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Meibomian gland dysfunction (MGD) is a group of disorders linked by functional abnormalities of the meibomian glands. Current studies on MGD pathogenesis focus on meibomian gland cells, providing information on a single cell's response to experimental manipulation, and do not maintain the architecture of an intact meibomian gland acinus and the acinar epithelial cells' secretion state in vivo. In this study, rat meibomian gland explants were cultured by a Transwell chamber-assisted method under an air-liquid interface (airlift) in vitro for 96 h. Analyses for tissue viability, histology, biomarker expression, and lipid accumulation were performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and TUNEL assays, hematoxylin and eosin (H&E) staining, immunofluorescence, Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), transmission electron microscopy (TEM), and western blotting (WB). MTT, TUNEL, and H&E staining indicated better tissue viability and morphology than the submerged conditions used in previous studies. Levels of MGD biomarkers, including keratin 1 (KRT1) and 14 (KRT14) and peroxisome proliferator-activated receptor-gamma (PPAR-γ), along with oxidative stress markers, including reactive oxygen species, malondialdehyde, and 4-hydroxy-2-nonenal, gradually increased over culture time. The MGD pathophysiological changes and biomarker expression of meibomian gland explants cultured under airlift conditions were similar to those reported by previous studies, indicating that abnormal acinar cell differentiation and glandular epithelial cell hyperkeratosis may contribute to obstructive MGD occurrence. [ABSTRACT FROM AUTHOR]

Regulatory T-cells (Tregs) play pivotal roles during infection, cancer, and autoimmunity. In our previous study, we demonstrated a role for the PD-1:PD-L1 pathway in controlling cytolytic responses of CD8+ T lymphocytes against microglial cells presenting viral peptides. In this study, we investigated the role of Tregs in suppressing CD8+ T-cell-mediated cytotoxicity against primary microglial cells. Using in vitro cytotoxicity assays and flow cytometry, we demonstrated a role for Tregs in suppressing antigen-specific cytotoxic T-lymphocyte (CTL) responses against microglia loaded with a model peptide (SIINFEKL). We went on to show a significant decrease in the frequency of IFN-γ- and TNF-producing CD8+ T-cells when cultured with Tregs. Interestingly, a significant increase in the frequency of granzyme B- and Ki67-producing CTLs was observed. We also observed a significant decrease in the production of interleukin (IL)-6 by microglia. On further investigation, we found that Tregs significantly reduced MHC class 1 (MHC-1) expression on IFN-γ-treated microglial cells. Taken together, these studies demonstrate an immunosuppressive role for Tregs on CTL responses generated against primary microglia. Hence, modulation of Treg cell activity in combination with negative immune checkpoint blockade may stimulate anti-viral T-cell responses to more efficiently clear viral infection from microglial cell reservoirs. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Previous work from our laboratory has demonstrated in vivo persistence of CD103+CD69+ brain resident memory CD8+ T‐cells (bTRM) following viral infection, and that the PD‐1: PD‐L1 pathway promotes development of these TRM cells within the brain. Although glial cells express low basal levels of PD‐L1, its expression is upregulated upon IFN‐γ‐treatment, and they have been shown to modulate antiviral T‐cell effector responses through the PD‐1: PD‐L1 pathway. Methods: We performed flow cytometric analysis of cells from co‐cultures of mixed glia and CD8+ T‐cells obtained from wild type mice to investigate the role of glial cells in the development of bTRM. Results: In this study, we show that interactions between reactive glia and anti‐CD3 Ab‐stimulated CD8+ T‐cells promote development of CD103+CD69+ CD8+ T‐cells through engagement of the PD‐1: PD‐L1 pathway. These studies used co‐cultures of primary murine glial cells obtained from WT animals along with CD8+ T‐cells obtained from either WT or PD‐1 KO mice. We found that αCD3 Ab‐stimulated CD8+ T‐cells from WT animals increased expression of CD103 and CD69 when co‐cultured with primary murine glial cells. In contrast, significantly reduced expression of CD103 and CD69 was observed using CD8+ T‐cells from PD‐1 KO mice. We also observed that reactive glia promoted high levels of CD127, a marker of memory precursor effector cells (MPEC), on CD69+ CD8+ T‐cells, which promotes development of TRM cells. Interestingly, results obtained using T‐cells from PD‐1 KO animals showed significantly reduced expression of CD127 on CD69+ CD8+ cells. Additionally, blocking of glial PD‐L1 resulted in decreased expression of CD103, along with reduced CD127 on CD69+ CD8+ T‐cells. Conclusions: Taken together, these results demonstrate a role for activated glia in promoting development of bTRM through the PD‐1: PD‐L1 pathway. [ABSTRACT FROM AUTHOR]

Objectives miR-375 is one of the highly expressed micro RNAs (mi RNAs) found in pancreatic islets of both humans and mice. In this study, we investigated functions of mi RNA miR-375 in porcine pancreatic stem cells ( PSC). Materials and methods We transfected mimic and inhibitor of miR-375 in PSCs to measure functional roles of the micro RNA and its effects on cell cycle proliferation and cell differentiation were determined. Luciferase assays were also performed to reveal the target gene of miR-375. Results Overexpression of miR-375 suppressed proliferation, promoted apoptosis and inhibited differentiation into islet-like cells. PDK1 was identified as being a target of miR-375. Furthermore, we found that overexpression of miR-375 inhibited activation of the PDK1- AKT signalling pathway. Conclusion miR-375 directly targeted PDK1 in porcine PSCs, suppressing cell proliferation and differentiation into islet-like cells. [ABSTRACT FROM AUTHOR]

Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8+ and CD4+ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8+ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4+ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8+ and CD4+ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8+ T-cells were CD127hi KLRG1- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (TRM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of TRM is impaired upon Treg depletion. Moreover, the effector function of TRM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as TRM cells. [ABSTRACT FROM AUTHOR]

The ability of neural stem/progenitor cells (NSCs) to self-renew, migrate to damaged sites, and differentiate into neurons has renewed interest in using them in therapies for neurodegenerative disorders. Neurological diseases, including viral infections of the brain, are often accompanied by chronic inflammation, whose impact on NSC function remains unexplored. We have previously shown that chronic neuroinflammation, a hallmark of experimental herpes simplex encephalitis (HSE) in mice, is dominated by brain-infiltrating activated CD8 T-cells. In the present study, activated CD8 lymphocytes were found to suppress NSC proliferation profoundly. Luciferase positive (luc+) NSCs co-cultured with activated, MHC-matched, CD8+ lymphocytes (luc−) showed two- to five-fold lower luminescence than co-cultures with un-stimulated lymphocytes. On the other hand, similarly activated CD4+ lymphocytes did not suppress NSC growth. This differential lymphocyte effect on proliferation was confirmed by decreased BrdU uptake by NSC cultured with activated CD8 T-cells. Interestingly, neutralizing antibodies to interferon-gamma (IFN-γ) reversed the impact of CD8 lymphocytes on NSCs. Antibodies specific to the IFN-γ receptor-1 subunit complex abrogated the inhibitory effects of both CD8 lymphocytes and IFN-γ, indicating that the inhibitory effect of these cells was mediated by IFN-γ in a receptor-specific manner. In addition, activated CD8 lymphocytes decreased levels of nestin and Sox2 expression in NSCs while increasing GFAP expression, suggesting possible induction of an altered differentiation state. Furthermore, NSCs obtained from IFN-γ receptor-1 knock-out embryos were refractory to the inhibitory effects of activated CD8+ T lymphocytes on cell proliferation and Sox2 expression. Taken together, the studies presented here demonstrate a role for activated CD8 T-cells in regulating NSC function mediated through the production of IFN-γ. This cytokine may influence neuro-restorative processes and ultimately contribute to the long-term sequelae commonly seen following herpes encephalitis. [ABSTRACT FROM AUTHOR]

