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Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV's evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

Vascular injury and pathologies underlie common diseases including ischemic stroke and cerebral small vessel disease (CSVD). Prior work has identified a key role for glial cells, including microglia, in the multifaceted and temporally evolving neuroimmune response to both stroke and CSVD. Transcriptional profiling has led to important advances including identification of distinct gene expression signatures in ischemia-exposed, flow cytometrically sorted microglia and more recently single cell RNA sequencing-identified microglial subpopulations or clusters. There is a reassuring degree of overlap in the results from these two distinct methodologies with both identifying a proliferative and a separate type I interferon responsive microglial element. Similar patterns were later seen using multimodal and spatial transcriptomal profiling in ischemia-exposed microglia and astrocytes. Methodological advances including enrichment of specific neuroanatomic/functional regions (such as the neurovascular unit) prior to single cell RNA sequencing has led to identification of novel cellular subtypes and generation of new credible hypotheses as to cellular function based on the enhanced cell sub-type specific gene expression patterns. A ribosomal tagging strategy focusing on the cellular translatome analyses carried out in the acute phases post stroke has revealed distinct inflammation-regulating roles for microglia and astrocytes in this setting. Early spatial transcriptomics experiments using cerebral ischemia models have identified regionally distinct microglial cell clusters in ischemic core versus penumbra. There is great potential for combination of these methods for multi-omics approaches to further elucidate glial responses in the context of both acute ischemic stroke and chronic CSVD., (© 2024 Wiley Periodicals LLC.)

Pharmaceutical-grade medium chain triglycerides (MCTs) are common excipients for in vivo pharmacological studies in laboratory animals, and as an experimental therapeutic in certain metabolic and neurological disorders. In this study, we examined the tolerability of repeated administration of a pharmaceutical-grade formulation of three MCTs-caprylic, capric, and lauric acid - in mice via the oral (PO) and intraperitoneal (IP) routes. We administered either 8 or 4 µL of 100% MCTs or saline/gram of body weight twice daily for seven days. During administration and for seven days after, we monitored weight change and clinical presentation. On day 14, or upon meeting euthanasia criteria, animals were sacrificed for gross necropsy, histology, and complete blood count. We observed significant weight loss, clinical decline and 100% mortality in animals receiving 8 µL/g of MCTs via the IP route of administration. Gross necropsy revealed serosanguinous fluid in the thoracic cavity, dark red mottled lungs, and adhesions in the abdominal cavity. Histology confirmed inflammation of the lungs, mediastinum, and peritoneum. Mild gross lesions and initial weight loss (through day 3) were also present in mice receiving 4 µL/g of MCTs IP. However, these animals regained weight by day seven and exhibited no clinical decline or mortality. None of these adverse effects were seen in animals receiving either 8 µL/g of MCTs PO or 8 µL/g of saline IP. These findings suggest repeated IP administration of MCTs may cause dose-dependent toxicity, and mortality at high doses, but confers no adverse effects when administered via the PO route., Significance Statement: Medium chain triglycerides (MCTs) are commonly used as an excipient in pharmacological studies involving laboratory animals. Our work provides much needed safety information regarding adverse effects of repeated MCTs administration via the intraperitoneal, but not the oral, route in mice.

Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC 50 of 7 nM, reduced Ca 2+ signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke., (© 2023. The Author(s).)

Ischemic preconditioning (IPC) is a phenomenon whereby a brief, non-injurious ischemic exposure enhances tolerance to a subsequent ischemic challenge. The mechanism of IPC has mainly been studied in rodent stroke models where gray matter (GM) constitutes about 85% of the cerebrum. In humans, white matter (WM) is 50% of cerebral volume and is a critical component of stroke damage. We developed a novel CNS WM IPC model using the mouse optic nerve (MON) and identified the involved immune signaling pathways. Here we tested the hypothesis that microglia are necessary for WM IPC. Microglia were depleted by treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. MONs were exposed to transient ischemia in vivo, acutely isolated 72 h later, and subjected to oxygen-glucose deprivation (OGD) to simulate a severe ischemic injury (i.e., stroke). Functional and structural axonal recovery was assessed by recording compound action potentials (CAPs) and by microscopy using quantitative stereology. Microglia depletion eliminated IPC-mediated protection. In control mice, CAP recovery was improved in preconditioned MONs compared with non-preconditioned MONs, however, in PLX5622-treated mice, we observed no difference in CAP recovery between preconditioned and non-preconditioned MONs. Microgliadepletion also abolished IPC protective effects on axonal integrity and survival of mature (APC + ) oligodendrocytes after OGD. IPC-mediated protection was independent of retinal injury suggesting it results from mechanistic processes intrinsic to ischemia-exposed WM. We conclude that preconditioned microglia are critical for IPC in WM. The "preconditioned microglia" phenotype might protect against other CNS pathologies and is a neurotherapeutic horizon worth exploring., (© 2021 Wiley Periodicals LLC.)

