Peripheral Immune Profiles Predict ALS Progression in an Age- and Sex-Dependent Manner.

BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease whose pathobiology associates with peripheral blood immune cell levels and activation patterns in an age and sex-dependent manner. This study's objective was to identify immune profile associations with ALS progression, whether the associations are age and sex-specific, and whether immune profiles can predict a future disease course. METHODS: Flow cytometry immune profiles (a combination of 22 peripheral blood immune markers) were generated for 241 participants with ALS and linked to ALS progression, using progression-free survival, which is a composite combining the revised ALS Functional Rating Scale and survival. Participants were first grouped by immune profiles using unsupervised hierarchical clustering, and clusters were associated with subsequent progression-free survival. Next, individual immune markers were associated with progression-free survival using least absolute shrinkage and selection operator-Cox regression. Analyses were stratified by age and sex to identify demographic-specific immune mechanisms. Finally, random forest determined the predictive power of immune profiles on ALS progression in the whole population and again stratified by age and sex. RESULTS: Progression-free survival differed between clusters of participants with similar immune profiles, particularly reduced natural killer (NK)-cell activation associated with slower progression. Individual markers such as neutrophil levels and NK-cell NKp46 expression associated with faster ALS progression while overall NK-cell levels and NK-cell subpopulations associated with slower progression; the strength of these associations varied by age and sex. Adding these immune markers to prediction models dramatically increased short-term prediction compared with routine clinical prognostic variables alone, and the addition of NK-cell markers further improved the prediction accuracy in female participants. DISCUSSION: Specific immune profiles likely contribute to ALS progression in an age and sex-dependent manner, and peripheral immune markers enhance the prediction of short-term clinical outcomes. These findings suggest a complex milieu of immune profiles associated with ALS progression, and more detailed immunophenotyping in ALS will facilitate personalized immunotherapeutics in ALS.

The Evolving Role of Postgraduate Year 7 in Neurological Surgery Residency.

BACKGROUND AND OBJECTIVES: In 2013, all neurosurgery programs were mandated to adopt a 7-year structure. We sought to characterize how programs use the seventh year of training (postgraduate year 7 [PGY7]). METHODS: We surveyed all accredited neurosurgery programs in the United States regarding the PGY7 residents' primary role and the availability of enfolded fellowships. We compiled responses from different individuals in each program: chair, program director, program coordinator, and current chiefs. RESULTS: Of 120 accredited neurological surgery residency programs within the United States, 91 (76%) submitted responses. At these programs, the primary roles of the PGY7 were chief of service (COS, 71%), enfolded fellowships (EFF, 18%), transition to practice (10%), and elective time (1%). Most residencies have been 7-year programs for >10 years (52, 57%). Sixty-seven programs stated that they offer some form of EFF (73.6%). The most common EFFs were endovascular (57, 62.6%), spine (49, 53.9%), critical care (41, 45.1%), and functional (37, 40.7%). These were also the most common specialties listed as Committee on Advanced Subspecialty Training accredited by survey respondents. Spine and endovascular EFFs were most likely to be restricted to PGY7 (24.2% and 23.1%, respectively), followed by neuro-oncology (12, 13.2%). The most common EFFs reported as Committee on Advanced Subspecialty Training accredited but not restricted to PGY7 were endovascular (24, 26.4%) and critical care (23, 25.3%). CONCLUSION: Most accredited neurological surgery training programs use the COS as the primary PGY7 role. Programs younger in their PGY7 structure seem to maintain the traditional COS role. Those more established seem to be experimenting with various roles the PGY7 year can fill, including enfolded fellowships and transition-to-practice years, predominantly. Most programs offer some form of enfolded fellowship. This serves as a basis for characterization of how neurological surgery training may develop in years to come.

Cytokine Modification of Adoptive Chimeric Antigen Receptor Immunotherapy for Glioblastoma.

