Role of CD23 in astrocytes inflammatory reaction during HIV-1 related encephalitis

Soluble factors released by intra-cerebral activated cells are implicated in neuronal alterations during central nervous system inflammatory diseases. In this study, the role of the CD23 pathway in astrocyte activation and its participation in human immunodeficiency virus-1 (HIV-1)-induced neuropathology were evaluated. In human primary astrocytes, CD23 protein membrane expression was dose-dependently upregulated by gp120. It was also upregulated by gamma-interferon (gamma-IFN) and modulated by interleukin-1-beta (IL-1beta) whereas microglial cells in these stimulation conditions did not express CD23. Cell surface stimulation of CD23 expressed by astrocytes induced production of nitric oxide (NO) and IL-1beta which was inhibited by a specific inducible NO-synthase (iNOS) inhibitor (aminoguanidine), indicating the implication of this receptor in the astrocyte inflammatory reaction. On brain tissues from five out of five patients with HIV-1-related encephalitis, CD23 was expressed by astrocytes and by some microglial cells, whereas it was not detectable on brain tissue from five of five HIV-1-infected patients without central nervous system (CNS) disease or from two of two control subjects. In addition, CD23 antigen was co-localized with iNOS and nitrotyrosine on brain tissue from patients with HIV1-related encephalitis, suggesting that CD23 participates in iNOS activation of astrocytes in vivo. In conclusion, CD23 ligation is an alternative pathway in the induction of inflammatory product synthesis by astrocytes and participates in CNS inflammation.     

Neurovirulent simian immunodeficiency virus infection induces neuronal, endothelial, and glial apoptosis.

BACKGROUND: Studies of human immunodeficiency virus type 1 (HIV-1) associated dementia have shown neuronal loss in discrete areas. The presence and mechanism of neuronal death, however, has remained quite elusive. One mechanism of cell death, apoptosis, has been clearly demonstrated outside the central nervous system (CNS) in HIV-1 infection but has not been firmly established within the CNS. Therefore, we set out to ascertain whether neuronal cell loss in simian immunodeficiency virus (SIV) encephalitis, an animal model of HIV-1-associated dementia, is a result of apoptosis. MATERIALS AND METHODS: With the aid of an in situ technique for identifying the 3'-OH ends of newly fragmented DNA characteristic of apoptosis, in conjunction with specific detected morphological criteria via light microscopy, we have examined encephalitic and nonencephalitic brains of macaques infected with a neurovirulent, neuroendotheliotropic strain of SIV to see if virus is spatially associated with apoptosis of neurons and non-neuronal cell types. RESULTS: We demonstrate the presence of DNA damage, indicative of apoptosis, in neurons, endothelial cells, and glial cells of the CNS of SIV-infected macaques. Furthermore, we observe an association between the localization of cells with significant DNA fragmentation and perivascular inflammatory cell infiltrates containing SIV-infected macrophages and multinucleated giant cells. Quantitative analysis reveals significantly more cells with DNA fragmentation in the CNS of macaques infected with neurovirulent, neuroendotheliotropic SIV strains as compared with strictly lymphocyte-tropic SIV strains and SIV negative controls. CONCLUSIONS: Our findings of apoptosis in SIV-infected CNS may potentially lead to a better understanding of the AIDS dementia complex, ultimately providing a basis for better treatments.     

Neutralization sensitivity of cell culture-passaged simian immunodeficiency virus.

CEMx174- and C8166-45-based cell lines which contain a secreted alkaline phosphatase (SEAP) reporter gene under the control of a tat-responsive promoter derived from either SIVmac239 or HIV-1(NL4-3) were constructed. Basal levels of SEAP activity from these cell lines were low but were greatly stimulated upon transfection of tat expression plasmids. Infection of these cell lines with simian immunodeficiency virus (SIV) or human immunodeficiency virus type 1 (HIV-1) resulted in a dramatic increase in SEAP production within 48 to 72 h that directly correlated with the amount of infecting virus. When combined with chemiluminescent measurement of SEAP activity in the cell-free supernatant, these cells formed the basis of a rapid, sensitive, and quantitative assay for SIV and HIV infectivity and neutralization. Eight of eight primary isolates of HIV-1 that were tested induced readily measurable SEAP activity in this system. While serum neutralization of cloned SIVmac239 was difficult to detect with other assays, neutralization of SIVmac239 was readily detected at low titers with this new assay system. The neutralization sensitivities of two stocks of SIVmac251 with different cell culture passage histories were tested by using sera from SIV-infected monkeys. The primary stock of SIVmac251 had been passaged only twice through primary cultures of rhesus monkey peripheral blood mononuclear cells, while the laboratory-adapted stock had been extensively passaged through the MT4 immortalized T-cell line. The primary stock of SIVmac251 was much more resistant to neutralization by a battery of polyclonal sera from SIV-infected monkeys than was the laboratory-adapted virus. Thus, SIVmac appears to be similar to HIV-1 in that extensive laboratory passage through T-cell lines resulted in a virus that is much more sensitive to serum neutralization.     

