Temporal Immune Profiling in the CSF and Blood of Patients with Aneurysmal Subarachnoid Hemorrhage.

Background: Delayed cerebral ischemia (DCI) is a significant complication of aneurysmal subarachnoid hemorrhage (aSAH). This study profiled immune responses after aSAH and evaluated their association with DCI onset.

Methods: Twelve aSAH patients were enrolled.

FACS to Identify Immune Subsets in Mouse Brain and Spleen.

Flow cytometry enables the multi-parametric quantification of cell types, especially in immunophenotyping of unique immune cell subsets that can either contribute to or ameliorate pathology. For tissues to be used in such analyses, single-cell suspensions must be created. Here we describe protocols for preparing single-cell suspensions of mouse spleen and brain tissue, as well as the steps for fluorescently activated cell staining/sorting (FACS).

Plasma protein alterations during human large vessel stroke: A controlled comparison study.

Background: Stroke is a major cause of death and disability in the United States. Mechanical thrombectomy (MT) and tissue plasminogen activator are the current treatments for ischemic stroke, which have improved clinical outcomes. Despite these treatments, functional and cognitive deficits still occur demonstrating a need for predictive biomarkers for beneficial clinical outcomes which can be used as therapeutic targets for pharmacotherapy.

Influence of BMI on adenosine deaminase and stroke outcomes in mechanical thrombectomy subjects.

Background: Emergent Large Vessel Occlusion (ELVO) strokes are ischemic vascular events for which novel biomarkers and therapies are needed. The purpose of this study is to investigate the role of Body Mass Index (BMI) on protein expression and signaling at the time of ELVO intervention. Additionally, we highlight the protein adenosine deaminase (ADA), which is a deaminating enzyme that degrades adenosine, which has been shown to be neuroprotective in ischemia.

Antimicrobial protein REG3A and signaling networks are predictive of stroke outcomes.

Regenerating Family Member 3 Alpha (REG3A) is a multifunctional protein with antimicrobial activity, and primarily secreted by the intestine and pancreas. Studies have shown an increased expression of REG3A in systemic inflammatory responses to acute injury and infection, but studies investigating REG3A during the pathogenesis of ischemic stroke are limited. The aims of this study were to examine the associations between arterial expression of REG3A and other arterial inflammatory proteins implicated in stroke pathogenesis, as well as associations between REG3A and markers of poor outcome for ischemic stroke.

Isolation and identification of leukocyte populations in intracranial blood collected during mechanical thrombectomy.

Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (NCT03153683) isolates intracranial arterial blood distal to the thrombus and proximal systemic blood in the carotid artery. We augmented the current protocol to study leukocyte subpopulations both distal and proximal to the thrombus during human stroke (n = 16 patients), and from patients with cerebrovascular disease (CVD) undergoing angiography for unrelated conditions (e.g.

Intracranial VCAM1 at time of mechanical thrombectomy predicts ischemic stroke severity.

Background: Emergent large vessel occlusion (ELVO) strokes are devastating ischemic vascular events for which novel treatment options are needed. Using vascular cell adhesion molecule 1 (VCAM1) as a prototype, the objective of this study was to identify proteomic biomarkers and network signaling functions that are potential therapeutic targets for adjuvant treatment for mechanical thrombectomy.

Methods: The blood and clot thrombectomy and collaboration (BACTRAC) study is a continually enrolling tissue bank and registry from stroke patients undergoing mechanical thrombectomy.

Delayed diapedesis of CD8 T cells contributes to long-term pathology after ischemic stroke in male mice.

Objective: Stroke is a debilitating disorder with significant annual mortality and morbidity rates worldwide. Immune cells are recruited to the injured brain within hours after stroke onset and can exhibit either protective or detrimental effects on recovery. However, immune cells, including CD8 T cells, persist in the injured brain for weeks, suggesting a longer-term role for the adaptive immune system during functional recovery.

Proteomic changes in intracranial blood during human ischemic stroke.

Background: Since 2015, mechanical thrombectomy has been the standard treatment for emergent large vessel occlusion ischemic stroke.

Objective: To investigate, using the previously published Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683), how the protein expression of a patient's intracranial blood during ischemic stroke compares with the protein expression of their systemic arterial blood in order to better understand and treat stroke.

Methodology and effects of repeated intranasal delivery of DNSP-11 in awake Rhesus macaques.

