Nebulized platelet-derived extracellular vesicles attenuate chronic cigarette smoke-induced murine emphysema.

Chronic obstructive pulmonary disease (COPD) is a prevalent lung disease usually resulting from cigarette smoking (CS). Cigarette smoking induces oxidative stress, which causes inflammation and alveolar epithelial cell apoptosis and represents a compelling therapeutic target for COPD. Purified human platelet-derived exosome product (PEP) is endowed with antioxidant enzymes and immunomodulatory molecules that mediate tissue repair. In this study, a murine model of CS-induced emphysema was used to determine whether nebulized PEP can influence the development of CS-induced emphysema through the mitigation of oxidative stress and inflammation in the lung. Nebulization of PEP effectively delivered the PEP vesicles into the alveolar region, with evidence of their uptake by type I and type II alveolar epithelial cells and macrophages. Lung function testing and morphometric assessment showed a significant attenuation of CS-induced emphysema in mice treated with nebulized PEP thrice weekly for 4 weeks. Whole lung immuno-oncology RNA sequencing analysis revealed that PEP suppressed several CS-induced cell injuries and inflammatory pathways. Validation of inflammatory cytokines and apoptotic protein expression on the lung tissue revealed that mice treated with PEP had significantly lower levels of S100A8/A9 expressing macrophages, higher levels of CD4+/FOXP3+ Treg cells, and reduced NF-κB activation, inflammatory cytokine production, and apoptotic proteins expression. Further validation using in vitro cell culture showed that pretreatment of alveolar epithelial cells with PEP significantly attenuated CS extract-induced apoptotic cell death. These data show that nebulization of exosomes like PEP can effectively deliver exosome cargo into the lung, mitigate CS-induced emphysema in mice, and suppress oxidative lung injury, inflammation, and apoptotic alveolar epithelial cell death.

Use of heparin to rescue immunosuppressive monocyte reprogramming by glioblastoma-derived extracellular vesicles.

OBJECTIVE: The profound immunosuppression found in glioblastoma (GBM) patients is a critical barrier to effective immunotherapy. Multiple mechanisms of tumor-mediated immune suppression exist, and the induction of immunosuppressive monocytes such as myeloid-derived suppressor cells (MDSCs) is increasingly appreciated as a key part of this pathology. GBM-derived extracellular vesicles (EVs) can induce the formation of MDSCs. The authors sought to identify the molecular consequences of these interactions in myeloid cells in order to identify potential targets that could pharmacologically disrupt GBM EV-monocyte interaction as a means to ameliorate tumor-mediated immune suppression. Heparin-sulfate proteoglycans (HSPGs) are a general mechanism by which EVs come into association with their target cells, and soluble heparin has been shown to interfere with EV-HSPG interactions. The authors sought to assess the efficacy of heparin treatment for mitigating the effects of GBM EVs on the formation of MDSCs. METHODS: GBM EVs were collected from patient-derived cell line cultures via staged ultracentrifugation and cocultured with monocytes collected from apheresis cones from healthy blood donors. RNA was isolated from EV-conditioned and unconditioned monocytes after 72 hours of coculture, and RNA-sequencing analysis performed. For the heparin treatment studies, soluble heparin was added at the time of EV-monocyte coculture and flow cytometry analysis was performed 72 hours later. After the initial EV-monocyte coculture period, donor-matched T-cell coculture studies were performed by adding fluorescently labeled and stimulated T cells for 5 days of coculture. RESULTS: Transcriptomic analysis of GBM EV-treated monocytes demonstrated downregulation of several important immunological and metabolic pathways, with upregulation of the pathways associated with synthesis of cholesterol and HSPG. Heparin treatment inhibited association between GBM EVs and monocytes in a dose-dependent fashion, which resulted in a concomitant reduction in MDSC formation (p < 0.01). The authors further demonstrated that reduced MDSC formation resulted in a partial rescue of immune suppression, as measured by effects on activated donor-matched T cells (p < 0.05). CONCLUSIONS: The authors demonstrated that GBM EVs induce broad but reproducible reprogramming in monocytes, with enrichment of pathways that may portend an immunosuppressive phenotype. The authors further demonstrated that GBM EV-monocyte interactions are potentially druggable targets for overcoming tumor-mediated immune suppression, with heparin inhibition of EV-monocyte interactions demonstrating proof of principle.