Genetic diversity of simian immunodeficiency virus

We have demonstrated that the genetic diversity of simian immunodeficiency virus from African green monkeys (SIVagm) is much greater than that observed previously for individual HIV-1, HIV-2, or SIVmac isolates. Extensive genetic variation among SIVagm isolates and the high prevalence of green monkey infection without disease suggest that the virus has been in the green monkey population for a long time. We have also demonstrated that SIV from a sooty mangabey monkey (isolate SMM-7) is closer to SIVmac and HIV-2 than to HIV-1 and SIVagm. The extensive genetic diversity of SIVagm and the relatedness of SIVsmm to HIV-2 warrant continued examination of SIVagm and SIVsmm isolates from dispersed geographic regions. SIV strains much more closely related to HIV-1, HIV-2, or SIVmac may be found which would be reasonable candidates for recent cross-species transmission.     

Involvement of phosphatidylinositol 3-kinase-mediated up-regulation of I kappa B alpha in anti-inflammatory effect of gemfibrozil in microglia

The present study underlines the importance of PI3K in mediating the anti-inflammatory effect of gemfibrozil, a prescribed lipid-lowering drug for humans, in mouse microglia. Gemfibrozil inhibited LPS-induced expression of inducible NO synthase (iNOS) and proinflammatory cytokines in mouse BV-2 microglial cells and primary microglia. By overexpressing wild-type and dominant-negative constructs of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in microglial cells and isolating primary microglia from PPAR-alpha-/- mice, we have demonstrated that gemfibrozil inhibits the activation of microglia independent of PPAR-alpha. Interestingly, gemfibrozil induced the activation of p85alpha-associated PI3K (p110beta but not p110alpha) and inhibition of that PI3K by either chemical inhibitors or dominant-negative mutants abrogated the inhibitory effect of gemfibrozil. Conversely, overexpression of the constitutively active mutant of p110 enhanced the inhibitory effect of gemfibrozil on LPS-induced expression of proinflammatory molecules. Similarly, gemfibrozil also inhibited fibrillar amyloid beta (Abeta)-, prion peptide (PrP)-, dsRNA (poly IC)-, HIV-1 Tat-, and 1-methyl-4-phenylpyridinium (MPP+)-, but not IFN-gamma-, induced microglial expression of iNOS. Inhibition of PI3K also abolished the inhibitory effect of gemfibrozil on Abeta-, PrP-, poly IC-, Tat-, and MPP+-induced microglial expression of iNOS. Involvement of NF-kappaB activation in LPS-, Abeta-, PrP-, poly IC-, Tat-, and MPP+-, but not IFN-gamma-, induced microglial expression of iNOS and stimulation of IkappaBalpha expression and inhibition of NF-kappaB activation by gemfibrozil via the PI3K pathway suggests that gemfibrozil inhibits the activation of NF-kappaB and the expression of proinflammatory molecules in microglia via PI3K-mediated up-regulation of IkappaBalpha.     

Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta: involvement of reactive oxygen species

AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS.     

Interleukin-1beta-induced nitric oxide production in rat aortic endothelial cells: inhibition by estradiol in normal and high glucose cultures.

Expression of inducible nitric oxide synthase (iNOS) and the resultant increased nitric oxide (NO) production are associated with septic shock, atherosclerosis, and cytokine-induced vascular injury. Estrogen is known to impact vascular injury and vascular tone, in part through regulation of NO production. In the current study, we examined the effect of physiological concentrations of estradiol on interleukin-1beta (IL-1beta)-induced NO production in rat aortic endothelial cells (RAECs). 17Beta-estradiol significantly decreased IL-1beta-induced iNOS protein levels and reduced NO production in RAECs. High glucose (25 mM) elevated the increase in IL-1beta-induced iNOS protein and NO production. Nevertheless, estradiol still inhibited IL-1beta-induced iNOS and NO production even in the presence of high glucose. These data suggest that estradiol may exert its beneficial effects in part by inhibiting induction of endothelial iNOS, a possible mechanism for the protective effect of estradiol against diabetes-associated cardiovascular complications.     

Platelet-activating factor: a candidate human immunodeficiency virus type 1-induced neurotoxin.

