Alcohol and e-cigarette damage alveolar-epithelial barrier by activation of P2X7r and provoke brain endothelial injury via extracellular vesicles.

BACKGROUND: Use of nicotine containing products like electronic cigarettes (e-Cig) and alcohol are associated with mitochondrial membrane depolarization, resulting in the extracellular release of ATP, and mitochondrial DNA (mtDNA), mediating inflammatory responses. While nicotine effects on lungs is well-known, chronic alcohol (ETH) exposure also weakens lung immune responses and cause inflammation. Extracellular ATP (eATP) released by inflammatory/stressed cells stimulate purinergic P2X7 receptors (P2X7r) activation in adjacent cells. We hypothesized that injury caused by alcohol and e-Cig to pulmonary alveolar epithelial cells (hPAEpiC) promote the release of eATP, mtDNA and P2X7r in circulation. This induces a paracrine signaling communication either directly or via EVs to affect brain cells (human brain endothelial cells - hBMVEC). METHODS: We used a model of primary human pulmonary alveolar epithelial cells (hPAEpiC) and exposed the cells to 100 mM ethanol (ETH), 100 µM acetaldehyde (ALD), or e-Cig (1.75 µg/mL of 1.8% or 0% nicotine) conditioned media, and measured the mitochondrial efficiency using Agilent Seahorse machine. Gene expression was measured by Taqman RT-qPCR and digital PCR. hPAEpiC-EVs were extracted from culture supernatant and characterized by flow cytometric analysis. Calcium (Ca2+) and eATP levels were quantified using commercial kits. To study intercellular communication via paracrine signaling or by EVs, we stimulated hBMVECs with hPAEpiC cell culture medium conditioned with ETH, ALD or e-cig or hPAEpiC-EVs and measured Ca2+ levels. RESULTS: ETH, ALD, or e-Cig (1.8% nicotine) stimulation depleted the mitochondrial spare respiration capacity in hPAEpiC. We observed increased expression of P2X7r and TRPV1 genes (3-6-fold) and increased intracellular Ca2+ accumulation (20-30-fold increase) in hPAEpiC, resulting in greater expression of endoplasmic reticulum (ER) stress markers. hPAEpiC stimulated by ETH, ALD, and e-Cig conditioned media shed more EVs with larger particle sizes, carrying higher amounts of eATP and mtDNA. ETH, ALD and e-Cig (1.8% nicotine) exposure also increased the P2X7r shedding in media and via EVs. hPAEpiC-EVs carrying P2X7r and eATP cargo triggered paracrine signaling in human brain microvascular endothelial cells (BMVECs) and increased Ca2+ levels. P2X7r inhibition by A804598 compound normalized mitochondrial spare respiration, reduced ER stress and diminished EV release, thus protecting the BBB function. CONCLUSION: Abusive drugs like ETH and e-Cig promote mitochondrial and endoplasmic reticulum stress in hPAEpiC and disrupts the cell functions via P2X7 receptor signaling. EVs released by lung epithelial cells against ETH/e-cig insults, carry a cargo of secondary messengers that stimulate brain cells via paracrine signals.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Effects of Electronic Nicotine Delivery Systems and Cigarettes on Systemic Circulation and Blood-Brain Barrier: Implications for Cognitive Decline.

Electronic nicotine delivery systems (often known as e-cigarettes) are a novel tobacco product with growing popularity, particularly among younger demographics. The implications for public health are twofold, as these products may represent a novel source of tobacco-associated disease but may also provide a harm reduction strategy for current tobacco users. There is increasing recognition that e-cigarettes impact vascular function across multiple organ systems. Herein, we provide a comparison of evidence regarding the role of e-cigarettes versus combustible tobacco in vascular disease and implications for blood-brain barrier dysfunction and cognitive decline. Multiple non-nicotinic components of tobacco smoke have been identified in e-cigarette aerosol, and their involvement in vascular disease is discussed. In addition, nicotine and nicotinic signaling may modulate peripheral immune and endothelial cell populations in a highly context-dependent manner. Direct preclinical evidence for electronic nicotine delivery system-associated neurovascular impairment is provided, and a model is proposed in which non-nicotinic elements exert a proinflammatory effect that is functionally antagonized by the presence of nicotine.

let-7g counteracts endothelial dysfunction and ameliorating neurological functions in mouse ischemia/reperfusion stroke model.

