HIV mediates a productive infection of the brain.

BACKGROUND: Approximately one quarter of patients with AIDS develop severe cognitive deficits called HIV-associated dementia complex. There is some controversy regarding the importance of viral load and distribution in mediating this neurologic disease. OBJECTIVE: Brain HIV proviral and RNA loads were compared to define the molecular nature of HIV infection of the brain. METHOD: Neuropathologic examination was performed on brains from 10 autopsies of patients with AIDS that had short post-mortem intervals and no evidence of opportunistic infection. Viral DNA and RNA were extracted and quantified from multiple brain regions. These findings were compared with triple-label immunofluorescence for viral and cell markers. RESULTS: Brains with histopathologic evidence of HIV encephalitis contained abundant HIV RNA and DNA. Regions without productive HIV infection showed minimal proviral load. By immunocytochemistry, only brain macrophages/microglia double labeled for viral proteins. CONCLUSIONS: HIV mediates a productive infection of brain macrophages/microglia. There was no evidence supporting the hypothesis of substantial neuronal or macroglial infection, or evidence of substantial proviral burden prior to the development of productive infection.

HIV-associated Waldeyer's ring lymphoid hyperplasias: characterization of multinucleated giant cells and the role of Epstein-Barr virus.

Lymphoid hyperplasia of Waldeyer's ring (WR) is an often-symptomatic complication of human immunodeficiency virus (HIV) infection. A characteristic but not well explained finding is the presence of multinucleated giant cells (MNGCs) adjacent to crypt or surface epithelium. To further elucidate the MNGCs and assess their relationship to HIV and Epstein-Barr virus (EBV), 12 specimens from 11 HIV-positive patients were stained with antibodies to HIV-1 p24, EBV (latent membrane protein, LMP-1), histiocytes (CD68), and other antigen-presenting cells: S-100 protein, the Langerhans cell (LC) marker CD1a, and the follicular dendritic cell (FDC) marker (CD21). Double immunofluorescent staining to assess co-expression of p24 and cell-specific markers was performed and analyzed by laser-scanning confocal microscopy with 3-dimensional reconstruction. In situ hybridization for EBV-encoded small RNA (EBER) was performed in all cases. Immunostains showed MNGCs labeled for p24, S-100, and CD68, but not CD1a. In 1 case, rare MNGCs were CD21-positive. EBV LMP-1 was uniformly negative, although EBER-positive lymphocytes were seen by in situ hybridization in 9 of 12 specimens (numerous in only 3 specimens). Double immunofluorescent staining showed co-localization of p24 with CD68 and S-100. Our results suggest that MNGCs are generally HIV-infected, EBV-negative, and most likely represent an unusual S-100-positive histiocyte subset (not LC or FDC). Their exact pathophysiologic role remains uncertain. EBV does not appear to play a major role in the pathogenesis of WR lymphoid hyperplasias in HIV infection.

Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy.

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.

Absence of brain-derived neurotrophic factor and trkB receptor immunoreactivity in glia of Alzheimer's disease.

Alterations in the neuronal expression of some neurotrophins have been shown in various neurodegenerative processes, particularly Alzheimer's disease (AD). Glia may up-regulate neurotrophins and their high-affinity tyrosine kinase (trk) receptors in response to neural injury. In human immunodeficiency virus type 1 (HIV-1) encephalitis, activated microglia were shown to express brain-derived neurotrophic factor (BDNF), while reactive astrocytes expressed trkB receptor. This observation has suggested the existence of local neurotrophic regulation between different glial populations. To characterize the glial cellular distribution of BDNF and trkB receptor proteins in AD, we studied selected regions of postmortem brains from four AD and three age-matched control patients by double-immunofluorescence confocal microscopy. In both groups, BDNF immunoreactivity was distributed in neuronal perikarya and neuritic processes in the neocortex and hippocampus. No BDNF immunoreactivity was observed in microglia or astrocytes within and between senile plaques of AD. Catalytic trkB receptor immunoreactivity was present in neuronal perikarya in the neocortex and hippocampus. Reactive astrocytes and microglia were not immunoreactive for catalytic trkB. The absence of BDNF and trkB proteins in glia in AD patients is in contrast to the finding in patients with HIV-1 encephalitis. This difference suggests that glial expression of BDNF and trkB proteins may be characteristic of particular disease processes, rather than merely representing a stereotyped response to any type of neural injury.