The pathogenesis of central nervous system disease during human immunodeficiency virus type 1 (HIV-1) infection revolves around productive viral infection of brain macrophages and microglia. Neuronal losses in the cortex and subcortical gray matter accompany macrophage infection. The question of how viral infection of brain macrophages ultimately leads to central nervous system (CNS) pathology remains unanswered. Our previous work demonstrated high-level production of tumor necrosis factor alpha, interleukin 1 beta, arachidonic acid metabolites, and platelet-activating factor (PAF) from HIV-infected monocytes and astroglia (H. E. Gendelman, P. Genis, M. Jett, and H. S. L. M. Nottet, in E. Major, ed., Technical Advances in AIDS Research in the Nervous System, in press; P. Genis, M. Jett, E. W. Bernton, H. A. Gelbard, K. Dzenko, R. Keane, L. Resnick, D. J. Volsky, L. G. Epstein, and H. E. Gendelman, J. Exp. Med. 176:1703-1718, 1992). These factors, together, were neurotoxic. The relative role(s) of each of these candidate neurotoxins in HIV-1-related CNS dysfunction was not unraveled by these initial experiments. We now report that PAF is produced during HIV-1-infected monocyte-astroglia interactions. PAF was detected at high levels in CSF of HIV-1-infected patients with immunosuppression and signs of CNS dysfunction. The biologic significance of the results for neurological disease was determined by addition of PAF to cultures of primary human fetal cortical or rat postnatal retinal ganglion neurons. Here, PAF at concentrations of > or = 300 pg/ml produced neuronal death. The N-methyl-D-aspartate receptor antagonist MK-801 or memantine partially blocked the neurotoxic effects of PAF. The identification of PAF as an HIV-1-induced neurotoxin provides new insights into how HIV-1 causes neurological impairment and how it may ultimately be ameliorated.     

The evolution of HIV-1 and the origin of AIDS

The major cause of acquired immune deficiency syndrome (AIDS) is human immunodeficiency virus type 1 (HIV-1). We have been using evolutionary comparisons to trace (i) the origin(s) of HIV-1 and (ii) the origin(s) of AIDS. The closest relatives of HIV-1 are simian immunodeficiency viruses (SIVs) infecting wild-living chimpanzees (Pan troglodytes troglodytes) and gorillas (Gorilla gorilla gorilla) in west central Africa. Phylogenetic analyses have revealed the origins of HIV-1: chimpanzees were the original hosts of this clade of viruses; four lineages of HIV-1 have arisen by independent cross-species transmissions to humans and one or two of those transmissions may have been via gorillas. However, SIVs are primarily monkey viruses: more than 40 species of African monkeys are infected with their own, species-specific, SIV and in at least some host species, the infection seems non-pathogenic. Chimpanzees acquired from monkeys two distinct forms of SIVs that recombined to produce a virus with a unique genome structure. We have found that SIV infection causes CD4⁺ T-cell depletion and increases mortality in wild chimpanzees, and so the origin of AIDS is more ancient than the origin of HIV-1. Tracing the genetic changes that occurred as monkey viruses adapted to infect first chimpanzees and then humans may provide insights into the causes of the pathogenicity of these viruses.     

On the origin and evolution of the human immunodeficiency virus (HIV)

The human AIDS viruses--HIV-1 and HIV-2--impose major burdens on the health and economic status of many developing countries. Surveys of other animal species have revealed that related viruses--the SIVs are widespread in a large number of African simian primates where they do not appear to cause disease. Phylogenetic analyses indicate that these SIVs are the reservoirs for the human viruses, with SIVsm from the sooty mangabey monkey the most likely source of HIV-2, and SIVcpz from the common chimpanzee the progenitor population for HIV-1. Although it is clear that AIDS has a zoonotic origin, it is less certain when HIV-1 and HIV-2 first entered human populations and whether cross-species viral transmission is common among primates. Within infected individuals the process of HIV evolution takes the form of an arms race, with the virus continually fixing mutations by natural selection which allow it to escape from host immune responses. The arms race is less intense in SIV-infected monkeys, where a weaker immune response generates less selective pressure on the virus. Such a difference in virus-host interaction, along with a broadening of co-receptor usage such that HIV strains are able to infect cells with both CCR5 and CXCR4 chemokine receptors, may explain the increased virulence of HIV in humans compared to SIV in other primates.     

Quantitation of a lentivirus in its natural host: simian immunodeficiency virus in African green monkeys.