Stroke is a debilitating disease, accounting for almost 20% of all hospital visits, and 8% of all fatalities in the United States in 2017. Following an ischemic attack, inflammatory processes originating from endothelial cells within the brain microvasculature can induce many toxic effects into the impacted area, from both sides of the blood brain barrier (BBB). In addition to increased BBB permeability, impacted brain microvascular endothelial cells can recruit macrophages and other immune cells from the periphery and can also trigger the activation of microglia and astrocytes within the brain. We have identified a key microRNA, let-7g, which levels were drastically diminished as consequence of transient middle cerebral artery occlusion (tMCAO) in vivo and oxygen-glucose deprivation (OGD) in vitro ischemia/reperfusion conditions, respectively. We have observed that let-7g* liposome-based delivery is capable of attenuating inflammation after stroke, reducing BBB permeability, limiting brain infiltration by CD3+CD4+ T-cells and Ly6G+ neutrophils, lessening microglia activation and neuronal death. These effects consequently improved clinical outcomes, shown by mitigating post-stroke gait asymmetry and extremity motor function. Due to the role of the endothelium in propagating the effects of stroke and other inflammation, treatments which can reduce endothelial inflammation and limit ischemic damage and improving recovery after a stroke are required. Our findings demonstrate a critical link between the CNS inflammation and the immune system reaction and lay important groundwork for future stroke pharmacotherapies.

Electronic cigarette exposure disrupts blood-brain barrier integrity and promotes neuroinflammation.

Electronic cigarette (e-cigarette) use has grown substantially since inception, particularly among adolescents and combustible tobacco users. Several cigarette smoke constituents with known neurovascular effect are present in e-cigarette liquids or formed during the vapor generation. The present study establishes inhaled models of cigarette and e-cigarette use with normalized nicotine delivery, then characterizes the impact on blood-brain barrier (BBB) function. Sequencing of microvessel RNA following exposure revealed downregulation of several genes with critical roles in BBB function. Reduced protein expression of Occludin and Glut1 is also observed at the tight junction in all groups following exposure. Pro-inflammatory changes in leukocyte-endothelial cell interaction are also noted, and mice exposed to nicotine-free e-cigarettes have impaired novel object recognition performance. On this basis, it is concluded that long term e-cigarette use may adversely impact neurovascular health. The observed effects are noted to be partly independent of nicotine content and nicotine may even serve to moderate the effects of non-nicotinic components on the blood-brain barrier.

Combination of HIV-1 and Diabetes Enhances Blood Brain Barrier Injury via Effects on Brain Endothelium and Pericytes.

Despite combined antiretroviral therapy (ART) achieving efficient HIV replication control, HIV-associated neurocognitive disorders (HAND) continue to be highly prevalent in HIV-infected patients. Diabetes mellitus (DM) is a well-known comorbidity of HAND in HIV-infected patients. Blood brain barrier (BBB) dysfunction has been linked recently to dementia development, specifically in DM patients. BBB injury exists both in HIV and DM, likely contributing to cognitive decline. However, its extent, exact cellular targets and mechanisms are largely unknown. In this report, we found a decrease in pericyte coverage and expression of tight junction proteins in human brain tissues from HIV patients with DM and evidence of HAND when compared to HIV-infected patients without DM or seronegative DM patients. Using our in vitro BBB models, we demonstrated diminution of barrier integrity, enhanced monocyte adhesion, changes in cytoskeleton and overexpression of adhesion molecules in primary human brain endothelial cells or human brain pericytes after exposure to HIV and DM-relevant stimuli. Our study demonstrates for the first-time evidence of impaired BBB function in HIV-DM patients and shows potential mechanisms leading to it in brain endothelium and pericytes that may result in poorer cognitive performance compared to individuals without HIV and DM.

Activation of GPR55 induces neuroprotection of hippocampal neurogenesis and immune responses of neural stem cells following chronic, systemic inflammation.

