Choroid plexus selectively accumulates T-lymphocytes in normal controls and after peripheral immune activation.

We determined T-lymphocyte migration into brain and choroid plexus (CPx) after enterotoxin-induced systemic immune activation. CPx T-lymphocytes/mm2 in control mice were > 3 logs more numerous than brain and increased by as much as 150-fold by post-enterotoxin Day 3 (p < 0.01). Flow cytometry of pooled CPx confirmed post-enterotoxin increases. Brain T-lymphocytes increased up to 17-fold after SEB and accumulated in subependymal and periventricular brain. T cell apoptosis was absent. These results show preferential T-lymphocyte migration to CPx over brain and suggest that brain T cells may be derived from the CPx by direct migration or by cerebrospinal fluid dissemination.

Analytic approaches to differential gene expression in AIDS versus control brains.

We previously showed that specific strains of human immunodeficiency virus (HIV)-1 infect the brain and contribute to Neuropathology, Cognitive Distress, and Neuropsychiatric Disease. To study further brain disease that results from HIV-1 infection, we commenced analysis of changes in gene expression in brain. We analyzed RNA purified from Frontal Cortex of 5 HIV-1 infected and 4 HIV-1 negative control subjects RNA was amplified and Affymetrix technology was used to analyze gene expression using the 12,585 gene Affymetrix Human Genome U95A chip. The expressed genes showed highly significant Pearsons correlations with each other within the two groups. Expression intensities were transferred to Microsoft Excel and Spotfire was used to analyze the results. Twenty-group K-means cluster analysis was done for HIV+ and HIV- subjects. Genes that were expressed in the same cluster numbers in the two groups were removed from further analysis. Analysis of Gene expression in the top 13 HIV+ clusters showed expression in the 40 gene categories designated in our prior studies. Genes from several categories occurred in more than one K-means cluster. Genes identified in these lists included several genes that have been previously studied: MBP, Myelin-PLP, NMDA receptor, MAG, astrocytic protein, Notch 3, APP, Senescence, proteasome, Ferritin, signaling, cell cycle, iNOS, Chemokine, splicing, synapse, protein tags, and ribosomal proteins. The first (primary significant) axis of both Principal Component Analyses ordered the genes in the same patient groups as the K-means cluster analysis for the respective patient groups. PCA was thus not more informative than K-Means cluster analysis. Ratios of HIV+ to HIV- intensities were calculated for all the averaged gene expression intensities. The ratio range was 0.14 to 9.26. The genes at the extremes (ad extrema) did not correspond to the gene order by K-means clustering (or PCA). The genes in the top 13 K-means clusters showed low-level changes by expression ratio. Genes ad extrema by ratio were in clusters with very large memberships. Mann-Whitney analysis confirmed expression ratio results. Several inferences result from our preliminary study. First, study design will be different in future studies involving additional replicates. Second, ratios inform us of the extent of changes in gene expression quantitatively. Third, Cluster methodology provides us with more subtle information, how bunches (clusters) of genes behave in terms of their centroids (attractors). Fourth, genes that change extensively by ratio tend to be in the larger k-Means clusters. We conclude that ranking gene expression with the use of expression ratio or by K-means clustering, yield different representations of the data.

CD4+ and CD8+ cells accumulate in the brains of acquired immunodeficiency syndrome patients with human immunodeficiency virus encephalitis.

