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 (

Summary: Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA pre cedes morphological changes in the nucleus and, later, in the cytoplasm, In ischemia, biochemical studies have detected oli gonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than ap optosis, To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by I to 14 days of reperfusion, Parameters evaluated in the CAl region of the hippocampus included morphology, in situ end labeling (ISEL) of fr'l-gmented DNA, and expression of p53, Neurons were indistinguishable from controls at postischemic day I but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appearedThe neuronal response to brain ischemia depends on the severity and duration of the ischemic insult as well as on intrinsic neuronal factors that impart selective vulner ability to specific brain regions (Scholz, 1953), Brief periods of global ischemia kill vulnerable neurons in the CAl region of the hippocampus whereas longer intervals of vascular occlusion kill neurons in the cerebral cortex and corpus striatum as well (Ito et aI., 1975; Pulsinelli et aI., 1982a), Although seemingly undamaged, neurons in other brain regions such as the CA3 region of the hip pocampus or the paramedian cerebral cortex undergo transient alterations as evidenced by reversible changes 967normaL In situ end labeling was absent At days 3 and 5, approximately 40 to 60% of CAl neurons had shrunken eosin ophilic 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 frag mentation occurs after the development of delayed neuronal death in CA I neurons subjected to 10 minutes of global isch emia, They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CAl neurons in this model. Key Words: Global ischemia-Apoptosis Necrosis-Delayed neuronal death-In situ end labeling-Rat in their subcellular organelles (Petito and Pulsinelli, 1984a,b), The postischemic interval required for the ap parent development of selective neuronal necrosis is in versely proportional to the severity or duration of the ischemic insult-the so-called maturation phenomena described by Ito et al. (Ito et at, 1975), This interval is especially prolonged in the CAl neurons of the hippo campus (Ito et aI., 1975; Pulsinelli et aI, 1982a). The term delayed neuronal death has been applied to this process (Kirino, 1982).During the past few years, the concept of cell death via apoptosis has become increasingly important. This mechanism of cell death is mediated by regulated path ways involving defined gene product...

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.

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CD8+ T cells infiltrate brains with human immunodeficiency virus type-1 (HIV-1) encephalitis (HIVE) and related animal models; their perineuronal localization suggests cytotoxic T cell (CTL)-mediated neuronal killing. Because CTLs have not been identified in acquired immunodeficiency syndrome (AIDS) brains, the authors identified their cytotoxic granules in autopsy AIDS brains with HIVE and without HIVE (HIVnE) plus controls (7 to 13 cases/group) and determined gene expression profiles of CTL-associated genes in a separate series of cases. CD3+ and CD8+ T cells were significantly increased (P < .01) in perivascular spaces and inflammatory nodules in HIVE but were rare or absent in brain parenchyma in HIVnE and control brains. Eight HIVE brains contained granzyme B+ T cells and five contained perforin+ T cells. Their T-cell origin was confirmed by colocalization of CD8 and granzyme B in the same cell and the absence of CD56+ natural killer cells. The CTLs directly contacted with neurons, as the authors showed previously for CD3+ and CD8+ T cells. CTLs were rare or absent in HIV nonencephalitis (HIVnE) and controls. Granzyme B and H precursor gene expression was up-regulated and interleukin (IL)-12A precursor, a maturation factor for natural killer cells and CTLs, was down-regulated in HIVE versus HIVnE brain. This study demonstrates, for the first time, CTLs in HIVE and shows that parenchymal T cells and CTLs are sensitive biomarkers for HIVE. Consequently, CD8+ T cells and CTLs could mediate brain injury in HIVE and may represent an important biomarker for productive brain infection by HIV-1.

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This study shows that several genes related to laminin are overexpressed in angiotropic melanoma areas vs. avascular melanoma areas. Since extravascular migration of melanoma cells along vessels has been demonstrated in the CAM model, taken together these results suggests that some laminins and laminin receptors may play a role in extravascular migratory metastasis. This model may represent a promising strategy to analyze differential gene expression in EVMM.

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.