New neurons are continuously produced by neural stem cells (NSCs) within the adult hippocampus. Numerous diseases, including major depressive disorder and HIV-1 associated neurocognitive disorder, are associated with decreased rates of adult neurogenesis. A hallmark of these conditions is a chronic release of neuroinflammatory mediators by activated resident glia. Recent studies have shown a neuroprotective role on NSCs of cannabinoid receptor activation. Yet, little is known about the effects of GPR55, a candidate cannabinoid receptor, activation on reductions of neurogenesis in response to inflammatory insult. In the present study, we examined NSCs exposed to IL-1ß in vitro to assess inflammation-caused effects on NSC differentiation and the ability of GPR55 agonists to attenuate NSC injury. NSC differentiation and neurogenesis was determined via immunofluorescence and flow cytometric analysis of NSC markers (Nestin, Sox2, DCX, S100ß, ßIII Tubulin, GFAP). GPR55 agonist treatment protected against IL-1ß induced reductions in neurogenesis rates. Moreover, inflammatory cytokine receptor mRNA expression was down regulated by GPR55 activation in a neuroprotective manner. To determine inflammatory responses in vivo, we treated C57BL/6 and GPR55-/- mice with LPS (0.2 mg/kg/day) continuously for 14 days via osmotic mini-pump. Reductions in NSC survival (as determined by BrdU incorporation), immature neurons, and neuroblast formation due to LPS were attenuated by concurrent direct intrahippocampal administration of the GPR55 agonist, O-1602 (4 µg/kg/day). Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55-/- animals manifesting in a prolonged inflammatory response (IL-1ß, IL-6, TNFα) after chronic, systemic inflammation as compared to C57BL/6 animals. Taken together, these results suggest a neuroprotective role of GPR55 activation on NSCs in vitro and in vivo and that GPR55 provides a novel therapeutic target against negative regulation of hippocampal neurogenesis by inflammatory insult.

Secoisolariciresinol diglucoside is a blood-brain barrier protective and anti-inflammatory agent: implications for neuroinflammation.

BACKGROUND: Secoisolariciresinol diglucoside (SDG), the main lignan in flaxseed, is known for its beneficial effects in inflammation, oxidative stress, heart disease, tumor progression, atherosclerosis, and diabetes. SDG might be an attractive natural compound that protects against neuroinflammation. Yet, there are no comprehensive studies to date investigating the effects of SDG on brain endothelium using relevant in vivo and in vitro models. METHODS: We evaluated the effects of orally administered SDG on neuroinflammatory responses using in vivo imaging of the brain microvasculature during systemic inflammation and aseptic encephalitis. In parallel, the anti-inflammatory actions of SDG on brain endothelium and monocytes were evaluated in vitro blood-brain barrier (BBB) model. Multiple group comparisons were performed by one-way analysis of variance with Dunnet's post hoc tests. RESULTS: We found that SDG diminished leukocyte adhesion to and migration across the BBB in vivo in the setting of aseptic encephalitis (intracerebral TNFα injection) and prevented enhanced BBB permeability during systemic inflammatory response (LPS injection). In vitro SDG pretreatment of primary human brain microvascular endothelial cells (BMVEC) or human monocytes diminished adhesion and migration of monocytes across brain endothelial monolayers in conditions mimicking CNS inflammatory responses. Consistent with our in vivo observations, SDG decreased expression of the adhesion molecule, VCAM1, induced by TNFα, or IL-1ß in BMVEC. SDG diminished expression of the active form of VLA-4 integrin (promoting leukocyte adhesion and migration) and prevented the cytoskeleton changes in primary human monocytes activated by relevant inflammatory stimuli. CONCLUSION: This study indicates that SDG directly inhibits BBB interactions with inflammatory cells and reduces the inflammatory state of leukocytes. Though more work is needed to determine the mechanism by which SDG mediates these effects, the ability of SDG to exert a multi-functional response reducing oxidative stress, inflammation, and BBB permeability makes it an exciting potential therapeutic for neuroinflammatory diseases. SDG can serve as an anti-inflammatory and barrier-protective agent in neuroinflammation.

PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Mitochondrial dysfunction, oxidative stress, inflammation, and metabolic reprograming are crucial contributors to hepatic injury and subsequent liver fibrosis. Poly(ADP-ribose) polymerases (PARP) and their interactions with sirtuins play an important role in regulating intermediary metabolism in this process. However, there is little research into whether PARP inhibition affects alcoholic and non-alcoholic steatohepatitis (ASH/NASH). METHODS: We investigated the effects of genetic deletion of PARP1 and pharmacological inhibition of PARP in models of early alcoholic steatohepatitis, as well as on Kupffer cell activation in vitro using biochemical assays, real-time PCR, and histological analyses. The effects of PARP inhibition were also evaluated in high fat or methionine and choline deficient diet-induced steatohepatitis models in mice. RESULTS: PARP activity was increased in livers due to excessive alcohol intake, which was associated with decreased NAD+ content and SIRT1 activity. Pharmacological inhibition of PARP restored the hepatic NAD+ content, attenuated the decrease in SIRT1 activation and beneficially affected the metabolic-, inflammatory-, and oxidative stress-related alterations due to alcohol feeding in the liver. PARP1-/- animals were protected against alcoholic steatohepatitis and pharmacological inhibition of PARP or genetic deletion of PARP1 also attenuated Kupffer cell activation in vitro. Furthermore, PARP inhibition decreased hepatic triglyceride accumulation, metabolic dysregulation, or inflammation and/or fibrosis in models of NASH. CONCLUSION: Our results suggests that PARP inhibition is a promising therapeutic strategy in steatohepatitis with high translational potential, considering the availability of PARP inhibitors for clinical treatment of cancer. LAY SUMMARY: Poly(ADP-ribose) polymerases (PARP) are the most abundant nuclear enzymes. The PARP inhibitor olaparib (Lynparza) is a recently FDA-approved therapy for cancer. This study shows that PARP is overactivated in livers of subjects with alcoholic liver disease and that pharmacological inhibition of this enzyme with 3 different PARP inhibitors, including olaparib, attenuates high fat or alcohol induced liver injury, abnormal metabolic alteration, fat accumulation, inflammation and/or fibrosis in preclinical models of liver disease. These results suggest that PARP inhibition is a promising therapeutic strategy in the treatment of alcoholic and non-alcoholic liver diseases.

PARP inhibition in leukocytes diminishes inflammation via effects on integrins/cytoskeleton and protects the blood-brain barrier.

BACKGROUND: Blood-brain barrier (BBB) dysfunction/disruption followed by leukocyte infiltration into the brain causes neuroinflammation and contributes to morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. The identification of pathways that decreases the inflammatory potential of leukocytes would prevent such injury. Poly(ADP-ribose) polymerase 1 (PARP) controls various genes via its interaction with myriad transcription factors. Selective PARP inhibitors have appeared lately as potent anti-inflammatory tools. Their effects are outside the recognized PARP functions in DNA repair and transcriptional regulation. In this study, we explored the idea that selective inhibition of PARP in leukocytes would diminish their engagement of the brain endothelium. METHODS: Cerebral vascular changes and leukocyte-endothelium interactions were surveyed by intravital videomicroscopy utilizing a novel in vivo model of localized aseptic meningitis when TNFα was introduced intracerebrally in wild-type (PARP+/+) and PARP-deficient (PARP-/-) mice. The effects of selective PARP inhibition on primary human monocytes ability to adhere to or migrate across the BBB were also tested in vitro, employing primary human brain microvascular endothelial cells (BMVEC) as an in vitro model of the BBB. RESULTS: PARP suppression in monocytes diminished their adhesion to and migration across BBB in vitro models and prevented barrier injury. In monocytes, PARP inactivation decreased conformational activation of integrins that plays a key role in their tissue infiltration. Such changes were mediated by suppression of activation of small Rho GTPases and cytoskeletal rearrangements in monocytes. In vitro observations were confirmed in vivo showing diminished leukocyte-endothelial interaction after selective PARP suppression in leukocytes accompanied by BBB protection. PARP knockout animals demonstrated a substantial diminution of inflammatory responses in brain microvasculature and a decrease in BBB permeability. CONCLUSIONS: These results suggest PARP inhibition in leukocytes as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation-induced leukocyte engagement.