Platelet-derived exosomes induce cell proliferation and wound healing in human endometrial cells.

Aim: To investigate the regenerative effects of a platelet-derived purified exosome product (PEP) on human endometrial cells. Materials & methods: Endometrial adenocarcinoma cells (HEC-1A), endometrial stromal cells (T HESC) and menstrual blood-derived stem cells (MenSC) were assessed for exosome absorption and subsequent changes in cell proliferation and wound healing properties over 48 h. Results: Cell proliferation increased in PEP treated T HESC (p < 0.0001) and MenSC (p < 0.001) after 6 h and in HEC-1A (p < 0.01) after 12 h. PEP improved wound healing after 6 h in HEC-1A (p < 0.01) and MenSC (p < 0.0001) and in T HESC between 24 and 36 h (p < 0.05). Conclusion: PEP was absorbed by three different endometrial cell types. PEP treatment increased cell proliferation and wound healing capacity.

The uterus has a remarkable ability to heal itself. Every month the inside lining of the uterus grows in preparation for pregnancy and sheds if no pregnancy occurs. Unfortunately, this cycle of growth, shedding and repair can be injured and lead to menstrual changes, pain or even infertility. In this study, we looked how special cell messengers ­ called exosomes ­ could help uterine cells. Exosomes are special messengers that contain substances to help the body heal and regenerate injured cells and tissues. We obtained exosomes created from human transfusion-grade platelets. We studied the exosomes' effects in three different cell types that all are important inside the uterine lining. Specifically, we studied the ability of the exosomes to help cells proliferate and migrate into a wound. In this study, exosomes were recognized by the human endometrial cells and were absorbed. Once they were inside the cells, they increased cell proliferation as well as the ability of the cells to heal a scratch wound. Furthermore, the more exosomes we presented to the cells, the more the cells were able to proliferate and move into a wound for healing. These findings lay the groundwork for future studies in animal models of uterine injury.

Phase I trial of adjuvant mature autologous dendritic cell/allogeneic tumor lysate vaccines in combination with temozolomide in newly diagnosed glioblastoma.

Background: Glioblastoma (GBM) has poor prognosis despite aggressive treatment. Dendritic cell (DC) vaccines are promising, but widespread clinical use has not been achieved, possibly reflecting manufacturing issues of antigen choice and DC potency. We previously optimized vaccine manufacture utilizing allogeneic human GBM tumor cell lysate and potent, mature autologous DCs. Here, we report a phase I study using this optimized DC vaccine in combination with standard therapy. Methods: Following surgical resection and radiation with concurrent temozolomide (TMZ), newly diagnosed adult GBM patients received intradermal DC vaccines plus TMZ. Primary endpoints were safety and feasibility. Immune and treatment responses were recorded. Results: Twenty-one patients were enrolled in this study. One progressed between leukapheresis and vaccine manufacture. Twenty patients received treatment per protocol. Vaccine doses (≥15) were generated following a single leukapheresis for each patient. No dose-limiting vaccine toxicities were encountered. One patient had symptomatic, histologically proven pseudoprogression. Median progression-free survival was 9.7 months. Median overall survival was 19 months. Overall survival was 25% at 2 years and 10% at 4 years. One patient remains progression-free 5 years after enrollment. Specific CD8 T-cell responses for the tumor-associated antigen gp100 were seen post-vaccination. Patients entered the trial with a leukocyte deficit compared to healthy donors which partly normalized over the course of therapy. Conclusions: This vaccine platform is safe and highly feasible in combination with standard therapy for newly diagnosed patients. Imaging, histological, survival, and immunological data suggest a positive biological response to therapy that warrants further investigation.

STAT3 is a biologically relevant therapeutic target in H3K27M-mutant diffuse midline glioma.

