Apoptotic neurons in brains from paediatric patients with HIV-I encephalitis and progressive encephalopathy

The pathogenesis of human immunodeficiency virus type 1 (HIV-1) associated dementia in adults involves neuronal loss from discrete areas of the neocortex and subcortical regions, but the mechanism for neuronal death is poorly understood. Gene-directed cell death resulting in apoptosis is thought to be a normal feature of neuronal development, but little is known about neuronal apoptosis in disease states. We investigated whether HIV-1 infection of the central nervous system is spatially associated with apoptosis of neurons. Using an in situ technique to identify newly cleaved 3'-OH ends of DNA as a marker for apoptosis, we demonstrate the presence of apoptotic neurons in cerebral cortex and basal ganglia of children that had HIV-1 encephalitis with progressive encephalopathy. Furthermore, an association was observed between the localization of apoptotic neurons and perivascular inflammatory cell infiltrates containing HIV-1 infected macrophages and multinucleated giant cells. Apoptotic neurons and p24–positive macrophages were observed infrequently in cerebral cortex and basal ganglia in children with HIV-1 infection without encephalitis or clinical encephalopathy. In nine control (HIV-1 negative) brains, ranging from the first post-natal month of life to 16.5 years of age, infrequent neuronal apoptosis was observed in three cases. These findings suggest that neuronal apoptosis is unlikely to be associated with post-natal development except in early post-natal germinal matrix, and that it may instead represent the end result of specific pathological processes, such as HIV-1 encephalitis.     

Expression of pro‐ and anti‐apoptosis gene products in brains from paediatric patients with HIV‐1 encephalitis

We have previously demonstrated the presence of DNA fragmentation in neurons, macrophages and microglia consistent with apoptosis, but not in reactive astrocytes in brain tissue from paediatric patients with HIV‐1 encephalitis (HIVE). To further understand the underlying mechanism(s) for these findings as they relate to gene‐directed neural cell death, we studied the in‐situ expression of the Bcl‐2 family of proteins, including the pro‐apoptosis gene product Bax, the anti‐apoptosis gene product Bcl‐2, and Bcl‐x. We demonstrate significantly elevated numbers of Bax‐positive microglia and macrophages immunoreactive in basal ganglia and cerebral cortex of children who had HIVE, in comparison to HIV‐1 infected children without encephalitis or children who were seronegative for HIV‐1. In contrast, patients with HIVE, but not HIV‐1 without encephalitis, or seronegative controls, had increased expression of Bcl‐2 and Bcl‐x in reactive astrocytes in cortex and basal ganglia. In vitro studies using Western blot analysis demonstrated an up‐regulation in the levels of Bax, and phosphorylated (i.e. inactive) Bcl‐2 in HIV‐1 infected macrophages, and in LPS‐activated macrophages, relative to levels in virus‐negative unstimulated macrophages. These results suggest that productive HIV‐1 infection, or cellular activation, renders macrophages more vulnerable to apoptosis. Taken together, these findings suggest that brain‐resident macrophages and microglia in patients with HIV‐1 encephalitis are more prone to undergo apoptosis and that astrocytes in contrast may be resistant to apoptosis. This may represent a mechanism to limit microglial activation and the spread of productive HIV‐1 infection in the CNS of children with HIV‐1 encephalitis.     

Modulation of innate immunity by copolymer-1 leads to neuroprotection in murine HIV-1 encephalitis

Virus-infected and immune-competent mononuclear phagocytes (MP; perivascular macrophages and microglia) drive the neuropathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. Modulation of the MP phenotype from neurodestructive to neuroprotective underlies adjunctive therapeutic strategies for human disease. We reasoned that, as Copolymer-1 (Cop-1) can induce neuroprotective activities in a number of neuroinflammatory and neurodegenerative disorders, it could directly modulate HIV-1-infected MP neurotoxic activities. We now demonstrate that, in laboratory assays, Cop-1-stimulated virus-infected human monocyte-derived macrophages (MDM) protect against neuronal injury. Severe combined immune-deficient (SCID) mice were stereotactically injected with HIV-1-infected human MDM, into the basal ganglia, to induce HIV-1 encephalitis (HIVE). Cop-1 was administered subcutaneously for 7 days. In HIVE mice, Cop-1 treatment led to anti-inflammatory and neuroprotective responses. Reduced micro- and astrogliosis, and conserved NeuN/MAP-2 levels were observed in virus-affected brain regions in Cop-1-treated mice. These were linked to interleukin-10 and brain-derived neurotrophic factor expression and downregulation of inducible nitric oxide synthase. The data, taken together, demonstrate that Cop-1 can modulate innate immunity and, as such, improve disease outcomes in an animal model of HIVE.     

