Aberrant mitochondrial DNA synthesis in macrophages exacerbates inflammation and atherosclerosis.

There is a large body of evidence that cellular metabolism governs inflammation, and that inflammation contributes to the progression of atherosclerosis. However, whether mitochondrial DNA synthesis affects macrophage function and atherosclerosis pathology is not fully understood. Here we show, by transcriptomic analyzes of plaque macrophages, spatial single cell transcriptomics of atherosclerotic plaques, and functional experiments, that mitochondrial DNA (mtDNA) synthesis in atherosclerotic plaque macrophages are triggered by vascular cell adhesion molecule 1 (VCAM-1) under inflammatory conditions in both humans and mice. Mechanistically, VCAM-1 activates C/EBPα, which binds to the promoters of key mitochondrial biogenesis genes - Cmpk2 and Pgc1a. Increased CMPK2 and PGC-1α expression triggers mtDNA synthesis, which activates STING-mediated inflammation. Consistently, atherosclerosis and inflammation are less severe in Apoe-/- mice lacking Vcam1 in macrophages. Downregulation of macrophage-specific VCAM-1 in vivo leads to decreased expression of LYZ1 and FCOR, involved in STING signalling. Finally, VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative damage to DNA. Collectively, our study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis pathology and proposes a self-acerbating pathway involving increased mtDNA synthesis.

Infection and chronic disease activate a systemic brain-muscle signaling axis.

Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show nonneural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood. We developed multiple models to investigate the impact of CNS stressors on motor function and found that Escherichia coli infections and SARS-CoV-2 protein expression caused reactive oxygen species (ROS) to accumulate in the brain. ROS induced expression of the cytokine Unpaired 3 (Upd3) in Drosophila and its ortholog, IL-6, in mice. CNS-derived Upd3/IL-6 activated the JAK-STAT pathway in skeletal muscle, which caused muscle mitochondrial dysfunction and impaired motor function. We observed similar phenotypes after expressing toxic amyloid-ß (Aß42) in the CNS. Infection and chronic disease therefore activate a systemic brain-muscle signaling axis in which CNS-derived cytokines bypass the connectome and directly regulate muscle physiology, highlighting IL-6 as a therapeutic target to treat disease-associated muscle dysfunction.

Myoglobin Inhibits Breast Cancer Cell Fatty Acid Oxidation and Migration via Heme-dependent Oxidant Production and Not Fatty Acid Binding.

The monomeric heme protein myoglobin (Mb), traditionally thought to be expressed exclusively in cardiac and skeletal muscle, is now known to be expressed in approximately 40% of breast tumors. While Mb expression is associated with better patient prognosis, the molecular mechanisms by which Mb limits cancer progression are unclear. In muscle, Mb's predominant function is oxygen storage and delivery, which is dependent on the protein's heme moiety. However, prior studies demonstrate that the low levels of Mb expressed in cancer cells preclude this function. Recent studies propose a novel fatty acid binding function for Mb via a lysine residue (K46) in the heme pocket. Given that cancer cells can upregulate fatty acid oxidation (FAO) to maintain energy production for cytoskeletal remodeling during cell migration, we tested whether Mb-mediated fatty acid binding modulates FAO to decrease breast cancer cell migration. We demonstrate that the stable expression of human Mb in MDA-MB-231 breast cancer cells decreases cell migration and FAO. Site-directed mutagenesis of Mb to disrupt Mb fatty acid binding did not reverse Mb-mediated attenuation of FAO or cell migration in these cells. In contrast, cells expressing Apo-Mb, in which heme incorporation was disrupted, showed a reversal of Mb-mediated attenuation of FAO and cell migration, suggesting that Mb attenuates FAO and migration via a heme-dependent mechanism rather than through fatty acid binding. To this end, we show that Mb's heme-dependent oxidant generation propagates dysregulated gene expression of migratory genes, and this is reversed by catalase treatment. Collectively, these data demonstrate that Mb decreases breast cancer cell migration, and this effect is due to heme-mediated oxidant production rather than fatty acid binding. The implication of these results will be discussed in the context of therapeutic strategies to modulate oxidant production and Mb in tumors. Highlights: Myoglobin (Mb) expression in MDA-MB-231 breast cancer cells slows migration.Mb expression decreases mitochondrial respiration and fatty acid oxidation.Mb-dependent fatty acid binding does not regulate cell migration or respiration.Mb-dependent oxidant generation decreases mitochondrial metabolism and migration.Mb-derived oxidants dysregulate migratory gene expression.

