair machinery in skeletal muscle. Interestingly, recombinant human MG53 (rhMG53) can be applied exogenously to increase membrane repair capacity both in vitro and in vivo. Increasing the membrane repair capacity of neurons could potentially minimize the death of these cells and affect the progression of various neuronal diseases. In this study we assess the therapeutic potential of rhMG53 to increase membrane repair in cultured neurons and in an in vivo mouse model of neurotrauma. We found that a robust repair response exists in various neuronal cells and that rhMG53 can increase neuronal membrane repair both in vitro and in vivo. These findings provide direct evidence of conserved membrane repair responses in neurons and that these repair mechanisms can be targeted as a potential therapeutic approach for neuronal injury.

Multiple poloxamers increase plasma membrane repair capacity in muscle and nonmuscle cells.

Various previous studies established that the amphiphilic tri-block copolymer known as poloxamer 188 (P188) or Pluronic-F68 can stabilize the plasma membrane following a variety of injuries to multiple mammalian cell types. This characteristic led to proposals for the use of P188 as a therapeutic treatment for various disease states, including muscular dystrophy. Previous studies suggest that P188 increases plasma membrane integrity by resealing plasma membrane disruptions through its affinity for the hydrophobic lipid chains on the lipid bilayer. P188 is one of a large family of copolymers that share the same basic tri-block structure consisting of a middle hydrophobic propylene oxide segment flanked by two hydrophilic ethylene oxide moieties [poly(ethylene oxide)80-poly(propylene oxide)27-poly(ethylene oxide)80]. Despite the similarities of P188 to the other poloxamers in this chemical family, there has been little investigation into the membrane-resealing properties of these other poloxamers. In this study we assessed the resealing properties of poloxamers P181, P124, P182, P234, P108, P407, and P338 on human embryonic kidney 293 (HEK293) cells and isolated muscle from the mdx mouse model of Duchenne muscular dystrophy. Cell membrane injuries from glass bead wounding and multiphoton laser injury show that the majority of poloxamers in our panel improved the plasma membrane resealing of both HEK293 cells and dystrophic muscle fibers. These findings indicate that many tri-block copolymers share characteristics that can increase plasma membrane resealing and that identification of these shared characteristics could help guide design of future therapeutic approaches.

Modeling integration: co-teaching basic and clinical sciences medicine in the classroom.

PURPOSE: Calls for revision in undergraduate medical education frequently cite the importance of integrating basic and clinical sciences and the use of active pedagogies. One under-appreciated approach to accomplishing both is interactive co-teaching, defined as two instructors with complementary expertise engaging students and each other instead of lecturing. This study sought to determine if interactive co-teaching helped students integrate and learn basic and clinical sciences, as well as to explore potential advantages and barriers to co-teaching. METHODS: The comparative success of solo- and co-teaching in a microbiology/infectious disease course was determined by surveying student perceptions at the end of the course and examination scores for questions based on either solo- or co-taught content. The advantages and barriers to co-teaching were explored by thematic analysis of student responses to open-ended survey questions. RESULTS: Results suggest that co-teaching supported content integration as a significant majority of students (92%, n=112) reported they understood the connection between basic and clinical sciences better when content was co-taught. In addition, a plurality of students indicated that co-teaching provided a better overall learning experience (81%, n=99), was more engaging (74%, n=90), and made it easier to apply content (74%, n=90). These positive perceptions were reflected in better exam outcomes for materials covered in co-taught over solo-taught sessions. CONCLUSION: Results suggest students value co-teaching as a means to integrate basic and clinical sciences. However, interactive co-teaching pedagogies require careful planning and collaboration among faculty. Co-teaching requires the commitment of both faculty members to this pedagogy.

Trusting early learners with critical professional activities through emergency medical technician certification.

