Differential modulation of cell death proteins in human brain cells by tumor necrosis factor alpha and platelet activating factor.

Programmed cell death contributes to the morbidity and mortality of several neurological disorders including stroke, Alzheimer's disease and human immunodeficiency virus (HIV)-associated dementia. Patients with HIV dementia show evidence of programmed cell death in brain. In vitro data demonstrates several neurotoxic products of macrophage infection that cause neural cell death, including tumor necrosis factor alpha (TNFalpha) and platelet activating factor (PAF). We treated human brain aggregate cultures with these cytokines and determined their effect on the mRNA and protein levels for Bcl-2, Bcl(x) and Bax alpha. TNFalpha and PAF differentially regulate the Bcl-2 family of proteins at a post-transcriptional level. Following TNFalpha treatment, Bcl-2 protein is significantly decreased, and at least one additional Bax isomer emerges. Bcl(xL) protein is slightly increased after treatment with either cytokine. We demonstrated that overexpression of Bcl-2 in brain aggregate cultures protects cells from TNFalpha-induced damage but has no effect on cell damage induced by PAF. We conclude that Bcl-2 and Bax alpha proteins play significant roles in modulating neural cell death from TNFalpha- but not from PAF-induced cell damage.

Quantification of neurotoxicity and identification of cellular subsets in a three-dimensional brain model.

Imaging of cells in a large intact three-dimensional tissue remains difficult. Quantification and identification of cell damage in a mixed culture system has been limited by the inability of fluorescent probes to discriminate types of cellular death and penetrate tissue more that 100 microm thick. We have investigated several probes in combination with neural cell-specific antibodies to quantify cell damage in the presence of several toxins. Acridine orange and ethidium bromide were excellent for determination of cell viability, death by necrosis, or apoptosis in thick brain tissue aggregates. Calcein and ethidium homodimer were effective on live/ dead stains, and the Syto dyes 11 and 13 worked well for quantification of all cells in the brain aggregate model. By using these combinations of dyes in conjunction with confocal microscopy, we were able to quantify neural cell damage without disrupting the three-dimensional environment.

Unique monocyte subset in patients with AIDS dementia.

BACKGROUND: 15-30% of patients infected with HIV will develop a debilitating dementia. Whilst HIV enters the brain soon after infection, presumably within monocyte-derived macrophages, not all patients with HIV become demented. Blood monocytes probably cross the blood-brain barrier and give rise ultimately to parenchyma macrophages. We looked for a specific monocyte subset in AIDS patients with dementia. METHODS: Peripheral blood monocytes from three groups were compared: AIDS patients with (n = 12) and without (n = 11) dementia, and ten HIV seronegative healthy controls. We used flow cytometry to analyse monocytes, and cell lysis and apoptosis assays to examine monocyte effects on human brain cells in vitro. FINDINGS: We found a unique subset of monocytes in patients with AIDS dementia. These monocytes were more dense and granular and expressed CD14/CD16 and CD14/CD69. Means (SD) for CD14/CD16 in HIV-negative controls and in AIDS non-dementia and AIDS dementia patients were 6.5% (4), 16% (13), and 37% (21), respectively (p = 0.008 between the two groups of patients). The corresponding means for CD14/CD69 were 7% (6), 8% (10), and 69% (18) (p < 0.0001). INTERPRETATION: CD69 is a member of the natural-killer-cell gene complex that is expressed after activation. Supernatants from cultures containing these dense cells can trigger apoptosis of human brain cells in vitro. The monocyte subset we found in patients with AIDS dementia might enter the brain and expose neural cells to toxic factors.

A class I antigen, HLA-G, expressed in human trophoblasts.

The alpha chain of the human histocompatibility antigen HLA-G was identified as an array of five 37- to 39-kilodalton isoforms by the use of two-dimensional gel electrophoresis. Both cell-associated and secreted HLA-G antigens are prominent in first trimester villous cytotrophoblasts and are greatly reduced in third trimester cytotrophoblasts. Allelic variation was not detected, an indication that HLA-G is not obviously polymorphic in cytotrophoblasts. Among the following choriocarcinoma cell lines studied, HLA-G is expressed in JEG but not in Jar or BeWo. Expression of endogenous HLA-G genes has not been found in normal lymphoid cells. Thus, HLA-G is subject to both cell type-specific and developmental regulation and is expressed in early gestation human cytotrophoblasts.