Abstract: Neural stem cells (NSCs) respond to inflammatory cues induced during brain injury and are thought to be involved in recovery from brain damage. Little is known about NSC response during brain infections. The present study evaluated NSC proliferation during Herpes Simplex Virus-1 brain infection. Total numbers of nestin(+) NSCs increased significantly in infected brains at 6days post infection (p.i.). However, by 15days p.i. the nestin(+) population decreased significantly below levels observed in uninfected brains and remained depressed through 30days p.i. This initial increase in NSC population occurred concurrently with increased brain cell proliferation, which peaked at 3days p.i. On closer examination, we found that while actively proliferating Sox2(+) NSCs increased in number at 6days p.i., proliferating DCX(+) neuroblasts contributed to the increased response at 3days p.i. However, overall proliferation decreased steadily from 15days p.i. to below control levels. To determine the mechanisms involved in altering NSC proliferation, neurotrophin and growth factor expression profiles were assessed. FGF-2 gene expression increased at 5days p.i. and was robustly down-regulated at 15days p.i. (>1000-fold), which was further confirmed by increased FGF-2 immunostaining around the lateral ventricles. Furthermore, supplementing infected animals with recombinant FGF-2, at 15days p.i., significantly increased the number of proliferating brain cells. These findings demonstrate that the temporal changes in NSC proliferation are mediated through the regulation of FGF-2 and that the NSC niche may benefit from supplementation with FGF-2 during HSV-1 brain infection. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) have been shown to be a contributor to aging and disease. ROS also serve as a trigger switch for signaling cascades leading to corresponding cellular and molecular events. In the central nervous system (CNS), microglial cells are likely the main source of ROS production. However, activated astrocytes also appear to be capable of generating ROS. In this study we investigated ROS production in human astrocytes stimulated with interleukin (IL)-1β and interferon (IFN)-γ and its potential harmful effects. Although IFN-γ alone had no effect, it potentiated IL-1β-induced ROS production in a time-dependent manner. One of the sources of ROS in IL-1β-activated astrocytes was from increased superoxide production in mitochondria accompanied by enhanced manganese superoxide dismutase and inhibited catalase expression. NADPH oxidase (NOX) may also contribute to ROS production as astrocytes express NOX isoforms. Glutamate uptake, which represents one of the most important methods of astrocytes to prevent excitotoxicity, was down-regulated in IL-1β-activated astrocytes, and was further suppressed in the presence of IFN-γ; IFN-γ itself exerted minimal effect. Elevated levels of 8-isoprostane in IL-1β ± IFN-γ-activated human astrocytes indicate downstream lipid peroxidation. Pretreatment with diphenyleneiodonium abolished the IL-1β ± IFN-γ-induced ROS production, restored glutamate uptake function and reduced 8-isoprostane to near control levels suggesting that ROS contributes to the dysfunction of activated astrocytes. These results support the notion that dampening activated human astrocytes to maintain the redox homeostasis is vital to preserve their neuroprotective potential in the CNS. [ABSTRACT FROM AUTHOR]

Murine cytomegalovirus (MCMV) brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. Here, we show that in response to MCMV brain infection, antigen-specific CD8(+) T cells migrated into the brain and persisted as long-lived memory cells. The role of these persistent T cells in the brain is unclear because most of our understanding of antimicrobial T cell responses comes from analyses of lymphoid tissue. Strikingly, memory T cells isolated from the brain exhibited an effector phenotype and produced IFN-γ upon restimulation with viral peptide. Furthermore, we observed time-dependent and long-term activation of resident microglia, indicated by chronic MHC class II up-regulation and TNF-α production. The immune response in this immunologically restricted site persisted in the absence of active viral replication. Lymphocyte infiltrates were detected until 30 days post-infection (p.i.), with CD8(+) and CD4(+) T cells present at a 3:1 ratio, respectively. We then investigated the role of IFN-γ in chronic microglial activation by using IFN-γ-knockout (GKO) mice. At 30 days p.i., GKO mice demonstrated a similar phenotypic brain infiltrate when compared to wild-type mice (Wt), however, MHC class II expression on microglia isolated from these GKO mice was significantly lower compared to Wt animals. When IFN-γ producing CD8(+) T cells were reconstituted in GKO mice, MHC class II up-regulation on microglial cells was restored. Taken together, these results suggest that MCMV brain infection results in long-term persistence of antigen-specific CD8(+) T cells which produce IFN-γ and drive chronic microglial cell activation. This response was found to be dependent on IFN-γ production by viral Ag-specific T cells during the chronic phase of disease. [ABSTRACT FROM AUTHOR]

Long-term neurological sequela is common among herpes simplex encephalitis (HSE) survivors. Animal models for HSE are used to investigate mechanisms of acute disease, but little has been done to model chronic manifestations of HSE. The current study presents a detailed, systematic analysis of chronic neuropathology, including characterization of topography and sequential progression of degenerative lesions and inflammation. Subsequent to intranasal HSV-1 infection, inflammatory responses that were temporally and spatially distinct persisted in infected cortical and brain stem regions. Neutrophils were present exclusively within the olfactory bulb and brain stem regions during the acute phase of infection, while the chronic inflammation was marked by plasma cells, lymphocytes and activated microglia. The chronic lymphocytic infiltrate, cytokine production, and activated microglia were associated with the loss of cortical neuropile in the entorhinal cortex and hippocampus. Animals surviving the acute infection showed a spectrum of chronic lesions from decreased brain volume, neuronal loss, activated astrocytes, and glial scar formation to severe atrophy and cavitations of the cortex. These lesions were also associated with severe spatial memory deficits in surviving animals. Taken together, this model can be utilized to further investigate the mechanisms of neurological defects that follow in the wake of HSE. [ABSTRACT FROM AUTHOR]