Microglia maintain brain health and play important roles in disease and injury. Despite the known ability of microglia to proliferate, the precise nature of the population or populations capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (Prom1; also known as CD133) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1-expressing cells isolated from mixed cortical cultures will generate new microglia in vitro To determine whether Prom1-expressing cells generate new microglia in vivo , we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1-expressing cells leads to the expression of TdTomato in all Prom1-expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato-expressing microglia at 6 months of age that increased in size at 9 months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1-expressing progenitors was observed with the majority of new microglia derived from Prom1-negative cells. Together, these findings reveal that Prom1-expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1-expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain but not in response to ischemia/reperfusion. SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the CNS, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia., (Copyright © 2021 the authors.)

Background: The majority of pediatric human immunodeficiency virus (HIV) cases in Africa reflect maternal-to-child transmission. HIV exposed but uninfected (HEU) children have increased rates of morbidity and mortality when compared to HIV unexposed and uninfected (HUU) children. The mechanisms behind these unexpected trends are only partially understood but could be explained by the differences in the immune response to infections triggered by an altered immune system state., Methods: Using quantitative reverse transcription polymerase chain reaction, we compared the nasopharyngeal carriage prevalence and density of Streptococcus pneumoniae (SP) and Pneumocystis jirovecii (PJ) between children living with HIV and HEU or HUU cases (pneumonia) and controls (without pneumonia)., Results: The cohort included 1154 children (555 cases and 599 matched controls). The SP carriage prevalence rates were similar between cases and controls. Among SP carriers with pneumonia, carriage density was increased among children living with HIV, versus HEU or HUU children (15.8, 4.7, and 3.6 × 105 copies/mL, respectively). The rate of PJ carriage was significantly higher among children living with HIV than among HEU and HUU children (31%, 15%, and 10%, respectively; P < .05), as was carriage density (63.9, 20.9, and 4.8 × 103 copies/mL, respectively; P < .05)., Conclusions: Carriage prevalences and densities for SP and PJ show different kinetics in terms of their relationship with HIV exposure and clinical status, particularly for Pneumocystis jirovecii. This supports the theory that the increased morbidity and mortality observed among HEU children may reflect deficits not just in humoral immunity but in cell-mediated immunity as well., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic stimulus confers protection against a subsequent prolonged ischemic event. Initially thought to be due to mechanistic changes in neurons, our understanding of IPC has evolved to encompass a global reprogramming of the Central Nervous System (CNS) after transient ischemia/reperfusion that requires innate immune signaling pathways including Toll-like receptors (TLRs) and Type I interferons. Microglia are the CNS resident neuroimmune cells that express these key innate immune receptors. Studies suggest that microglia are required for IPC-mediated neuronal and axonal protection. Multiple paradigms targeting TLRs have converged on a distinctive Type I interferon response in microglia that is critical for preconditioning-mediated protection against ischemia. These pathways can be targeted through administration of TLR agonists, cytokines including interferon-β, and pharmaceutical agents that induce preconditioning through cross-tolerance mechanisms. Transcriptomic analyses and single cell RNA studies point to specific gene expression signatures in microglia that functionally shift these mutable cells to an immunomodulatory or protective phenotype. Although there are technological challenges and gaps in knowledge to overcome, the targeting of specific molecular signaling pathways in microglia is a promising direction for development of novel and effective pharmacotherapies for stroke. Studies on preconditioning in animal models, including nonhuman primates, show promise as prophylactic preconditioning treatments for selected at risk patient populations. In addition, our growing understanding of the mechanisms of IPC-mediated protection is identifying novel cellular and molecular targets for therapeutic interventions that could apply broadly to both acute stroke and chronic vascular cognitive impairment patients., (© 2019 Wiley Periodicals, Inc.)

Background: Household air pollution from solid fuels is a leading risk factor for morbidity and mortality worldwide. Pregnant women's exposure to polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs), two components of solid-fuel smoke, is associated with adverse birth outcomes. Even with improved solid-fuel stoves, exposure to PAHs and VOCs remains high. Therefore, cleaner cooking fuels need to be prioritized., Objective: We aimed to quantify exposure reduction to PAHs and VOCs among pregnant women in rural Guatemala with a liquefied petroleum gas (LPG) stove intervention., Materials and Methods: Urine from pregnant women (N = 50) was collected twice: at <20 weeks gestation, when women cooked exclusively with wood, and 6-8 weeks after receiving an LPG stove. Metabolites of four PAHs and eight VOCs were analyzed. Concurrent with urine collection, personal 48-h PM 2.5 exposure was measured., Results: Women cooking exclusively with wood were exposed to high levels of particulate matter (PM 2.5 ), which was reduced by 57% with the LPG stove. Urinary concentrations of total PAH metabolites (-37%), PMA (benzene metabolite; -49%), and CNEMA (acrylonitrile metabolite; -51%) were reduced. However, recent use of a wood-fired sauna bath led to large increases in excretion of urinary toxicant metabolites (+66-135%)., Conclusions: This is the first study to report PAH and VOC reductions from an LPG stove intervention introduced during pregnancy. However, other sources of air pollution minimized the gains seen from using an LPG stove. Thus, all sources of air pollution must be addressed in concert to reduce exposures to levels that protect health.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a subthreshold ischemic insult applied to the brain reduces damage caused by a subsequent more severe ischemic episode. Identifying key molecular and cellular mediators of IPC will provide critical information needed to develop novel therapies for stroke. Here we report that the transcriptomic response of acutely isolated preconditioned cortical microglia is dominated by marked upregulation of genes involved in cell cycle activation and cellular proliferation. Notably, this transcriptional response occurs in the absence of cortical infarction. We employed ex vivo flow cytometry, immunofluorescent microscopy, and quantitative stereology methods on brain tissue to evaluate microglia proliferation following IPC. Using cellular colocalization of microglial (Iba1) and proliferation (Ki67 and BrdU) markers, we observed a localized increase in the number of microglia and proliferating microglia within the preconditioned hemicortex at 72, but not 24, hours post-IPC. Our quantification demonstrated that the IPC-induced increase in total microglia was due entirely to proliferation. Furthermore, microglia in the preconditioned hemisphere had altered morphology and increased soma volumes, indicative of an activated phenotype. Using transgenic mouse models with either fractalkine receptor (CX3CR1)-haploinsufficiency or systemic type I interferon signaling loss, we determined that microglial proliferation after IPC is dependent on fractalkine signaling but independent of type I interferon signaling. These findings suggest there are multiple distinct targetable signaling pathways in microglia, including CX3CR1-dependent proliferation that may be involved in IPC-mediated protection., (© 2019 Wiley Periodicals, Inc.)