BACKGROUND: H3K27M-mutant diffuse midline glioma (DMG) is a lethal brain tumor that usually occurs in children. Despite advances in our understanding of its underlying biology, efficacious therapies are severely lacking. METHODS: We screened a library of drugs either FDA-approved or in clinical trial using a library of patient-derived H3K27M-mutant DMG cell lines with cell viability as the outcome. Results were validated for clinical relevance and mechanistic importance using patient specimens from biopsy and autopsy, patient-derived cell lines, inhibition by gene knockdown and small molecule inhibitors, and patient-derived xenografts. RESULTS: Kinase inhibitors were highly toxic to H3K27M-mutant DMG cells. Within this class, STAT3 inhibitors demonstrated robust cytotoxic activity in vitro. Mechanistic analyses revealed one form of activated STAT3, phospho-tyrosine- 705 STAT3 (pSTAT3), was selectively upregulated in H3K27M-mutant cell lines and clinical specimens. STAT3 inhibition by CRISPR/Cas9 knockout, shRNA or small molecule inhibition reduced cell viability in vitro, and partially restored expression of the polycomb repressive mark H3K27me3, which is classically lost in H3K27M-mutant DMG. Putative STAT3-regulated genes were enriched in an H3K27M-knockout DMG cell line, indicating relative gain of STAT3 signaling in K27M-mutant cells. Treatment of patient-derived intracranial xenografts with WP1066, a STAT3 pathway inhibitor currently in clinical use for pediatric brain tumors, resulted in stasis of tumor growth, and increased overall survival. Finally, pSTAT3(Y705) was detected in circulating plasma extracellular vesicles of patients with H3K27M-mutant DMG. CONCLUSIONS: STAT3 is a biologically relevant therapeutic target in H3K27M-mutant DMG. STAT3 inhibition should be considered in future clinical trials.

Pediatric brain tumor cell lines exhibit miRNA-depleted, Y RNA-enriched extracellular vesicles.

BACKGROUND: Medulloblastoma (MB) and diffuse infiltrative pontine glioma (DIPG) are malignant pediatric tumors. Extracellular vesicles (EVs) and their bioactive cargoes have been implicated in tumorigenesis. Most studies have focused on adult tumors, therefore the role of EVs and the noncoding RNA (ncRNA) landscape in pediatric brain tumors is not fully characterized. The overall aim of this pilot study was to isolate EVs from MB and DIPG patient-derived cell lines and to explore the small ncRNA transcriptome. METHODS: EVs from 3 DIPG and 4 MB patient-derived cell lines were analyzed. High-throughput next generation sequencing interrogated the short non-coding RNA (ncRNA) transcriptome. Known and novel miRNAs were quantified. Differential expression analysis, in silico target prediction, and functional gene enrichment were performed. RESULTS: EV secretomes from MB and DIPG patient-derived cell lines demonstrated discrete ncRNA biotypes. Notably, miRNAs were depleted and Y RNAs were enriched in EV samples. Hierarchical cluster analysis revealed high discrimination in miRNA expression between DIPG and MB cell lines and RNA-Seq identified novel miRNAs not previously implicated in MB or DIPG pathogenesis. Known and putative target genes of dysregulated miRNAs were identified. Functional annotation analysis of the target genes for differentially expressed EV-and parental-derived miRNAs revealed significant cancer-related pathway involvement. CONCLUSIONS: This hypothesis-generating study demonstrated that pediatric brain tumor-derived cell lines secrete EVs comprised of various ncRNA cargoes. Validation of these findings in patient samples may provide new insights into the pediatric brain tumor microenvironment and identification of novel therapeutic candidates.

Administration of Purified Exosome Product in a Rat Sciatic Serve Reverse Autograft Model.

BACKGROUND: The nerve autograft remains the gold standard when reconstructing peripheral nerve defects. However, although autograft repair can result in useful functional recovery, poor outcomes are common, and better treatments are needed. The purpose of this study was to evaluate the effect of purified exosome product on functional motor recovery and nerve-related gene expression in a rat sciatic nerve reverse autograft model. METHODS: Ninety-six Sprague-Dawley rats were divided into three experimental groups. In each group, a unilateral 10-mm sciatic nerve defect was created. The excised nerve was reversed and used to reconstruct the defect. Group I animals received the reversed autograft alone, group II animals received the reversed autograft with fibrin glue, and group III animals received the reversed autograft with purified exosome product suspended in the fibrin glue. The animals were killed at 3 and 7 days and 12 and 16 weeks after surgery. Evaluation included compound muscle action potentials, isometric tetanic force, tibialis anterior muscle wet weight, nerve regeneration-related gene expression, and nerve histomorphometry. RESULTS: At 16 weeks, isometric tetanic force was significantly better in group III (p = 0.03). The average axon diameter of the peroneal nerve was significantly larger in group III at both 12 and 16 weeks (p = 0.015 at 12 weeks; p < 0.01 at 16 weeks). GAP43 and S100b gene expression was significantly up-regulated by purified exosome product. CONCLUSIONS: Local administration of purified exosome product demonstrated improved nerve regeneration profiles in the reverse sciatic nerve autograft rat model. Thus, purified exosome product may have beneficial effects on nerve regeneration, gene profiles, and motor outcomes.