Insights into end-organ injury in HIV infection: dynamics of monocyte trafficking to the brain in SIV encephalitis.

This commentary highlights the article by Nowlin et al, which explores the dynamic roles of macrophages in early and late central nervous system lentiviral disease.

Immune activation of human brain microvascular endothelial cells inhibits HIV replication in macrophages.

There is limited information about the role of blood-brain barrier (BBB) endothelial cells (ECs) in the central nervous system (CNS) and their innate immunity against HIV. We examined whether brain ECs can be immunologically activated to produce antiviral factors that inhibit HIV replication in macrophages. Human brain microvascular ECs expressed functional toll-like receptor 3 (TLR3) that could be activated by polyinosinic-polycytidylic acid (PolyI:C), resulting in the induction of endogenous interferon-ß (IFN-ß) and IFN-λ. The TLR3 activation of ECs also induced the phosphorylation of interferon regulatory transcription factor 3 (IRF3) and IRF7, the key regulators of IFN signaling pathway. When supernatant (SN) of PolyI:C-activated EC cultures was applied to infected macrophage cultures, HIV replication was significantly suppressed. This SN action of ECs on HIV was mediated through both IFN-ß and IFN-λ because antibodies to their receptors could neutralize the SN-mediated anti-HIV effect. The role of IFNs in EC-mediated anti-HIV activity is further supported by the observation that treatment with SN from EC cultures induced the expression of IFN-stimulated genes (ISGs: ISG56, OAS-1, and MxA) in macrophages. These observations indicate that brain microvascular ECs may be a key regulatory bystander, playing a crucial role in the BBB innate immunity against HIV infection.

Dysregulation of claudin-5 in HIV-induced interstitial pneumonitis and lung vascular injury. Protective role of peroxisome proliferator-activated receptor-γ.

RATIONALE: HIV-1-induced interstitial pneumonitis (IP) is a serious complication of HIV-1 infection, characterized by inflammation and cellular infiltration in lungs, often leading to respiratory failure and death. The barrier function of the pulmonary endothelium is caused in part by tight junction (TJ) proteins, such as claudin-5. Peroxisome proliferator-activated receptor (PPAR)-γ is expressed in lung tissues and regulates inflammation. We hypothesize that HIV-1 induces vascular lung injury, and HIV-1-mediated damage of the pulmonary endothelium and IP is associated with dysregulation of PPAR-γ. OBJECTIVES: Investigate the effects of HIV-1 infection on the pulmonary microvasculature and the modulatory effects of the PPAR-γ ligands. METHODS: Using human lung tissues, we demonstrated down-regulation of claudin-5 (marker of pulmonary barrier integrity), down-regulation of PPAR-γ transcription, and expression in lung tissues of HIV-1-infected humans with IP. MEASUREMENTS AND MAIN RESULTS: Human lung microvascular endothelial cells expressed the TJ proteins claudin-5, ZO-1, and ZO-2; HIV-1 decreased TJ proteins expression and induced nuclear factor-κB promoter activity, which was reversed by PPAR-γ agonist. Using two murine HIV/AIDS models, we demonstrated decreased claudin-5 expression and increased macrophage infiltration in the lungs of HIV-1-infected animals. Activation of PPAR-γ prevented HIV-1-induced claudin-5 down-regulation and significantly reduced viremia and pulmonary macrophage infiltration. CONCLUSIONS: HIV-induced IP is associated with injury to the lung vascular endothelium, with decreased TJ and PPAR-γ expression, and increased pulmonary macrophage infiltration. PPAR-γ ligands abrogated these effects. Thus, regulation of PPAR-γ can be a therapeutic approach against HIV-1-induced vascular damage and IP in infected humans. Removal of Expression of Concern: Issues leading to the previous expression of concern for this article have been resolved after further revisions and editorial review. No further concerns exist.

Selective activation of cannabinoid receptor 2 in leukocytes suppresses their engagement of the brain endothelium and protects the blood-brain barrier.

