Global research priorities for infections that affect the nervous system.

Infections that cause significant nervous system morbidity globally include viral (for example, HIV, rabies, Japanese encephalitis virus, herpes simplex virus, varicella zoster virus, cytomegalovirus, dengue virus and chikungunya virus), bacterial (for example, tuberculosis, syphilis, bacterial meningitis and sepsis), fungal (for example, cryptococcal meningitis) and parasitic (for example, malaria, neurocysticercosis, neuroschistosomiasis and soil-transmitted helminths) infections. The neurological, cognitive, behavioural or mental health problems caused by the infections probably affect millions of children and adults in low- and middle-income countries. However, precise estimates of morbidity are lacking for most infections, and there is limited information on the pathogenesis of nervous system injury in these infections. Key research priorities for infection-related nervous system morbidity include accurate estimates of disease burden; point-of-care assays for infection diagnosis; improved tools for the assessment of neurological, cognitive and mental health impairment; vaccines and other interventions for preventing infections; improved understanding of the pathogenesis of nervous system disease in these infections; more effective methods to treat and prevent nervous system sequelae; operations research to implement known effective interventions; and improved methods of rehabilitation. Research in these areas, accompanied by efforts to implement promising technologies and therapies, could substantially decrease the morbidity and mortality of infections affecting the nervous system in low- and middle-income countries.

Central nervous system tuberculosis: pathogenesis and clinical aspects.

Tuberculosis of the central nervous system (CNS) is a highly devastating form of tuberculosis, which, even in the setting of appropriate antitubercular therapy, leads to unacceptable levels of morbidity and mortality. Despite the development of promising molecular diagnostic techniques, diagnosis of CNS tuberculosis relies largely on microbiological methods that are insensitive, and as such, CNS tuberculosis remains a formidable diagnostic challenge. Insights into the basic neuropathogenesis of Mycobacterium tuberculosis and the development of an appropriate animal model are desperately needed. The optimal regimen and length of treatment are largely unknown, and with the rising incidence of multidrug-resistant strains of M. tuberculosis, the development of well-tolerated and effective antibiotics remains a continued need. While the most widely used vaccine in the world largely targets this manifestation of tuberculosis, the BCG vaccine has not fulfilled the promise of eliminating CNS tuberculosis. We put forth this review to highlight the current understanding of the neuropathogenesis of M. tuberculosis, to discuss certain epidemiological, clinical, diagnostic, and therapeutic aspects of CNS tuberculosis, and also to underscore the many unmet needs in this important field.

Preferential sensitivity of human dopaminergic neurons to gp120-induced oxidative damage.

The dopamine (DA)-rich midbrain is known to be a key target of human immunodeficiency virus (HIV)-1. Studies of simian immunodeficiency virus (SIV)-induced neuropathogenesis recently established that there is a major disruption within the nigrostriatal dopaminergic system characterized by marked depletion of dopaminergic neurons, microglial cell activation, and reactive astrocytes. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, experiments were performed to characterize the damage to dopaminergic neurons induced by HIV-1 gp120. Functional impairment was assessed by DA uptake, and neurotoxicity was measured by apoptosis and oxidative damage. Through the use of this mesencephalic neuronal/glial culture model, we were able to identify the relative sensitivity of dopaminergic neurons to gp120-induced damage, manifested as reduced function (decreased DA uptake), morphological changes, and reduced viability. We also showed that gp120-induced oxidative damage is involved in this neuropathogenic process.

Morphine stimulates CCL2 production by human neurons.

BACKGROUND: Substances of abuse, such as opiates, have a variety of immunomodulatory properties that may influence both neuroinflammatory and neurodegenerative disease processes. The chemokine CCL2, which plays a pivotal role in the recruitment of inflammatory cells in the nervous system, is one of only a few chemokines produced by neurons. We hypothesized that morphine may alter expression of CCL2 by human neurons. METHODS: Primary neuronal cell cultures and highly purified astrocyte and microglial cell cultures were prepared from human fetal brain tissue. Cell cultures were treated with morphine, and cells were examined by RNase protection assay for mRNA. Culture supernatants were assayed by ELISA for CCL2 protein. beta-funaltrexamine (beta-FNA) was used to block mu-opioid receptor (MOR)s. RESULTS: Morphine upregulated CCL2 mRNA and protein in neuronal cultures in a concentration- and time-dependent fashion, but had no effect on CCL2 production in astrocyte or microglial cell cultures. Immunocytochemical analysis also demonstrated CCL2 production in morphine-stimulated neuronal cultures. The stimulatory effect of morphine was abrogated by beta-FNA, indicating an MOR-mediated mechanism. CONCLUSION: Morphine stimulates CCL2 production by human neurons via a MOR-related mechanism. This finding suggests another mechanism whereby opiates could affect neuroinflammatory responses.