A Vanishing Cecal Mass: A Rare Gastrointestinal Manifestation of Systemic Mastocytosis.

Systemic mastocytosis (SM) is a heterogeneous disease that often involves the gastrointestinal (GI) tract. Activation and accumulation of mast cells in GI organs can result in symptoms of abdominal pain, nausea and diarrhea along with organ damage with more aggressive disease. Mast cell degranulation can also result in anaphylactic reactions, which can be life-threatening. Recognition of GI manifestations is important for gastroenterologists to aid in diagnosis and management when GI involvement is suspected. Edema, small nodules, urticarial lesions and occasionally ulceration in the small bowel and colon are the most commonly described endoscopic findings. Here we describe a case of SM presenting as a large colonic mass and provide a brief review of the literature on GI involvement of SM.

Novel strategy for manufacturing autologous dendritic cell/allogeneic tumor lysate vaccines for glioblastoma.

BACKGROUND: Glioblastoma, the most common primary malignant brain tumor, is nearly universally fatal by 5 years. Dendritic cell vaccines are promising but often limited clinically by antigen choice, dendritic cell potency, and/or manufacturing yield. We optimized vaccine manufacture, generating potent mature autologous dendritic cells pulsed with allogeneic glioblastoma lysates. METHODS: Platelet lysate-based supplement was used to establish human glioblastoma cell lines. Phenotype and genotype were assessed. An improved culture technique to generate mature dendritic cells from glioblastoma patients' monocytes was developed. The ability of T cells stimulated with autologous dendritic cells pulsed with allogeneic glioblastoma cell lysate to kill HLA-A2-matched glioblastoma cells was assessed. RESULTS: Glioblastoma cell lines established with platelet lysate supplement grew faster and expressed more stem-like markers than lines grown in neural stem cell media or in the presence of serum. They expressed a variety of glioma-associated antigens and had genomic abnormalities characteristic of glioblastoma stable up to 15 doublings. Unlike standard culture techniques, our optimized technique produced high levels of mature dendritic cells from glioblastoma patients' monocytes. Autologous T cells stimulated with mature dendritic cells pulsed with allogeneic glioblastoma cell line lysate briskly killed HLA-A2-matched glioblastoma cells. CONCLUSIONS: Our glioblastoma culture method provides a renewable source for a broad spectrum glioblastoma neoantigens while our dendritic cell culture technique results in more mature dendritic cells in glioblastoma patients than standard techniques. This broadly applicable strategy could be easily integrated into patient care.

The role of extracellular vesicles and PD-L1 in glioblastoma-mediated immunosuppressive monocyte induction.

BACKGROUND: Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. METHODS: Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA-mediated knockdown was used to determined the role of altered PD-L1 expression. RESULTS: GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. CONCLUSION: These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

Short non-coding RNA sequencing of glioblastoma extracellular vesicles.

INTRODUCTION: Like all nucleated cells, glioblastoma (GBM) cells shed small membrane-encapsulated particles called extracellular vesicles (EVs). EVs can transfer oncogenic components and promote tumor growth by transferring short non-coding RNAs, altering target cell gene expression. Furthermore, GBM-derived EVs can be detected in blood and have potential to serve as liquid biopsies. METHODS: EVs were harvested from culture supernatants from human GBM cell lines, purified via sequential centrifugation, and quantified by nanoparticle tracking. RNA was isolated and short non-coding RNA was sequenced. Data was analyzed via the OASIS-2.0 platform using HG38. MirTarBase and MirDB interrogated validated/predicted miRNA-gene interactions respectively. RESULTS: Many short non-coding RNA's were identified within GBM EV's. In keeping with earlier reports utilizing GBM EV micro-RNA (miRNA) arrays, these included abundant micro-RNA's including miR-21. However, RNA sequencing revealed a total of 712 non-coding RNA sequences most of which have not been associated with GBM EV's previously. These included many RNA species (piRNA, snoRNA, snRNA, rRNA and yRNAs) in addition to miRNA's. miR-21-5p, let-7b-5p, miR-3182, miR-4448, let-7i-5p constituted highest overall expression. Top genes targeted by non-coding RNA's were highly conserved and specific for cell cycle, PI3K/Akt signaling, p53 and Glioma curated KEGG pathways. CONCLUSIONS: Next generation short non-coding RNA sequencing on GBM EV's validates findings from earlier studies using miRNA arrays but also demonstrates expression of many additional non-coding RNA sequences and classes previously unassociated with GBM. This may yield important insights into pathophysiology, point to new therapeutic targets, and help develop new biomarkers for disease burden and treatment response.