Cannabinoid receptor 2 (CB2) is highly expressed in immune cells and stimulation decreases inflammatory responses. We tested the idea that selective CB2 activation in human monocytes suppresses their ability to engage the brain endothelium and migrate across the blood-brain barrier (BBB), preventing consequent injury. Intravital videomicroscopy was used to quantify adhesion of leukocytes to cortical vessels in lipopolysaccharide-induced neuroinflammation, after injection of ex vivo CB2-activated leukocytes into mice; CB2 agonists markedly decreased adhesion of ex vivo labeled cells in vivo. In an in vitro BBB model, CB2 activation in monocytes largely attenuated adhesion to and migration across monolayers of primary human brain microvascular endothelial cells and diminished BBB damage. CB2 stimulation in monocytes down-regulated active forms of integrins, lymphocyte function-associated antigen 1 (LFA-1), and very late antigen 4 (VLA-4). Cells treated with CB2 agonists exhibited increased phosphorylation levels of inhibitory sites of the actin-binding proteins cofilin and VASP, which are upstream regulators of conformational integrin changes. Up-regulated by relevant stimuli, Rac1 and RhoA were suppressed by CB2 agonists in monocytes. CB2 stimulation decreased formation of lamellipodia, which play a key role in monocyte migration. These results indicate that selective CB2 activation in leukocytes decreases key steps in monocyte-BBB engagement, thus suppressing inflammatory leukocyte responses and preventing neuroinflammation.

CB2 receptor activation inhibits melanoma cell transmigration through the blood-brain barrier.

During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.

Neuroinflammatory Responses and Blood-Brain Barrier Injury in Chronic Alcohol Exposure: Role of Purinergic P2X7 Receptor Signaling.

Alcohol consumption leads to neuroinflammation and blood-brain barrier (BBB) damage, resulting in neurological impairment. We previously demonstrated that ethanol-induced disruption of barrier function in human brain endothelial cells was associated with mitochondrial injury, increased ATP and extracellular vesicle (EV) release, and purinergic receptor P2X7R activation. Therefore, we aimed to evaluate the effect of P2X7r blockade on peripheral and neuro-inflammation in EtOH-exposed mice. In a chronic intermittent ethanol (CIE)-exposed mouse model, P2X7R was inhibited by two different methods: Brilliant Blue G (BBG) or gene knockout. We assessed blood ethanol concentration (BEC), plasma P2X7R and P-gp, number of extra-cellular vesicles (EV), serum ATP and EV-ATP levels. Brain microvessel gene expression and EV mtDNA copy numbers were measured by RT2 PCR array and digital PCR, respectively. A RT2 PCR array of brain microvessels revealed significant upregulation of proinflammatory genes involved in apoptosis, vasodilation, and platelet activation in CIE-exposed animals, which were decreased 15-50-fold in BBG-treated CIE-exposed animals. Plasma P-gp levels and serum P2X7R shedding were significantly increased in CIE-exposed animals. Pharmacological or genetic suppression of P2X7R decreased P2X7R shedding to levels equivalent to those in control group. The increase in EV number and EV-ATP content in the CIE-exposed mice was significantly reduced by P2X7R inhibition. CIE mice showed augmented EV-mtDNA copy numbers which were reduced in EVs after P2X7R inhibition or receptor knockout. These observations suggested that P2X7R signaling plays a critical role in ethanol-induced brain injury. Increased eATP, EV-ATP, EV numbers, and EV-mtDNA copy numbers highlight a new mechanism of brain injury during alcohol exposure via P2X7R and biomarkers of such damage. In this study, for the first time, we report the in vivo involvement of P2X7R signaling in CIE-induced brain injury.

Activation of cannabinoid receptor 2 attenuates leukocyte-endothelial cell interactions and blood-brain barrier dysfunction under inflammatory conditions.

