Jorge Torres-Munoz

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.

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.

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.

Brain CD8+ and cytotoxic T lymphocytes are associated with, and may be specific for, human immunodeficiency virus type 1 encephalitis in patients with acquired immunodeficiency syndrome

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.

Expression of the Antiapoptotic Proteins Clusterin and Bcl-2 in Laryngeal Squamous Cell Carcinomas

Bcl-2 and clusterin genes have been related to the inhibition of apoptosis, an event that plays a key role in malignant transformation and in invasive disease. In this work, we determine the significance of clusterin and bcl-2 expression in a large series of laryngeal carcinomas. We used immunohistochemical methods and in situ hybridization to examine the expression of these proteins. Nontumoral epithelial laryngeal tissues did not express clusterin and bcl-2 proteins. However, 9% (14 out of 154) and 25% of these tumors (39 of 154) had positive clusterin and bcl-2 staining, respectively. Clusterin expression was significantly related to the degree of local invasion and higher bcl-2 expression was found in these clusterin-positive tumors (p < 0.05). Bcl-2 expression was significantly correlated with supraglottic localization, nodal metastases, invasion in depth, and poorly differentiated tumors. However, by multivariate analysis, bcl-2 was shown to be an independent predictor of good prognosis in these tumors (OR = 0.12, 95% CI = 0.02–0.91). These findings indicate that clusterin and bcl-2 are upregulated in laryngeal carcinomas and their expression is related to the invasiveness of these tumors.

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 malignancy‐associated laminins and laminin receptors by angiotropic human melanoma cells in a chick chorioallantoic membrane model

Background: As distinct from intravascular/lymphatic dissemination, extravascular migratory metastasis (EVMM) has been described as a potential additional mechanism of melanoma spread in which tumor cells migrate along the external surfaces of vessels. Angiotropic melanoma cells are linked to the endothelium by a matrix containing laminin. In addition, it has been shown that C16 laminin‐derived peptide increases extravascular migration of human green fluorescent protein (GFP) melanoma cells along vessels in a chicken chorioallantoic membrane model (CAM). In this study, we have tested the hypothesis that expression levels of some genes related to lamimin and metastasis are differentially expressed in vascularized angiotropic melanoma areas vs. avascular melanoma areas from the same tumor.

Successful Application of Hyperbranched Multidisplacement Genomic Amplification to Detect HIV-1 Sequences in Single Neurons Removed from Autopsy Brain Sections by Laser Capture Microdissection

To confirm studies suggesting that HIV-1 infects neurons and to determine whether CD8(+) T lymphocytes traffic to HIV-1-infected neurons, we used laser capture microdissection to remove hippocampal neurons with and without perineuronal CD8(+) T cells from AIDS patients with HIV-1 encephalitis (HIVE) or without HIVE and from normal controls. We used hyperbranched multidisplacement amplification for whole gene amplification (MDA-WGA) plus two rounds of PCR to amplify housekeeping sequences (HK(+)) and, in HK(+) samples, to amplify HIV-1 gag, nef, and pol sequences. Sample size and, in single neurons, MDA-WGA correlated with housekeeping gene amplification (P < 0.05), whereas patient group and postmortem interval did not (P > 0.05). Neuronal viral sequences correlated with HIVE (43% vs. 13% and 0 in non-HIVE and controls, respectively) and, in HIVE cases, with perineuronal CD8(+) T lymphocytes (70% in CD8(+) samples vs. 37% of CD8(-) samples). Our results suggest that MDA-WGA is a useful technique when analyzing DNA from single cells from autopsy brains, supporting prior studies that show that neurons may contain HIV-1 neuronal sequences in vivo. The association between neuronal infection and perineuronal CD8(+) T cells supports our hypothesis that these cells specifically traffic to infected neurons but raises the possibility that CD8(+) T cells, if infected, could transmit virus to neurons.

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.

Clinical translation of a novel microfilter technology Capture, characterization and culture of circulating tumor cells

The most important determinant of prognosis and management of cancer is the presence or absence of metastasis [1]. The road to metastasis involves tumor cells to become detached from the primary tumor and travel in the blood to distant sites, causing secondary tumors. These tumor cells traveling in blood are termed Circulating tumor cells (CTC). Capture of CTC from whole blood has been a challenging feat. The fact that these CTC are few in number, to effectively and efficiently isolate them from whole blood can be thought of as looking for a needle in a haystack. Our microfilter technology exploits the use of size based capture of the larger CTC from the smaller white blood cells and components of whole blood. The effective capture potential of the microfilter platform has driven the area of CTC analysis into a new age of research in the field of cancer. The ability to finally analyze CTC at a molecular level, leads to a deeper understanding of metastatic process, while providing an opportunity to evaluate, monitor and manage treatment options as well as the adherent possibility of having an “on-chip” drug sensitivity assay for focused treatment options. We have demonstrated through clinical trials the ability to effectively identify, enumerate and characterize CTC based on immunfluorescence and FISH assays and provide a companion endpoint for monitoring and evaluating treatment management. Our work on viable CTC capture has resulted in successfully capturing and culturing CTC from blood in mouse models that have been inoculated with breast cancer cell lines to form primary and secondary metastatic cancer sites. The future potential within the microfilter technology to capture viable CTC for culture, will catapult therapeutic interventions to a new level of personalized medicine in cancer management.