We have examined the viral load in the peripheral blood of simian immunodeficiency virus (SIV)-infected African green monkeys with a view to the unexplained apathogenicity of African green monkey SIV (SIVagm) in its natural host. By using polymerase chain reaction, viral DNA was detected in fresh peripheral blood mononuclear cells (PBMC) of each of nine seropositive animals. The virus DNA load was variable among the monkeys tested, ranging from 5 to 50 (mean = 15) copies per 10(5) PBMC, which is comparable to that of human immunodeficiency virus type 1 (HIV-1) in humans. The level of infectious SIVagm in PBMC was measured by endpoint dilution cultures. SIVagm was recovered from PBMC from 14 of 17 antibody-positive monkeys (82%), and the mean SIVagm titer in PBMC of seropositive African green monkeys was 10 tissue culture infectious doses per 10(6) cells, similar to the titer shown for HIV in asymptomatic carriers. Free infectious virus was isolated from the plasma of 4 of 17 monkeys (24%), and SIVagm expression in peripheral blood in vivo, as demonstrated by in situ hybridization, was detectable only in those animals which were viremic. SIVagm replication is therefore not totally suppressed in vivo, and SIVagm has a viral load equivalent to that seen for HIV-1 in asymptomatic humans.     

Suppression of simian immunodeficiency virus replication in vitro by CD8+ lymphocytes

The AIDS-like disease in rhesus monkeys induced by the simian immunodeficiency virus (SIV) has been used as a model to explore the nature of the T lymphocyte response after infection with viruses of the human immunodeficiency virus family. Activated CD8+ lymphocytes are present in increased numbers in the paracortex of lymph nodes of SIV-infected rhesus monkeys with a lymphadenopathy syndrome. We demonstrate that SIV is more readily isolated from CD8+ lymphocyte-depleted PBL of SIV-infected animals than from their unfractionated PBL. Rather than reflecting the fact that the CD8+ lymphocyte-depleted cell populations are simply enriched for CD4+ lymphocytes, this indicates that CD8+ cells themselves are critical in this regulatory interaction. In fact, CD8+ lymphocytes from SIV-infected but not uninfected rhesus monkeys can block SIV replication in vitro in PBL populations. A T lymphocyte population that blocks replication of viruses of the HIV family may contribute to containing the progression of AIDS.     

恒河猴Mamu-A^＊01基因与SIV／SHIV感染相关的研究进展

SIV／SHIV感染的恒河猴是研究艾滋病及艾滋病药物筛选、疫苗评价较理想的动物模型。MHC在细胞免疫中起着关键作用,研究表明,MHC-I类分子的多态性与SIV／SHIV感染者的疾病进展有着明显的关联作用,Mamu-A^＊01是恒河猴中的一种MHC-I类分子,它可以呈递特定的病毒蛋白片段到细胞的表面,从而激发CTL反应。国外发现Mamu-A^＊01阳性的猴艾滋病恒河猴会出现疾病进展缓慢,存活时间长等特征。本文就恒河猴Mamu-A^＊01基因与SIV／SHIV感染相关的研究进展做一综述,以期进一步加深对MHC在疫苗研究中的作用的了解,并促进更行之有效地对HIV／AIDS疫苗进行评价。     

Anti-α4 Antibody Treatment Blocks Virus Traffic to the Brain and Gut Early,and Stabilizes CNS Injury Late in Infection

Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28⁺, RNA⁺) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV – RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity.     

Effect of cyclosporin-A on the blood--retinal barrier permeability in streptozotocin-induced diabetes

BACKGROUND: Our previous results showed that in retinas from streptozotocin (STZ)-induced diabetic rats there is an increased level of interleukin-1beta (IL-1beta). This cytokine may be involved in the expression of the inducible isoform of the nitric oxide synthase (iNOS), with consequent synthesis of large amounts of NO and blood-retinal barrier (BRB) breakdown. AIMS: The aim of this work was to examine whether the administration of cyclosporin-A (Cs-A) to STZ-induced diabetic rats inhibits the synthesis of IL-1beta and the expression of the inducible proteins, iNOS and cyclo-oxygenase-2 (COX-2) in retinal cells, and whether the activity of these proteins contribute to BRB breakdown. METHODS: The level of IL-1beta was evaluated by ELISA and the NO production by L-[3H]-citrulline formation. Expression of iNOS and COX-2 proteins was determined by two methods, western blot and immunohistochemistry. The permeability of the BRB was assessed by quantification of the vitreous protein. RESULTS AND DISCUSSION: Our results indicated that the levels of IL-1beta and NO in retinas from Cs-A-treated diabetic rats are significantly reduced, as compared to that in non-treated diabetic rats. The treatment of diabetic rats with Cs-A also significantly inhibited the expression of the inducible proteins, iNOS and COX-2. The evaluation of the vitreous protein content revealed that Cs-A also reduces the BRB permeability. Taken together, these results suggest that the increased production of the inflammatory mediators, IL-1beta and NO, in diabetes may affect the BRB permeability and therefore contribute to the development of diabetic retinopathy.