HIV-1 non-specifically stimulates production of transforming growth factor-β1 transfer in primary astrocytes

HIV-1 expression in monocytes/macrophages can be controlled by transforming growth factor-beta l (TGF-β1). TGF-β1 is present in astrocytes surrounding HIV-1-infected monocyte/macrophages in brain tissue from patients with AIDS but not from seronegative, normal individuals. We sought to determine whether or not production of TGF-β1 can be directly stimulated by HIV-1 in astrocytes. Astrocytes from neonatal rat cortex grown in primary culture were exposed to HIV-1 virions for 24 h. One day later, TGF-β1 was measured in culture supernatants by a biological assay. HIV-1 caused 1.7-2.1-fold increase in extracellular concentration of TGF-β1. TGF-β1 production also was stimulated by recombinant HIV-1 proteins gp120, p66 and p24. Gpl20 labeled with fluorescein was visualized inside astrocytes and its stimulatory effect was not blocked by antibodies against rat CD4. The effect was not specific to HIV-1 and its proteins, because non-opsonized Latex particles and leucine methyl ester (LME) (known to be phagocytosed and endocytosed, respectively, by astrocytes) also stimulated TGF-β1 production. The effect was inhibited by two inhibitors of the phago/endocytotic pathway, chloroquine and leupeptin. These results may be relevant to the neuropathogenesis of HIV-1 infection.     

高血糖对大鼠局灶性脑缺血再灌注后神经细胞凋亡及caspase-9、caspase-3表达的影响

目的探讨高血糖对大鼠局灶性脑缺血再灌注后神经细胞凋亡及半胱氨酸天冬氨酸蛋白酶-9(caspase-9)、caspase-3表达的影响,进一步探讨其加重脑缺血损伤的作用机制。方法将30只雄性Sprague-Dawley(SD)大鼠随机分为3组:高血糖组、正常血糖组和假手术组。按体质量4g/kg给予大鼠尾静脉注射25%(质量浓度)的葡萄糖,造成大鼠高血糖状态;继而制作大脑中动脉阻塞脑缺血再灌注模型。于缺血2h再灌注24h进行神经功能评分,采用脱氧核糖核苷酸末端转移酶介导的dUTP缺口末端标记法(terminal-deoxy-nucleotidyl transferase mediated dUTP nick end labeling,TUNEL)检测大鼠脑组织细胞凋亡数,采用免疫组化染色检测大鼠脑组织caspase-9和caspase-3表达。结果 (1)高血糖组神经功能评分为(4.20±0.63)分,正常血糖组为(3.50±0.70)分,假手术组为0分。高血糖组及正常血糖组的神经功能评分较假手术组均显著增加(P<0.05),且高血糖组神经功能评分较正常血糖组增加(P<0.05)。(2)高血糖组凋亡细胞数为(16.30±3.30)个,正常血糖组为(14.60±3.27)个,假手术组为(0.50±0.53)个。高血糖组及正常血糖组的凋亡细胞数较假手术组增多(P<0.05),且高血糖组细胞凋亡数较正常血糖组增多(P<0.05)。(3)高血糖组caspase-9和caspase-3灰度值分别为114.30±3.83、111.50±3.17,正常血糖组分别为117.20±4.34、117.40±3.24,假手术组分别为146.20±1.98、146.80±0.74。高血糖组及正常血糖组的caspase-9和caspase-3表达水平较假手术组均增强(P<0.05),且高血糖组caspase-9和caspase-3表达水平较正常血糖组增强(P<0.05)。结论高血糖可增加缺血再灌注后脑神经细胞凋亡;caspase-9、caspase-3的激活及表达增强可能是高血糖加重脑缺血再灌注损伤的机制之一。     

Caspase-3 is activated following axotomy of neonatal facial motoneurons and caspase-3 gene deletion delays axotomy-induced cell death in rodents

In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.     

Induction of immunoreactive interleukin-1β and tumor necrosis factor-α in the brains of rabies virus infected rats

Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) are important cytokines in the development of brain inflammation during pathological process. During rabies virus infection, the level of these proinflammatory cytokines are enhanced in the brain. In the present study we determined the cellular localization of these two cytokines by immunocytochemistry in brains of rats infected with rabies virus, at different time-intervals of the disease (day 1, 3, 4, 5 and at final stage day 6 post-infection (p.i.)). Cellular identification of IL-1β (irIL-1β) and TNFα (irTNFα) immunopositive cells was studied using a polyclonal antibody against these cytokines and against glial fibrillary acidic protein (GFAP) to detect astrocytes and GSA-I-B4 isolectin to detect microglial cells and/or infiltrating macrophages. In brains of control and early infected rats, irIL-1β was only detected in fibers located in the hypothalamus, supraoptic and tractus optic nuclei and infundibular nucleus. From day 4 onwards until day 6 p.i., enhanced irIL-1β was found and identified either in activated ameboid and/or infiltrated macrophages (amygdala, thalamus, internal capsula, subtantia nigra, septal nuclei and around blood vessels), or in activated ramified cells (hypothalamus and periventricular nucleus, piriformis and cingulate cortex, hippocampus). IrTNFα was observed in the brains of rats at a final stage of disease (day 5 and 6 p.i.): in the hypothalamus, the amygdala, the internal capsula, the thalamus, the septal nuclei, the hippocampus, the habenular nuclei and around the blood vessels. Ir-TNFα was detected in round cells identified as ameboid microglia and/or infiltrated macrophages. A marked activation of microglial and astroglial cells was observed mainly in the hypothalamus, the thalamus and hippocampus and around the blood vessels, at day 4 p.i. and later, revealing a high central inflammatory reaction in brains of rabies virus infected rats. These results showed that IL-1β and TNFα are produced in the brain both by local microglial cells and infiltrating macrophages during rabies infection. Thus, these cytokines may play an important role in coordinating the dramatic inflammatory response associated with the rabies-encephalopathy as well as in the neural modification and alteration of brain functions.     

Stavudine and the peripheral nerve in HIV-1 infected patients

Stavudine (2′,3′-didehydro-3′deoxythymidine) is a pyrimidine analogue that may be of great value in combination antiretroviral therapy (ART) for treating patients infected with human immunodeficiency virus type 1 (HIV-1). We assessed potential neurotoxic side effects by comparing peripheral nerve function in patients receiving ART including stavudine (n = 107) with that of patients receiving ART with zidovudine (n = 103). A cross-sectional analysis of electroneurographic data revealed no significant differences. In a follow-up examination of 31 patients newly started on ART with stavudine we observed no significant effects of the drug on electrophysiological measures. At a daily dose of 1.0 mg/kg the incidence of peripheral nervous system disease in our patients was about 10%. Repeated follow-up analysis of 13 patients on stavudine showed a significant reduction in sural nerve amplitude. Quantitative sensory testing in 13 patients revealed no systematic effect of stavudine on small nerve fibers. Peripheral nerve function in HIV-1 seropositive patients on ART with stavudine did not differ significantly from that in patients on ART with zidovudine. Therefore stavudine at a daily dose of 1.0 mg/kg is an alternative for patients who do not tolerate, or who have become resistant to zidovudine and can be recommended as a first-line drug in combination ART. Received: 31 March 1998 Received in revised form: 9 July 1998 Accepted: 19 August 1998     

Degeneration of the cerebellar dentate nucleus and the inferior olivary nuclei in HIV-1-infected brains: a morphometric analysis

Motor dysfunction is frequently noted in human immunodeficiency virus type 1 (HIV-1)-infected patients. Until recently, neuropathological changes found in the basal ganglia were advanced as pathogenetic mechanisms. In the present study, further brain structures involved in motor control were analyzed morphometrically. The volume density, numerical density, and the size of neurons in the cerebellar dentate nucleus and in both inferior olivary nuclei were determined. In both regions of HIV-1-infected brains, a significant reduction in the volume density, the numerical density of neurons and neuronal size was apparent. The morphometric data from the present study disclose involvement of both types of nuclei investigated during the course of HIV-1 infection, and might constitute a possible morphological substrate for the motor dysfunction seen in HIV-1-infected patients. Received: 22 February 1995 / Revised: 15 December 1995 / Accepted: 26 February 1996     

Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell cocultures. In the present study, we tested the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when cocultured with lipopolysaccharide-stimulated human fetal brain cells. Marked upregulation of HIV-1 expression was observed in these cocultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants), and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-α by microglial cells.     

Programmed cell death in brains of HIV-1-positive AIDS and pre-AIDS patients

Neuropathological studies have revealed that the brains of HIV-1-infected AIDS patients show the typical encephalitis and, in addition, neuronal loss. More recently, this neuronal cell loss has been thought to take place via programmed cell death (apoptosis) which has been demonstrated by an in situ end labelling (ISEL) technique. In this study 54 brains of HIV-1-positive patients were investigated by the ISEL technique to investigate whether apoptosis is also present in the brains of patients at the asymptomatic stage. Of these, 10 patients suffered from HIV encephalitis (HIVE), 8 had AIDS without neuropathological disorders and 36 were HIV-1-positive pre-AIDS patients. Apoptotic cells were detected in 6 of the 10 HIVE, 1 of the 8 AIDS without central nervous system (CNS) disease and 4 of the 36 asymptomatic individuals. A difference seen between the AIDS and pre-AIDS cases was that, in the latter, apoptotic cells were found in the white matter in all 4 cases, while only 2 of these 4 showed apoptotic neurons. The presence of apoptotic cells in a number, albeit small, of brains of HIV-1-positive pre-AIDS individuals, combined with abnormalities described previously in the same group of patients gives further support to the opinion that brain damage already occurs during the early stages of HIV infection. Received: 19 June 1995 / Revised: 31 July 1995 / Accepted: 6 September 1995     