The neuropathology of a chromosome 17-linked autosomal dominant parkinsonism and dementia ("pallido-ponto-nigral degeneration").

A group of similar autosomal dominant hereditary neurodegenerative disorders have been linked to chromosome 17 in thirteen kindreds. One of these disorders, known as pallido-ponto-nigral degeneration (PPND), is characterized by extensive degeneration of the globus pallidus and substantia nigra as well as accumulation of abnormally phosphorylated tau proteins. The authors now present comprehensive data on the cellular and molecular pathology of PPND, allowing its classification among chromosome 17-linked neurodegenerative disorders as well as its classification among sporadic and other familial tauopathies. First, we showed that PPND is characterized by abundant ballooned neurons in neocortical and subcortical regions as well as by tau-rich inclusions in the cytoplasm of neurons and oligodendroglia morphologically similar to those seen in corticobasal degeneration (CBD), but in a distribution pattern resembling progressive supranuclear palsy (PSP). Second, we demonstrated that antibodies to phosphorylation-independent (Alz50, 133, 304, Tau-2, T-46) as well as phosphorylation-dependent (AT8, PHF-6, 12E8, PHF-1, T3P, pS422) epitopes in human tau proteins stain these glial and neuronal inclusions as intensely as they stain CBD or PSP inclusions. Third, we probed PPND brain by Western blots using some of the same anti-tau antibodies to reveal 2 tau immunobands with molecular weights of 69 kD and 64 kD in gray and white matter extracts, as reported for both PSP and CBD. Finally, electron microscopy showed that these abnormal tau proteins formed flat twisted ribbons with a maximum diameter of 20 nanometers (nm) and a periodicity of about 200 nm, resembling those reported in CBD. Based on this, we conclude that PPND is a hereditary neurodegenerative disorder characterized by neuronal and glial tau-rich inclusions formed from aggregated filaments and hyperphosphorylated tau proteins and, hence, can be subcategorized into the tauopathy group of chromosome 17-linked neurodegenerative disorders. Further, since the morphologic and biochemical lesions of PPND overlap with those seen in sporadic CBD and PSP, we speculate that these disorders share common pathogenetic mechanisms.

Confocal microscopy assessment of lymphoid tissues with follicular hyperplasia from patients infected with human immunodeficiency virus type 1.

OBJECTIVE: To characterize human immunodeficiency virus (HIV) infection of lymphoid tissues during follicular hyperplasia. METHODS: We examined 10 tonsil/adenoid, 3 parotid lymphoepithelial cyst, and 7 lymph node specimens that had been surgically removed from 13 patients infected with HIV-1. Characteristics of productive HIV-1 infection were assessed using immunocytochemistry for HIV-1 p24. Cellular colocalization was determined with the aid of a confocal microscope using double immunofluorescent staining for HIV-1 p24 and cell-specific markers. RESULTS: All specimens showed follicular hyperplasia. Using confocal microscopy with three-dimensional reconstruction, HIV-1 p24 was seen to be "intimately" colocalized with CD21 within the germinal centers. While lymphoid follicles were generally hyperplastic, only a subset of these follicles contained HIV-1 p24. Occasional HIV-1-expressing mononuclear cells identified outside follicles stained for CD68 or CD3. CONCLUSIONS: The differential involvement of hyperplastic follicles by HIV-1 within individual lymphoid tissues and the intimate colocalization of HIV-1 p24 and CD21 suggest that infected follicular dendritic cells may be an important reservoir of HIV-1 during follicular hyperplasia.