Household air pollution is the sixth leading risk factor for premature mortality in Guatemala. Households in Guatemala are gradually adopting liquefied petroleum gas (LPG) stoves, but a strong tradition of woodstove use persists. We conducted a mixed-methods study of LPG stove use in peri-urban Guatemala. We used knowledge, attitudes and practices surveys with 187 LPG stove owners who also used woodstoves to identify perceptions of stove and cooking practices. Barriers to sustained use of LPG stoves were evaluated through focus groups, participant observations with stove users, and key informant interviews with community leaders. Seven themes emerged that explain household decisions to use LPG stoves: (1) The "new technology" should be framed in terms of what the "old technology" lacks, (2) income is not a predictor of gas stove acquisition but may predict sustained use, (3) men are key decision-makers but messages about LPG do not target them, (4) when stoves are viewed as "prize possessions" they may not be used, (5) collective fear about gas stoves is not based on personal experience, but on "stories we hear," (6) sustained LPG use is hampered by two major factors, seasonally available wood and LPG retailers who are perceived as dishonest, and (7) wood fuel collection is a time to enjoy the company of friends and family and is not "drudgery." National policies should promote the use of clean cookstove technologies in peri-urban and rapidly urbanizing areas in Guatemala where LPG stoves are in use, but used intermittently, instead of the current plan to install 100,000 "improved" woodstoves by 2032. This could be done by improving dependable cylinder distribution services, targeting gas safety and promoting positive health messages that appeal to men, as well as women.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Research on IPC has implicated cellular, molecular, and systemic elements of the immune response in this phenomenon. Potent molecular mediators of IPC include innate immune signaling pathways such as Toll-like receptors and type 1 interferons. Brain ischemia results in release of pro- and anti-inflammatory cytokines and chemokines that orchestrate the neuroinflammatory response, resolution of inflammation, and transition to neurological recovery and regeneration. Cellular mediators of IPC include microglia, the resident central nervous system immune cells, astrocytes, and neurons. All of these cell types engage in cross-talk with each other using a multitude of signaling pathways that modulate activation/suppression of each of the other cell types in response to ischemia. As the postischemic neuroimmune response evolves over time there is a shift in function toward provision of trophic support and neuroprotection. Peripheral immune cells infiltrate the central nervous system en masse after stroke and are largely detrimental, with a few subtypes having beneficial, protective effects, though the role of these immune cells in IPC is largely unknown. The role of neural progenitor cells in IPC-mediated neuroprotection is another active area of investigation as is the role of microglial proliferation in this setting. A mechanistic understanding of these molecular and cellular mediators of IPC may not only facilitate more effective direct application of IPC to specific clinical scenarios, but also, more broadly, reveal novel targets for therapeutic intervention in stroke.

Background: Preterm birth is the leading cause of death among children <5 years of age. Accurate determination of prematurity is necessary to provide appropriate neonatal care and guide preventive measures. To estimate the most accurate method to identify infants at risk for adverse outcomes, we assessed the validity of two widely available methods-last menstrual period (LMP) and the New Ballard (NB) neonatal assessment-against ultrasound in determining gestational age and preterm birth in highland Guatemala., Methods: Pregnant women (n = 188) were recruited with a gestational age <20 weeks and followed until delivery. Ultrasound was performed by trained physicians and LMP was collected during recruitment. NB was performed on infants within 96 hours of birth by trained study nurses. LMP and NB accuracy at determining gestational age and identifying prematurity was assessed by comparing them to ultrasound., Results: By ultrasound, infant mean gestational age at birth was 38.3 weeks (SD = 1.6) with 16% born at less than 37 gestation. LMP was more accurate than NB (mean difference of +0.13 weeks for LMP and +0.61 weeks for NB). However, LMP and NB estimates had low agreement with ultrasound-determined gestational age (Lin's concordance<0.48 for both methods) and preterm birth (κ<0.29 for both methods). By LMP, 18% were judged premature compared with 6% by NB. LMP underestimated gestational age among women presenting later to prenatal care (0.18 weeks for each additional week). Gestational age for preterm infants was overestimated by nearly one week using LMP and nearly two weeks using NB. New Ballard neuromuscular measurements were more predictive of preterm birth than those measuring physical criteria., Conclusion: In an indigenous population in highland Guatemala, LMP overestimated prematurity by 2% and NB underestimated prematurity by 10% compared with ultrasound estimates. New, simple and accurate methods are needed to identify preterm birth in resource-limited settings worldwide.