Background: To determine if the intranasal delivery of neuroactive compounds is a viable, long-term treatment strategy for progressive, chronic neurodegenerative disorders, such as Parkinson's disease (PD), intranasal methodologies in preclinical models comparable to humans are needed.

New Method: We developed a methodology to evaluate the repeated intranasal delivery of neuroactive compounds on the non-human primate (NHP) brain, without the need for sedation. We evaluated the effects of the neuroactive peptide, DNSP-11 following repeated intranasal delivery and dose-escalation over the course of 10-weeks in Rhesus macaques.

Distinctly perturbed metabolic networks underlie differential tumor tissue damages induced by immune modulator β-glucan in a two-case ex vivo non-small-cell lung cancer study.

Cancer and stromal cell metabolism is important for understanding tumor development, which highly depends on the tumor microenvironment (TME). Cell or animal models cannot recapitulate the human TME. We have developed an ex vivo paired cancerous (CA) and noncancerous (NC) human lung tissue approach to explore cancer and stromal cell metabolism in the native human TME.

Methodology and effects of repeated intranasal delivery of DNSP-11 in a rat model of Parkinson's disease.

Background: To circumvent the challenges associated with delivering large compounds directly to the brain for the treatment of Parkinson's disease (PD), non-invasive procedures utilizing smaller molecules with protective and/or restorative actions on dopaminergic neurons are needed.

New Method: We developed a methodology for evaluating the effects of a synthetic neuroactive peptide, DNSP-11, on the nigrostriatal system using repeated intranasal delivery in both normal and a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD.

Results: Normal rats repeatedly administered varying doses of DNSP-11 intranasally for 3 weeks exhibited a significant increase in dopamine (DA) turnover in both the striatum and substantia nigra (SN) at 300μg, suggestive of a stimulative effect of the dopaminergic system.

Dynamic changes in dopamine neuron function after DNSP-11 treatment: effects in vivo and increased ERK 1/2 phosphorylation in vitro.

Glial cell-line derived neurotrophic factor (GDNF) has demonstrated robust effects on dopamine (DA) neuron function and survival. A post-translational processing model of the human GDNF proprotein theorizes the formation of smaller, amidated peptide(s) from the proregion that exhibit neurobiological function, including an 11-amino-acid peptide named dopamine neuron stimulating peptide-11 (DNSP-11). A single treatment of DNSP-11 was delivered to the substantia nigra in the rat to investigate effects on DA-neuron function.

A synthetic five amino acid propeptide increases dopamine neuron differentiation and neurochemical function.

A major consequence of Parkinson's disease (PD) involves the loss of dopaminergic neurons in the substantia nigra (SN) and a subsequent loss of dopamine (DA) in the striatum. We have shown that glial cell line-derived neurotrophic factor (GDNF) shows robust restorative and protective effects for DA neurons in rats, non-human primates and possibly in humans. Despite GDNF's therapeutic potential, its clinical value has been questioned due to its limited diffusion to target areas from its large size and chemical structure.

Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence.

Recently, a small 11-amino acid amidated peptide, dopamine neuron stimulating peptide-11 (DNSP-11), was shown to exert neurotrophic-like actions on primary dopaminergic neurons and in parkinsonian rat models. This suggests smaller neurotrophic-like molecules may be deliverable and modifiable for therapeutic use. Here we evaluate the molecular and cellular protection properties of DNSP-11 and two other amidated-peptides, a 5-mer (DNSP-5) and a 17-mer (DNSP-17), hypothesized to be endoproteolytically processed from the pro- and mature glial cell line-derived neurotrophic factor (GDNF) protein sequence, respectively.

Dopamine neuron stimulating actions of a GDNF propeptide.

Background: Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some Parkinson's disease (PD) clinical trials. However, the delivery of neurotrophic factors to the brain is difficult due to their large size and poor bio-distribution. In addition, developing more efficacious trophic factors is hampered by the difficulty of synthesis and structural modification.

Estrogen receptor alpha inhibits the estrogen-mediated suppression of HIV transcription in astrocytes: implications for estrogen neuroprotection in HIV dementia.