Previous studies have shown that modulation of the receptor-mediated cannabinoid system during neuroinflammation can produce potent neuroprotective and anti-inflammatory effects. However, in this context, little is known about how selective activation of the cannabinoid type-2 receptor (CB2R) affects the activated state of the brain endothelium and blood-brain barrier (BBB) function. Using human brain tissues and primary human brain microvascular endothelial cells (BMVECs), we demonstrate that the CB2R is highly upregulated during inflammatory insult. We then examined whether the CB2R agonists could attenuate inflammatory responses at the BBB using a mouse model of LPS-induced encephalitis and highly selective CB2R agonists. Visualization by intravital microscopy revealed that administration of JWH133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran] or a novel resorcinol-based compound, O-1966 (1-[4-(1,1-dimethyl-heptyl)-2,6-dimethoxy-phenyl]-3-methyl-cyclohexanol), greatly attenuated leukocyte adhesion in surface pial vessels and in deep ascending cortical postcapillary venules. BBB permeability assessments with small and large fluorescent tracers showed that CB2R agonists were effective at preventing barrier leakiness after LPS administration. To determine whether the effects by CB2R agonists on barrier protection are not only due to the CB2R modulation of immune cell function, we tested the agonists in vitro with barrier-forming primary BMVECs. Remarkably, the addition of CB2R agonist increased transendothelial electrical resistance and increased the amount of tight junction protein present in membrane fractions. Furthermore, CB2R agonists decreased the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 surface expression in BMVECs exposed to various proinflammatory mediators. Together, these results suggest that pharmacological CB2R ligands offer a new strategy for BBB protection during neuroinflammation.

SIV infection of rhesus macaques of Chinese origin: a suitable model for HIV infection in humans.

Simian immunodeficiency virus (SIV) infection of Indian-origin rhesus macaques (RM) has been widely used as a well-established nonhuman primate (NHP) model for HIV/AIDS research. However, there have been a growing number of studies using Chinese RM to evaluate immunopathogenesis of SIV infection. In this paper, we have for the first time reviewed and discussed the major publications related to SIV or SHIV infection of Chinese RM in the past decades. We have compared the differences in the pathogenesis of SIV infection between Chinese RM and Indian RM with regard to viral infection, immunological response, and host genetic background. Given AIDS is a disease that affects humans of diverse origins, it is of importance to study animals with different geographical background. Therefore, to examine and compare results obtained from RM models of Indian and Chinese origins should lead to further validation and improvement of these animal models for HIV/AIDS research.

Glycogen synthase kinase 3ß inhibition prevents monocyte migration across brain endothelial cells via Rac1-GTPase suppression and down-regulation of active integrin conformation.

Glycogen synthase kinase (GSK) 3ß has been identified as a regulator of immune responses. We demonstrated previously that GSK3ß inhibition in human brain microvascular endothelial cells (BMVECs) reduced monocyte adhesion/migration across BMVEC monolayers. Herein, we tested the idea that GSK3ß inhibition in monocytes can diminish their ability to engage the brain endothelium and migrate across the blood-brain barrier. Pretreatment of primary monocytes with GSK3ß inhibitors resulted in a decrease in adhesion (60%) and migration (85%), with similar results in U937 monocytic cells. Monocyte-BMVEC interactions resulted in diminished barrier integrity that was reversed by GSK3ß suppression in monocytic cells. Because integrins mediate monocyte rolling/adhesion, we detected the active conformational form of very late antigen 4 after stimulation with a peptide mimicking monocyte engagement by vascular cell adhesion molecule-1. Peptide stimulation resulted in a 14- to 20-fold up-regulation of the active form of integrin in monocytes that was suppressed by GSK3ß inhibitors (40% to 60%). Because small GTPases, such as Rac1, control leukocyte movement, we measured active Rac1 after monocyte activation with relevant stimuli. Stimulation enhanced the level of active Rac1 that was diminished by GSK3ß inhibitors. Monocytes treated with GSK3ß inhibitors showed increased levels of inhibitory sites of the actin-binding protein, cofilin, and vasodilator-stimulated phosphoprotein-regulating conformational changes of integrins. These results indicate that GSK3ß inhibition in monocytes affects active integrin expression, cytoskeleton rearrangement, and adhesion via suppression of Rac1-diminishing inflammatory leukocyte responses.

Full genomic sequence of the HLA-DRB3*02:32 allele by Single Molecule Real-time Sequencing Technology.

Full-length sequence covers the 5'-untranslated region (UTR), all introns and exons and the 3' UTR.