Background: Three billion people use solid cooking fuels, and 4 million people die from household air pollution annually. Shifting households to clean fuels, like liquefied petroleum gas (LPG), may protect health only if stoves are consistently used. Few studies have used an implementation science framework to systematically assess "de-implementation" of traditional stoves, and none have done so with pregnant women who are more likely to adopt new behaviors. We evaluated an introduced LPG stove coupled with a phased behavioral intervention to encourage exclusive gas stove use among pregnant women in rural Guatemala., Methods: We enrolled 50 women at < 20 weeks gestation in this prospective cohort study. All women received a free 3-burner LPG stove and ten tank refills. We conducted formative research using COM-B Model and Theoretical Domains Framework (TDF). This included thematic analysis of focus group findings and classes delivered to 25 pregnant women (Phase 1). In Phase 2, we complemented classes with a home-based tailored behavioral intervention with a different group of 25 pregnant women. We mapped 35 TDF constructs onto survey questions. To evaluate stove use, we placed temperature sensors on wood and gas stoves and estimated fraction of stove use three times during pregnancy and twice during the first month after infant birth., Results: Class attendance rates were above 92%. We discussed feasible ways to reduce HAP exposure, proper stove use, maintenance and safety. We addressed food preferences, ease of cooking and time savings through cooking demonstrations. In Phase 2, the COM-B framework revealed that other household members needed to be involved if the gas stove was to be consistently used. Social identity and empowerment were key in decisions about stove repairs and LPG tank refills. The seven intervention functions included training, education, persuasion, incentivization, modelling, enablement and environmental restructuring. Wood stove use dropped upon introduction of the gas stove from 6.4 h to 1.9 h., Conclusions: This is the first study using the COM-B Model to develop a behavioral intervention that promotes household-level sustained use of LPG stoves. This study lays the groundwork for a future LPG stove intervention trial coupled with a behavioral change intervention., Trial Registration: NCT02812914, registered 3 June 2016, retrospectively registered.

A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Microglia, the resident immune cells of the CNS, are primary regulators of the neuroimmune response to injury. Type I interferons (IFNs), including the IFNαs and IFNβ, are key cytokines in the innate immune system. Their activity is implicated in the regulation of microglial function both during development and in response to neuroinflammation, ischemia, and neurodegeneration. Data from numerous studies in multiple sclerosis (MS) and stroke suggest that type I IFNs can modulate the microglial phenotype, influence the overall neuroimmune milieu, regulate phagocytosis, and affect blood-brain barrier integrity. All of these IFN-induced effects result in numerous downstream consequences on white matter pathology and microglial reactivity. Dysregulation of IFN signaling in mouse models with genetic deficiency in ubiquitin specific protease 18 (USP18) leads to a severe neurological phenotype and neuropathological changes that include white matter microgliosis and pro-inflammatory gene expression in dystrophic microglia. A class of genetic disorders in humans, referred to as pseudo-TORCH syndrome (PTS) for the clinical resemblance to infection-induced TORCH syndrome, also show dysregulation of IFN signaling, which leads to severe neurological developmental disease. In these disorders, the excessive activation of IFN signaling during CNS development results in a destructive interferonopathy with similar induction of microglial dysfunction as seen in USP18 deficient mice. Other recent studies implicate "microgliopathies" more broadly in neurological disorders including Alzheimer's disease (AD) and MS, suggesting that microglia are a potential therapeutic target for disease prevention and/or treatment, with interferon signaling playing a key role in regulating the microglial phenotype.

Stroke is the leading cause of serious long-term disability and the fifth leading cause of death in the United States. Treatment options for stroke are few in number and limited in efficacy. Neuroinflammation mediated by microglia and infiltrating peripheral immune cells is a major component of stroke pathophysiology. Interfering with the inflammation cascade after stroke holds the promise to modulate stroke outcome. The calcium activated potassium channel K Ca 3.1 is expressed selectively in the injured CNS by microglia. K Ca 3.1 function has been implicated in pro-inflammatory activation of microglia and there is recent literature suggesting that this channel is important in the pathophysiology of ischemia/reperfusion (stroke) related brain injury. Here we describe the potential of repurposing Senicapoc, a K Ca 3.1 inhibitor, to intervene in the inflammation cascade that follows ischemia/reperfusion.