BACKGROUND: Two dominant themes face medical education: developing integrated curricula and improving the undergraduate medical education (UME) to graduate medical education (GME) transition. An innovative solution to both of these challenges at the Zucker School of Medicine has been the application of the cognitive apprenticeship framework in requiring emergency medical technician (EMT) certification during the first course in medical school as the core on which to build an integrated curriculum and provide entrustable clinical skills. METHODS: Beginning with the Class of 2011, student feedback about the short-term impact of the experience was collected annually. In addition, perceptions of near graduates and alumni were surveyed in 2017 to explore the long-term impact of the experience. Theme analysis was conducted via inductive coding. RESULTS: Both first-year and more experienced learners report the value of the EMT curriculum as an integrated component of the first course of medical school. Reported positive long-term impacts included the first-hand observation of social determinants of health and interprofessionalism. Negative comments by early learners focused on course logistics, whereas older learners recalled the variability of clinical experiences during ambulance runs. CONCLUSIONS: The integration of the EMT curriculum as a core component of the first course serves multiple purposes: 1) it provides the foundation of a spiral learning approach; 2) it contextualizes the basic sciences within clinical practice; 3) it provides opportunities for students to engage in authentic clinical activities under the guidance of mentors; 4) it introduces students to the interdisciplinary nature of medicine; and 5) it serves as the first entrustable professional activity (EPA) for our students.

Conserved structural and functional aspects of the tripartite motif gene family point towards therapeutic applications in multiple diseases.

The tripartite motif (TRIM) gene family is a highly conserved group of E3 ubiquitin ligase proteins that can establish substrate specificity for the ubiquitin-proteasome complex and also have proteasome-independent functions. While several family members were studied previously, it is relatively recent that over 80 genes, based on sequence homology, were grouped to establish the TRIM gene family. Functional studies of various TRIM genes linked these proteins to modulation of inflammatory responses showing that they can contribute to a wide variety of disease states including cardiovascular, neurological and musculoskeletal diseases, as well as various forms of cancer. Given the fundamental role of the ubiquitin-proteasome complex in protein turnover and the importance of this regulation in most aspects of cellular physiology, it is not surprising that TRIM proteins display a wide spectrum of functions in a variety of cellular processes. This broad range of function and the highly conserved primary amino acid sequence of family members, particularly in the canonical TRIM E3 ubiquitin ligase domain, complicates the development of therapeutics that specifically target these proteins. A more comprehensive understanding of the structure and function of TRIM proteins will help guide therapeutic development for a number of different diseases. This review summarizes the structural organization of TRIM proteins, their domain architecture, common and unique post-translational modifications within the family, and potential binding partners and targets. Further discussion is provided on efforts to target TRIM proteins as therapeutic agents and how our increasing understanding of the nature of TRIM proteins can guide discovery of other therapeutics in the future.

Contextualizing the relevance of basic sciences: small-group simulation with debrief for first- and second-year medical students in an integrated curriculum.

AIM: There has been a call for increased integration of basic and clinical sciences during preclinical years of undergraduate medical education. Despite the recognition that clinical simulation is an effective pedagogical tool, little has been reported on its use to demonstrate the relevance of basic science principles to the practice of clinical medicine. We hypothesized that simulation with an integrated science and clinical debrief used with early learners would illustrate the importance of basic science principles in clinical diagnosis and management of patients. METHODS: Small groups of first- and second-year medical students were engaged in a high-fidelity simulation followed by a comprehensive debrief facilitated by a basic scientist and clinician. Surveys including anchored and open-ended questions were distributed at the conclusion of each experience. RESULTS: The majority of the students agreed that simulation followed by an integrated debrief illustrated the clinical relevance of basic sciences (mean ± standard deviation: 93.8% ± 2.9% of first-year medical students; 96.7% ± 3.5% of second-year medical students) and its importance in patient care (92.8% of first-year medical students; 90.4% of second-year medical students). In a thematic analysis of open-ended responses, students felt that these experiences provided opportunities for direct application of scientific knowledge to diagnosis and treatment, improving student knowledge, simulating real-world experience, and developing clinical reasoning, all of which specifically helped them understand the clinical relevance of basic sciences. CONCLUSION: Small-group simulation followed by a debrief that integrates basic and clinical sciences is an effective means of demonstrating the relationship between scientific fundamentals and patient care for early learners. As more medical schools embrace integrated curricula and seek opportunities for integration, our model is a novel approach that can be utilized.

