CXCL9 recombinant adeno-associated virus (AAV) virotherapy sensitizes glioblastoma (GBM) to anti-PD-1 immune checkpoint blockade.

The promise of immunotherapy to induce long-term durable responses in conventionally treatment resistant tumors like glioblastoma (GBM) has given hope for patients with a dismal prognosis. Yet, few patients have demonstrated a significant survival benefit despite multiple clinical trials designed to invigorate immune recognition and tumor eradication. Insights gathered over the last two decades have revealed numerous mechanisms by which glioma cells resist conventional therapy and evade immunological detection, underscoring the need for strategic combinatorial treatments as necessary to achieve appreciable therapeutic effects. However, new combination therapies are inherently difficult to develop as a result of dose-limiting toxicities, the constraints of the blood-brain barrier, and the suppressive nature of the GBM tumor microenvironment (TME). GBM is notoriously devoid of lymphocytes driven in part by a paucity of lymphocyte trafficking factors necessary to prompt their recruitment, infiltration, and activation. We have developed a novel recombinant adeno-associated virus (AAV) gene therapy strategy that enables focal and stable reconstitution of the GBM TME with C-X-C motif ligand 9 (CXCL9), a powerful call-and-receive chemokine for cytotoxic T lymphocytes (CTLs). By precisely manipulating local chemokine directional guidance, AAV-CXCL9 increases tumor infiltration by CD8-postive cytotoxic lymphocytes, sensitizing GBM to anti-PD-1 immune checkpoint blockade (ICB). These effects are accompanied by immunologic signatures evocative of an inflamed and responsive TME. These findings support targeted AAV gene therapy as a promising adjuvant strategy for reconditioning GBM immunogenicity given its excellent safety profile, TME-tropism, modularity, and off-the-shelf capability, where focal delivery bypasses the constrains of the blood-brain barrier, further mitigating risks observed with high-dose systemic therapy.

Soluble brain homogenates from diverse human and mouse sources preferentially seed diffuse Aß plaque pathology when injected into newborn mouse hosts.

Background: Seeding of pathology related to Alzheimer's disease (AD) and Lewy body disease (LBD) by tissue homogenates or purified protein aggregates in various model systems has revealed prion-like properties of these disorders. Typically, these homogenates are injected into adult mice stereotaxically. Injection of brain lysates into newborn mice represents an alternative approach of delivering seeds that could direct the evolution of amyloid-ß (Aß) pathology co-mixed with either tau or α-synuclein (αSyn) pathology in susceptible mouse models. Methods: Homogenates of human pre-frontal cortex were injected into the lateral ventricles of newborn (P0) mice expressing a mutant humanized amyloid precursor protein (APP), human P301L tau, human wild type αSyn, or combinations thereof. The homogenates were prepared from AD and AD/LBD cases displaying variable degrees of Aß pathology and co-existing tau and αSyn deposits. Behavioral assessments of APP transgenic mice injected with AD brain lysates were conducted. For comparison, homogenates of aged APP transgenic mice that preferentially exhibit diffuse or cored deposits were similarly injected into the brains of newborn APP mice. Results: We observed that lysates from the brains with AD (Aß+, tau+), AD/LBD (Aß+, tau+, αSyn+), or Pathological Aging (Aß+, tau-, αSyn-) efficiently seeded diffuse Aß deposits. Moderate seeding of cerebral amyloid angiopathy (CAA) was also observed. No animal of any genotype developed discernable tau or αSyn pathology. Performance in fear-conditioning cognitive tasks was not significantly altered in APP transgenic animals injected with AD brain lysates compared to nontransgenic controls. Homogenates prepared from aged APP transgenic mice with diffuse Aß deposits induced similar deposits in APP host mice; whereas homogenates from APP mice with cored deposits induced similar cored deposits, albeit at a lower level. Conclusions: These findings are consistent with the idea that diffuse Aß pathology, which is a common feature of human AD, AD/LBD, and PA brains, may arise from a distinct strain of misfolded Aß that is highly transmissible to newborn transgenic APP mice. Seeding of tau or αSyn comorbidities was inefficient in the models we used, indicating that additional methodological refinement will be needed to efficiently seed AD or AD/LBD mixed pathologies by injecting newborn mice.

Obesity and STING1 genotype associate with 23-valent pneumococcal vaccination efficacy.