Cocaine-induced HIV-1 expression in microglia involves sigma-1 receptors and transforming growth factor-beta1.

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)-beta1 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-beta1 (SB-431542 and anti-TGF-beta1 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.

TNF-alpha-induced chemokine production and apoptosis in human neural precursor cells.

Recent studies have shown that proinflammatory cytokines damage rodent neural precursor cells (NPCs), a source of self-renewing, multipotent cells that play an important role in the developing as well as adult brain. In this study, the effects of tumor necrosis factor alpha (TNF-alpha) on cytokine and chemokine production by human NPCs (>98% nestin- and >90% A2B5-positive), obtained from 6- to 8-week-old fetal brain specimens, were evaluated. NPCs stimulated with this proinflammatory cytokine were found to produce abundant amounts of the chemokines monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL2) and interferon-inducible protein 10 (IP-10)/CXC chemokine ligand 10 (CXCL10) in a time- and concentration-dependent manner. TNF-alpha treatment also induced NPC apoptosis. Receptors for TNF [TNFRI (p55) and TNFRII (p75)] mRNA were constitutively expressed on NPCs. However, only TNFRI was involved in TNF-alpha-induced chemokine production and apoptosis by NPCs, as anti-TNFRI but not anti-TNFRII antibodies blocked the stimulatory effect. TNF-alpha treatment induced p38 mitogen-activated protein kinase (MAPK) phosphorylation in NPCs, and SB202190, an inhibitor of p38 MAPK, blocked TNF-alpha-induced chemokine production. Thus, this study demonstrated that NPCs constitutively express receptors for TNF-alpha, which when activated, trigger via a p38 MAPK signaling pathway production of two chemokines, MCP-1/CCL2 and IP-10/CXCL10, which are involved in infectious and inflammatory diseases of the brain.

Anti-HIV-1 activity of propolis in CD4(+) lymphocyte and microglial cell cultures.

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 66.6 microg/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.

Early detection of central nervous system tuberculosis with the gen-probe nucleic Acid amplification assay: utility in an inner city hospital.

Central nervous system tuberculosis is a serious clinical problem, the treatment of which is sometimes hampered by delayed diagnosis. We investigated the utility of the Gen-Probe nucleic acid amplification assay for the rapid diagnosis of tuberculous meningitis and as a noninvasive method of identifying intracranial tuberculoma.

Morphine induces apoptosis of human microglia and neurons.

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.

Kappa-opioid receptor agonist inhibition of HIV-1 envelope glycoprotein-mediated membrane fusion and CXCR4 expression on CD4(+) lymphocytes.

Our previous studies have shown that the suppressive effect of kappa-opioid receptor (KOR) ligand treatment on HIV-1(AT) (a T-tropic strain) expression in acutely infected CD4(+) lymphocytes is time-dependent. This finding implied that the inhibition observed following treatment with KOR agonists such as U50,488 (trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl]benzeneaceamide methanesulfonate) occurs at an early step in the viral replication cycle, perhaps as early as viral entry. In the present study, we examined the hypothesis that U50,488 treatment of CD4(+) lymphocytes inhibits HIV-1 envelope (Env) glycoprotein-mediated membrane fusion. We used a vaccinia virus-based assay to measure the effects of U50,488 treatment of CD4(+) lymphocytes on HIV-1 IIIB Env glycoprotein-mediated fusogenic activity, based on the cytoplasmic activation of a reporter gene. The results show that U50,488 inhibited Env-mediated cell fusion in a bell-shaped concentration-response manner with suppression ranging between 31 and 98% at concentrations of 10(-8) and 10(-10)M (N=9 experiments). U50,488 was also found to inhibit cell fusion when monitored in situ with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining. Blockade of the inhibitory activity of U50,488 by the KOR antagonist nor-bialtorphimine (nor-BNI) suggested that U50,488 was acting via a KOR-related mechanism. Using flow cytometry, we demonstrated that the chemokine co-receptor CXCR4, but not CD4, is down-regulated as a consequence of KOR activation, with 44.2+/-3.5% suppression at 10(-10)M U50,488. These findings support the hypothesis that KOR-related activation of CD4(+) lymphocytes inhibits HIV-1 entry via down-regulation of CXCR4.