Codeveloping theories of change for improved community-based violence intervention evaluation.

BACKGROUND: Community-based violence intervention (CVI) programs are considered important strategies for preventing community violence and promoting health and safety. Mixed and inconclusive results from some prior CVI evaluations, as well as our general lack of understanding about the reasons for such varied findings, may be explained in part by misalignment of program theories of change and evaluation measures. Furthermore, most prior evaluations have focused solely on deficit-based outcomes; this narrow focus is inconsistent with the premise of CVI and may fail to capture improvements in health and well-being that are on the hypothesized pathway from intervention to violence reduction. METHODS: This article describes the process and results of codeveloping a theory of change for community-based youth firearm violence intervention and prevention programs in Washington state through a community-researcher partnership. We followed a multistep iterative process, involving (1) CVI program documentation review, (2) individual meetings, and (3) a day-long workshop. RESULTS: The theory of change included six key domains: (1) root causes, (2) promotive factors, (3) activities, (4) intermediate outcomes, (5) longer-term outcomes, and (6) multilevel context (youth/family, staff/organizational, community, and societal). Root causes were social and structural drivers of community violence. Promotive factors were assets and resources among the community, youth/their families, and community organizations that promote health and safety. Activities were supports and services the program provided to youth and their families, staff, and, potentially, the broader community. Intermediate and longer-term outcomes were the changes among youth, their families, staff, and the community that resulted from program activities. Intermediate outcomes may be felt within 6 months to 1 year, and longer-term outcomes may be felt after 1 to 2 years and beyond. CONCLUSION: The theory of change we codeveloped provides a common lens to conceptualize, compare, and evaluate CVI programs in Washington state and may support more rigorous and equity-centered evaluations. LEVEL OF EVIDENCE: Diagnostic Test/Criteria; Level V.

The Impact of Preoperative Weight Loss Timing on Surgical Outcomes in Total Hip Arthroplasty.

BACKGROUND: Elevated body mass index (BMI) increases surgical complications post-total hip arthroplasty (THA). However, the effects of rapid weight loss pre-THA remain unclear. This study evaluated patients who had initial BMIs between 40 and 50, and then achieved a BMI under 35 at various intervals before their THA. Comparisons were made with consistent obese and nonobese groups to understand potential complications. METHODS: Using a national database, we categorized THA patients based on initial BMI and weight loss timing before the surgery. These were contrasted with those maintaining a steady BMI of 20 to 30 or 40 to 50. We monitored outcomes like periprosthetic joint infections (PJI), surgical site infections (SSI), and noninfectious revisions for 2 years postsurgery, incorporating demographic considerations. Statistical analyses utilized Chi-square tests for categorical outcomes and Student's t-tests for continuous variables. RESULTS: Among patients who had a BMI of 45 to 50, weight loss 3 to 9 months presurgery increased PJI risks at 90 days (Odds Ratios [OR]: 2.15 to 5.22, P < .001). However, weight loss a year before the surgery lowered the PJI risk (OR: 0.14 to 0.27, P < .005). Constantly obese patients faced heightened PJI risks 1 to 2 years postsurgery (OR: 1.64 to 1.95, P < .015). Regarding SSI, risks increased with weight loss 3 to 9 months before surgery, but decreased when weight loss occurred a year earlier. In the BMI 40 to 45 group, weight loss 3 to 6 months presurgery showed higher PJI and SSI at 90 days (P < .001), with obese participants consistently at greater risk. CONCLUSIONS: While high BMI poses THA risks, weight loss timing plays a crucial role in postoperative complications. Weight loss closer to the surgery (0 to 9 months) can heighten risks, but shedding weight a year in advance seems beneficial. Conversely, initiating weight loss approximately a year before surgery offers potential protective effects against postoperative issues. This highlights the importance of strategic weight management guidance for patients considering THA, ensuring optimal surgical results and reducing potential adverse outcomes.