Innate immune signaling is important in the pathophysiology of ischemia/reperfusion (stroke)-induced injury and recovery. Several lines of evidence support a central role for microglia in these processes. Recent work has identified Toll-like receptors (TLRs) and type I interferon (IFN) signaling in both ischemia/reperfusion-induced brain injury and ischemic preconditioning-mediated neuroprotection. To determine the effects of "ischemia/reperfusion-like" conditions on microglia, we performed genomic analyses on wild-type (WT) and TLR4 -/- cultured microglia after sequential exposure to hypoxia/hypoglycemia and normoxia/normoglycemia (H/H-N/N). We observed increased expression of type 1 IFN-stimulated genes (ISGs) as the predominant transcriptomal feature of H/H-N/N-exposed WT, but not TLR4 -/- , microglia. Microarray analysis on ex vivo sorted microglia from ipsilateral male mouse cortex after a transient in vivo ischemic pulse also demonstrated robust expression of ISGs. Type 1 IFNs, including the IFN-αs and IFN-β, activate the interferon-α/β receptor (IFNAR) complex. We confirmed both in vitro H/H-N/N- and in vivo ischemia/reperfusion-induced microglial ISG responses by quantitative real-time PCR and demonstrated that both were dependent on IFNAR1. We characterized the effects of hypoxia/hypoglycemia on phosphorylation of signal transducer and activator of transcription 1 (STAT1), release of type 1 IFNs, and surface expression of IFNAR1 in microglia. We demonstrated that IFN-β induces dose-dependent secretion of ISG chemokines in cultured microglia and robust ISG expression in microglia both in vitro and in vivo Finally, we demonstrated that the microglial ISG chemokine responses to TLR4 agonists were dependent on TLR4 and IFNAR1. Together, these data suggest novel ischemia/reperfusion-induced pathways for both TLR4-dependent and -independent, IFNAR1-dependent, type 1 IFN signaling in microglia. SIGNIFICANCE STATEMENT Stroke is the fifth leading cause of death in the United States and is a leading cause of serious long-term disability worldwide. Innate immune responses are critical in stroke pathophysiology, and microglia are key cellular effectors in the CNS response to ischemia/reperfusion. Using a transcriptional analysis approach, we identified a robust interferon (IFN)-stimulated gene response within microglia exposed to ischemia/reperfusion in both in vitro and in vivo experimental paradigms. Using a number of complementary techniques, we have demonstrated that these responses are dependent on innate immune signaling components including Toll-like receptor-4 and type I IFNs. We have also elucidated several novel ischemia/reperfusion-induced microglial signaling mechanisms., (Copyright © 2017 the authors 0270-6474/17/378292-17$15.00/0.)

Background: Household air pollution is a major contributor to death and disability worldwide. Over 95% of rural Guatemalan households use woodstoves for cooking or heating. Woodsmoke contains carcinogenic or fetotoxic polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOCs). Increased PAHs and VOCs have been shown to increase levels of oxidative stress., Objective: We examined PAH and VOC exposures among recently pregnant rural Guatemalan women exposed to woodsmoke and compared exposures to levels seen occupationally or among smokers., Methods: Urine was collected from 23 women who were 3 months post-partum three times over 72h: morning (fasting), after lunch, and following dinner or use of wood-fired traditional sauna baths (samples=68). Creatinine-adjusted urinary concentrations of metabolites of four PAHs and eight VOCs were analyzed by liquid chromatography-mass spectrometry. Creatinine-adjusted urinary biomarkers of oxidative stress, 8-isoprostane and 8-OHdG, were analyzed using enzyme-linked immunosorbent assays (ELISA). Long-term (pregnancy through 3 months prenatal) exposure to particulate matter and airborne PAHs were measured., Results: Women using wood-fueled chimney stoves are exposed to high levels of particulate matter (median 48h PM 2.5 105.7μg/m 3 ; inter-quartile range (IQR): 77.6-130.4). Urinary PAH and VOC metabolites were significantly associated with woodsmoke exposures: 2-naphthol (median (IQR) in ng/mg creatinine: 295.9 (74.4-430.9) after sauna versus 23.9 (17.1-49.5) fasting; and acrolein: 571.7 (429.3-1040.7) after sauna versus 268.0 (178.3-398.6) fasting. Urinary PAH (total PAH: ρ=0.89, p<0.001) and VOC metabolites of benzene (ρ=0.80, p<0.001) and acrylonitrile (ρ=0.59, p<0.05) were strongly correlated with long-term exposure to particulate matter. However urinary biomarkers of oxidative stress were not correlated with particulate matter (ρ=0.01 to 0.05, p>0.85) or PAH and VOC biomarkers (ρ=-0.20 to 0.38, p>0.07). Urinary metabolite concentrations were significantly greater than those of heavy smokers (mean cigarettes/day=18) across all PAHs. In 15 (65%) women, maximum 1-hydroxypyrene concentrations exceeded the occupational exposure limit of coke-oven workers., Conclusions: The high concentrations of urinary PAH and VOC metabolites among recently pregnant women is alarming given the detrimental fetal and neonatal effects of prenatal PAH exposure. As most women used chimney woodstoves, cleaner fuels are critically needed to reduce smoke exposure., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Tissue plasminogen activator (tPA) was first approved in the USA 25 years ago for those who had experienced a recent occlusion (<3 h) of a cerebral vessel. Now, advances in clot retrieval (stentriever), in concert with tPA, heralds new optimism for ischemic stroke victims, but adds more pressure to identify therapies that will minimize hypoxic damage, protect compromised cells, and promote rehabilitation. In the past preclinical investigations have been poor at predicting potential clinical therapy, but they have contributed enormously to understanding post-stroke pathology. Current clinical trials ( www.strokecenter.org/trials ) anticipate a broad range of approaches: from hypothermia, to cell therapy, to neuroprotection.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Research on IPC has implicated cellular, molecular, and systemic elements of the immune response in this phenomenon. Potent molecular mediators of IPC include innate immune signaling pathways such as Toll-like receptors and type 1 interferons. Brain ischemia results in release of pro- and anti-inflammatory cytokines and chemokines that orchestrate the neuroinflammtory response, resolution of inflammation, and transition to neurological recovery and regeneration. Cellular mediators of IPC include microglia, the resident central nervous system immune cells, astrocytes, and neurons. All of these cell types engage in cross-talk with each other using a multitude of signaling pathways that modulate activation/suppression of each of the other cell types in response to ischemia. As the postischemic neuroimmune response evolves over time there is a shift in function toward provision of trophic support and neuroprotection. Peripheral immune cells infiltrate the central nervous system en masse after stroke and are largely detrimental, with a few subtypes having beneficial, protective effects, though the role of these immune cells in IPC is largely unknown. The role of neural progenitor cells in IPC-mediated neuroprotection is another active area of investigation as is the role of microglial proliferation in this setting. A mechanistic understanding of these molecular and cellular mediators of IPC may not only facilitate more effective direct application of IPC to specific clinical scenarios, but also, more broadly, reveal novel targets for therapeutic intervention in stroke.