To test the hypothesis that CD4+ T lymphocytes accumulate in brains of end-stage acquired immunodeficiency syndrome (AIDS) patients, we examined T-lymphocyte subsets in the CA1, CA3, and CA4 regions of the hippocampus of AIDS patients with (n = 10) and without (n = 11) human immunodeficiency virus encephalitis (HIVE) plus controls (n = 7). HIV p24 antigen was common in monocytic cells and rare in activated/memory CD45RO+ lymphocytes. Hippocampal activated/memory CD45RO+ T lymphocytes significantly increased (P <.001) in seven of the eight hippocampal subregions with hippocampal HIVE (1.14 +/- 1.4 T cells/high-power field [hpf]), but AIDS hippocampus without HIVE were similar to controls (0.03 +/- 0.07 T cells/hpf and 0.03 +/- 0.09 T cells/hpf, respectively). CD45RO+ and CD3+ lymphocytes were similar in numbers and distribution, whereas CD4+ and CD8+ lymphocytes were weakly immunoreactive and less frequent. All four lymphocyte subtypes were present in perivascular spaces and microglial nodules of HIVE, and had direct contact with neurons. Monocytes, microglia, and multinucleated giant cells were immunoreactive for CD4 in AIDS cases with hippocampal HIVE but microglia in remaining AIDS cases and controls were CD4-. CD68+ macrophages significantly increased in hippocampus of HIVE patients (P <.05) and were predominately perivascular in the absence of local HIVE. These studies show that CD4+ T lymphocytes, as well as CD8+ T lymphocytes, participate in the local inflammatory response of HIVE in end-stage AIDS patients, and suggest that their recruitment requires local HIV infection. The perineuronal location of CD4+ cells provides the potential for lymphocyte-mediated neuronal injury or trans-receptor-mediated neuronal infection.

Detection of HIV-1 gene sequences in hippocampal neurons isolated from postmortem AIDS brains by laser capture microdissection.

We employed laser capture microdissection to remove individual pyramidal neurons from the CA1, CA3, and CA4 regions of formalin-fixed, paraffin-embedded hippocampus from 8 AIDS brains and 2 HIV-1-seronegative normal brains. We amplified HIV-1 gag and nef gene sequences using separate, double round PCR reactions for each of the primer sets. In all 3 hippocampal regions, amplification efficiency was best with sequence length between 284 and 324 bp; HIV-1 nef gene sequences were more common than HIV-1 gag sequences; and rank order for percent positive amplification was CA3 > CA4 > CA1 samples. These results are the first to detect HIV-1 gene sequences in microdissected human tissue. They indicate that brain neurons in vivo contain HIV-1 DNA sequences consistent with latent infection by this virus, and suggest that neurons display a selective vulnerability for HIV infection. Neuronal HIV infection could contribute to neuronal injury and death or act as a potential viral reservoir if reactivated.

Hippocampal injury and alterations in neuronal chemokine co-receptor expression in patients with AIDS.

Hippocampal neurons express high levels of HIV chemokine co-receptors, activation of which causes injury or death in vitro. To determine if their in vivo expression correlates with injury, we evaluated neuronal CXCR4 and CCR5 immunoreactivity and reactive gliosis in autopsy hippocampus of 10 control cases, 11 AIDS cases without HIV encephalitis (HIVnE) or opportunistic infections/lymphomas (OI/L), and 11 AIDS cases with HIV encephalitis (HIVE). All groups had higher CXCR4 and CCR5 expression in CA3 and CA4 neurons than CA1 neurons (p < 0.05). HIVE cases had increased neuronal CXCR4 and decreased neuronal CCR5 expression as well as increased numbers of hippocampal GFAP-positive astrocytes and LN3-positive microglia. Changes were most severe in CA3 and CA4 and lowest in CA1 regions. These findings also were noted in the 4 HIVE cases with neither hippocampal HIVE nor brain OI/L and in the HIVnE groups. This study quantitates the regional distribution of hippocampal neuronal CXCR4 and CCR5 and shows their respective increase and decrease in AIDS. It suggests a relationship between neuronal loss and gliosis with intensity of neuronal chemokine expression and raises the possibility of a selective vulnerability of hippocampal neurons to AIDS-related injury.

Upregulation of glial clusterin in brains of patients with AIDs.

Since clusterin (CLU) production in reactive astrocytes may be neuroprotective, we examined its distribution in AIDS brains where brain injury and reactive astrocytosis are common. The relative area and number of CLU-positive astrocytes, as well as their percent total of all white matter glia, significantly increased in AIDS brains with and without HIV encephalitis (P<0.05). Proliferation markers were absent. In contrast, the relative area and number of GFAP-positive astrocytes and their percent of all white matter glia, increased in some cases but the mean increases were not significant. Clusterin is sensitive marker of glial reactivity in AIDS brains and its enhanced expression was not dependent on increases in GFAP.

Overexpression of clusterin in human breast carcinoma.