U50,488 inhibits HIV-1 Tat-induced monocyte chemoattractant protein-1 (CCL2) production by human astrocytes.

Kappa-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 kappaB (NF-kappaB) 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-kappaB activation, it potently suppressed Tat-induced NF-kappaB 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.

High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells.

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-1alpha, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1alpha. These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development.

Changes in the NMR metabolic profile of human microglial cells exposed to lipopolysaccharide or morphine.

Microglial cells play a major role in host defense of the central nervous system. Once activated, several functional properties are up-regulated including migration, phagocytosis, and secretion of inflammatory mediators such as cytokines and chemokines. Little, if anything, is known about the metabolic changes that occur during the activation process. High-resolution (1)H nuclear magnetic resonance spectra obtained from perchloric acid extracts of human microglial cell cultures exposed to lipopolysaccharide (LPS) or morphine were used to both identify and quantify the metabolites. We found that human microglia exposed to LPS had increased concentrations of glutamate and lactate, whereas the cells exposed to morphine had decreased concentrations in creatinine, taurine, and thymine. Glutamate and creatinine were the key metabolites differentiating between the two stimuli. These results are discussed in terms of activation and differences in the inflammatory response of human microglial cells to LPS and morphine.

Serial testing of refugees for latent tuberculosis using the QuantiFERON-gold in-tube: effects of an antecedent tuberculin skin test.

Screening for latent tuberculosis infection (LTBI) in refugee populations immigrating to low-incidence countries remains a challenge. We assessed the characteristics of the QuantiFERON-Gold In-Tube (QFT-GIT) compared with the tuberculin skin test (TST) in 198 refugees of all ages from tuberculosis-endemic countries. Diagnostic agreement between the first QFT-GIT and simultaneous TST was 78% (kappa = 0.56) and between serial QFT-GITs was 89% (kappa = 0.76). In serial QFT-GIT testing, 70% of subjects had an increased QFT-GIT value, perhaps the result of an antecedent TST in the setting of previous TB exposure. This boosting seemed to become less prevalent with time from TST and occurred less frequently in those with negative first QFT-GIT readings. Despite small changes in the quantitative results caused by nonspecific variation and boosting, the diagnostic result of the QFT-GIT was reliable. The QFT-GIT shows the potential to replace the TST for LTBI screening in refugees from tuberculosis-endemic areas.

Potentiation of HIV-1 expression in microglial cells by nicotine: involvement of transforming growth factor-beta 1.

HIV-1 infection and nicotine addiction are global public health crises. In the central nervous system, HIV-1 causes a devastating neurodegenerative disease. It is well recognized that microglial cells play a pivotal role in the neuropathogenesis of HIV-1 and that drugs of abuse not only contribute to the spread of this agent but may facilitate viral expression in these brain macrophages. Nicotine has been shown to stimulate the production of HIV-1 by in vitro-infected alveolar macrophages, and the HIV-1 protein gp120 binds to nicotinic receptors. In this study, we demonstrated the constitutive expression of nicotinic acetylcholine receptor mRNA in primary human microglial cells and showed that the pretreatment of microglia with nicotine increased HIV-1 expression in a concentration-dependent manner, as measured by p24 antigen levels in culture supernatants. We also found that nicotine robustly altered the gene expression profile of HIV-1-infected microglia and that the transforming growth factor-beta1 is involved in the enhanced expression of HIV-1 by nicotine.

Role of nitric oxide in defense of the central nervous system against Mycobacterium tuberculosis.