Low-Value Care and Clinician Engagement in a Large Medicare Shared Savings Program ACO: a Survey of Frontline Clinicians.

BACKGROUND: Although the Medicare Shared Savings Program (MSSP) created new incentives for organizations to improve healthcare value, Accountable Care Organizations (ACOs) have achieved only modest reductions in the use of low-value care. OBJECTIVE: To assess ACO engagement of clinicians and whether engagement was associated with clinicians' reported difficulty implementing recommendations against low-value care. DESIGN: Cross-sectional survey of ACO clinicians in 2018. PARTICIPANTS: 1289 clinicians in the Physician Organization of Michigan ACO, including generalist physicians (18%), internal medicine specialists (16%), surgeons (10%), other physician specialists (27%), and advanced practice providers (29%). Response rate was 34%. MAIN MEASURES: Primary exposures included clinicians' participation in ACO decision-making, awareness of ACO incentives, perceived influence on practice, and perceived quality improvement. Our primary outcome was clinicians' reported difficulty implementing recommendations against low-value care. RESULTS: Few clinicians participated in the decision to join the ACO (3%). Few clinicians were aware of ACO incentives, including knowing the ACO was accountable for both spending and quality (23%), successfully lowered spending (9%), or faced upside risk only (3%). Few agreed (moderately or strongly) the ACO changed compensation (20%), practice (19%), or feedback (15%) or that it improved care coordination (17%) or inappropriate care (13%). Clinicians reported they had difficulty following recommendations against low-value care 18% of the time; clinicians reported patients had difficulty accepting recommendations 36% of the time. Increased ACO awareness (1 standard deviation [SD]) was associated with decreased difficulty (- 2.3 percentage points) implementing recommendations (95% confidence interval [CI] - 3.8, - 0.7), as was perceived quality improvement (1 SD increase, - 2.1 percentage points, 95% CI, - 3.4, - 0.8). Participation in ACO decision-making and perceived influence on practice were not associated with recommendation implementation. CONCLUSIONS: Clinicians participating in a large Medicare ACO were broadly unaware of and unengaged with ACO objectives and activities. Whether low clinician engagement limits ACO efforts to reduce low-value care warrants further longitudinal study.

Isolation and Analysis of Plasma-Derived Exosomes in Patients With Glioma.

Gliomas including glioblastoma (GBM) are the most common primary malignant brain tumors. Glioma extracellular vesicles (EVs) including exosomes have biological effects (e.g., immunosuppression) and contain tumor-specific cargo that could facilitate liquid biopsies. We aimed to develop a simple, reproducible technique to isolate plasma exosomes in glioma patients. Glioma patients' and normal donors' plasma exosomes underwent brief centrifugation to remove cells/debris followed by serial density gradient ultracentrifugation (DGU). EV size/concentration was determined by nanoparticle tracking. Protein cargo was screened by array, western blot, and ELISA. Nanoscale flow cytometry analysis quantified exosome and microvesicle populations pre- and post-DGU. One-step DGU efficiently isolates exosomes for nanoparticle tracking. Wild type isocitrate dehydrogenase glioma patients' (i.e., more aggressive tumors) plasma exosomes are smaller but higher concentration than normal donors. A second DGU efficiently concentrates exosomes for subsequent cargo analysis but results in vesicle aggregation that skews nanoparticle tracking. Cytokines and co-stimulatory molecules are readily detected but appeared globally reduced in GBM patients' exosomes. Surprisingly, immunosuppressive programmed death-ligand 1 (PD-L1) is present in both patients' and normal donors' exosomes. Nanoscale flow cytometry confirms efficient exosome (<100 nm) isolation post-DGU but also demonstrates increase in microvesicles (>100 nm) in GBM patients' plasma pre-DGU. Serial DGU efficiently isolates plasma exosomes with distinct differences between GBM patients and normal donors, suggesting utility for non-invasive biomarker assessment. Initial results suggest global immunosuppression rather than increased circulating tumor-derived immunosuppressive exosomes, though further assessment is needed. Increased glioma patients' plasma microvesicles suggest these may also be a key source for biomarkers.