BACKGROUNDObesity has been associated with attenuated vaccine responses and an increased risk of contracting pneumococcal pneumonia, but no study to our knowledge has assessed the impact of obesity and genetics on 23-valent pneumococcal vaccine (PPSV23) efficacy. We assessed the relationship of obesity (primary analysis) and stimulator of interferon genes (STING1) genotype (secondary analysis) on PPSV23 efficacy.METHODSNonobese (BMI 22-25 kg/m2) and obese participants (BMI ≥30 kg/m2) were given a single dose of PPSV23. Blood was drawn immediately prior to and 4-6 weeks after vaccination. Serum samples were used to assess PPSV23-specific antibodies. STING1 genotypes were identified using PCR on DNA extracted from peripheral blood samples.RESULTSForty-six participants were categorized as nonobese (n = 23; 56.5% women; mean BMI 23.3 kg/m2) or obese (n = 23; 65.2% women; mean BMI 36.3 kg/m2). Obese participants had an elevated fold change in vaccine-specific responses compared with nonobese participants (P < 0.0001). The WT STING1 group (R232/R232) had a significantly higher PPSV23 response than individuals with a single copy of HAQ-STING1 regardless of BMI (P = 0.0025). When WT was assessed alone, obese participants had a higher fold serotype-specific response compared with nonobese participants (P < 0.0001), but no difference was observed between obese and nonobese individuals with 1 HAQ allele (P = 0.693).CONCLUSIONSThese observations demonstrate a positive association between obesity and PPSV23 efficacy specifically in participants with the WT STING1 genotype.TRIAL REGISTRATIONClinicalTrials.gov NCT02471014.FUNDINGThis research was supported by the NIH and the University of Florida MD-PhD Training Program.

Utilizing minimally purified secreted rAAV for rapid and cost-effective manipulation of gene expression in the CNS.

BACKGROUND: Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. METHODS: We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated. RESULTS: There was ~ 40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~ 4.7 × 1010 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~ 2.0 × 1012 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. CONCLUSIONS: Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.

An anti-CRF antibody suppresses the HPA axis and reverses stress-induced phenotypes.

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction contributes to numerous human diseases and disorders. We developed a high-affinity monoclonal antibody, CTRND05, targeting corticotropin-releasing factor (CRF). In mice, CTRND05 blocks stress-induced corticosterone increases, counteracts effects of chronic variable stress, and induces other phenotypes consistent with suppression of the HPA axis. CTRND05 induces skeletal muscle hypertrophy and increases lean body mass, effects not previously reported with small-molecule HPA-targeting pharmacologic agents. Multiorgan transcriptomics demonstrates broad HPA axis target engagement through altering levels of known HPA-responsive transcripts such as Fkbp5 and Myostatin and reveals novel HPA-responsive pathways such as the Apelin-Apelin receptor system. These studies demonstrate the therapeutic potential of CTRND05 as a suppressor of the HPA axis and serve as an exemplar of a potentially broader approach to target neuropeptides with immunotherapies, as both pharmacologic tools and novel therapeutics.

Solitaire Stent Retriever Mechanical Thrombectomy in a 6-Month-Old Patient with Acute Occlusion of the Internal Carotid Artery Terminus: Case Report.

BACKGROUND: Endovascular mechanical thrombectomy (MT) performed with stent retriever devices have proven efficacious in the treatment of adult patients with acute cerebral ischemia resulting from large-vessel occlusion. Recently, reports describing the use of MT in pediatric large-vessel occlusion have increased. However, these case reports are generally limited to older children and teenagers. CASE DESCRIPTION: We report the use of a Solitaire (Medtronic, Minneapolis, Minnesota, USA) stent retriever for thrombectomy and revascularization of the right middle cerebral artery) in a 6-month-old patient. CONCLUSIONS: To our knowledge, this is the youngest patient reported to be treated with MT using a stent retriever. This case adds to the growing literature that supports the use of MT as a stroke intervention in pediatric patients.

Targeting psychologic stress signaling pathways in Alzheimer's disease.

Alzheimer's Disease (AD) is the most prevalent progressive neurodegenerative disease; to date, no AD therapy has proven effective in delaying or preventing the disease course. In the search for novel therapeutic targets in AD, it has been shown that increased chronic psychologic stress is associated with AD risk. Subsequently, biologic pathways underlying psychologic stress have been identified and shown to be able to exacerbate AD relevant pathologies. In this review, we summarize the literature relevant to the association between psychologic stress and AD, focusing on studies investigating the effects of stress paradigms on transgenic mouse models of Amyloid-ß (Aß) and tau pathologies. In recent years, a substantial amount of research has been done investigating a key stress-response mediator, corticotropin-releasing hormone (CRH), and its interactions with AD relevant processes. We highlight attempts to target the CRH signaling pathway as a therapeutic intervention in these transgenic mouse models and discuss how targeting this pathway is a promising avenue for further investigation.