Chimeric antigen receptor (CAR) cell-based therapies have demonstrated limited success in solid tumors, including glioblastoma (GBM). GBMs exhibit high heterogeneity and create an immunosuppressive tumor microenvironment (TME). In addition, other challenges exist for CAR therapy, including trafficking and infiltration into the tumor site, proliferation, persistence of CARs once in the tumor, and reduced functionality, such as suboptimal cytokine production. Cytokine modification is of interest, as one can enhance therapy efficacy and minimize off-target toxicity by directly combining CAR therapy with cytokines, antibodies, or oncolytic viruses that alter cytokine response pathways. Alternatively, one can genetically modify CAR T-cells or CAR NK-cells to secrete cytokines or express cytokines or cytokine receptors. Finally, CARs can be genetically altered to augment or suppress intracellular cytokine signaling pathways for a more direct approach. Codelivery of cytokines with CARs is the most straightforward method, but it has associated toxicity. Alternatively, combining CAR therapy with antibodies (e.g., anti-IL-6, anti-PD1, and anti-VEGF) or oncolytic viruses has enhanced CAR cell infiltration into GBM tumors and provided proinflammatory signals to the TME. CAR T- or NK-cells secreting cytokines (e.g., IL-12, IL-15, and IL-18) have shown improved efficacy within multiple GBM subtypes. Likewise, expressing cytokine-modulating receptors in CAR cells that promote or inhibit cytokine signaling has enhanced their activity. Finally, gene editing approaches are actively being pursued to directly influence immune signaling pathways in CAR cells. In this review, we summarize these cytokine modification methods and highlight any existing gaps in the hope of catalyzing an improved generation of CAR-based therapies for glioblastoma.

Bi-Specific Killer Cell Engager Enhances NK Cell Activity against Interleukin-13 Receptor Alpha-2 Positive Gliomas.

Glioblastoma (GBM) is a lethal brain tumor with limited therapeutic options. Bi-specific killer cell engagers (BiKEs) are novel immunotherapies designed to engage natural killer (NK) cells against cancer. We designed a BiKE molecule consisting of a single-domain CD16 antibody, an interleukin-15 linker, and a single-chain variable antibody against the glioma-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2). Recombinant BiKE protein was expressed in HEK cells and purified. Flow cytometric analysis of co-cultures of peripheral blood-derived NK cells with GBM6 and GBM39 patient-derived xenograft lines revealed significantly increased activation of NK cells (CD25+CD69+) and increased glioma cell killing following BiKE treatment compared to controls (n = 4, p < 0.01). Glioma cell killing was also confirmed via immunofluorescence staining for cleaved caspase-3 (p < 0.05). In vivo, intracranial delivery of NK cells with BiKE extended median survival in mice bearing GBM6 (p < 0.01) and GBM12 (p < 0.01) tumors compared to controls. Finally, histological analysis of brain tissues revealed a higher frequency of peritumoral NK cells in mice treated with BiKE than with NK cells alone (p < 0.05). In conclusion, we demonstrate that a BiKE generated in a mammalian expression system is functional in augmenting NK cell targeting of IL13Rα2-positive gliomas.

Modeling glioblastoma complexity with organoids for personalized treatments.

Glioblastoma (GBM) remains a fatal diagnosis despite the current standard of care of maximal surgical resection, radiation, and temozolomide (TMZ) therapy. One aspect that impedes drug development is the lack of an appropriate model representative of the complexity of patient tumors. Brain organoids derived from cell culture techniques provide a robust, easily manipulatable, and high-throughput model for GBM. In this review, we highlight recent progress in developing GBM organoids (GBOs) with a focus on generating the GBM microenvironment (i.e., stem cells, vasculature, and immune cells) recapitulating human disease. Finally, we also discuss the use of organoids as a screening tool in drug development for GBM.