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR -null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator ( CFTR ) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl - transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR -null pig airways., Competing Interests: KJDAE is a member of the scientific advisory board, and DVS is the cofounder, acting CSO, and cochairman of 4D Molecular Therapeutics. MJW is a founder of Exemplar Genetics. DAS, MJW, and the University of Iowa Research Foundation have applied for a patent related to genetically modified pigs. MJW, JFE, and JZ are founders of and hold equity in Talee Bio.

Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon whereby brief ischemic exposure confers tolerance to a subsequent ischemic challenge. IPC has not been studied selectively in CNS white matter (WM), although stroke frequently involves WM. We determined whether IPC is present in WM and, if so, its mechanism. We delivered a brief in vivo preconditioning ischemic insult (unilateral common carotid artery ligation) to 12- to 14-week-old mice and determined WM ischemic vulnerability [oxygen-glucose deprivation (OGD)] 72 h later, using acutely isolated optic nerves (CNS WM tracts) from the preconditioned (ipsilateral) and control (contralateral) hemispheres. Functional and structural recovery was assessed by quantitative measurement of compound action potentials (CAPs) and immunofluorescent microscopy. Preconditioned mouse optic nerves (MONs) showed better functional recovery after OGD than the non-preconditioned MONs (31 ± 3 vs 17 ± 3% normalized CAP area, p < 0.01). Preconditioned MONs also showed improved axon integrity and reduced oligodendrocyte injury compared with non-preconditioned MONs. Toll-like receptor-4 (TLR4) and type 1 interferon receptor (IFNAR1), key receptors in innate immune response, are implicated in gray matter preconditioning. Strikingly, IPC-mediated WM protection was abolished in both TLR4(-/-) and IFNAR1(-/-) mice. In addition, IPC-mediated protection in WM was also abolished in IFNAR1(fl/fl) LysM(cre), but not in IFNAR1(fl/fl) control, mice. These findings demonstrated for the first time that IPC was robust in WM, the phenomenon being intrinsic to WM itself. Furthermore, WM IPC was dependent on innate immune cell signaling pathways. Finally, these data demonstrated that microglial-specific expression of IFNAR1 plays an indispensable role in WM IPC., Significance Statement: Ischemic preconditioning (IPC) has been studied predominantly in gray matter, but stroke in humans frequently involves white matter (WM) as well. Here we describe a novel, combined in vivo/ex vivo mouse model to determine whether IPC occurs in WM. It does. Using genetically altered mice, we identified two innate immune cell receptors, Toll-like receptor 4 and type 1 interferon receptor (IFNAR1), that are required for IPC-mediated protection in WM. Furthermore, using microglia-targeted IFNAR1 knockdown, we demonstrate that interferon signaling specifically in microglia is essential for this protection. The discovery of IPC as an intrinsic capability of WM is novel and important. This is also the first in vivo demonstration that cell-type-specific expression of an individual gene plays an indispensable role in IPC-mediated protection., (Copyright © 2015 the authors 0270-6474/15/3515599-13$15.00/0.)