Brain HIV burden and length of survival after AIDS diagnosis.

Patients with AIDS in the late stages of disease can develop dementia. Previous studies have suggested HIV encephalitis is the pathological substrate of HIV-associated dementia. We hypothesized that patients who survive longer after the initial diagnosis of AIDS would have a higher brain HIV burden and consequently manifest dementia. We examined the relationship between length of survival after AIDS diagnosis and the presence of HIV encephalitis or HIV-associated dementia. We studied retrospectively the following parameters in 74 consecutive AIDS autopsies: length of survival after AIDS diagnosis, clinical diagnosis of dementia, and neuropathologic findings (including HIV burden assessment). Multinucleated giant cells (MNGC) were identified in 20% of the brains studied. HIV gp41 was detected by immunocytochemistry in 54%, approximately half of which had abundant HIV burden. Brains from all 4 patients who were clinically diagnosed with dementia and had no opportunistic neuropathologic changes contained MNGC and abundant HIV burden. Survival after AIDS diagnosis was significantly longer in patients with MNGC (p = 0.03) or abundant HIV burden (p = 0.02). A trend toward longer survival after AIDS diagnosis was apparent in patients with dementia, but did not reach statistical significance. These findings suggest that prolonged survival with immunosuppression may be a prerequisite for the development of HIV encephalitis.

Distribution of brain HIV load in AIDS.

Approximately one quarter of patients with AIDS develop severe cognitive deficits called HIV-associated dementia complex (ADC). There is some controversy regarding the importance of viral load in mediating neurologic disease. With the advent of sensitive, quantitative and reproducible RNA assays for HIV load in plasma and CSF, we quantified viral load in brains from 10 autopsied HIV-infected subjects and 2 non-infected controls. The new quantitative HIV RNA assays showed general agreement with previously used semi-quantitative immunocytochemical assessments of HIV envelope protein, and were performed without professional subjective interpretation. All cases with very high levels of HIV in the CSF, had high overall levels in the brain, suggesting that CSF viral loads exceeding 10(6) copies per mL may be a surrogate marker of high viral load in the brain. Levels of virus in the spleen showed no clear association with those found in the brain. HIV RNA was not uniformly distributed throughout the brain. Selective regions, including basal ganglia and hippocampus, showed higher levels of virus than the cerebellar cortex and mid-frontal cortical gray matter. Assessment of overall brain viral load requires careful attention to regional quantitation.

Expression of brain-derived neurotrophic factor protein in activated microglia of human immunodeficiency virus type 1 encephalitis.

The role of neurotrophic factors and their therapeutic potential have been investigated in various neurodegenerative disorders. In neurodegeneration associated with human immunodeficiency virus (HIV) infection, neuronal function and survival may be affected by abnormal neurotrophic regulation involving HIV-infected microglia and reactive astrocytes. To characterize the cellular localization of brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor, trkB, proteins in HIV-1 encephalitis, we examined post-mortem brains from patients with acquired immunodeficiency syndrome and brains from non-HIV-infected controls. Using double immunofluorescent confocal microscopy, we found that BDNF immunoreactivity was distributed in neocortical neuronal perikarya and neuritic processes, while in the striatum only neurites were BDNF-immunoreactive. Additionally, the striatum with HIV infection was characterized by BDNF immunoreactivity in infiltrating activated microglia/macrophages and multinucleated giant cells. Catalytic trkB receptor immunoreactivity was observed in neuronal perikarya in the neocortex and striatum, as well as in reactive astrocytes within HIV-infected regions. Our findings suggest that expression of BDNF by activated microglia in HIV-1 encephalitis may affect neuronal survival and astroglial response through corresponding trkB receptors.

Giant cell arteritis in association with cerebral amyloid angiopathy: immunohistochemical and molecular studies.