Seroreactivity to human T cell leukemia/lymphoma virus type 1 and related retroviruses in multiple sclerosis patients from Denmark and the Faroes

A total of 40 multiple sclerosis (MS) patients from Denmark and 10 from the Faroes were examined for antibodies with affinity to human T cell leukemia/lymphoma virus type 1 (HTLV-I) and human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2). Using ELISA, MS patients and a group of healthy controls did not differ significantly in their reactivities to HTLV-I. However, elevated reactivities were recorded with 5 MS sera, whereas only 2 of the sera from the controls produced highly values. Ten patients with other neurological diseases all seemed to exhibit low reactivity in HTLV-I ELISA. The reactivities of 2 MS sera decreased considerably by absorption with an HTLV-I lysate. In immunofluorescence assay, two other MS sera reacted with HTLV-I transformed cell lines as well as with non-infected cells. Examined by Western blotting (WB), a single MS serum produced a distinct HTLV-I p19 band. With ELISA for detection of HIV-1 and HIV-2 antibodies, 2 MS sera exhibited borderline reactions. Further examination of these two sera by WB revealed weak reactivities against p24 and p53 of HIV-1. One the whole, the present observations do not suggest that a putative MS retrovirus would be closely related with HTLV-I, HIV-1 or HIV-2.     

Expression of active caspase-3 in mitotic and postmitotic cells of the rat forebrain

Active caspase-3 immunoreactivity was detected in the rat forebrain proliferative regions at birth and remained high in these areas for about 2 weeks, during which period labeled cells were present centroperipherally across the olfactory bulb. By the end of the third postnatal week, only a small number of immunolabeled cells remained in these forebrain structures. Active caspase-3 immunolabeling was localized mostly to cell nuclei and co-localized partially with TuJ1 and NeuN immunoreactivity, but not with glial fibrially acidic protein, OX-42, gamma-aminobutyric acid, or terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-positive labeling. Active caspase-3 and 5-bromo-2'-deoxyuridine (BrdU) double-labeled nuclei were seen in the proliferative regions after 2 hours and in the periglomerular region of the bulb after 7 days following BrdU injections. Examination of the cells with electron microscopy confirmed that the active caspase-3-containing nuclei in the proliferative regions often had infoldings and appeared to be undergoing division. Some of the cells with active caspase-3-labeled nuclei in the bulb had synapses on their somata or dendrites. Labeled dendritic spines and a few axon terminals were also observed in the olfactory bulb. Taken together, it appears that a wave of active caspase-3-positive cells are dividing in the proliferative zones and then migrating to the bulb as they differentiate into neurons. Therefore, active caspase-3 may play a role in cellular processes such as neuronal differentiation, migration, and plasticity, in addition to its role in cell death.     

Simultaneous detection of ferritin and HIV-1 in reactive microglia

Summary Using ferritin as a marker of reactive microglia, we demonstrated a close association between proliferation of reactive microglia and expression of human immunodeficiency virus type 1 (HIV-1) in brain tissue from autopsied cases of acquired immunodeficiency syndrome (AIDS). An increased number of ferritin-positive reactive microglia was observed in formalin-fixed paraffin-embedded brain sections from all 13 AIDS cases examined. Similar findings were observed in brain tissue from other neurological diseases (subacute sclerosing penencephalitis, herpes simplex encephalitis and multiple sclerosis). Multinucleated giant cells were found in 7 of the AIDS cases which were also intensely labeled for ferritin. Dual-label immunohistochemistry using anti-ferritin and cell-specific markers showed that ferritin-positive cells were distinct from astrocytes, neurons and endothelia using anti-glial fibrillary acidic protein (anti-GFAP), anti-neurofilament protein and Ulex europaeus agglutinin 1, respectively. In 5 AIDS brains, only ferritin-positive cells were shown to contain HIV-1 gp41 antigen using dual-label immunohistochemistry. In addition, HIV-1 RNA was localized in territin-positive reactive microglia but not in GFAP-positive astrocytes using immunohistochemistry combined with in situ hybridization. Ferritin-positive reactive microglia and multinucleated giant cells were colabeled with the microglial marker, Ricinus communis agglutinin 1 (RCA-1). Howerver, RCA-1 also extensively stained resting microglia only a few of which were colabeled for ferritin. The density of ferritin-positive cells was correlated with the presence of HIV-1 RNA-positive cells in AIDS brain. Thus, ferritin immunoreactivity can be used as an activation marker of microglia in archival paraffin sections and reflects the extent of inflammation in HIV-1-infected brain.Supported in part by NIH Grants RO1 DA04787, RO1 HD26621, PO1 NS25569, the Biopsychosocial Center for the Study of AIDS (NIMH P50 MH 43455), the Department of Veterans Affairs, the Mary Jane Crowe Foundation, the Swedish Society of Medicine (Stockholm, Sweden), and the Multiple Sclerosis Society of Göteborg (Göteborg, Sweden)     