Antitumor activity of novel pyrazole-based small molecular inhibitors of the STAT3 pathway in patient derived high grade glioma cells.

Abnormal activation of signal transducer and activator of transcription 3 (STAT3) transcription factor has been observed in many human cancers with roles in tumor initiation, progression, drug resistance, angiogenesis and immunosuppression. STAT3 is constitutively activated in a variety of cancers including adult high grade gliomas (aHGGs) such as glioblastoma (GBM), and pediatric high grade gliomas (pHGG). Inhibiting STAT3 is a promising target-specific chemotherapeutic strategy for tumors with aberrant STAT3 signaling. Here we investigated the antitumor effects of novel pyrazole-based STAT3 pathway inhibitors named MNS1 (Mayo Neurosurgery 1) in both pediatric and adult HGG tumor cells. MNS1 compounds selectively decreased cell viability and proliferation in patient-derived HGG cells with minimal toxicity on normal human astrocytes. These inhibitors selectively blocked IL-6-induced STAT3 phosphorylation and nuclear localization of pSTAT3 without affecting other signaling molecules including Akt, STAT1, JAK2 or ERK1/2 phosphorylation. Functional analysis showed that MNS1 compounds induced apoptosis and decrease tumor migration. The anti-tumor effects extended into a murine pHGG (diffuse intrinsic pontine glioma) patient derived xenograft, and systemic toxicity was not evident during dose escalation in mice. These results support further development of STAT3 inhibitors for both pediatric and adult HGG.

Current Murine Models and New Developments in H3K27M Diffuse Midline Gliomas.

Diffuse Midline Gliomas with Histone 3-Lysine-27-Methionine (H3K27M) mutation constitute the majority of Diffuse Intrinsic Pontine Glioma (DIPG), which is the most aggressive form of pediatric glioma with a dire prognosis. DIPG are lethal tumors found in younger children with a median survival <1 year from diagnosis. Discovery of the characteristic H3K27M mutations offers opportunity and hope for development of targeted therapies for this deadly disease. The H3K27M mutation, likely through epigenetic alterations in specific H3 lysine trimethylation levels and subsequent gene expression, plays a significant role in pathogenesis of DIPG. Animal models accurately depicting molecular characteristics of H3K27M DIPG are important to elucidate underlying pathologic events and for preclinical drug evaluation. Here we review the past and present DIPG models and describe our efforts developing patient derived cell lines and xenografts from pretreated surgical specimens. Pre-treated surgical samples retain the characteristic genomic and phenotypic hallmarks of DIPG and establish orthotopic tumors in the mouse brainstem that recapitulate radiographic and morphological features of the original human DIPG tumor. These models that contain the H3K27M mutation constitute a valuable tool to further study this devastating disease and ultimately may uncover novel therapeutic vulnerabilities.

Intermittent hypoxia regulates vasoactive molecules and alters insulin-signaling in vascular endothelial cells.

Vascular dysfunction and insulin resistance (IR) are associated with obstructive sleep apnea (OSA), which is characterized by frequent episodes of nocturnal intermittent hypoxia (IH). While it is recognized that the balance between vasoconstrictive (endothelin-1) and vasodilatory molecules (nitric oxide, NO) determine vascular profile, molecular mechanisms contributing to vascular dysfunction and IR in OSA are not completely understood. Caveolin-1 is a membrane protein which regulates endothelial nitric oxide synthase (eNOS) activity which is responsible for NO generation and cellular insulin-signaling. Hence, we examined the effects of IH on caveolin-1, eNOS, and endothelin-1 in human coronary artery endothelial cells in the context of IR. Chronic 3-day IH exposure up-regulated caveolin-1 and endothelin-1 expression while reducing NO. Also, IH altered insulin-mediated activation of AKT but not ERK resulting in increased endothelin-1 transcription. Similarly, caveolin-1 overexpression attenuated basal and insulin-stimulated NO synthesis along with impaired insulin-dependent activation of AKT and eNOS, with no effect on insulin-stimulated ERK1/2 phosphorylation and endothelin-1 transcription. Our data suggest that IH contributes to a vasoconstrictive profile and to pathway-selective vascular IR, whereby insulin potentiates ET-1 expression. Moreover, IH may partly mediate its effects on NO and insulin-signaling via upregulating caveolin-1 expression.

Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia.

Plasticity is an important aspect of the neural control of breathing. One well-studied form of respiratory plasticity is phrenic long-term facilitation (pLTF) induced by acute intermittent but not sustained hypoxia. Okadaic acid-sensitive protein phosphatases (PPs) differentially regulate phrenic nerve activity with intermittent vs. sustained hypoxia, at least partially accounting for pLTF pattern sensitivity. However, okadaic acid inhibits multiple serine/threonine phosphatases, and the relevant phosphatase (PP1, PP2A, PP5) for pLTF pattern sensitivity has not been identified. Here, we demonstrate that sustained hypoxia (25 min, 9-10.5% O2) elicits phrenic motor facilitation in rats pretreated with bilateral intrapleural injections of small interfering RNAs (siRNAs; Accell-modified to preferentially transfect neurons, 3.33 µM, 3 days) targeting PP1 mRNA (48 ± 14% change from baseline, n = 6) but not PP2A (14 ± 9% baseline, n = 6) or nontargeting siRNAs (4 ± 10% baseline, n = 7). In time control rats (no hypoxia) treated with siRNAs ( n = 6), no facilitation was evident (-9 ± 9% baseline). siRNAs had no effect on the hypoxic phrenic response. Immunohistochemistry revealed PP1 and PP2A protein in identified phrenic motoneurons. Although PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, we were not able to verify "knockdown" in vivo after siRNA treatment. On the other hand, PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, verifying the intended siRNA effects. In conclusion, PP1 (not PP2A) is the relevant okadaic acid-sensitive phosphatase constraining phrenic motor facilitation after sustained hypoxia and likely contributing to pLTF pattern sensitivity. NEW & NOTEWORTHY This study demonstrates that the relevant okadaic acid-sensitive Ser/Thr protein phosphatase (PP) constraining facilitation after sustained hypoxia is PP1 and not PP2A. It suggests that PP1 may be critical in the pattern sensitivity of hypoxia-induced phrenic motor plasticity.

Modulating glioma-mediated myeloid-derived suppressor cell development with sulforaphane.

Glioblastoma is the most common primary tumor of the brain and has few long-term survivors. The local and systemic immunosuppressive environment created by glioblastoma allows it to evade immunosurveillance. Myeloid-derived suppressor cells (MDSCs) are a critical component of this immunosuppression. Understanding mechanisms of MDSC formation and function are key to developing effective immunotherapies. In this study, we developed a novel model to reliably generate human MDSCs from healthy-donor CD14+ monocytes by culture in human glioma-conditioned media. Monocytic MDSC frequency was assessed by flow cytometry and confocal microscopy. The resulting MDSCs robustly inhibited T cell proliferation. A cytokine array identified multiple components of the GCM potentially contributing to MDSC generation, including Monocyte Chemoattractive Protein-1, interleukin-6, interleukin-8, and Macrophage Migration Inhibitory Factor (MIF). Of these, Macrophage Migration Inhibitory Factor is a particularly attractive therapeutic target as sulforaphane, a naturally occurring MIF inhibitor derived from broccoli sprouts, has excellent oral bioavailability. Sulforaphane inhibits the transformation of normal monocytes to MDSCs by glioma-conditioned media in vitro at pharmacologically relevant concentrations that are non-toxic to normal leukocytes. This is associated with a corresponding increase in mature dendritic cells. Interestingly, sulforaphane treatment had similar pro-inflammatory effects on normal monocytes in fresh media but specifically increased immature dendritic cells. Thus, we have used a simple in vitro model system to identify a novel contributor to glioblastoma immunosuppression for which a natural inhibitor exists that increases mature dendritic cell development at the expense of myeloid-derived suppressor cells when normal monocytes are exposed to glioma conditioned media.