Improving Transfer Times for Acute Ischemic Stroke Patients to a Comprehensive Stroke Center.

BACKGROUND AND OBJECTIVE: The transfer of acute ischemic stroke (AIS) patients to a comprehensive stroke center (CSC) must be rapid. Delays pose an obstacle to time-sensitive stroke treatments and, therefore, increase the likelihood of exclusion from endovascular stroke therapy. This study aims to evaluate the impact of the Stroke Rescue Program, with its goal of minimizing interfacility transfer delays and increasing the number of transport times completed within 60 minutes. METHODS: The Stroke Rescue Program was initiated to facilitate the rapid transfer of AIS patients from regional primary stroke centers (PSCs) to the network's CSC. The transfer process was divided into 3 time elements: transport 1 time (initial phone call from the PSC until emergency medical service [EMS] arrival at the PSC), emergency department (ED) time (EMS PSC arrival to PSC departure), and transport 2 time (PSC departure to CSC arrival). The total transport time target was set at less than 60 minutes. Protocols and procedures were implemented with a focus on decreasing the ED time. RESULTS: Comparing baseline (preimplementation) quarter (n = 21) to postproject quarter (1 year later, n = 31), the percent transported within 60 minutes increased from 62% to 81%. A statistically significant improvement was seen for both median ED time (23 minutes versus 14 minutes; U = 171, P < .01) and median total transport time (56 minutes versus 44 minutes; U = 199, P < .05). CONCLUSION: Interfacility transfer protocols minimizing the time paramedics spend in a PSC ED can significantly reduce total transfer time to a comprehensive stroke center.

Not for industry only: medical students and office-based academic detailing the PIVOT (Pregnant women Influenza Vaccine Optimization Team) initiative.

Academic detailing is a method of educational outreach that utilizes individualized encounters with physicians to broach specific medical issues in an evidence-based and quality-driven manner. Medical students utilized the matter of influenza vaccination during pregnancy as a lens through which to explore the methods of academic detailing in a community setting. Structured and customized dialogues between North Shore-LIJ affiliated obstetricians and Hofstra North Shore-LIJ medical students were conducted regarding the disparity between the proportion of providers that recommend the vaccine and the percentage of pregnant women being vaccinated annually. Ultimately the project aimed to increase vaccine-carrying rates throughout office based practices in the community, while establishing a viable method for up-to-date information exchange between practicing physicians and academic medicine. While the extent of affected change is currently being quantified, the project proved successful insofar as academic detailing allowed the students to gain access to physicians, and engage in compelling and educational conversations. Both the physicians and students felt these interactions were valuable and well worth continuing. The goal for the future is to expand these practices to other pressing public health issues while continuing to refine the technique.

Medical students as EMTs: skill building, confidence and professional formation.