Ventricular Volume Change as a Predictor of Shunt-Dependent Hydrocephalus in Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Hydrocephalus is a common complication of aneurysmal subarachnoid hemorrhage (aSAH) that often requires acute placement of an external ventricular drain (EVD). The current systems available for determining which patients will require long-term cerebrospinal fluid diversion remain subjective. We investigated the ventricular volume change (ΔVV) after EVD clamping as an objective predictor of shunt dependence in patients with aSAH. METHODS: We performed a retrospective medical record review and image analysis of patients treated for aSAH at a single academic institution who had required EVD placement for acute hydrocephalus and had undergone 1 EVD weaning trial. Head computed tomography (CT) scans obtained before and after EVD clamping were analyzed using a custom semiautomated MATLAB program (MathWorks, Natick, Massachusetts, USA), which segments each CT scan into 5 tissue types using k-means clustering. Differences in the pre- and postclamp ventricular volumes were calculated. RESULTS: A total of 34 patients with an indwelling shunt met the inclusion criteria and were sex- and age-matched to 34 controls without a shunt. The mean ΔVV was 19.8 mL in the shunt patients and 3.8 mL in the nonshunt patients (P < 0.0001). The area under the receiver operating characteristic curve was 0.84. The optimal ΔVV threshold was 11.4 mL, with a sensitivity of 76.5% and specificity of 88.2% for predicting shunt dependence. The mean ΔVV was significantly greater for the patients readmitted for shunt placement compared with the patients not requiring cerebrospinal fluid diversion (18.69 mL vs. 3.84 mL; P = 0.005). Finally, 70% of the patients with delayed shunt dependence had ΔVV greater than the identified threshold. CONCLUSIONS: The ΔVV volume between head CT scans taken before and after EVD clamping was predictive of early and delayed shunt dependence.

NK cells associate with ALS in a sex- and age-dependent manner.

NK cells are innate immune cells implicated in ALS; whether NK cells impact ALS in a sex- and age-specific manner was investigated. Herein, NK cells were depleted in male and female SOD1G93A ALS mice, survival and neuroinflammation were assessed, and data were stratified by sex. NK cell depletion extended survival in female but not male ALS mice with sex-specific effects on spinal cord microglia. In humans, NK cell numbers, NK cell subpopulations, and NK cell surface markers were examined in prospectively blood collected from subjects with ALS and control subjects; longitudinal changes in these metrics were correlated to revised ALS functional rating scale (ALSFRS-R) slope and stratified by sex and age. Expression of NK cell trafficking and cytotoxicity markers was elevated in subjects with ALS, and changes in CXCR3+ NK cells and 7 trafficking and cytotoxicity markers (CD11a, CD11b, CD38, CX3CR1, NKG2D, NKp30, NKp46) correlated with disease progression. Age affected the associations between ALSFRS-R and markers NKG2D and NKp46, whereas sex impacted the NKp30 association. Collectively, these findings suggest that NK cells contribute to ALS progression in a sex- and age-specific manner and demonstrate that age and sex are critical variables when designing and assessing ALS immunotherapy.

Temporal evolution of the microbiome, immune system and epigenome with disease progression in ALS mice.

Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease. Genetic predisposition, epigenetic changes, aging and accumulated life-long environmental exposures are known ALS risk factors. The complex and dynamic interplay between these pathological influences plays a role in disease onset and progression. Recently, the gut microbiome has also been implicated in ALS development. In addition, immune cell populations are differentially expanded and activated in ALS compared to healthy individuals. However, the temporal evolution of both the intestinal flora and the immune system relative to symptom onset in ALS is presently not fully understood. To better elucidate the timeline of the various potential pathological factors, we performed a longitudinal study to simultaneously assess the gut microbiome, immunophenotype and changes in ileum and brain epigenetic marks relative to motor behavior and muscle atrophy in the mutant superoxide dismutase 1 (SOD1G93A) familial ALS mouse model. We identified alterations in the gut microbial environment early in the life of SOD1G93A animals followed by motor dysfunction and muscle atrophy, and immune cell expansion and activation, particularly in the spinal cord. Global brain cytosine hydroxymethylation was also altered in SOD1G93A animals at disease end-stage compared to control mice. Correlation analysis confirmed interrelationships with the microbiome and immune system. This study serves as a starting point to more deeply comprehend the influence of gut microorganisms and the immune system on ALS onset and progression. Greater insight may help pinpoint novel biomarkers and therapeutic interventions to improve diagnosis and treatment for ALS patients.This article has an associated First Person interview with the joint first authors of the paper.