Many human immunodeficiency virus (HIV) proteins including Tat are produced by HIV-infected astrocytes and secreted into the brain resulting in extensive neuronal damage that contributes to the pathogenesis of HIV dementia. The neuroprotective hormone 17beta-estradiol (E2) is known to negatively regulate the HIV transcriptional promoter in human fetal astrocytes (SVGA cell line) in a Tat-dependent manner. In the present study we extended our investigation in HIV-infected SVGA cells and found a reduction in HIV p24 levels following E2 treatment in comparison to control.

Morphine and HIV-Tat increase microglial-free radical production and oxidative stress: possible role in cytokine regulation.

Opiate abuse alters the progression of human immunodeficiency virus and may increase the risk of neuroAIDS. As neuroAIDS is associated with altered microglial reactivity, the combined effects of human immunodeficiency virus-Tat and morphine were determined in cultured microglia. Specifically, experiments determined the effects of Tat and morphine on microglial-free radical production and oxidative stress, and on cytokine release.

NADPH oxidase drives cytokine and neurotoxin release from microglia and macrophages in response to HIV-Tat.

Previous reports have shown that the human immunodeficiency virus (HIV) regulatory protein Tat has both pro-oxidant and pro-inflammatory properties, suggesting that Tat might contribute to the neurological complications of HIV. However, the intracellular mechanisms whereby Tat triggers free radical production and inflammation, and the relationship between Tat-induced free radicals and inflammatory reactions, are still subject to debate. The present study was undertaken to evaluate the specific effects of Tat on NADPH oxidase in microglia and macrophages, and to determine the specific role of NADPH oxidase in Tat-induced cytokine/chemokine release and neurotoxicity.

Morphine causes rapid increases in glial activation and neuronal injury in the striatum of inducible HIV-1 Tat transgenic mice.

HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV-related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3-nitrotyrosine (3-NT) positive macrophages/microglia, was determined following a 2-day exposure to morphine (5 mg/kg/day via time-release, subcutaneous implant) and doxycycline in GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure.

Cell-specific actions of HIV-Tat and morphine on opioid receptor expression in glia.

HIV-1 patients who abuse opiate-based drugs, including heroin and morphine, are at a higher risk of developing HIV dementia. The effects of opiates are mediated predominantly through opioid receptors, which are expressed on glial cells. As HIV-1 infection in the CNS is restricted to glial cells, experiments were designed to measure the cell-specific effects of HIV Tat and morphine exposure on opioid receptor expression in both astrocytes and microglia.

Interaction of HIV Tat and matrix metalloproteinase in HIV neuropathogenesis: a new host defense mechanism.

Tat, the HIV transactivating protein, and matrix metalloproteinases (MMPs), a family of extracellular matrix (ECM) endopeptidases, have been implicated in the pathogenesis of HIV-associated dementia. However, the possibility that MMPs interact with viral proteins has remained unexplored. We therefore treated mixed human fetal neuronal cultures with recombinant Tat and select MMPs.

Increased vulnerability of ApoE4 neurons to HIV proteins and opiates: protection by diosgenin and L-deprenyl.

Human immunodeficiency virus (HIV) infection continues to rise in drug-abusing populations and causes a dementing illness in a subset of individuals. Factors contributing to the development of dementia in this population remain unknown. We found that HIV-infected individuals with the E4 allele of Apolipoprotein E (ApoE) or history of intravenous drug abuse had increased oxidative stress in the CNS.

Gonadal steroids differentially modulate neurotoxicity of HIV and cocaine: testosterone and ICI 182,780 sensitive mechanism.

Background: HIV Associated Dementia (HAD) is a common complication of human immunodeficiency virus (HIV) infection that erodes the quality of life for patients and burdens health care providers. Intravenous drug use is a major route of HIV transmission, and drug use is associated with increased HAD. Specific proteins released as a consequence of HIV infection (e.

14-3-3 protein in CSF: an early predictor of SIV CNS disease.

In neurons, 14-3-3 proteins regulate diverse processes, including signal transduction, neurotransmitter production, and apoptosis by binding to target proteins, but the role 14-3-3 proteins play in the pathogenesis of central nervous system (CNS) disease remains unclear. To examine the relationship between presence of 14-3-3 protein in cerebrospinal fluid (CSF) and encephalitis in the SIV/macaque model of HIV CNS disease, CSF levels of 14-3-3 protein were measured by quantitative immunoblotting throughout infection in 6 SIV-infected pigtailed macaques. Beginning during asymptomatic infection and continuing until death, CSF levels of 14-3-3 were elevated in 4 of 6 SIV-infected animals.