Immediate weightbearing is safe after revision total hip arthroplasty for Vancouver B2/B3 periprosthetic femur fractures.

INTRODUCTION: Modular fluted, tapered stems provide a reliable treatment for Vancouver B2/B3 fractures. Historically, these patients had weightbearing restrictions postoperatively. Although full immediate postoperative weightbearing may provide benefits in this patient population, stem subsidence is a concern. QUESTIONS/PURPOSES: The objective of this study was to investigate the effect of post-operative weight-bearing status on stem subsidence in patients treated with modular tapered stems for Vancouver B2 and B3 periprosthetic fractures. We sought to answer two questions: (1) Does full immediate postoperative weightbearing after revision total hip arthroplasty for periprosthetic femur fracture lead to increased stem subsidence compared to protected weightbearing? (2) Is there a mortality difference between these two groups of patients with different weightbearing restrictions? METHODS: From 2009 to 2015 all patients who underwent revision for Vancouver B2/B3 fractures were made non-weightbearing (NWB) for six weeks postoperatively. After 2015, immediate weightbearing as tolerated (WBAT) was allowed postoperatively. We compared stem subsidence between immediate postoperative and final radiographs. Additionally, we performed a Kaplan-Meijer analysis with one-year mortality as an endpoint. RESULTS: The final cohort included forty-seven patients with an average follow-up of 254 days. The average stem subsidence was 1.0 mm (95 % CI, 0.5-1.5 mm) in the NWB cohort and 0.3 mm (95 % CI, 0-0.7 mm) in the WBAT cohort (P = 0.10). In our survivorship analysis, we noted no deaths in the WBAT cohort compared to 17 % mortality in the NWB cohort at the one-year timepoint. CONCLUSION: Allowing patients to weight bear immediately after revision does not increase stem subsidence. Further studies are needed to determine whether early weightbearing provides a mortality benefit.

The Location of Biofilms on Chronic Prosthetic Joint Infections and the Ramifications for Clinical Practice.

Revision surgery is paramount to cure chronic prosthetic joint infections because these infections are associated with biofilms on prosthetics that conventional antibiotics cannot eradicate. However, there is a paucity of research on where in vivo biofilms are located on infected prosthetics. Consequently, the objective of this pilot study was to address this gap in knowledge by staining 5 chronically infected prosthetics, that were removed at the time of revision surgery, with methylene blue. Scanning electron microscopic images were then taken of the methylene blue-stained areas to visualize biofilms. The findings show that all chronically infected prosthetics had biofilms located on the bone-prosthetic interface, yet only 2 had biofilms also located on the prosthetic interface exposed to synovial fluid. Subsequently, this pilot study provides a pathophysiological understanding of why the current treatment paradigm for chronic periprosthetic joint infection requires a revision surgery and not debridement and an implant retention surgery.

The stability of Staphylococcal bacteriophage in presence of local vancomycin concentrations used in clinical practice.