Abstract: Murine cytomegalovirus (MCMV) brain infection induces a transient increase in chemokine production, which precedes the infiltration of CD3+ lymphocytes. In this study, we hypothesized that an absence of anti-inflammatory cytokines would result in sustained proinflammatory neuroimmune responses. Direct intracerebroventricular injection of MCMV into IL-10 knockout (KO) mice produced an unexpected result: while wild-type animals controlled MCMV, the infection was lethal in IL-10 KO animals. Identical infection of IL-4 KO animals did not produce lethal disease. To further characterize the role of IL-10, infected brain tissue from both wild-type and IL-10 KO animals was assessed for cytokine and chemokine levels, as well as viral gene expression. These data show vastly elevated levels of interferon (IFN)-γ, and the IFN-γ-inducible chemokines CXCL9 and CXCL10, as well as IL-6 in brain homogenates obtained from IL-10 KO animals. However, MCMV viral load, glycoprotein B mRNA levels, and titers of infectious virus were similar in both IL-10 KO and wild-type animals. Separation of cells isolated from murine brain tissue into distinct populations using FACS, along with subsequent quantitative RT real-time PCR, showed that brain-infiltrating CD45(hi)/CD11b(−) and CD45(hi)/CD11b(int) were the cellular source of IL-10 in the brain. Taken together, these data demonstrate that MCMV brain infection of IL-10-deficient mice causes lethal disease, which occurs in the presence of a dysregulated IFN-γ-mediated neuroimmune response. [Copyright &y& Elsevier]

Abstract: Central nervous system (CNS) infection with herpes simplex virus (HSV)-1 triggers neuroinflammatory responses leading to peripheral immune cell infiltration into the brain. Previous in vitro studies from our laboratory, using primary human brain cells, implicated microglia as the cellular source of infection-induced chemokines, such as CXC ligand 10 (CXCL10) and CC ligand 2 (CCL2). Here, we evaluated the role of microglial cells in HSV-induced neuroimmune responses using an in vivo murine model of herpes encephalitis. Data obtained during this study demonstrated robust levels of CXCL10, CCL2 and CXCL9 detectable in the brains of infected BALB/c mice between 5 and 8 days post-infection (p.i.). Microglial cells were identified as a source of this HSV-induced chemokine production. Additional experiments established that induction of these immune mediators preceded the presence of CD3, CD4, CD8, and CD45 mRNA in the brain, and immunohistochemical analysis confirmed the presence of infiltrating CD3+ cells. Further analysis suggested that microglia-derived chemokines drive peripheral immune cell chemotaxis, as antibodies to CXCL10 and CCL2 blocked the migration of murine splenocytes toward HSV-infected microglia by approximately 59.3±4.1% and 17.5±1.4%, respectively. Taken together, these results demonstrate that a vigorous microglia-driven cascade of pro-inflammatory immune responses is not sufficient to protect susceptible mice from HSV-1 brain infection. [Copyright &y& Elsevier]

Abstract: The neuropharmacological properties of cocaine are known to be associated with the activation of sigma-1 receptors. Cocaine also has been shown to alter both cytokine production and HIV-1 expression in mononuclear phagocytes, including microglial cells. This study tested the hypothesis that sigma-1 receptors and transforming growth factor (TGF)-β1 are involved in cocaine-induced up-regulation of HIV-1 expression in microglial cell cultures. Treatment of microglial cells with cocaine resulted in a concentration-dependent increase in viral expression assessed by measurement of p24 antigen levels in culture supernatants. This cocaine-mediated stimulation of HIV-1 expression was blocked by treatment of microglia with inhibitors of sigma-1 receptors (BD1047) and TGF-β1 (SB-431542 and anti-TGF-β1 antibodies). Microglia were also shown to constitutively express sigma-1 receptor mRNA. Thus, the results of this study support the notion that neuroimmunopharmacological properties of cocaine involve sigma-1 receptors and cytokines. [Copyright &y& Elsevier]

Although corticosteroids are recommended as adjunctive therapy for tuberculous meningitis, the mechanism underlying their beneficial effect is poorly understood. In this study, human microglia and astrocytes were infected with Mycobacterium tuberculosis H37Rv, and cytokine and chemokine expression was examined with and without dexamethasone treatment. Microglia were the principal cells infected by tubercle bacilli, which elicited robust amounts of several cytokines and chemokines. Treatment with dexamethasone markedly suppressed production of these mediators. The results of this study support the concept that micro glia play an important role in neuropathogenesis of tuberculosis and that dexamethasone could operate via modulation of the production of proinflammatory cytokines and chemokines by these brain macrophages. [ABSTRACT FROM AUTHOR]

Abstract: An urgent need for additional agents to treat human immunodeficiency virus type 1 (HIV-1) infection led us to assess the anti-HIV-1 activity of the natural product propolis in CD4+ lymphocytes and microglial cell cultures. Propolis inhibited viral expression in a concentration-dependent manner (maximal suppression of 85 and 98% was observed at 66.6μg/ml propolis in CD4+ and microglial cell cultures, respectively). Similar anti-HIV-1 activity was observed with propolis samples from several geographic regions. The mechanism of propolis antiviral property in CD4+ lymphocytes appeared to involve, in part, inhibition of viral entry. While propolis had an additive antiviral effect on the reverse transcriptase inhibitor zidovudine, it had no noticeable effect on the protease inhibitor indinavir. The results of this in vitro study support the need for clinical trials of propolis or one or more of its components in the treatment of HIV-1 infection. [Copyright &y& Elsevier]

To investigate the effect of opiates on human immunodeficiency virus type 1 (HIV-1)-related neuronal apoptosis, primary human fetal neuronal/glial cell cultures were exposed to gp120IIB in the absence and the presence of morphine. Although morphine by itself had little effect on neuronal survival, the combination of morphine (fl07 mol/L) and gp120IIBnmol/L) significantly increased neuronal apoptosis. The mechanism whereby morphine potentiates gp120IIB minduced neuronal apoptosis appears to involve activation of the p38 mitogen-activated protein kinase intracellular signaling pathway and micro glial cells dispersed within the neuronal/glial cell cultures. These results provide additional in- sight into the molecular basis whereby opiate abuse could promote HIV- 1 -associated dementia. [ABSTRACT FROM AUTHOR]

Neural precursor cells (NPCs) are self-renewing, multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). Fetal NPCs have attracted attention for their potential use in studying normal CNS development. Several studies of rodent neural progenitors have suggested that chemokines and their receptors are involved in directing NPC migration during CNS development. In this study, we established a consistent system to culture human NPCs and examined the expression of chemokine receptors on these cells. NPCs were found to express the markers nestin and CD133 and to differentiate into neurons, astrocytes and oligodendrocytes at the clonal level. Flow cytometry and RNase protection assay (RPA) indicated that NPCs express high levels of CXCR4 and low levels of several other chemokine receptors. When examined using a chemotaxis assay, NPCs were able to respond to CXCL12/SDF-1α, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1α.  These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development. [Copyright &y& Elsevier]