Clusterin has been implicated in numerous processes including active cell death, immune regulation, cell adhesion and morphological transformation. The purpose of this study was to examine clusterin expression in a large series of breast carcinomas by immunohistochemistry and in situ hybridization. The study included 40 samples of non-neoplastic glandular epithelia, 42 benign lesions, 15 atypical intraductal hyperplasias, 35 carcinomas in situ, 114 invasive carcinomas, and lymph node metastases from 40 patients. Epithelial normal cells were always negative for clusterin expression and only 19% of the benign lesions presented positive staining. In contrast to the benign lesions, however, the frequency of clusterin positive samples increased in atypical hyperplasias (47%, P = 0.08), intraductal carcinomas (49%, P = 0.01) and invasive carcinomas (53%, P < 0.001). Positive staining presented a cytoplasmic pattern, except in 3 cases of invasive carcinomas which had nuclear staining. Clusterin mRNA by in situ hybridization confirmed the specific cellular pattern of clusterin expression by immunohistochemistry. Clusterin expression was associated with large tumor size (P = 0.04), estrogen and progesterone receptor negative status (P = 0.02 and P = 0.001, respectively) and with the progression from primary carcinoma to metastatic carcinoma in lymph nodes (80% metastatic nodes had positive expression) (P = 0.004). Ten of 15 (67%) primary carcinomas without clusterin expression became positive in lymph node metastases, while most (22 of 25, 88%) of the clusterin-positive primary carcinomas were also immunoreactive in metastases. In survival analysis, clusterin expression did not represent a prognostic indicator by uni- or multivariate analysis. The increased clusterin expression in breast carcinomas tended to correlate inversely with the apoptotic index (P = 0.09) which indicates that clusterin gene expression is not a prerequisite to cellular death by apoptosis. From these results, we suggest that clusterin may have a role in tumorigenesis and progression of human breast carcinomas.

Comparisons of HIV-1 viral sequences in brain, choroid plexus and spleen: potential role of choroid plexus in the pathogenesis of HIV encephalitis.

Possible mechanisms of HIV transmission to the brain include direct viral infection of cerebral endothelium and hematogeneous dissemination of viral-infected lymphocytes and monocytes. Cerebrospinal fluid dissemination from a primary infection of choroid plexus (CPx) is an alternative mechanism supported by recent studies in our laboratory. We showed that HIV-infected asymptomatic patients as well as AIDS patients have HIV infection of the CPx; the cell types so infected included stromal monocytes and dendritic cells. To further explore the potential role of CPx in the pathogenesis of HIV encephalitis, we analyzed HIV sequences from brain, CPx, and spleen of four AIDS patients by extracting DNA from paraffin sections and amplifying the V3 region of the HIV env gene by PCR. Several different clones from each tissue were characterized. We found that viruses from the brain and spleen grouped into two distinct clusters, while viruses of the CPx contained viral strains that were a mixture of those found in the brain or spleen. Net charge analysis of the V3 tip region showed that the brain viral sequences had fewer positive charges than blood viral sequences. Our results support the hypothesis that CPx may be one of the sites where HIV-1 gains access to the brain from the blood and therefore contains viruses that are of both genotypes.

HIV infection of choroid plexus in AIDS and asymptomatic HIV-infected patients suggests that the choroid plexus may be a reservoir of productive infection.

The choroid plexus (CPx) may be an important site of viral dissemination since monocytes and dendritic cells in its stroma are infected with HIV in AIDS patients and since the ratio of CPx to brain infection is more than 2 : 1. In order to see if CPx infection also develops in asymptomatic (ASY) HIV-infected patients, we examined archival formalin-fixed brain and CPx from 14 AIDS and seven ASY cases, using routine histology, immunohistochemistry for HIV gp41, and DNA extraction and gene amplification for HIV DNA. Eight of 14 AIDS (57%) had HIV-positive cells in the CPx and four (29%) had HIV encephalitis. Two of seven ASY cases (29%) had HIV-positive cells in the CPx but none had HIV encephalitis. Extracted DNA from brain, CPx and systemic organs of five ASY cases was amplified by nested PCR with or without Southern blotting for HIV env gene. It was positive in systemic organs in five cases; in CPx in four cases; and in brain in one case. This study shows that the CPx is a site of HIV infection in ASY patients and that the frequency of CPx infection is higher than seen in brain in both AIDS and ASY cases. The results are consistent with the hypothesis that the CPx may be a site for hematogeneous spread and a reservoir for HIV infection during the period of clinical latency.

