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This study aimed to investigate the influence of stroke lesions in pre-defined highly interconnected (rich club) brain regions on functional outcome post-stroke, determine their spatial specificity and explore the effects of biological sex on their relevance.We analyzed MRI data recorded at index stroke and ∼3-months modified Rankin Scale (mRS) data from patients with acute ischemic stroke (AIS) enrolled in the multisite MRI-GENIE study. Structural stroke lesions were spatially normalized and parcellated into 108 atlas-defined bilateral (sub)cortical brain regions. Unfavorable outcome (mRS>2) was modeled in a Bayesian logistic regression framework that relied on both lesion location, as well as the covariates: age, sex, total DWI lesion volume and comorbidities. Effects of individual brain regions were captured as two compound effects for (i) six bilateral rich club and (ii) all further non-rich club regions. Via model comparisons, we first tested whether the rich club region model was superior to a baseline model considering clinical covariates and lesion volume only. In spatial specificity analyses, we randomized the split into “rich club” and “non-rich club” regions and compared the effect of the actual rich club regions to the distribution of effects from 1,000 combinations of six random regions. In sex-specific analyses, we introduced an additional hierarchical level in our model structure to compare male and female-specific rich club region effects.A total of 822 patients (age: 64.7 (standard deviation: 15.0), 39% women, 27.7% with mRS>2) were analyzed. The rich club model substantially outperformed the baseline model (weights of model comparison: rich club model: 0.96; baseline: 0.04). Rich club regions had substantial relevance in explaining unfavorable functional outcome (mean of posterior distribution: 0.08, area under the curve: 0.8). In particular, the rich club-combination had a higher relevance than 98.4% of random constellations (15/1,000 random constellations with higher mean posterior values). Among the these 15 random constellations with higher means, the most frequently selected regions were the inferior temporal gyrus (posterior division, 8/15), the putamen (8/15), the cingulate gyrus (7/15) and the superior parietal lobule (6/15). Rich club regions were substantially more important in explaining long-term outcome in women than in men (mean of the difference distribution:-0.107, 90%-HDPI:-0.193 to -0.0124).Lesions in rich club regions were associated with increased odds of unfavorable outcome. These effects were spatially specific, i.e., the majority of random combinations of six regions had comparably smaller effects on long-term outcome. Effects were substantially more pronounced in women.

BACKGROUND: Studies of neurologic outcomes have found conflicting results regarding differences between patients with substance-related cardiac arrests (SRCA) and non-SRCA. We investigate the effects of SRCA on severe cerebral edema development, a neuroimaging intermediate endpoint for neurologic injury. METHODS: 327 out-of-hospital comatose cardiac arrest patients were retrospectively analyzed. Demographics and baseline clinical characteristics were examined. SRCA categorization was based on admission toxicology screens. Severe cerebral edema classification was based on radiology reports. Poor clinical outcomes were defined as discharge Cerebral Performance Category scores>3. RESULTS: SRCA patients (N=86) were younger (P

Background and ObjectivesTo examine whether high white matter hyperintensity (WMH) burden is associated with greater stroke severity and worse functional outcomes in lesion pattern–specific ways.MethodsMR neuroimaging and NIH Stroke Scale data at index stroke and the modified Rankin Scale (mRS) score at 3–6 months after stroke were obtained from the MRI–Genetics Interface Exploration study of patients with acute ischemic stroke (AIS). Individual WMH volume was automatically derived from fluid-attenuated inversion recovery images. Stroke lesions were automatically segmented from diffusion-weighted imaging (DWI) images, parcellated into atlas-defined brain regions and further condensed to 10 lesion patterns via machine learning–based dimensionality reduction. Stroke lesion effects on AIS severity and unfavorable outcomes (mRS score >2) were modeled within purpose-built Bayesian linear and logistic regression frameworks. Interaction effects between stroke lesions and a high vs low WMH burden were integrated via hierarchical model structures. Models were adjusted for age, age2, sex, total DWI lesion and WMH volumes, and comorbidities. Data were split into derivation and validation cohorts.ResultsA total of 928 patients with AIS contributed to acute stroke severity analyses (age: 64.8 [14.5] years, 40% women) and 698 patients to long-term functional outcome analyses (age: 65.9 [14.7] years, 41% women). Stroke severity was mainly explained by lesions focused on bilateral subcortical and left hemispherically pronounced cortical regions across patients with both a high and low WMH burden. Lesions centered on left-hemispheric insular, opercular, and inferior frontal regions and lesions affecting right-hemispheric temporoparietal regions had more pronounced effects on stroke severity in case of high compared with low WMH burden. Unfavorable outcomes were predominantly explained by lesions in bilateral subcortical regions. In difference to the lesion location–specific WMH effects on stroke severity, higher WMH burden increased the odds of unfavorable outcomes independent of lesion location.DiscussionHigher WMH burden may be associated with an increased stroke severity in case of stroke lesions involving left-hemispheric insular, opercular, and inferior frontal regions (potentially linked to language functions) and right-hemispheric temporoparietal regions (potentially linked to attention). Our findings suggest that patients with specific constellations of WMH burden and lesion locations may have greater benefits from acute recanalization treatments. Future clinical studies are warranted to systematically assess this assumption and guide more tailored treatment decisions.