κ-Opioid receptor (KOR) ligands have been reported to alter many cell functions and to exert an immunomodulatory role in the CNS. Astrocytes, the predominant brain cell type, have been implicated in the neuropathogenesis of human immunodeficiency virus type 1 (HIV-1). HIV-1 nuclear protein Tat has been reported to induce production of the chemokine monocyte chemoattractant protein-1 (MCP-1 or CCL2) and to activate nuclear factor κB (NF-κB) in human astrocytes. In the present study, we investigated whether the synthetic KOR ligand trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate (U50,488) would down-regulate MCP-1 production in primary human astrocytes stimulated by Tat. Treatment of astrocytes with U50,488 inhibited Tat-induced MCP-1 production in a concentration-dependent manner. The KOR-selective antagonist nor-binaltrophimine (nor-BNI) completely blocked the inhibitory effect of U50,488, indicating involvement of KOR. While U50,488 alone had a partial inhibitory effect on constituent NF-κB activation, it potently suppressed Tat-induced NF-κB activation. These findings suggest that KOR ligands could have an anti-inflammatory effect in the CNS and thereby be beneficial in the treatment of HIV-1-associated brain disease. [Copyright &y& Elsevier]

Apoptosis plays a critical role in normal brain development and in a number of neurodegenerative diseases. Recently, opiates have been shown to promote apoptotic death of cells of the immune and nervous systems. In this study, we investigated the effect of morphine on apoptosis of primary human fetal microglial cell, astrocyte and neuronal cell cultures. Exposure of microglia and neurons to 10−6 M morphine potently induced apoptosis of these brain cells (approximately fourfold increase above untreated control cells). In contrast to microglia and neurons, astrocytes were completely resistant to morphine-induced apoptosis. Concentration–response and time-course studies indicated that neurons were more sensitive than microglia to morphine’s effect on apoptosis. Naloxone blocked morphine-induced apoptosis suggesting involvement of an opiate receptor mechanism. Potent inhibition (>70%) of apoptosis by an inhibitor of caspase-3 as well as co-localization of active caspase-3 and DNA fragmentation in microglia or neurons treated with morphine indicated that caspase-3 is involved in the execution phase of morphine-induced apoptosis. The results of these in vitro studies have implications regarding the potential effect of opiates on fetal brain development and on the course of certain neurodegenerative diseases. [Copyright &y& Elsevier]

The beta-chemokine RANTES has recently been implicated in the neuropathogenesis of the human immunodeficiency virus. Based upon previous studies of the effects of morphine on microglial cell production of cytokines and chemotaxis towards the activated complement component C5a, we tested the hypothesis that this opiate would alter the production of and migration towards RANTES by human microglia. Treatment of highly purified microglial cell cultures with morphine (10(-8)-10(-6) M) potently inhibited RANTES production by lipopolysaccharide- and interleukin-1beta-stimulated cells. Using a chemotaxis chamber to assess directed migration towards RANTES, treatment of microglial cells with morphine (10(-10)-10(-6) M) was found to suppress chemotaxis. The inhibitory effects of morphine on RANTES production and on chemotaxis were blocked by naloxone and beta-funaltrexamine, indicating that morphine mediated its suppressive effects via activation of microglial p-opioid receptors. Morphine's inhibitory effect on chemotaxis did not appear to be associated with an alteration in RANTES-induced [Ca2+]i mobilization. While the clinical significance of these in-vitro findings is unknown, they suggest that mu-opioid receptor agonists could alter certain neurodegenerative and inflammatory processes within the brain. [ABSTRACT FROM AUTHOR]

Different bioinks have been used to produce cell-laden alginate-based hydrogel constructs for cell replacement therapy but some of these approaches suffer from issues with print quality, long-term mechanical instability, and bioincompatibility. In this study, new alginate-based bioinks were developed to produce cell-laden grid-shaped hydrogel constructs with stable integrity and immunomodulating capacity. Integrity and printability were improved by including the co - block -polymer Pluronic F127 in alginate solutions. To reduce inflammatory responses, pectin with a low degree of methylation was included and tested for inhibition of Toll-Like Receptor 2/1 (TLR2/1) dimerization and activation and tissue responses under the skin of mice. The viscoelastic properties of alginate-Pluronic constructs were unaffected by pectin incorporation. The tested pectin protected printed insulin-producing MIN6 cells from inflammatory stress as evidenced by higher numbers of surviving cells within the pectin-containing construct following exposure to a cocktail of the pro-inflammatory cytokines namely, IL-1β, IFN-γ, and TNF-α. The results suggested that the cell-laden construct bioprinted with pectin-alginate-Pluronic bioink reduced tissue responses via inhibiting TLR2/1 and support insulin-producing β-cell survival under inflammatory stress. Our study provides a potential novel strategy to improve long-term survival of pancreatic islet grafts for Type 1 Diabetes (T1D) treatment. Diagram model of pectin molecules in alginate-based constructs allow revascularization, protect pancreatic β-cell from inflammatory damage and inhibit tissue responses by inhibit TLR2/1 activation (Created with BioRender.com). [Display omitted] • A tri-component bioink containing pectin was formulated. • Pectin-incorporated constructs support viability of pancreatic β-cells under inflammatory stress. • Pectin-incorporated constructs inhibit activation of Toll-Like Receptor 2/1. • Constructs containing pectin exhibit high biocompatibility in vivo. [ABSTRACT FROM AUTHOR]

Encapsulation of pancreatic islets in alginate-microcapsules is used to reduce or avoid the application of life-long immunosuppression in preventing rejection. Long-term graft function, however, is limited due to varying degrees of host tissue responses against the capsules. Major graft-longevity limiting responses include inflammatory responses provoked by biomaterials and islet-derived danger-associated molecular patterns (DAMPs). This paper reports on a novel strategy for engineering alginate microcapsules presenting immunomodulatory polymer pectin with varying degrees of methyl-esterification (DM) to reduce these host tissue responses. DM18-pectin/alginate microcapsules show a significant decrease of DAMP-induced Toll-Like Receptor-2 mediated immune activation in vitro , and reduce peri-capsular fibrosis in vivo in mice compared to higher DM-pectin/alginate microcapsules and conventional alginate microcapsules. By testing efficacy of DM18-pectin/alginate microcapsules in vivo , we demonstrate that low-DM pectin support long-term survival of xenotransplanted rat islets in diabetic mice. This study provides a novel strategy to attenuate host responses by creating immunomodulatory capsule surfaces that attenuate activation of specific pro-inflammatory immune receptors locally at the transplantation site. [ABSTRACT FROM AUTHOR]