Chronic systemic administration of tumor necrosis factor alpha and HIV gp120: effects on adult rodent brain and blood-brain barrier.

Since tumor necrosis factor alpha (TNF-alpha) and HIV gpl20 glycoprotein are both neurotoxic, the possibility that systemic sources of these two agents mediate AIDS-associated blood-brain barrier (BBB) breakdown and brain damage was tested in two murine models: (1) intramuscular implantation of a TNF-alpha-transfected tumor in nu/nu mice and (2) daily subcutaneous injections of HIV gpl20 in BALB/c mice. The BBB remained intact; brain damage was not found, and apoptotic cell numbers did not increase. These results show that normal adult brain and BBB is unaffected by exposure to TNF-alpha or HIV gpl20 and suggest that severity of brain disease is not directly affected by systemic levels of these compounds.

Independent evolution of HIV type 1 in different brain regions.

HIV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V1-V5 domains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, medial temporal lobe, and nonmedial temporal lobe) from three patients. We found that in each separate brain region HIV-1 forms different quasispecies and that there is little gene flow among these regions. In further support of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known amino acid signatures associated with macrophage tropism and the lack of syncytium formation, we found 15 majority amino acid signature patterns from the V1-V5 env sequences associated with the neuroanatomical regions analyzed from the three individuals. Furthermore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be much smaller than genetic distances between brain regions. Specific strains of HIV-1 may be neurotropic or neuroinvasive (replication preference in brain tissue) and may contribute to pathology, cognitive loss, and neuropsychiatric disease.

Cerebral aneurysmal arteriopathy in childhood AIDS.

OBJECTIVE: To identify cerebral aneurysmal arteriopathy in children with longstanding AIDS. BACKGROUND: Five cases are described from the authors' experience, and eight additional cases are reviewed from the literature. Details are presented in regard to the clinical picture as well as brain imaging with cerebral angiography and magnetic resonance angiography in some cases. Autopsy information is available in four cases, including one of the authors' patients. RESULTS: Seven patients acquired HIV infection perinatally, five patients were infected by blood transfusions, and one patient had both risk factors. In the four postmortem patients, the vascular pathology was similar, showing ectasia and aneurysmal dilatation confined to the large arteries of the circle of Willis. Medial fibrosis and loss of muscularis with destruction of the internal elastic lamina and intimal hyperplasia was common. The latency period following infection varied from 2 to 11 years. Once a diagnosis of aneurysmal arteriopathy was made, the patients deteriorated rapidly, with death occurring in less than 6 months. CONCLUSIONS: The development of cerebral aneurysmal arteriopathy in childhood AIDS tends to occur after a prolonged delay and is usually followed by death in a short period of time. The etiology for the vasculitis is unknown. Varicella zoster virus may be the etiology in some of the cases because of its potential to cause this pathology and the striking unilateral arterial involvement found in Case 5. HIV vasculitis is also a possibility, as suggested by the detection of HIV protein or genomic material in two of the four autopsy cases.

Selective glial vulnerability following transient global ischemia in rat brain.

Global cerebral ischemia selectively damages neurons, but its contribution to glial cell death is uncertain. Accordingly, adult male rats were sacrificed by perfusion fixation at 1, 2, 3, 5, and 14 days following 10 minutes of global ischemia. This insult produces CA1 hippocampal neuronal death at post-ischemic (PI) day 3, but minor or no damage to neurons in other regions. In situ end labeling (ISEL) and immunohistochemistry identified fragmented DNA of dead or dying glia and distinguished glial subtypes. Rare ISEL-positive oligodendroglia, astrocytes, and microglia were present in control brain. Apoptotic bodies and ISEL-positive glia significantly increased at PI day 1 in cortex and thalamus (p < 0.05), but were similar to controls in other regions and at other PI intervals. Most were oligodendroglia, although ISEL-positive microglia and astrocytes were also observed. These results show that oligodendroglia die rapidly after brief global ischemia and are more sensitive than neurons in certain brain regions. Their selective vulnerability to ischemia may be responsible for the delayed white matter damage following anoxia or CO poisoning or that associated with white matter arteriopathies. Glial apoptosis could contribute to the DNA ladders of apoptotic oligonucleosomes that have been found in post-ischemic brain.