OBJECTIVE: The first course of the medical curriculum at the Hofstra North Shore-LIJ School of Medicine, From the Person to the Professional: Challenges, Privileges and Responsibilities, provides an innovative early clinical immersion. The course content specific to the Emergency Medical Technician (EMT) curriculum was developed using the New York State Emergency Medical Technician curriculum. Students gain early legitimate clinical experience and practice clinical skills as team members in the pre-hospital environment. We hypothesized this novel curriculum would increase students' confidence in their ability to perform patient care skills and enhance students' comfort with team-building skills early in their training. METHODS: Quantitative and qualitative data were collected from first-year medical students (n=97) through a survey developed to assess students' confidence in patient care and team-building skills. The survey was completed prior to medical school, during the final week of the course, and at the end of their first year. A paired-samples t-test was conducted to compare self-ratings on 12 patient care and 12 team-building skills before and after the course, and a theme analysis was conducted to examine open-ended responses. RESULTS: Following the course, student confidence in patient care skills showed a significant increase from baseline (p<0.05) for all identified skills. Student confidence in team-building skills showed a significant increase (p<0.05) in 4 of the 12 identified skills. By the end of the first year, 84% of the first-year students reported the EMT curriculum had 'some impact' to 'great impact' on their patient care skills, while 72% reported the EMT curriculum had 'some impact' to 'great impact' on their team-building skills. CONCLUSIONS: The incorporation of EMT training early in a medical school curriculum provides students with meaningful clinical experiences that increase their self-reported level of confidence in the performance of patient care skills early in their medical education.

Stroke chameleons.

BACKGROUND: Many conditions called "stroke mimics" may resemble acute stroke. The converse of the "stroke mimic" is a presentation suggestive of another condition, which actually represents stroke. These would be "stroke chameleons." The recognition of a chameleon as stroke has implications for therapy and quality of care. METHODS: We performed a retrospective chart review, including all cases for 1 year in which patients had a stroke missed on hospital presentation. Initial erroneous diagnoses were compared for all patients correctly admitted with those diagnoses to determine positive predictive value (PPV) for each chameleon. RESULTS: Ninety-four cases were identified as chameleons where brain imaging revealed acute stroke. The common chameleons were initially diagnosed as altered mental status (AMS) (29, 31%), syncope (15, 16%), hypertensive emergency (12, 13%), systemic infection (10, 11%), and suspected acute coronary syndrome (ACS) (9, 10%). The total number of patients who were diagnosed with these conditions over the same year were AMS (393), syncope (326), hypertensive emergency (144), systemic infection (753), and suspected ACS (817) (total N = 2528). For each chameleon diagnosis, the PPV of each presentation for acute stroke was AMS (7%), syncope (4%), hypertensive emergency (8%), systemic infection (1%), and suspected ACS (1%). CONCLUSIONS: Stroke chameleons may result in patients not receiving appropriate care. The largest proportions of chameleons were AMS, syncope, hypertensive emergency, systemic infection, and suspected ACS. Patients diagnosed with hypertensive emergency or AMS had an 8% and 7% chance of having an acute stroke. Physicians should consider stroke in patients with these diagnoses with a lower threshold to obtain neuroimaging with subsequent appropriate management.

Increased A1C among adult emergency department patients with type 2 diabetes.

STUDY OBJECTIVE: Long-term glycemic control can prevent or delay complications of diabetes. Although diabetes is a common comorbidity in emergency department (ED) patients, the adequacy of long-term control is not known. Our objectives are to determine the frequency of poorly controlled type 2 diabetes among adults presenting to an ED and to identify characteristics associated with poor control. METHODS: An A1C level was obtained for diabetic patients 18 years and older who presented to the ED for acute medical problems and had blood tests performed for usual medical care. Consecutive patients were screened for a total of 58 24-hour periods during a 10-week period. A1C values were stratified, with less than 7% defined as good control and greater than or equal to 7% poor long-term control. Logistic regression analysis was used to identify factors associated with poor control. RESULTS: Of the 500 patients with type 2 diabetes, 53.4% had inadequate long-term control. An increased ED glucose level was independently associated with an increased A1C level. If the ED glucose level was 126 to 149 mg/dL, the odds ratio (OR) for an increased A1C level relative to a glucose level less than 100 mg/dL was 2.3 (95% confidence interval [CI] 0.95, 5.68); the OR was 6.4 (95% CI 2.9, 14.1) for glucose levels 150 to 199 mg/dL, and for glucose level of 200 mg/dL or above, the OR for an increased A1C level was 21.2 (9.1, 49.3). Other factors independently associated with increased A1C level were black race, aged 40 to 59 years, and Medicaid insurance. CONCLUSION: The high frequency of A1C levels more than 7% points to the ED as a potential source for identifying patients with poorly controlled type 2 diabetes.

Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules.

Mutations in the RNA-binding protein FUS (fused in sarcoma) are linked to amyotrophic lateral sclerosis (ALS), but the mechanism by which these mutants cause motor neuron degeneration is not known. We report a novel ALS truncation mutant (R495X) that leads to a relatively severe ALS clinical phenotype compared with FUS missense mutations. Expression of R495X FUS, which abrogates a putative nuclear localization signal at the C-terminus of FUS, in HEK-293 cells and in the zebrafish spinal cord caused a striking cytoplasmic accumulation of the protein to a greater extent than that observed for recessive (H517Q) and dominant (R521G) missense mutants. Furthermore, in response to oxidative stress or heat shock conditions in cultures and in vivo, the ALS-linked FUS mutants, but not wild-type FUS, assembled into perinuclear stress granules in proportion to their cytoplasmic expression levels. These findings demonstrate a potential link between FUS mutations and cellular pathways involved in stress responses that may be relevant to altered motor neuron homeostasis in ALS.

Phase IIB/III trial of tenecteplase in acute ischemic stroke: results of a prematurely terminated randomized clinical trial.

BACKGROUND AND PURPOSE: Intravenous alteplase (rtPA) remains the only approved treatment for acute ischemic stroke, but its use remains limited. In a previous pilot dose-escalation study, intravenous tenecteplase showed promise as a potentially safer alternative. Therefore, a Phase IIB clinical trial was begun to (1) choose a best dose of tenecteplase to carry forward; and (2) to provide evidence for either promise or futility of further testing of tenecteplase versus rtPA. If promise was established, then the trial would continue as a Phase III efficacy trial comparing the selected tenecteplase dose to standard rtPA. METHODS: The trial began as a small, multicenter, randomized, double-blind, controlled clinical trial comparing 0.1, 0.25, and 0.4 mg/kg tenecteplase with standard 0.9 mg/kg rtPA in patients with acute stroke within 3 hours of onset. An adaptive sequential design used an early (24-hour) assessment of major neurological improvement balanced against occurrence of symptomatic intracranial hemorrhage to choose a "best" dose of tenecteplase to carry forward. Once a "best" dose was established, the trial was to continue until at least 100 pairs of the selected tenecteplase dose versus standard rtPA could be compared by 3-month outcome using the modified Rankin Scale in an interim analysis. Decision rules were devised to yield a clear recommendation to either stop for futility or to continue into Phase III. RESULTS: The trial was prematurely terminated for slow enrollment after only 112 patients had been randomized at 8 clinical centers between 2006 and 2008. The 0.4-mg/kg dose was discarded as inferior after only 73 patients were randomized, but the selection procedure was still unable to distinguish between 0.1 mg/kg and 0.25 mg/kg as a propitious dose at the time the trial was stopped. There were no statistically persuasive differences in 3-month outcomes between the remaining tenecteplase groups and rtPA. Symptomatic intracranial hemorrhage rates were highest in the discarded 0.4-mg/kg tenecteplase group and lowest (0 of 31) in the 0.1-mg/kg tenecteplase group. Neither promise nor futility could be established. CONCLUSION: This prematurely terminated trial has demonstrated the potential efficiency of a novel design in selecting a propitious dose for future study of a new thrombolytic agent for acute stroke. Given the truncation of the trial, no convincing conclusions can be made about the promise of future study of tenecteplase in acute stroke.