• Pectins prevent pancreatic β-cell damage during oxidative and inflammatory stress. • Pectin with lower DM value is more efficacious than higher-DM pectins in protection of β-cell for stress. • Low-DM Pectin protects β-cell through protecting mitochondria for toxin and cytokine induced dysfunction. • The protective effect of pectin depends on the receptor galectin-3. Insufficient intake of dietary fibers in Western societies is considered a major contributing factor in the high incidence rates of diabetes. The dietary fiber pectin has been suggested to be beneficial for management of both Diabetes Type 1 and Type 2, but mechanisms and effects of pectin on insulin producing pancreatic β-cells are unknown. Our study aimed to determine the effects of lemon pectins with different degree of methyl-esterification (DM) on β-cells under oxidative (streptozotocin) and inflammatory (cytokine) stress and to elucidate the underlying rescuing mechanisms, including effects on galectin-3. We found that specific pectins had rescuing effects on toxin and cytokine induced stress on β-cells but effects depended on the pectin concentration and DM-value. Protection was more pronounced with low DM5 pectin and was enhanced with higher pectin-concentrations. Our findings show that specific pectins might prevent diabetes by making insulin producing β-cells less susceptible for stress. [ABSTRACT FROM AUTHOR]

Materials with zero thermal expansion (ZTE) properties demonstrate remarkable dimensional stability, making them crucial in aerospace, electronics, and various industrial sectors. However, these materials face challenges such as weak mechanical properties and narrow ZTE temperature range. In this study, a novel ZTE metallic glass-based composite (Cu 2 P 2 O 7 /La 65 Al 14 Cu 9.17 Ag 1.83 Ni 5 Co 5) with negative thermal expansion (NTE) reinforcement was prepared using gas pressure infiltration and water quenching. SEM and 3D CT results revealed the dense structure and the non-reactive interface structure. The NTE behaviors resulting from the coupling of CuO 5 and PO 4 polyhedra within the NTE Cu 2 P 2 O 7 are demonstrated to counterbalance the positive thermal expansion in metallic glass. Consequently, the resulting composites exhibit a wide range of zero thermal expansion and high strength properties, presenting a novel approach for creating high-performance ZTE metallic glass-based composites. [ABSTRACT FROM AUTHOR]

Islet dysfunction mediated by oxidative and mitochondrial stress contributes to the development of type 1 and 2 diabetes. Acetate and butyrate, produced by gut microbiota via fermentation, have been shown to protect against oxidative and mitochondrial stress in many cell types, but their effect on pancreatic β-cell metabolism has not been studied. Here, human islets and the mouse insulinoma cell line MIN6 were pre-incubated with 1, 2, and 4 mM of acetate or butyrate with and without exposure to the apoptosis inducer and metabolic stressor streptozotocin (STZ). Both short-chain fatty acids (SCFAs) enhanced the viability of islets and β-cells, but the beneficial effects were more pronounced in the presence of STZ. Both SCFAs prevented STZ-induced cell apoptosis, viability reduction, mitochondrial dysfunction, and the overproduction of reactive oxygen species (ROS) and nitric oxide (NO) at a concentration of 1 mM but not at higher concentrations. These rescue effects of SCFAs were accompanied by preventing reduction of the mitochondrial fusion genes MFN, MFN2, and OPA1. In addition, elevation of the fission genes DRP1 and FIS1 during STZ exposure was prevented. Acetate showed more efficiency in enhancing metabolism and inhibiting ROS, while butyrate had less effect but was stronger in inhibiting the SCFA receptor GPR41 and NO generation. Our data suggest that SCFAs play an essential role in supporting β-cell metabolism and promoting survival under stressful conditions. It therewith provides a novel mechanism by which enhanced dietary fiber intake contributes to the reduction of Western diseases such as diabetes. [ABSTRACT FROM AUTHOR]

Fcγ receptors (FcγRs) for IgG couple innate and adaptive immunity through activation of effector cells by antigen-antibody complexes. We investigated relative levels of activating and inhibitory FcγRs on brain-resident microglia following murine cytomegalovirus (MCMV) infection. Flow cytometric analysis of microglial cells obtained from infected brain tissue demonstrated that activating FcγRs were expressed maximally at 5 d post-infection (dpi), while the inhibitory receptor (FcγRIIB) remained highly elevated during both acute and chronic phases of infection. The highly induced expression of activating FcγRIV during the acute phase of infection was also noteworthy. Furthermore, in vitro analysis using cultured primary microglia demonstrated the role of interferon (IFN)γ and interleukin (IL)-4 in polarizing these cells towards a M1 or M2 phenotype, respectively. Microglial cell-polarization correlated with maximal expression of either FcγRIV or FcγRIIB following stimulation with IFNγ or IL-4, respectively. Finally, we observed a significant delay in polarization of microglia towards an M2 phenotype in the absence of FcγRs in MCMV-infected Fcer1g and FcgR2b knockout mice. These studies demonstrate that neuro-inflammation following viral infection increases expression of activating FcγRs on M1-polarized microglia. In contrast, expression of the inhibitory FcγRIIB receptor promotes M2-polarization in order to shut-down deleterious immune responses and limit bystander brain damage. [ABSTRACT FROM AUTHOR]

Despite the therapeutic impact of anti-retroviral therapy, HIV-1-associated neurocognitive disorder (HAND) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Recent work suggests that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAND and that human dopaminergic neurons have a particular sensitivity to gp120-induced damage, manifested as reduced function (decreased dopamine uptake), morphological changes, and reduced viability. Synthetic cannabinoids inhibit HIV-1 expression in human microglia, suppress production of inflammatory mediators in human astrocytes, and there is substantial literature demonstrating the neuroprotective properties of cannabinoids in other neuropathogenic processes. Based on these data, experiments were designed to test the hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV-1 protein gp120. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, we were able to show that the CB1/CB2 agonist WIN55,212-2 blunts gp120-induced neuronal damage as measured by dopamine transporter function, apoptosis and lipid peroxidation; these actions were mediated principally by the CB2 receptor. Adding supplementary human microglia to our cultures enhances gp120-induced damage; WIN55,212-2 is able to alleviate this enhanced damage. Additionally, WIN55,212-2 inhibits gp120-induced superoxide production by purified human microglial cells, inhibits migration of human microglia towards supernatants generated from gp120-stimulated human mesencephalic neuronal/glial cultures and reduces chemokine and cytokine production from the human mesencephalic neuronal/glial cultures. These data suggest that synthetic cannabinoids are capable of protecting human dopaminergic neurons from gp120 in a variety of ways, acting principally through the CB2 receptors and microglia. [ABSTRACT FROM AUTHOR]

Highly active and multifunctional non‐precious metal‐based hydrogen evolution reaction (HER) eletcrocatalysts have aroused great interest in the large‐scale conversion of sustainable electrical energy to fuels and feedstocks in different application scenarios. Here, a metal sulfide‐based super hybrid nanoarray composed of a metallic Co3S4 nanosheet, P doped WS2 nanosheet, and Co9S8 nanoparticles (Super‐Co3S4/P‐WS2/Co9S8) for all‐pH HER is reported. The Super‐Co3S4/P‐WS2/Co9S8 only requires 58, 70, and 129 mV in alkaline, acid, and neutral media, respectively, to reach 10 mA cm‐2, which also presents fast reaction kinetics and high long‐term stability. The experimental and density functional theory (DFT) results verify that the abundant active heterostructure in Super‐Co3S4/P‐WS2/Co9S8 can not only provide Pt‐like H*‐adsorption Gibbs free energy (ΔGH*) but also promote the H2O adsorption and dissociation kinetics for all‐pH HER. Moreover, the collaboration of favorable metallic components and special nanoarray morphology can also strengthen the electric conductivity for electron transfer and promote the maximum exposure of active heterointerfaces for mass transport in electrocatalysis. The interesting strategy herein is expected to inspire the future design of advanced metal sulfide‐based heterostructures for energy storage and conversion. [ABSTRACT FROM AUTHOR]