Introduction: Previous studies have shown that women have poorer neurological outcomes than men following out-of-hospital cardiac arrest (OHCA). However, those studies focused on survival, a measure that may be confounded by withdrawal of life-sustaining therapy (WLST) decisions. We sought to assess sex differences in severe cerebral edema development following cardiac arrest. Methods: Data from adult OHCA patients presenting between 2007-2019 were retrospectively analyzed. Patients with multiple concomitant acute neurologic dysfunctions were excluded. Severe cerebral edema was classified as herniation or more than minimal ventricular effacement in MRI or CT radiology reports. Arrest types were categorized as shockable (VT/VF) or non-shockable (PEA, asystole). Poor outcomes were defined as Cerebral Performance Category > 3 at discharge. Multivariable backward stepwise logistic regression was performed. Results: 359 patients met our inclusion criteria. Demographics, imaging and clinical outcomes are summarized in Table 1. Women were more likely than men to have a non-shockable initial rhythm (P=0.003) and develop severe cerebral edema (P=0.005). Backwards stepwise logistic regression of an initial model including age, sex, rhythm, witnessed arrest and TTM-treatment, produced a final model that showed younger age (P Conclusion: For OHCA patients who are initially comatose, women are more likely than men to have non-shockable rhythms and severe cerebral brain edema. These differences may be responsible for the poorer discharge outcomes that have been observed by other studies.

ObjectiveTo examine whether high white matter hyperintensity (WMH) burden is associated with greater stroke severity and worse functional outcomes in lesion pattern-specific ways.MethodsMR neuroimaging and National Institutes of Health Stroke Scale data at index stroke, as well as modified Rankin Scale (mRS) at 3-6 months post-stroke were obtained from MRI-GENIE study of acute ischemic stroke (AIS) patients. Individual WMH volume was automatically derived from FLAIR-images. Stroke lesions were automatically segmented from DWI-images, spatially normalized and parcellated into atlas-defined brain regions. Stroke lesion effects on AIS severity and unfavorable outcomes (mRS>2) were modeled within a purpose-built machine learning and Bayesian regression framework. In particular, interaction effects between stroke lesions and a high versus low WMH burden were integrated via hierarchical model structures. Models were adjusted for the covariates age, age2, sex, total DWI-lesion and WMH volumes, and comorbidities. Data were split into derivation and validation cohorts.ResultsA total of 928 AIS patients contributed to stroke severity analyses (mean age: 64.8(14.5), 40% women), 698 patients to functional outcome analyses (mean age: 65.9(14.7), 41% women). Individual stroke lesions were represented in five anatomically distinct left-hemispheric and five right-hemispheric lesion patterns. Across all patients, acute stroke severity was substantially explained by three of these patterns, that were particularly focused on bilateral subcortical and left-hemispherically pronounced cortical regions. In high WMH burden patients, two lesion patterns consistently emerged as more pronounced in case of stroke severity: the first pattern was centered on left-hemispheric insular, opercular and inferior frontal regions, while the second pattern combined right-hemispheric temporo-parietal regions. Bilateral subcortical regions were most relevant in explaining long term unfavorable outcome. No WMH-specific lesion patterns of functional outcomes were substantiated. However, a higher overall WMH burden was associated with higher odds of unfavorable outcomes.ConclusionsHigher WMH burden increases stroke severity in case of stroke lesions involving left-hemispheric insular, opercular and inferior frontal regions (potentially linked to language functions) and right-hemispheric temporo-parietal regions (potentially linked to attention). These findings may contribute to augment stroke outcome predictions and motivate a WMH burden and stroke lesion pattern-specific clinical management of AIS patients.