PURPOSE: To evaluate the stability of a clinically used Staphylococcal bacteriophage with doses of vancomycin that are encountered with local administration of vancomycin for musculoskeletal infections. METHODS: A Staphylococcal bacteriophage was evaluated for stability in different pH ranges. Then that same bacteriophage was evaluated for stability with different concentrations of vancomycin and with vancomycin biodegradable antibiotic beads. RESULTS: The bacteriophage had stability within a pH range of 4-10. There was a statistically significant (P < 0.05) decrease in the amount of bacteriophage over 24 h for vancomycin concentrations of 10 mg/mL and 100 mg/mL compared to lower vancomycin concentrations (1 mg/mL, 0.1 mg/mL and normal saline). However, no statistically significant decrease in the amount of bacteriophage was seen with biodegradable vancomycin beads over 24 h. CONCLUSION: These findings have important clinical ramifications in that they show local administration of bacteriophages with concomitant local vancomycin powder therapy should be avoided. Moreover, these findings should spearhead further research into bacteriophage stability in in vivo environments.

TREM2 mediates MHCII-associated CD4+ T cell response against gliomas.

BACKGROUND: Myeloid cells comprise up to 50% of the total tumor mass in glioblastoma (GBM) and have been implicated in promoting tumor progression and immunosuppression. Modulating the response of myeloid cells to the tumor has emerged as a promising new approach for cancer treatment. In this regard, we focus on the Triggering Receptor Expressed on Myeloid cells 2 (TREM2), which has recently emerged as a novel immune modulator in peripheral tumors. METHODS: We studied the TREM2 expression profile in various patient tumor samples and conducted single-cell transcriptomic analysis in both glioblastoma patients and the GL261 mouse glioma model. We utilized multiple mouse glioma models and employed state-of-the-art techniques such as in vivo two-photon imaging, spectrum flow cytometry, and in vitro co-culture assays to study TREM2 function in myeloid cell-mediated phagocytosis of tumor cells, antigen presentation, and response of CD4+ T cells within the tumor hemispheres. RESULTS: Our research revealed significantly elevated levels of TREM2 expression in brain tumors compared to other types of tumors in patients. TREM2 was predominantly localized in tumor-associated myeloid cells and was highly expressed in nearly all microglia, as well as various subtypes of macrophages. Surprisingly, in pre-clinical glioma models, TREM2 deficiency did not confer a beneficial effect; instead, it accelerated glioma progression. Through detailed investigations, we determined that TREM2 deficiency impaired the ability of tumor-myeloid cells to phagocytose tumor cells and led to reduced expression of MHCII. This deficiency further significantly decreased the presence of CD4+ T cells within the tumor hemispheres. CONCLUSIONS: Our study unveiled a previously unrecognized protective role of tumor-myeloid TREM2. Specifically, we found TREM2 enhance the phagocytosis of tumor cells and promote an immune response by facilitating MHCII-associated CD4+ T cell responses against gliomas.

Salphage: Salvage bacteriophage therapy for a chronic Enterococcus faecalis prosthetic joint infection.

Chronic prosthetic joint infections are difficult to treat without conducting revision surgery because conventional antibiotics cannot eradicate bacteria that reside in biofilms. Consequently, novel therapeutics are needed to help treat prosthetic joint infections with one being bacteriophage therapy given its innate biofilm activity. Herein a sixty-nine-year-old man with a recalcitrant Enterococcus faecalis prosthetic joint infection is discussed. The patient was successfully treated with personalized bacteriophage therapy and after two years of follow up he has not had a clinical recurrence. Overall, this case report supports that bacteriophage therapy for prosthetic joint infections has promise to reduce the morbidity that is associated with current treatments. However, more research is needed to assess whether this therapeutic is helping eradicate infections or if it is making bacteria less pathogenic. This is an important point which will need to be evaluated as this therapeutic continues to be developed for all infections.

Spectral flow cytometry identifies distinct nonneoplastic plasma extracellular vesicle phenotype in glioblastoma patients.