The role of select pro- and anti-inflammatory mediators in driving microglial cell polarization into classically (M1), or alternatively, (M2) activated states, as well as the subsequent differential responses of these induced phenotypes, was examined. Expression of PD-L1, MHC-II, MHC-I, arginase 1 (Arg-1), and inducible nitric oxide synthase (iNOS) was assessed using multi-color flow cytometry. We observed that both pro- and anti-inflammatory mediators induced PD-L1 expression on non-polarized microglia. Moreover, IFN-γ stimulated significant MHC class I and II expression on these cells. Interestingly, we observed that only IL-4 treatment induced Arg-1 expression, indicating M2 polarization. These M2 cells were refractory to subsequent depolarization and maintained their alternatively activated state. Furthermore, PD-L1 expression was significantly induced on these M2-polarized microglia after treatment with pro-inflammatory mediators, but not anti-inflammatory cytokines. In addition, we observed that only LPS induced iNOS expression in microglial cells, indicating M1 polarization. Furthermore, IFN-γ significantly increased the percentage of M1-polarized microglia expressing iNOS. Surprisingly, when these M1-polarized microglia were treated with either IL-6 or other anti-inflammatory cytokines, they returned to their non-polarized state, as demonstrated by significantly reduced expression of iNOS. Taken together, these results demonstrate differential responses of microglial cells to mediators present in dissimilar microenvironments. [ABSTRACT FROM AUTHOR]

Abstract: Wild-type mice control murine cytomegalovirus (MCMV) brain infection, but identical infection is lethal to animals deficient in interleukin (IL)-10. Here, we report that MCMV-infected IL-10 knockout (KO) mice displayed a marked increase in neutrophil infiltration into the infected, IL-10-deficient brain when compared to wild-type animals. Enhanced microglial cell activation, determined by MHC class II up-regulation, overexpression of CXCL2, and elevated P-selectin mRNA levels were observed. In vivo blocking of CXCL2 attenuated neutrophil infiltration and significantly improved the outcome of infection. Collectively, these data indicate that the absence of IL-10 results in pathologic neutrophil infiltration into MCMV-infected brains. [ABSTRACT FROM AUTHOR]

The brain's intrinsic immune system consists of glial cells that produce cytokines and chemokines in response to stimulation with cytomegalovirus (CMV). The present experiments were undertaken to determine whether this intrinsic glial cell response alone is sufficient to control CMV infection of the central nervous system (CNS) or whether effector cells from the somatic immune system are also required. Following stereotactic, intracerebroventricular (icv), injection of murine cytomegalovirus (MCMV) into immunocompetent (C.B-17) mice, viral spread in the brain was limited to the cells of the ventricular walls and the infection was resolved by 10 days post infection (p.i.). In contrast, icv infection of immunodeficient (C.B-17 SCID/bg) mice resulted in viral spread from the ventricles throughout the brain parenchyma and these mice succumbed to lethal disease. Adoptive transfer of total splenocytes from major histocompatibility complex (MHC)-matched, MCMV-primed animals restricted intracerebral viral infection to the periventricular cells and reduced levels of reporter gene expression from the viral genome. Peripheral immune cell transfer also protected immunodeficient animals from lethal disease. Depletion of Thy 1.2+ cells from MCMV-primed splenocytes abolished the protective effect of adoptive transfer. Viral expression was found to be fourfold greater in the brains of animals given Thy 1.2-depleted splenocytes than from those receiving total undepleted cells. As MCMV infection proceeded in the brains of immunodeficient mice, levels of the T-cell chemoattractants CXCL10 and CCL2 remained elevated, whereas CXCL10 levels waned in the brains of animals receiving transferred splenocytes. Taken together, these results demonstrate the ability of T lymphocytes to restrict intracerebral viral spread and indicate that intrinsic glial cell responses alone are insufficient to control MCMV brain infection. [ABSTRACT FROM AUTHOR]

The crystal structures of BiOI under different pressures are predicted, in which the phase transformation from tetragonal BiOI into orthorhombic BiOI is observed at 100 GPa due to the appearance of Bi–I bonds (Bi‐6s2 lone pair and I‐pz obital) between interlayer Bi and I atoms. Most interestingly, for orthorhombic BiOI, an isostructural electronic phase separation is observed at 160 GPa. The electronic structure of BiOI separates into semiconductor state (contributed by intralayer O–Bi–O) and metal state, which originate from the interlayer I–I bonds. More specifically, conversion from interlayer Bi–I interaction to interlayer I–I interaction under higher pressure, results in the continuous decline of the I‐dx2–y2 energy band, which then crosses the Fermi‐level. In comparison, the O–Bi–O layer shows almost no change, owing to its strong covalent bonds. These results demonstrate that the dramatic variation of the interaction between the van der Waals layers can lead to the metallic iodine character in BiOI. [ABSTRACT FROM AUTHOR]

Defects in metal oxides can significantly improve their physical and chemical properties. However, the conventional methods to generate defects often require complex procedures and harsh conditions. In this study, we design a simple and room-temperature preparation route to prepare defective metal oxide nanoparticles with high yield. The formation of defects is attributed to the generation of oxygen vacancies (VO) and hydrogenation caused by charge transfer at a Mg–metal oxide junction structure. Defective TiO2 exhibits excellent performance toward wastewater cleaning and water splitting. The proposed route is promising in terms of convenience, low cost, and large-scale production. [ABSTRACT FROM AUTHOR]