Acute ischemic stroke affects men and women differently. In particular, women are often reported to experience higher acute stroke severity than men. We derived a low-dimensional representation of anatomical stroke lesions and designed a Bayesian hierarchical modeling framework tailored to estimate possible sex differences in lesion patterns linked to acute stroke severity (National Institute of Health Stroke Scale). This framework was developed in 555 patients (38% female). Findings were validated in an independent cohort (n = 503, 41% female). Here, we show brain lesions in regions subserving motor and language functions help explain stroke severity in both men and women, however more widespread lesion patterns are relevant in female patients. Higher stroke severity in women, but not men, is associated with left hemisphere lesions in the vicinity of the posterior circulation. Our results suggest there are sex-specific functional cerebral asymmetries that may be important for future investigations of sex-stratified approaches to management of acute ischemic stroke., Acute ischemic stroke impacts men and women differently. Here, the authors show how different lesion patterns in men and women are linked to the extent of stroke severity.

Introduction: The incidence of substance-related cardiac arrests (SRCA) has increased due to the opioid epidemic. Previous studies have shown that SRCA present differently than non-SRCA. Weinvestigated differences in the clinical courses between the two groups for patients who were initially comatose post-resuscitation. Methods: Data from out-of-hospital arrest patients seen between 2007-2019 were retrospectively analyzed. Arrests were classified as SRCA if the patient had a positive toxicology screen, witnesses testified to substance use, or drug paraphernalia was present at the scene. Arrest types were categorized as shockable or non-shockable. Cerebral edema classifications were based on MRI or CT radiology reports. Poor outcomes were defined as Cerebral Performance Category (CPC) > 3 at discharge. Differences in demographics, presentation and outcomes were compared (Fisher’s Exact Test or Exact Wilcoxon-Mann-Whitney). Multivariable backward stepwise logistic regression was performed. Results: SRCA patients (N=90) were younger (p Discussion: SRCA patients are more likely to have non-shockable arrests which in turn leads to cerebral edema development and potentially worse outcomes.

Acute ischemic stroke affects men and women differently in many ways. In particular, women are oftentimes reported to experience a higher acute stroke severity than men. Here, we derived a low-dimensional representation of anatomical stroke lesions and designed a sex-aware Bayesian hierarchical modelling framework for a large-scale, well phenotyped stroke cohort. This framework was tailored to carefully estimate possible sex differences in lesion patterns explaining acute stroke severity (NIHSS) in 1,058 patients (39% female). Anatomical regions known to subserve motor and language functions emerged as relevant regions for both men and women. Female patients, however, presented a more widespread pattern of stroke severity-relevant lesions than male patients. Furthermore, particularly lesions in the posterior circulation of the left hemisphere underlay a higher stroke severity exclusively in women. These sex-sensitive lesion pattern effects could be discovered and subsequently robustly replicated in two large independent, multisite lesion datasets. The constellation of findings has several important conceptual and clinical implications: 1) suggesting sex-specific functional cerebral asymmetries, and 2) motivating a sex-stratified approach to management of acute ischemic stroke. To go beyond sex-averaged stroke research, future studies should explicitly test whether acute therapies administered on the basis of sex-specific cutoff volumes of salvageable tissue will lead to improved outcomes in women after acute ischemic stroke.