Reduced expression of the Kinesin-Associated Protein 3 (KIFAP3) gene increases survival in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a degenerative disorder of motor neurons that typically develops in the 6th decade and is uniformly fatal, usually within 5 years. To identify genetic variants associated with susceptibility and phenotypes in sporadic ALS, we performed a genome-wide SNP analysis in sporadic ALS cases and controls. A total of 288,357 SNPs were screened in a set of 1,821 sporadic ALS cases and 2,258 controls from the U.S. and Europe. Survival analysis was performed using 1,014 deceased sporadic cases. Top results for susceptibility were further screened in an independent sample set of 538 ALS cases and 556 controls. SNP rs1541160 within the KIFAP3 gene (encoding a kinesin-associated protein) yielded a genome-wide significant result (P = 1.84 x 10(-8)) that withstood Bonferroni correction for association with survival. Homozygosity for the favorable allele (CC) conferred a 14.0 months survival advantage. Sequence, genotypic and functional analyses revealed that there is linkage disequilibrium between rs1541160 and SNP rs522444 within the KIFAP3 promoter and that the favorable alleles of rs1541160 and rs522444 correlate with reduced KIFAP3 expression. No SNPs were associated with risk of sporadic ALS, site of onset, or age of onset. We have identified a variant within the KIFAP3 gene that is associated with decreased KIFAP3 expression and increased survival in sporadic ALS. These findings support the view that genetic factors modify phenotypes in this disease and that cellular motor proteins are determinants of motor neuron viability.

The combined approach to lysis utilizing eptifibatide and rt-PA in acute ischemic stroke: the CLEAR stroke trial.

BACKGROUND AND PURPOSE: Multiple approaches are being studied to enhance the rate of thrombolysis for acute ischemic stroke. Treatment of myocardial infarction with a combination of a reduced-dose fibrinolytic agent and a glycoprotein (GP) IIb/IIIa receptor antagonist has been shown to improve the rate of recanalization versus fibrinolysis alone. The combined approach to lysis utilizing eptifibatide and recombinant tissue-type plasminogen activator (rt-PA) (CLEAR) stroke trial assessed the safety of treating acute ischemic stroke patients within 3 hours of symptom onset with this combination. METHODS: The CLEAR trial was a National Institutes of Health/National Institute of Neurological Disorders and Stroke-funded multicenter, double-blind, randomized, dose-escalation and safety study. Patients were randomized 3:1 to either low-dose rt-PA (tier 1=0.3 mg/kg, tier 2=0.45 mg/kg) plus eptifibatide (75 microg/kg bolus followed by 0.75 microg/kg per min infusion for 2 hours) or standard-dose rt-PA (0.9 mg/kg). The primary safety end point was the incidence of symptomatic intracerebral hemorrhage within 36 hours. Secondary analyses were performed regarding clinical efficacy. RESULTS: Ninety-four patients (40 in tier 1 and 54 in tier 2) were enrolled. The combination group of the 2 dose tiers (n=69) had a median age of 71 years and a median baseline National Institutes of Health Stroke Scale (NIHSS) score of 14, and the standard-dose rt-PA group (n=25) had a median age of 61 years and a median baseline NIHSS score of 10 (P=0.01 for NIHSS score). Fifty-two (75%) of the combination treatment group and 24 (96%) of the standard treatment group had a baseline modified Rankin scale score of 0 (P=0.04). There was 1 (1.4%; 95% CI, 0% to 4.3%) symptomatic intracranial hemorrhage in the combination group and 2 (8.0%; 95% CI, 0% to 19.2%) in the rt-PA-only arm (P=0.17). During randomization in tier 2, a review by the independent data safety monitoring board demonstrated that the safety profile of combination therapy at the tier 2 doses was such that further enrollment was statistically unlikely to indicate inadequate safety for the combination treatment group, the ultimate outcome of the study. Thus, the study was halted. There was a trend toward increased clinical efficacy of standard-dose rt-PA compared with the combination treatment group. CONCLUSIONS: The safety of the combination of reduced-dose rt-PA plus eptifibatide justifies further dose-ranging trials in acute ischemic stroke.