Age-related behavioural deficits in transgenic mice expressing the HIV-1 coat protein gp120

Transgenic mice expressing HIV-1 coat glycoprotein gp120 in brain glial cells were previously shown to display AIDS dementia-like neuropathological changes and reduced hippocampal long-term potentiation. In this report, neuromotor and cognitive performance in 3- and 12-month-old gp120-expressing mice was compared with wildtype controls. Rotarod and cage activity measures showed no significant differences between transgenic animals and controls of either age. Open field activity was slightly altered in 12-month-old gp120 animals (reduced corner crossings and dwell in centre), but not in the 3-month-olds. Cognitive assessment using the Morris water maze showed unimpaired performance in 3-month-old mice during acquisition and (no-platform) probe trials. In 12-month-old gp120 animals, escape latency and swimming velocity during the acquisition trials were significantly reduced, but performance improved at roughly the same rate as in control animals. However, the probe trials revealed a highly significant reduction in spatial retention in transgenic mice of this age. This demonstration of age-dependent impairments in open field activity and spatial reference memory may relate to cognitive and neuromotor deficits seen in a proportion of HIV-1-infected individuals.     

Expression of allograft inflammatory factor-1 and haeme oxygenase-1 in brains of rats infected with the neurotropic Borna disease virus

Experimental infection of Lewis rats with Borna disease virus (BDV) causes an immune-mediated nonpurulent meningoencephalitis. Viral persistence in the central nervous system is accompanied by mononuclear infiltrates, activated monocytic/microglial cells and reactive astrocytes. The immune-mediated process was further characterized by expression analysis of allograft inflammatory factor-1 (AIF-1), a novel marker of monocyte/microglial activation and of glial fibrillary acid protein (GFAP) between day 3 and day 50 post infection (p.i.). Potential neuroprotective effects of these cells were studied by the induction of haeme oxygenase-1 (HO-1), a defensive molecule against oxidative stress in various brain insults. In BDV-infected rat brains, mononuclear infiltrates and AIF-1 expression increased up to day 28 p.i. During early time points p.i., AIF-1 expression was mainly found in inflammatory lesions and adjacent brain parenchyma. Already 24 days p.i., a widespread upregulation of AIF-1 was observed which declined only moderately beyond day 28 p.i. HO-1 induction was maximal between days 18 and 28 p.i. Increased amounts of GFAP-positive astrocytes were present beyond 24 days p.i. Viral antigen expression increased simultaneously to the inflammatory reaction and persisted up to 50 days p.i. Widespread upregulation of AIF-1 indicates an early, long-lasting microglial activation, which might be involved in the immunesurveillance of the immune-mediated inflammatory events. The early peak of HO-1 most likely represents a neuroprotective, anti-inflammatory response by invading monocytes, microglial cells and astrocytes during the formation of encephalitic lesions and acute viral replication.     

Characteristics of HIV-1 gp120 glycoprotein binding to glycolipids

We examined the binding of the gp120 envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV-1) to sulfatide (GalS), galactocerebroside (GalC), and GMI-ganglioside (GMI). The gp120 glycoprotein bound to GalS but not to GalC or GMI by enzyme-linked immunosorbent assay (ELISA) and by an immunospot assay on nitrocellulose paper. However, it bound to all three glycolipids by an immunospot assay on thin layer chromotography (TLC) plates. In studies to determine whether GalS could be a receptor for gp120 on the surface of cells, gp120 bound to GalS incorporated into the plasma membrane of lymphoid cells as determined by cytofluorometric analysis and immunofluorescence microscopy. These studies indicate that GalS may function as a receptor for gp120 and HIV-1. © 1994 Wiley-Liss, Inc.