Introduction: We developed a unique autologous cellular therapeutic strategy using a proprietary engineered leukocyte stimulator cell lines (ENLIST cells) that express an array of immunomodulatory proteins to activate and expand tumor cell killing effector cells from human peripheral blood mononuclear cells (PBMCs). Our clinical aim is to use these expanded cells, called SUPLEXA therapy in cancer patients. Results of prior immunophenotyping of SUPLEXA cells by mass cytometry (CyTOF) reveals that they are comprised of NK cells, CD8+ T cells and γδ T cells that express markers indicative of cytolytic function, e.g. granzymes and perforin. Using an unbiased approach to better define the specificity of SUPLEXA, we performed TCR repertoire analysis using the iRepertoire and 10X Genomics RNA sequencing platforms. Results: SUPLEXA was prepared from healthy donor PBMCs. RNA was subsequently prepared from SUPLEXA cells and donor PBMCs to compare TCR αβ and γδ chain clonality and diversity using the iRepertoire DAM-PCR sequencing platform. SUPLEXA cells showed dramatic and significantly increased clonality among the 4 TCR chains that were analyzed as compared to normal PBMCs. We also observed a diversification of TCRαβ and TCRγδ sequences in SUPLEXA as compared to PBMCs. In addition, to the increased diversity, clonal expansion was also observed. Single-cell RNA sequencing of flow-sorted CD3+ T cells using the 10X Genomics platform with paired TCRαβ and TCRγδ PCR sequencing feature overlays revealed high frequencies of T cells with paired TCR clonotypes. We discovered that the top 5 paired TCRαβ clonotype sequences accounted for 32.7% of T cells, while CD8+ T cells from a normal donor contains only 3.0% in the top 5 clonotypes. Similar high frequency clonotypes were observed for γδ T cells by 10X single-cell RNA sequencing. Conclusions: The creation of SUPLEXA from healthy adult PBMCs results in dramatic expansion of αβ and γδ TCR T cell clonotypes. Given that SUPLEXA cells demonstrate broad, dose-dependent tumor cytolytic activity, these findings suggest that the expanded αβ and γδ T cell clonotypes may react to tumor neoantigens that are present during the activation process by ENLIST cells, which are derived from the SK-MEL-2 melanoma cell line. Accordingly, TCR reagents made using the paired αβ and γδ T cell sequences from the expanded clonotypes will be useful for future tumor neoantigen discovery. Plans for a phase 1 clinical trial are currently underway. Citation Format: Frank Borriello, Joshua W. Keegan, Brandon L. Hancock, Fei Guo, James R. Lederer. A novel approach for autologous pan-cancer cellular immunotherapy reveals dramatic expansion of ab and gd TCR T cell clonotypes indicative of an antigen-driven response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1772.

Background: For cardiac arrest survivors who are initially comatose after restoration of spontaneous circulation, the extent of brain injury and expected recovery are crucial for management decisions. Advanced diffusion imaging approaches may provide additional insight into microstructural integrity and potential for arousal recovery (AR). Methods: Multi-shell diffusion imaging was acquired in a prospective study. Neurite orientation dispersion (OD), intracellular volume fraction (ICVF), free water fraction (ISO), mean kurtosis (MK), axial kurtosis (AK), radial kurtosis (RK), mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD) and fractional anisotropy (FA) were calculated (see Figure). Median whole-brain values in patients with AR by discharge were compared with those with no AR and to controls (ANOVA, post-hoc 2-tailed Wilcoxon test). Multivariable backward stepwise logistic regression was performed to predict AR. Results: 19 patients (mean ±SD 48±23 y, 42% men) and 5 controls (37±19 y, 40% men) were analyzed (10 patients with AR, 9 without). Median [IQR] time-to-MRI was 5 [4-8] days. Patients with no AR had higher AK (P=0.037), and RK (P=.045) than those with AR. Controls exhibited higher FA than either AR (P=.009) or no AR (P=.02), lower MK than no AR (P=.02), lower AK than no AR (P=.02), lower RK than no AR (P=0.02), lower OD than no AR (P=.008), and lower ICVF than no AR (P=.046). AK (P=.049) and ICVF (P=.01) were found in multivariable regression to be significant predictors of AR with 81% area under the receiver operating curve (AUC). Discussion: Advanced diffusion imaging metrics, AK and ICVF, were able to predict which patients were likely to regain consciousness, with 81% AUC. Despite the small sample size, we note statistically significant differences using more sensitive measures of microstructural injury compared to more commonly used diffusivity metrics. Further investigation of advanced diffusion imaging methods is warranted.