Toll-like receptor-4 (TLR4) is important in neuroinflammation. Single nucleotide polymorphisms (SNPs) in TLR4, including 1063 A/G [Asp299Gly] and 1363 C/T [Thr399Ile], are associated with altered immune responses but their effect on acute ischemic stroke (AIS) outcome is unknown. We collected demographic, clinical, laboratory, radiologic, and genotype data on 113 AIS patients and performed multivariate analyses to assess associations between TLR4 SNP haplotype and either neurological outcome, infection, or inflammatory markers. In adjusted analyses, TLR4 SNPs were associated with worse outcome as well as increases in circulating leukocytes, C-reactive protein, and interleukin-1 receptor antagonist. In AIS, variations in TLR4 may influence neurological outcome (for video abstract, please see Supplemental digital content 1 file, http://links.lww.com/WNR/A274).

Thrombin is a multifunctional serine proteinase that induces a variety of responses from neural cells by cleavage of proteinase-activated receptors (PARs) including PAR1 and PAR4. Thrombin/PAR signaling has been implicated in the neuroinflammatory response that occurs in the brain following stroke and other central nervous system pathologies. The neuroinflammatory response involves astrocytes and results in induction of proinflammatory chemokines including interleukin-8 (IL-8 or CXCL8) and interferon-γ-induced protein-10 (IP-10 or CXCL10) in these cells. Astroctyes are known to express PARs, however the effect of thrombin on astrocytic chemokine secretion is unknown. Here we characterize the ability of thrombin to induce proliferation/metabolic activity and chemokine secretion in primary human fetal astrocytes. Thrombin induces dose-dependent astrocyte proliferation as well as release of both IL-8 and IP-10, but not IL-6 or the chemokine regulated and normal T cell expressed and secreted (RANTES). The chemokine responses were mimicked by PAR1, but not PAR4, activating peptides. Our data indicate that astrocytic chemokine release is part of the neuroinflammatory response triggered by the exposure of the central nervous system to thrombin.

RCVS is a clinical condition of recurrent severe headaches that may be associated with ischemic or hemorrhagic stroke and that is defined by the presence of segmental vasoconstriction in multiple cerebral arteries. The angiographic appearance resembles vasculitis, except that the abnormalities resolve during the course of several months. Because the treatment of RCVS differs from that for vasculitis, radiologists must understand the clinical and radiologic features so as to better guide imaging algorithms and facilitate diagnosis. We present a series of 6 cases of RCVS that highlight the imaging features across multiple modalities.

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution-the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest-offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties.

Objectives: Statistical models predicting outcome after intraparenchymal hemorrhage include patients irrespective of do-not-attempt-resuscitation orders. We built a model to explore how the inclusion of patients with do-not-attempt-resuscitation orders affects intraparenchymal hemorrhage prognostic models., Design: Retrospective, observational cohort study from May 2001 until September 2003., Setting: University-affiliated tertiary referral hospital in Seattle, WA., Patients: Four hundred twenty-four consecutive patients with spontaneous intraparenchymal hemorrhage., Measurements and Main Results: We retrospectively abstracted information from medical records of intraparenchymal hemorrhage patients admitted to a single hospital. Using multivariate logistic regression of presenting clinical characteristics, but not do-not-attempt-resuscitation status, we generated a prognostic score for favorable outcome (defined as moderate disability or better at discharge). We compared observed probability of favorable outcome with that predicted, stratified by do-not-attempt-resuscitation status. We then generated a modified prognostic score using only non-do-not-attempt-resuscitation patients. Records of 424 patients were reviewed: 44% had favorable outcome, 43% had a do-not-attempt-resuscitation order, and 38% died in hospital. The observed and predicted probability of favorable outcome agreed well with all patients taken together. The observed probability of favorable outcome was significantly higher than predicted in non-do-not-attempt-resuscitation patients and significantly lower in do-not-attempt-resuscitation patients. Results were similar when applying a previously published and validated prognostic score. Our modified prognostic score was no longer pessimistic in non-do-not-attempt-resuscitation patients but remained overly optimistic in do-not-attempt-resuscitation patients., Conclusions: Although our prognostic model was well-calibrated when assessing all intraparenchymal hemorrhage patients, predictions were significantly pessimistic in patients without and optimistic in those with do-not-attempt-resuscitation orders. Such pessimism may drive decisions not to attempt resuscitation in patients in whom a favorable outcome may have been possible, thereby creating a self-fulfilling prophecy. To be most useful in clinical decision making, intraparenchymal hemorrhage prognostic models should be calibrated to large intraparenchymal hemorrhage cohorts in whom do-not-attempt-resuscitation orders were not used.