Background: Peripheral neuropathy is currently the most common neurological complication in HIV-infected individuals, occurring in 35-50% of patients undergoing combination anti-retroviral therapy. Data have shown that distal symmetric polyneuropathy develops in mice by 6 weeks following infection with the LP-BM5 retrovirus mixture. Previous work from our laboratory has demonstrated that glial cells modulate antiviral T-cell effector responses through the programmed death (PD)-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation.Methods: Using the MouseMet electronic von Frey system, we assessed hind-paw mechanical hypersensitivity in LP-BM5-infected wild-type (WT) and PD-1 KO animals. Using multi-color flow cytometry, we quantitatively assessed cellular infiltration and microglial activation. Using real-time RT-PCR, we assessed viral load, expression of IFN-γ, iNOS, and MHC class II. Using western blotting, we measured protein nitrosylation within the lumbar spinal cord (LSC) and dorsal root ganglion (DRG). Histochemical staining was performed to analyze the presence of CD3, ionized calcium binding adaptor molecule (Iba)-1, MHCII, nitrotyrosine, isolectin B4 (IB4) binding, and neurofilament 200 (NF200). Statistical analyses were carried out using graphpad prism.Results: Hind-paw mechanical hypersensitivity observed in LP-BM5-infected animals was associated with significantly increased lymphocyte infiltration into the spinal cord and DRG. We also observed elevated expression of IFN-γ (in LSC and DRG) and MHC II (on resident microglia in LSC). We detected elevated levels of 3-nitrotyrosine within the LSC and DRG of LP-BM5-infected animals, an indicator of nitric oxide (NO)-induced protein damage. Moreover, we observed 3-nitrotyrosine in both small (IB4+) and large (NF200+) DRG sensory neurons. Additionally, infected PD-1 KO animals displayed significantly greater mechanical hypersensitivity than WT or uninfected mice at 4 weeks post-infection (p.i.). Accelerated onset of hind-paw hypersensitivity in PD-1 KO animals was associated with significantly increased infiltration of CD4+ and CD8+ T lymphocytes, macrophages, and microglial activation at early time points. Importantly, we also observed elevated levels of 3-nitrotyrosine and iNOS in infected PD-1 KO animals when compared with WT animals.Conclusions: Results reported here connect peripheral immune cell infiltration and reactive gliosis with nitrosative damage. These data may help elucidate how retroviral infection-induced neuroinflammatory networks contribute to nerve damage and neuropathic pain. [ABSTRACT FROM AUTHOR]

Background: Previous work from our laboratory has demonstrated that during acute viral brain infection, glial cells modulate antiviral T cell effector responses through the PD-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation. Here, we evaluated the PD-1: PD-L1 pathway in development of brain-resident memory T cells (bTRM) following murine cytomegalovirus (MCMV) infection.Methods: Flow cytometric analysis of immune cells was performed at 7, 14, and 30 days post-infection (dpi) to assess the shift of brain-infiltrating CD8+ T cell populations from short-lived effector cells (SLEC) to memory precursor effector cells (MPEC), as well as generation of bTRMs.Results: In wild-type (WT) animals, we observed a switch in the phenotype of brain-infiltrating CD8+ T cell populations from KLRG1+ CD127- (SLEC) to KLRG1- CD127+ (MPEC) during transition from acute through chronic phases of infection. At 14 and 30 dpi, the majority of CD8+ T cells expressed CD127, a marker of memory cells. In contrast, fewer CD8+ T cells expressed CD127 within brains of infected, PD-L1 knockout (KO) animals. Notably, in WT mice, a large population of CD8+ T cells was phenotyped as CD103+ CD69+, markers of bTRM, and differences were observed in the numbers of these cells when compared to PD-L1 KOs. Immunohistochemical studies revealed that brain-resident CD103+ bTRM cells were localized to the parenchyma. Higher frequencies of CXCR3 were also observed among WT animals in contrast to PD-L1 KOs.Conclusions: Taken together, our results indicate that bTRMs are present within the CNS following viral infection and the PD-1: PD-L1 pathway plays a role in the generation of this brain-resident population. [ABSTRACT FROM AUTHOR]

Porcine pancreatic stem cells (PSCs) are one kind of the potential cells for treatment of human diabetes. Autophagy is a highly conserved cellular degradation process in which it helps to maintain the balance between the synthesis, degradation and subsequent recycling of cellular components. However, how autophagy contributes to PSCs has not yet been investigated. Here, we established GFP-LC3 transfected porcine PSC lines in which the accumulation of autophagosomes can be efficiently visualized to evaluate the autophagic activity. Moreover, we observed that starved PSCs which showed increased autophagic activity exhibited an increased tendency to proliferate through the results of BrdU, flow cytometry and western blotting. Furthermore, increased expression of active β-catenin after inducing autophagy indicated that it might be the canonical Wnt signaling that autophagy activated to exert the function on the stimulation of PSCs proliferation. Collectively, these results demonstrated that autophagy stimulated proliferation of PSCs might be regulated by the canonical Wnt signaling pathway. Our results for the first time shed light on a role of autophagy for stimulating the proliferation of porcine PSCs. [ABSTRACT FROM AUTHOR]

Background: Chemokines produced by reactive glia drive migration of immune cells and previous studies from our laboratory have demonstrated that CD19(+) B cells infiltrate the brain. In this study, in vivo and in vitro experiments investigated the role of reactive glial cells in recruitment and survival of B-lineage cells in response to (murine cytomegalovirus) MCMV infection.Methods: Flow cytometric analysis was used to assess chemokine receptor expression on brain-infiltrating B cells. Real-time RT-PCR and ELISA were used to measure chemokine levels. Dual-immunohistochemical staining was used to co-localize chemokine production by reactive glia. Primary glial cell cultures and migration assays were used to examine chemokine-mediated recruitment. Astrocyte: B cell co-cultures were used to investigate survival and proliferation.Results: The chemokine receptors CXCR3, CXCR5, CCR5, and CCR7 were detected on CD19(+) cells isolated from the brain during MCMV infection. In particular, CXCR3 was found to be elevated on an increasing number of cells over the time course of infection, and it was the primary chemokine receptor expressed at 60 days post infection Quite different expression kinetics were observed for CXCR5, CCR5, and CCR7, which were elevated on the highest number of cells early during infection and decreased by 14, 30, and 60 days post infection Correspondingly, elevated levels of CXCL9, CXCL10, and CXCL13, as well as CCL5, were found within the brains of infected animals, and only low levels of CCL3 and CCL19 were detected. Differential expression of CXCL9/CXCL10 and CXCL13 between microglia and astrocytes was apparent, and B cells moved towards supernatants from MCMV-infected microglia, but not astrocytes. Pretreatment with neutralizing Abs to CXCL9 and CXCL10 inhibited this migration. In contrast, neutralizing Abs to the ligand of CXCR5 (i.e., CXCL13) did not significantly block chemotaxis. Proliferation of brain-infiltrating B cells was detected at 7 days post infection and persisted through the latest time tested (60 days post infection). Finally, astrocytes produce BAFF (B cell activating factor of the TNF family) and promote proliferation of B cells via cell-to-cell contact.Conclusions: CXCR3 is the primary chemokine receptor on CD19(+) B cells persisting within the brain, and migration to microglial cell supernatants is mediated through this receptor. Correspondingly, microglial cells produce CXCL9 and CXCL10, but not CXCL13. Reactive astrocytes promote B cell proliferation. [ABSTRACT FROM AUTHOR]