Background: Glioblastoma (GBM) is the most common malignant brain tumor and has a poor prognosis. Imaging findings at diagnosis and in response to treatment are nonspecific. Developing noninvasive assays to augment imaging would be helpful. Plasma extracellular vesicles (EVs) are a promising biomarker source for this. Here, we develop spectral flow cytometry techniques that demonstrate differences in bulk plasma EV phenotype between GBM patients and normal donors that could serve as the basis of a liquid biopsy. Methods: Plasma EVs were stained for EV-associated tetraspanins (CD9/CD63/CD81), markers indicating cell of origin (CD11b/CD31/CD41a/CD45), and actin/phalloidin (to exclude cell debris). EVs were analyzed using spectral flow cytometry. Multiparametric analysis using t-distributed stochastic neighbor embedding (t-SNE) and self-organizing maps on flow cytometry data (FlowSOM) was performed comparing GBM and normal donor (ND) plasma EVs. Results: Size exclusion chromatography plus spectral-based flow cytometer threshold settings enriched plasma EVs while minimizing background noise. GBM patients had increased CD9+, CD63+, CD81+, and myeloid-derived (CD11b+) EVs. Multiparametric analysis demonstrated distinct surface marker expression profiles in GBM plasma EVs compared to ND EVs. Fifteen plasma EV sub-populations differing in size and surface marker expression were identified, six enriched in GBM patients and two in normal donors. Conclusions: Multiparametric analysis demonstrates that GBM patients have a distinct nonneoplastic plasma EV phenotype compared to ND. This simple rapid analysis can be performed without purifying tumor EVs and may serve as the basis of a liquid biopsy.

A Study of Physical Resilience and Aging (SPRING): Conceptual framework, rationale, and study design.

Understanding the physiological basis of physical resilience to clinical stressors is crucial for the well-being of older adults. This article presents a novel framework to discover the biological underpinnings of physical resilience in older adults as part of the "Characterizing Resiliencies to Physical Stressors in Older Adults: A Dynamical Physiological Systems Approach" study, also known as The Study of Physical Resilience and Aging (SPRING). Physical resilience, defined as the capacity of a person to withstand clinical stressors and quickly recover or improve upon a baseline functional level, is examined in adults aged 55 years and older by studying the dynamics of stress response systems. The hypothesis is that well-regulated stress response systems promote physical resilience. The study employs dynamic stimulation tests to assess energy metabolism, the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, and the innate immune system. Baseline characteristics influencing resilience outcomes are identified through deep phenotyping of physical and cognitive function, as well as of biological, environmental, and psychosocial characteristics. SPRING aims to study participants undergoing knee replacement surgery (n = 100), bone and marrow transplantation (n = 100), or anticipating dialysis initiation (n = 60). Phenotypic and functional measures are collected pre-stressor and at multiple times after stressor for up to 12 months to examine resilience trajectories. By improving our understanding of physical resilience in older adults, SPRING has the potential to enhance resilient outcomes to major clinical stressors. The article provides an overview of the study's background, rationale, design, pilot phase, implementation, and implications for improving the health and well-being of older adults.

Chemogenetic manipulation of CX3CR1+ cells transiently induces hypolocomotion independent of microglia.

Chemogenetic approaches using Designer Receptors Exclusively Activated by Designer Drugs (DREADD, a family of engineered GPCRs) were recently employed in microglia. Here, we used Cx3cr1CreER/+:R26hM4Di/+ mice to express Gi-DREADD (hM4Di) on CX3CR1+ cells, comprising microglia and some peripheral immune cells, and found that activation of hM4Di on long-lived CX3CR1+ cells induced hypolocomotion. Unexpectedly, Gi-DREADD-induced hypolocomotion was preserved when microglia were depleted. Consistently, specific activation of microglial hM4Di cannot induce hypolocomotion in Tmem119CreER/+:R26hM4Di/+ mice. Flow cytometric and histological analysis showed hM4Di expression in peripheral immune cells, which may be responsible for the hypolocomotion. Nevertheless, depletion of splenic macrophages, hepatic macrophages, or CD4+ T cells did not affect Gi-DREADD-induced hypolocomotion. Our study demonstrates that rigorous data analysis and interpretation are needed when using Cx3cr1CreER/+ mouse line to manipulate microglia.

Brain resident memory T cells rapidly expand and initiate neuroinflammatory responses following CNS viral infection.