Age-associated loss of kidney function has been recognized for decades. With aging, many subjects exhibit progressive decreases in glomerular filtration rate and renal blood flow, with wide variability among individuals. The fall in glomerular filtration rate is because of reductions in the glomerular capillary plasma flow rate and the glomerular capillary ultrafiltration coefficient. In addition, a primary reduction in afferent arteriolar resistance is associated with an increase in glomerular capillary hydraulic pressure. These hemodynamic changes occur in concert with structural changes, including loss of renal mass; hyalinization of afferent arterioles and in some cases, development of aglomerular arterioles; an increase in the percentage of sclerotic glomeruli; and tubulointerstitial fibrosis. Aging is associated with altered activity and responsiveness to vasoactive stimuli, such that responses to vasoconstrictor stimuli are enhanced, whereas vasodilatory responses are impaired. Changes in the activity of the renin-angiotensin and nitric oxide systems appear to be particularly important, as is the modulating effect of gender. These changes may predispose the older kidney to acute kidney injury, including normotensive ischemic nephropathy, as well as progressive chronic kidney disease., (2010 National Kidney Foundation, Inc. All rights reserved.)

Microglia are resident CNS immune cells that are active sensors in healthy brain and versatile effectors under pathological conditions. Cerebral ischemia induces a robust neuroinflammatory response that includes marked changes in the gene-expression profile and phenotype of a variety of endogenous CNS cell types (astrocytes, neurons and microglia), as well as an influx of leukocytic cells (neutrophils, macrophages and T-cells) from the periphery. Many molecules and conditions can trigger a transformation of surveying microglia to microglia of an alerted or reactive state. Here we review recent developments in the literature that relate to microglial activation in the experimental setting of in vitro and in vivo ischemia. We also present new data from our own laboratory demonstrating the direct effects of in vitro ischemic conditions on the microglial phenotype and genomic profile. In particular, we focus on the role of specific molecular signaling systems, such as hypoxia inducible factor-1 and Toll-like receptor-4, in regulating the microglial response in this setting. We then review histological and novel radiological data that confirm a key role for microglial activation in the setting of ischemic stroke in humans. We also discuss recent progress in the pharmacologic and molecular targeting of microglia in acute ischemic stroke. Finally, we explore how recent studies on ischemic preconditioning have increased interest in pre-emptively targeting microglial activation in order to reduce stroke severity.

Purpose: Angiotensin-converting enzyme (ACE) inhibitors are recommended for patients with chronic kidney disease (CKD) because they slow disease progression. But physicians' concerns about the risk of hyperkalemia (elevated serum potassium level), a potentially fatal adverse effect, may limit optimal management with ACE-inhibitors. We synthesized known predictors of hyperkalemia into a prognostic risk score to predict the risk of hyperkalemia., Methods: We assembled a retrospective cohort of adult patients with possible CKD (at least one estimated glomerular filtration rate (eGFR) value less than 60 ml/min/1.73 m(2)) who started an ACE-inhibitor (i.e., incident users) between 1998 and 2006 at a health maintenance organization. We followed patients for hyperkalemia: (1) potassium value >5.5 mmol/L; or (2) diagnosis code for hyperkalemia. Cox regression synthesized a priori predictors recorded in the electronic medical record into a risk score., Results: We followed 5171 patients and 145 experienced hyperkalemia, a 90-day risk of 2.8%. Predictors included: age, eGFR, diabetes, heart failure, potassium supplements, potassium-sparing diuretics, and a high dose for the ACE-inhibitor (lisinopril). The risk score separated high-risk patients (top quintile, observed risk of 6.9%) from low-risk patients (bottom quintile, observed risk of 0.7%). Predicted and observed risks agreed within 1% for each quintile. The risk increased gradually in relation to declining eGFR with no apparent threshold for contraindicating ACE-inhibitors., Conclusions: The risk score separated high-risk patients (who may need more intensive laboratory monitoring) from low-risk patients. The risk score should be validated in other populations before it is ready for use in clinical practice.

As resident macrophages in the CNS, microglia can transform from a surveillance state to an activated phenotype in response to brain injury. During this transition microglia become highly capable phagocytic cells. Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate F(c) receptors. In mice, both FcgammaRI and FcgammaRIIb receptors are involved in IgG-mediated phagocytosis of opsonzied pathogens. At sites of inflammation, microglial activity is regulated by T-cell derived cytokines. Here we first investigated the effects of IFN-gamma, IL-4, IL-13 and GM-CSF on expression of FcgammaRI and FcgammaRIIb mRNA levels in both primary microglia and microglial cell line N9. Using quantitative real-time PCR we show that IFN-gamma induced a 4-fold increase in the mRNA level of FcgammaRI but did not induce changes in FcgammaRIIb expression. IL-4 and IL-13 induced approximately 2-fold increases in expression of FcgammaRIIb mRNA, but had no effect on FcgammaRI expression. GM-CSF increased both FcgammaRI and FcgammaRIIb mRNA expression. We then characterized the ability of these same cytokines to regulate phagocytosis of immune complexes composed of IgG and the bacteria Staphylococcus aureus. IFN-gamma and GM-CSF both induced approximately 2-fold increases in IgG-mediated phagocytosis whereas IL-4 and IL-13 both decreased IgG-mediated phagocytosis by about one-third. None of the cytokines influenced basal levels of phagocytosis. These findings demonstrate a highly selective cytokine-induced regulation of both phagocytosis-related Fcgamma receptor subtypes and IgG-mediated phagocytosis itself in microglia. This selective regulation has implications for our understanding of the pathophysiology of CNS infection and autoimmune disease.