Murine models of tuberculous meningitis (TBM) have not reflected the severity of disease in humans. Based on reports that activated murine microglial cells, but not human microglial cells, express inducible nitric oxide synthase (iNOS), the objective of this study was to determine whether iNOS-knockout (iNOS(-/-)) mice would provide such a model. iNOS(-/-) mice infected with M. tuberculosis developed serious clinical manifestations and granulomatous lesions containing tubercle bacilli throughout the meninges, all of which were absent in wild-type mice. This study underscores the importance of nitric oxide in defense against TBM and suggests that iNOS(-/-) mice are an appropriate model for human TBM.

Toll-like receptors in defense and damage of the central nervous system.

Members of the Toll-like receptor (TLR) family play critical roles as regulators of innate and adaptive immune responses. TLRs function by recognizing diverse molecular patterns on the surface of invading pathogens. In the brain, microglial cells generate neuroimmune responses through production of proinflammatory mediators. The upregulation of cytokines and chemokines in response to microbial products and other stimuli has both beneficial and deleterious effects. Emerging evidence demonstrates a central role for TLRs expressed on microglia as a pivotal factor in generating these neuroimmune responses. Therefore, understanding the basis of TLR signaling in producing these responses may provide insights into how activated microglia attempt to strike a balance between defense against invading pathogens and inflicting irreparable brain damage. These insights may lead to innovative therapies for CNS infections and neuroinflammatory diseases based on the modulation of microglial cell activation through TLR signaling.

Morphine modulates gammadelta lymphocytes cytolytic activity following BCG vaccination.

Chronic opioid administration modulates lymphocytes' functional capabilities increasing susceptibility to infectious diseases. Bacille-Calmette-Guérin (BCG) vaccination initiates a non-specific and specific cell-mediated immunity orchestrated by T lymphocytes including gammadelta T lymphocytes. gammadelta T lymphocytes increase in natural killer and antigen-directed cytolytic response following BCG vaccination. The objective of this study was to determine morphine effects on gammadelta T lymphocytes' cytolytic activity. Pigs were chronically administered morphine and subsequently vaccinated with Mycobacterium bovis BCG. By administering morphine prior to BCG vaccination, natural killer response was significantly suppressed (p=.034). Furthermore, innate cytolytic response against M. bovis-infected monocytes (p=.002) as well as antigen specific cytolytic functions (p=.04) were significantly altered due to morphine administration. It was concluded that administering morphine prior to BCG vaccination significantly altered gammadelta T lymphocyte cytolytic responses.

Human neural precursor cells express functional kappa-opioid receptors.

Neural stem cells (NSCs) play an important role in the developing as well as adult brain. NSCs have been shown to migrate toward sites of injury in the brain and to participate in the process of brain repair. Like NSCs, cultured human neural precursor cells (NPCs) are self-renewing, multipotent cells capable of differentiating into neurons, astrocytes, and oligodendrocytes and of migrating toward chemotactic stimuli. Cellular and environmental factors are important for NPC proliferation and migration. Expression of kappa-opioid receptors (KORs) and mu-opioid receptors (MORs) in murine embryonic stem cells and of MORs and delta-opioid receptors in rodent neuronal precursors, as well as hippocampal progenitors has been reported by other investigators. In this study, we demonstrated robust expression of KORs in highly enriched (>90% nestin-positive) human fetal brain-derived NPCs. We found that KOR ligands, dynorphin(1-17) and trans-3,4-dichloro-N-methyl-N[2-(1-pyrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate (U50,488) but not dynorphin(2-17), stimulated proliferation and migration of NPCs in a concentration-dependent manner. NPC proliferation was maximally stimulated at 10(-14) M dynorphin(1-17) and 10(-12) M U50,488. The KOR selective antagonist, nor-binaltorphimine, partially blocked the migratory and proliferative effects of KOR agonists supporting, at least in part, the involvement of a KOR-related mechanism. As has been described for rodent P19 embryonal carcinoma stem cells, retinoic acid treatment markedly suppressed KOR mRNA expression in human NPCs. Taken together, the results of this study suggest that activation of KORs alters functional properties of NPCs/NSCs that are relevant to human brain development and repair.