The contribution of circulating verses tissue resident memory T cells (TRMs) to clinical neuropathology is an enduring question due to a lack of mechanistic insights. The prevailing view is TRMs are protective against pathogens in the brain. However, the extent to which antigen-specific TRMs induce neuropathology upon reactivation is understudied. Using the described phenotype of TRMs, we found that brains of naïve mice harbor populations of CD69+ CD103- T cells. Notably, numbers of CD69+ CD103- TRMs rapidly increase following neurological insults of various origins. This TRM expansion precedes infiltration of virus antigen-specific CD8 T cells and is due to proliferation of T cells within the brain. We next evaluated the capacity of antigen-specific TRMs in the brain to induce significant neuroinflammation post virus clearance, including infiltration of inflammatory myeloid cells, activation of T cells in the brain, microglial activation, and significant blood brain barrier disruption. These neuroinflammatory events were induced by TRMs, as depletion of peripheral T cells or blocking T cell trafficking using FTY720 did not change the neuroinflammatory course. Depletion of all CD8 T cells, however, completely abrogated the neuroinflammatory response. Reactivation of antigen-specific TRMs in the brain also induced profound lymphopenia within the blood compartment. We have therefore determined that antigen-specific TRMs can induce significant neuroinflammation, neuropathology, and peripheral immunosuppression. The use of cognate antigen to reactivate CD8 TRMs enables us to isolate the neuropathologic effects induced by this cell type independently of other branches of immunological memory, differentiating this work from studies employing whole pathogen re-challenge. This study also demonstrates the capacity for CD8 TRMs to contribute to pathology associated with neurodegenerative disorders and long-term complications associated with viral infections. Understanding functions of brain TRMs is crucial in investigating their role in neurodegenerative disorders including MS, CNS cancers, and long-term complications associated with viral infections including COVID-19.

Tobacco and Cannabis Use Have a Synergistic Association on Infection Risk Following Total Knee Arthroplasty.

BACKGROUND: Studies suggest an increase in the number of combined users of tobacco and cannabis. Therefore, we specifically assessed tobacco, cannabis, and combined users who underwent primary total knee arthroplasty (TKA) to determine 90-day to 2-year: (1) odds of periprosthetic joint infection; (2) odds of revision; and (3) medical complications. METHODS: We queried a national, all payer database of patients undergoing primary TKA between 2010 and 2020. Patients were stratified according to current use of tobacco products (n = 30,000), cannabis (n = 400), or a combination (n = 3,526). These were defined according to International Classification of Disease codes, Ninth and Tenth Editions. Patients were tracked from the 2 years before TKA through 2 years afterwards. A fourth group of TKA recipients who did not have tobacco nor cannabis use was used as a matching cohort. Periprosthetic joint infections (PJIs), revisions, and other medical/surgical complications from 90 days through 2 years were evaluated between these cohorts using bivariate analyses. Multivariate analyses assessed independent risk factors for PJI at 90 days through 2 years, adjusted for patient demographics and health metrics. RESULTS: Combined tobacco and cannabis use were associated with the highest rates of PJI following TKA. The odds of 90-day PJI risk among cannabis, tobacco, and combined users was 1.60, 2.14, and 3.39, respectively, as compared to the matched cohort (P < .001). Co-users had the highest and significantly increased revision odds at 2 years following TKA (odds ratio = 1.52, 95% confidence interval, 1.15 to 2.00). At 1 and 2 years following TKA, cannabis, tobacco, and co-users had higher rates of myocardial infarctions, respiratory failures, surgical site infections, and manipulations under anesthesia when compared to the matched cohort (all P < .001). CONCLUSION: Tobacco and cannabis use before primary TKA demonstrated a synergistic association on PJI risk from 90 days through 2 years. Although the harms of tobacco use are well-known, this additional knowledge about cannabis should be incorporated in the shared decision-making discussions in the pre-operative setting to best prepare for expected risks following primary TKA.