Giant cell arteritis (GCA) usually manifests as a transmural vascular infiltrate of mononuclear and multinucleated giant cells (MNGC). We describe six patients with GCA associated with severe cerebral amyloid angiopathy (CAA), all with cerebral hemorrhage or varying degrees of cerebral infarct, and histological evidence of Alzheimer's disease (cortical CAA often predominating over senile plaques and neurofibrillary tangles). One case showed mostly cortical involvement (with old microhemorrhages), and the others were primarily leptomeningeal (with involvement of the underlying cortex and extensive encephalomalacia of adjacent brain). Many vessels with CAA exhibited a pronounced adventitial and perivascular infiltrate of lymphocytes, histiocytes, and MNGC. Immunohistochemical staining showed deposition of beta/A4 peptide primarily in the thickened media of CAA vessels, and within the cytoplasm of MNGC--suggesting phagocytosis of insoluble peptide. Cystatin C antibody stained vascular amyloid and diffusely highlighted astrocytic and MNGC cytoplasm. HAM56-positive macrophages were frequently seen around amyloid-laden vessels. Anti-smooth muscle actin immunohistochemistry suggests the occurrence of medial destruction by amyloid, with relative preservation of intimal cells. Ultrastructural studies performed in one case confirmed the presence of intracytoplasmic amyloid in MNGC. The GCA seen in these cases of CAA most likely represents a foreign body response to amyloid proteins, causing secondary destruction of the vessel wall. DNA from brain tissues of five affected patients was examined to assess whether mutations were present in exon 17 of the APP gene or exon 2 of the cystatin C gene, a finding that might explain the foreign body giant cell response to amyloid proteins in these cases. However, restriction fragment mapping of amplified gene segments showed that previously described mutations were not present in these cases.

Pontine neurocytoma.

A case of neurocytoma arising in the rostral pontine region of an 18 year old man is reported. The patient developed a right trochlear nerve palsy and was shown to have a well circumscribed, contrast enhancing mass on magnetic resonance imaging. The tumour was characterised histologically by a uniform population of medium sized round nuclei and slightly eosinophilic cytoplasm or occasional perinuclear halos, with delicate branching capillaries, patches of fibrillary matrix, and occasional perivascular pseudorosettes. Immunohistochemical studies demonstrated strong reactivity for synaptophysin in the fibrillary processes and cytoplasm of tumour cells. The present tumour is an exceptional case of neurocytoma arising in the pons.

Fatal paraquat poisoning: a light microscopic study in eight autopsy cases.

Eight autopsy cases of paraquat poisoning from 1980 to 1990 were studied by light microscopy. An attempt was made to correlate the severity of poisoning, as assessed by the blood paraquat concentrations and the time between ingestion and treatment, with the survival periods and pathological changes. Six of the patients were male. The mean age was 21 years (range 12-33 years). The blood paraquat concentrations on admission ranged from 0.04 to 4.27 micrograms/ml. The survival periods were between 26 hours and 59 days. The main causes of death included circulatory collapse in one patient with 26 hours survival, and acute alveolar injury of the lungs and acute tubular necrosis or diffuse cortical necrosis of the kidneys in 4 patients who survived less than 7 days. Pulmonary proliferative changes leading to respiratory failure were detected in the remaining patients, who survived 11, 17, and 59 days. The liver revealed bile duct injury in the portal areas, centrolobular cholestasis, fatty metamorphosis, and inconspicuous centrolobular hepatic necrosis. The adrenal glands showed diffuse cortical necrosis in 3 severe cases. Mild acute pancreatitis was evident in one case. The brain was edematous with or without focal minimal hemorrhages. Toxic myocarditis, myositis, and aplasia of erythropoiesis, as previously described, were not present in this study. The severity of poisoning seems to correlate reversely with the survival periods and directly with degrees of pulmonary damage and adrenal cortical necrosis.