Neuron-astrocyte signaling is preserved in the aging brain.

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca2+ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte-neuron signaling is derived from studies with young animals; however, the features of astrocyte-neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte-neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month-old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter-dependent intracellular Ca2+ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG-induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte-neuron signaling in pathological conditions. Disruption of the astrocytic IP3 R2 mediated-signaling, which is required for neurotransmitter-induced astrocyte Ca2+ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte-neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca2+ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569-580.

The proof-of-concept of ASS234: Peripherally administered ASS234 enters the central nervous system and reduces pathology in a male mouse model of Alzheimer disease.

BACKGROUND: The heterogeneity of Alzheimer disease requires the development of multitarget drugs for treating the symptoms of the disease and its progression. Both cholinergic and monoamine oxidase dysfunctions are involved in the pathological process. Thus, we hypothesized that the development of therapies focused on these targets might be effective. We have developed and assessed a new product, coded ASS234, a multipotent acetyl and butyrylcholinesterase/monoamine oxidase A-B inhibitor with a potent inhibitory effect on amyloid-ß aggregation as well as antioxidant and antiapoptotic properties. But there is a need to reliably correlate in vitro and in vivo drug release data. METHODS: We examined the effect of ASS234 on cognition in healthy adult C57BL/6J mice in a model of scopolamine-induced cognitive impairment that often accompanies normal and pathological aging. Also, in a characterized transgenic APPswe/PS1ΔE9 mouse model of Alzheimer disease, we examined the effects of short-term ASS234 treatment on plaque deposition and gliosis using immunohistochemistry. Toxicology of ASS234 was assessed using a quantitative high-throughput in vitro cytotoxicity screening assay following the MTT assay method in HepG2 liver cells. RESULTS: In vivo, ASS234 significantly decreased scopolamine-induced learning deficits in C57BL/6J mice. Also, reduction of amyloid plaque burden and gliosis in the cortex and hippocampus was assessed. In vitro, ASS234 exhibited lesser toxicity than donepezil and tacrine. LIMITATIONS: The study was conducted in male mice only. Although the Alzheimer disease model does not recapitulate all features of the human disease, it exhibits progressive monoaminergic neurodegeneration. CONCLUSION: ASS234 is a promising alternative drug of choice to treat the cognitive decline and neurodegeneration underlying Alzheimer disease.

Adrenomedullin Expression in Alzheimer's Brain.

Adrenomedullin (AM) is a potent vasodilator peptide highly expressed throughout the brain and originally isolated from pheochromocytoma cells. In addition to its vasoactive properties, AM is considered a neuromodulator that possesses antiapoptotic and antioxidant properties that suggest that this peptide can protect the brain from damage. In a previous study, we found that AM exerts a neuroprotective action in the brain and that this effect may be mediated by regulation of nitric oxide synthases, matrix metalloproteases, and inflammatory mediators. AM upregulation contributes to neuroprotection, but understanding the precise roles played by AM and its receptor (RAMP2) in neurodegenerative diseases including Alzheimer's disease (AD), awaits further research. In search of Alzheimer's biomarkers, the expression levels of peptides with endothelial vasodilatory action, including AM, were found to be significantly altered in mild AD or during pre-dementia stage of mild cognitive impairment. These studies concluded that ratio of AM or its precursor fragment mid-regional proAM in blood hold promise as diagnostic marker for AD. We are now presenting a study regarding the hypothesis that the AMRAMP2 system might be implicated in the pathophysiology of AD.

Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis.

KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dnKif3a or inhibiting SMO signaling using cyclopamine, and the survival times of mice implanted with dnKif3a-expressing cells were increased. In a second line, expression of dnKif3a increased the cells' baseline proliferation while, surprisingly, sensitizing them to SHH-induced cell death. The survival times of mice implanted with these dnKif3a-expressing cells were decreased. Finally, expression of dnKif3a in a third cell line had no effect on cell proliferation, SHH sensitivity, or mouse survival times. These findings indicate that KIF3A is essential for GBM cell ciliogenesis, but its role in modulating GBM cell behavior is highly variable.

Differential connexin function enhances self-renewal in glioblastoma.

The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin 43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.