Systemic immune derangements are shared across various CNS pathologies and reflect novel mechanisms of immune privilege.

Background: The nervous and immune systems interact in a reciprocal manner, both under physiologic and pathologic conditions. Literature spanning various CNS pathologies including brain tumors, stroke, traumatic brain injury and de-myelinating diseases describes a number of associated systemic immunologic changes, particularly in the T-cell compartment. These immunologic changes include severe T-cell lymphopenia, lymphoid organ contraction, and T-cell sequestration within the bone marrow. Methods: We performed an in-depth systematic review of the literature and discussed pathologies that involve brain insults and systemic immune derangements. Conclusions: In this review, we propose that the same immunologic changes hereafter termed 'systemic immune derangements', are present across CNS pathologies and may represent a novel, systemic mechanism of immune privilege for the CNS. We further demonstrate that systemic immune derangements are transient when associated with isolated insults such as stroke and TBI but persist in the setting of chronic CNS insults such as brain tumors. Systemic immune derangements have vast implications for informed treatment modalities and outcomes of various neurologic pathologies.

Presumptive Diagnosis in Tele-Health Laryngology: A Multi-Center Observational Study.

OBJECTIVES: Early in the COVID-19 pandemic, outpatient visits were adapted for the virtual setting, forcing laryngologists to presume certain diagnoses without the aid of laryngoscopy, solely based on history and the limited physical exam available via video visit. This study aims to examine the accuracy of presumptive diagnoses made via telemedicine, compared to subsequent in-person follow up, where endoscopic examination could confirm or refute suspected diagnoses. METHODS: A retrospective chart review was conducted of 38 patients evaluated for voice-related issues at NYU Langone Health and the University of California-San Francisco. Presumptive diagnoses at the initial telemedicine encounter were noted, along with diagnostic cues used for clinical reasoning and recommended treatment plans. These presumptive diagnoses were compared to diagnoses and plans established following laryngoscopy at follow-up in-person visits. RESULTS: After laryngoscopy at the first in-person visit, 38% of presumptive diagnoses changed, as did 37% of treatment plans. The accuracy varied among conditions. Muscle tension dysphonia and Reinke's edema were accurately diagnosed without laryngoscopy, but other conditions, including vocal fold paralysis and subglottic stenosis, were not initially suspected, relying on laryngoscopy for diagnosis. CONCLUSIONS: While some laryngologic conditions may be reasonably identified without in-person examination, laryngoscopy remains central to definitive diagnosis and treatment. Telemedicine can increase access to care, but it may provide more utility as a screening tool, triaging which patients should present more urgently for in-person laryngoscopy. LEVEL OF EVIDENCE: 4.

TREM2 mediates MHCII-associated CD4+ T cell response against gliomas.

Triggering receptor expressed on myeloid cells 2 (TREM2) was recently highlighted as a novel immune suppressive marker in peripheral tumors. The aim of this study was to characterize TREM2 expression in gliomas and investigate its contribution in glioma progression by using Trem2-/- mouse line. Our results showed that higher TREM2 expression was correlated with poor prognosis in glioma patients. Unexpectedly, TREM2 deficiency did not have a beneficial effect in a pre-clinical model of glioma. The increased TREM2 expression in glioma was likely due to increased myeloid cell infiltration, as evidenced by our single-cell analysis showing that almost all microglia and macrophages in gliomas were TREM2+. Furthermore, we found that deficiency of TREM2 impaired tumor-myeloid phagocytosis and MHCII presentation, and significantly reduced CD4+ T cells in tumor hemispheres. Our results revealed a previously unrecognized protective role of tumor-myeloid TREM2 in promoting MHCII-associated CD4+ T cell response against gliomas.

Anti-PD-1 and Extended Half-life IL2 Synergize for Treatment of Murine Glioblastoma Independent of Host MHC Class I Expression.

Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM. This study aimed to determine whether supplementing anti-PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I-restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I-independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-derived Xenograft Mouse Model.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.