Exophytic malignant brainstem mixed glioma in an adult: a case report.

Anaplastic mixed gliomas are rare tumors that occur mostly in the cerebral hemispheres. They have a distinctive histological appearance characterized by the presence of two or more glial cellular constituents. The incidence of malignant mixed glioma of the brainstem and posterior fossa is extremely low. The authors report an unusual case of an exophytic malignant mixed glioma. Following subtotal resection, the patient received conventional radiotherapy, but continued to deteriorate, and died five months after surgery. The extensive literature review focuses on histopathology, clinical features, natural history, and possible treatment modalities of this unusual neoplasm.

HLA-DR-positive dendritic cells of the normal human choroid plexus: a potential reservoir of HIV in the central nervous system.

In a previous study of choroid plexus (CPx) from patients with the acquired immunodeficiency syndrome (AIDS), we found a population of stromal cells infected with the human immunodeficiency virus (HIV). To determine whether these represented antigen-presenting dendritic cells, we examined the phenotype of normal human choroid plexus by light and electron microscopy (EM) and established the HIV-infected cell type by immunohistochemistry in AIDS cases with HIV-infected CPx. Monoclonal antibodies were used to detect class II major histocompatibility antigens (MHC), S-100 and S-100beta protein, lymphocytes, monocytes/macrophages, and HIV glycoprotein. A variable number of stromal cells had slightly elongated nuclei and long branching processes that were strongly immunoreactive for class II MHCs, rarely reactive for S-100 and S-100beta and immunonegative for monocyte/macrophage markers. Phagocytic activity was absent by EM and immunomarkers. They were numerous in the subepithelial region, and their processes occasionally extended toward the stromal capillaries or between the CPx epithelial cells. The HIV-infected cells were intensely immunoreactive for class II MHC markers and often displayed a dendritic morphology. These results document the presence of dendritic cells in the normal human CPx whose morphology and immunophenotype closely resemble those of DCs elsewhere in the body. They also show that these immunoreactive MHC class II cells are the cell type infected by HIV. We suggest that the functional activity of the CPx DCs is similar to that of antigen-presenting dendritic cells elsewhere in the body. This includes the potential to harbor HIV during the prolonged period of clinical latency, acting as a central nervous system reservoir of infection before the onset of AIDS.

HIV-1 proviral DNA load across neuroanatomic regions of individuals with evidence for HIV-1-associated dementia.

A definitive relation between HIV-1 load and the clinical diagnosis of HIV-1-associated dementia (HAD) has not yet been established. Knowledge of the neuroanatomic distribution of HIV-1 load in the brain of individuals with HAD and HIV-1 encephalitis may facilitate elucidation of this relation. Nine individuals with AIDS were analyzed postmortem by three independent methods with each assessment performed blinded to the others: 1) a neuropsychiatric review of clinical records for evidence of possible HAD, 2) HIV-1 DNA load determination by quantitative polymerase chain reaction (PCR) across several neuroanatomic regions, and 3) a pathologic examination for diagnosis of HIV-1 encephalitis by immunohistochemical techniques. Of eight AIDS cases with clinical records sufficient for neuropsychiatric review, seven were shown to have evidence for HAD. HIV-1 DNA was detected and quantified in specimens from all of the medial temporal lobe regions analyzed but was not detectable in the frontal lobe at the same level of sensitivity in two of these cases (<1 per 1000 cellular genomes). HIV-1 DNA load in the medial temporal lobe region was significantly larger than that in the frontal lobe. Only four of seven cases with evidence for HAD were also diagnosed with HIV-1 encephalitis.

DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat.

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Parameters evaluated in the CA1 region of the hippocampus included morphology, in situ end labeling (ISEL) of fragmented DNA, and expression of p53. Neurons were indistinguishable from controls at postischemic day 1 but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appeared normal. In situ end labeling was absent. At days 3 and 5, approximately 40 to 60% of CA1 neurons had shrunken eosinophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL. By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL. Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis. These results show that DNA fragmentation occurs after the development of delayed neuronal death in CA1 neurons subjected to 10 minutes of global ischemia. They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CA1 neurons in this model.