Background and Purpose— We evaluated deep learning algorithms’ segmentation of acute ischemic lesions on heterogeneous multi-center clinical diffusion-weighted magnetic resonance imaging (MRI) data sets and explored the potential role of this tool for phenotyping acute ischemic stroke. Methods— Ischemic stroke data sets from the MRI-GENIE (MRI-Genetics Interface Exploration) repository consisting of 12 international genetic research centers were retrospectively analyzed using an automated deep learning segmentation algorithm consisting of an ensemble of 3-dimensional convolutional neural networks. Three ensembles were trained using data from the following: (1) 267 patients from an independent single-center cohort, (2) 267 patients from MRI-GENIE, and (3) mixture of (1) and (2). The algorithms’ performances were compared against manual outlines from a separate 383 patient subset from MRI-GENIE. Univariable and multivariable logistic regression with respect to demographics, stroke subtypes, and vascular risk factors were performed to identify phenotypes associated with large acute diffusion-weighted MRI volumes and greater stroke severity in 2770 MRI-GENIE patients. Stroke topography was investigated. Results— The ensemble consisting of a mixture of MRI-GENIE and single-center convolutional neural networks performed best. Subset analysis comparing automated and manual lesion volumes in 383 patients found excellent correlation (ρ=0.92; P 3 (0.9–16.6 cm 3 ). Patients with small artery occlusion stroke subtype had smaller lesion volumes ( P Conclusions— Automated accurate clinical diffusion-weighted MRI lesion segmentation using deep learning algorithms trained with multi-center and diverse data is feasible. Both lesion volume and topography can provide insight into stroke subtypes with sufficient sample size from big heterogeneous multi-center clinical imaging phenotype data sets.

The mechanism of cellulose synthesis has been studied by characterizing the motility of cellulose synthase complexes tagged with a fluorescent protein; however, this approach has been used exclusively on the hypocotyl of Arabidopsis thaliana. Here we characterize cellulose synthase motility in the model grass, Brachypodium distachyon. We generated lines in which mEGFP is fused N-terminal to BdCESA3 or BdCESA6 and which grew indistinguishably from the wild type (Bd21-3) and had dense fluorescent puncta at or near the plasma membrane. Measured with a particle tracking algorithm, the average speed of GFP-BdCESA3 particles in the mesocotyl was 164 ± 78 nm min−1 (error gives standard deviation [SD], n = 1451 particles). Mean speed in the root appeared similar. For comparison, average speed in the A. thaliana hypocotyl expressing GFP-AtCESA6 was 184 ± 86 nm min−1 (n = 2755). For B. distachyon, we quantified root diameter and elongation rate in response to inhibitors of cellulose (dichlorobenylnitrile; DCB), microtubules (oryzalin), or actin (latrunculin B). Neither oryzalin nor latrunculin affected the speed of CESA complexes; whereas, DCB reduced average speed by about 50% in B. distachyon and by about 35% in A. thaliana. Evidently, between these species, CESA motility is well conserved.

The polysaccharide cellulose is the main component of plant cell walls, so it is the most abundant polymer on the earth. While it is widely used in industry due to its remarkable properties, such as renewability and biodegradability, its biosynthesis is still not well understood. The large transmembrane protein complex responsible for synthesizing cellulose contains several cellulose synthase A (CESA) subunits that polymerize UDP glucose into the constituent glucan chains of cellulose. Here, we used variable angle epi-fluorescence microscopy in combination with single-particle tracking to characterize the motion of GFP labeled CESA complexes in the root and mesocotyl of Brachypodium distachyon seedlings that are a 3 to 4 days old. We show that CESA complexes move through the plasma membrane at approximately 165 nm/minute. Their motion is known to be guided by cortical microtubules, but no molecular motors are involved. Rather, the motion is thought to be driven by the polymerization and crystallization of the cellulose. A mean-squared displacement analysis shows that CESA complexes move diffusively on short time scales and undergo a transition to super-diffusive motion on a time scale of about 10 s. We also report on the effect of actin and microtubule inhibitors on CESA motion. This work was funded through NSF grant PHY- 1205989.