Objectives Porcine pancreatic stem cells ( PSCs) are highly valuable in transplantation applications for type II diabetes. However, there are still many problems to be solved before they can be used in the clinic, such as insufficient cell number availability and low secretion level of insulin. It has been reported that Wnt3a plays pivotal roles during cell proliferation and differentiation. Here, we have aimed to establish an ideal research platform using the T- REx™ system, to study mechanisms of Wnt3a during PSC proliferation and differentiation. Materials and methods Construction of the recombinant plasmid and cell transfection were used for establishment of a porcine PSC line. Related gene expressions were examined using quantitative real-time PCR ( QRT- PCR), western blotting, immunostaining and flow cytometry. BrdU incorporation assay and cell cycle analysis were used to investigate Wnt3a roles in PSCs. Results Wnt3a-expressing clones regulated by T- REx™ were successfully obtained. Wnt3a and GFP expression were strictly regulated by Dox in a time- and dose-dependent manner. Furthermore, we found that Wnt3a-expressing porcine PSCs induced by Dox exhibited raised proliferative potential. After Dox stimulation, expression of PCNA, C- MYC and active β-catenin were higher, but were down-regulated after Dkk1 addition. Conclusion We established a porcine PSC line that dynamically expressed Wnt3a, and we found that Wnt3a promoted PSC proliferative potential. This inducible expression system thus provides an important tool for further study on porcine PSC development and differentiation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Hierarchical CoNi 2 S 4 /WS 2 /Co 9 S 8 (NiCoWS) frameworks were constructed. • NiCoWS featured with highly exposed active multi-heterointerfaces. • NiCoWS exhibited robust electrocatalytic performance toward all-pH HER. • The synergy among geometric, electronic, and interfacial structures was achieved. Water dissociation, hydroxyl desorption, and hydrogen recombination are three major prerequisites for all-pH hydrogen evolution reaction (HER). Herein, a multi-interfacial engineering in hierarchical CoNi 2 S 4 /WS 2 /Co 9 S 8 (NiCoWS) hybrid frameworks was developed. The NiCoWS is composed of highly exposed active sites and abundant heterogeneous interfaces. The NiCoWS exhibits small overpotentials of 70 mV, 61 mV, and 146 mV at 10 mA cm−2 in alkaline, acid, and neutral medium, respectively, for HER. The robust performance of NiCoWS should originate from the collective synergy of special morphological, electronic, and interfacial structures. The hierarchical nanosheet framework can facilitate the transport of charge/mass as well as the exposure of more active interface sites. Density functional theory (DFT) calculations verify that the multi-interfacial engineering in NiCoWS can endow the electrocatalyst with enhanced electrical conductivity and favorable adsorption energies of H*, H 2 O* and H...OH*. [ABSTRACT FROM AUTHOR]

Although production of reactive nitrogen and reactive oxygen species (RNS and ROS) is a component of innate defense against viral infection, their overproduction in the brain may also lead to deleterious consequences. To investigate potential immunopathologic roles of oxidative stress during herpes encephalitis, the authors examined the expression kinetics of inducible nitric oxide synthase (iNOS) as well as heme oxygenase-1 (HO-1), a marker of oxidative stress, and evaluated infection-induced oxidative brain damage. Results from these studies showed that both iNOS and HO-1 gene expression were highly elevated in the brain within 7 days post infection (d.p.i.) and remained elevated through 21 d.p.i. Real-time bioluminescence imaging of HO-1 promoter-luciferase transgenic mice confirmed HO-1 promoter activity in the brains of HSV-1-infected animals within 3 d.p.i., which peaked between 5 and 7 d.p.i. Immunohistochemical staining for both 3-nitrotyrosine and 8-hydroxydeoxyguanosine (8-OH-dG), as well as quantitative assessment of 8-isoprostane levels, demonstrated the presence of viral infection-induced oxidative brain damage. In addition, when brain leukocytes obtained from animals with experimental herpes encephalitis were sorted using fluorescence-activated cell sorting (FACS) and the individual cell populations analyzed, CD45(int)/CD11b(+) resident microglia were found to be the major cellular source of iNOS expression. [ABSTRACT FROM AUTHOR]

For the development of lead-free Bi 0.5 Na 0.5 TiO 3 prototype materials, it seems conflicting to improve the piezoelectric response (d 33) and depolarization temperature (T d) simultaneously. Here, we propose a general chemical substitution method to simultaneously improve d 33 and T d of Bi 0.5 Na 0.5 TiO 3 -BaTiO 3 (BNT-BT) via introducing oxygen-defect perovskite. This has been demonstrated by introducing BaAlO 2.5 into BNT-BT perovskite crystal lattice and forming solid-solutions, in which a series of ceramics with P 4 mm phase after electrical poling can be achieved. Not only its d 33 but also T d can be much improved, which goes far beyond the performance boundary limit of d 33 vs. T d for all BNT-BT based systems. The large d 33 stems from the enhancement of lattice strain and domain switching confirmed by in situ synchrotron X-ray diffraction analysis. Critically, the improved T d originates from the increased tetragonality that caused by oxygen vacancies, as elucidated by the comparative experiments and density functional theory calculations. The present study provides a general method to tune the piezoelectric performance of BNT-based ceramics, and expects to benefit other piezoelectric materials. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Heme oxygenase (HO)-1 has been shown to attenuate oxidative injury and reduce apoptosis. HO-1 can be induced by various stimuli released during cellular injury, such as heme. Deleterious free heme is degraded by HO-1 to carbon monoxide, iron and biliverdin, which have potent anti-oxidant and anti-inflammatory properties. In this study, we tested the hypothesis that upregulation of HO-1 would inhibit production of the free radical (NO) by interleukin (IL)-1β-activated human astrocytes.Methods: To measure NO production, inducible NO synthase (iNOS), HO-1 expression and mitogen-activated protein (MAP) kinase activation we used hemin as an HO-1 inducer and tin protoporphyrin (SnPP) IX as an inhibitor of HO-1 activity in human astrocyte cultures prior to IL-1β exposure. Transfection of astrocyte cultures was performed using a pLEX expression vector carrying the human HO-1 sequence prior to IL-1β treatment. Supernatants of astrocyte cultures pretreated with inhibitors of p38 MAPK or MEK1/2 prior to IL-1β exposure were collected for NO assay.Results: IL-1β treatment of astrocytes alone induced undetectable amounts of HO-1 protein by western blot. However, HO-1 mRNA expression was modestly up-regulated in response to IL-1β stimulation. Pretreatment with hemin alone substantially induced both HO-1 mRNA and protein expression, and HO-1 mRNA expression was further enhanced when hemin was combined with IL-1β treatment. In contrast, IL-1β-induced iNOS mRNA expression and NO production were markedly inhibited by hemin treatment. When pretreated with SnPP, the inhibitory effect of hemin on IL-1β-induced NO production and iNOS expression was reversed, suggesting the involvement of HO-1. IL-1β-induced p38 MAPK activation, which is known to be required for NO production, was also down-regulated by hemin.Conclusion: These findings support the hypothesis that up-regulation of HO-1 in astrocytes is associated with down-regulation of iNOS expression and thereby NO production, an effect that involves the p38 MAPK signaling pathway, which suggests that this glial cell response could play an important protective role against oxidative stress in the brain. [ABSTRACT FROM AUTHOR]