Analytical Cellular Pathology - Volume 21, issue 1

Authors: Alaiya, Ayodele | Roblick, Uwe | Egevad, Lars | Carlsson, Adelaide | Franzén, Bo | Volz, Daniela | Huwendiek, Sören | Linder, Stig | Auer, Gert

Article Type: Research Article

Abstract: Cells were collected from prostate hyperplasias (n=6) and prostate carcinomas (n=6) and subjected to two‐dimensional gel electrophoresis (2‐DE). The resulting polypeptide patterns were analysed with the PDQUEST computer software. Malignant tumors showed significant increases in the level of expression of proliferating cell nuclear antigen (PCNA), calreticulin, HSP 90 and pHSP 60, oncoprotein 18(v), elongation factor 2, glutathione‐S‐transferase π (GST‐π), superoxide dismutase and triose phosphate isomerase. In addition, decreases in the levels of tropomyosin‐1 and 2 and cytokeratin 18 were observed in prostate carcinomas compared to prostate hyperplasias. This pattern of alterations is similar to that observed in other carcinomas in …our previous studies. All malignant tumors showed simultaneous alterations in 5 or more of 9 markers studied, whereas only one case of benign hyperplasia showed alterations in 5 markers. The EST‐data base for prostate tumors available from NCI (CGAP) was searched for the expression of the mRNAs corresponding to proteins identified in our gels. Large differences in the relative expression of mRNAs and proteins were observed. Our data show alterations in the pattern of polypeptide expression in prostate carcinomas which are similar to those observed in other carcinomas. Show more

Keywords: Prostate cancer, two‐dimensional gel electrophoresis, prognostic markers

Citation: Analytical Cellular Pathology, vol. 21, no. 1, pp. 1-9, 2000

Authors: Losa, Gabriele A. | Graber, Riccardo

Article Type: Research Article

Abstract: Peripheral blood mononuclear cells (PBMC) from 251 HIV‐positive drug abusers of known clinical stage and from 40 healthy donors were tested for conventional immunologic markers (CD3, CD4, CD8, CD19, CD14, CD16/CD56, CD45 and HLA‐DR). Additional cell parameters and the occurrence of spontaneous apoptosis (programmed cell death) were investigated on freshly isolated PBMC by flow cytometric measurement of either annexin‐V bound to plasma membrane phosphatidylserine or propidium iodide uptake. The activity of γ‐glutamyltransferase (γ‐GT), an ectoenzyme contributing to the synthesis of the intracellular antioxidant glutathione (GSH) and involved in early apoptosis, was also determined in these cells. Immunocompetent T‐cell counts were …lower in HIV+ patients, with the exception of CD8+ and HLA‐DR+ lymphocytes. The external binding of annexin‐V was significantly higher in HIV+ PBMC and occurred in both CD8+ and CD4+ T‐lymphocyte subsets. The activity of γ‐GT, was significantly lower in the PBMC from HIV+ patients, indicating that the redox status of PBMC may be affected in HIV+ individuals. Finally, the most dominant features characterising patients receiving antiretroviral therapy were greater long‐term stability in the distribution of various cell parameters excepted the level of apoptosis. Show more

Keywords: Apoptosis, AIDS, HIV stages A, B and C, blood lymphocytes, CD, γ‐glutamyltransferase, glutathione (GSH), surface markers, annexin‐V/phosphatidylserine, flow cytometry

Citation: Analytical Cellular Pathology, vol. 21, no. 1, pp. 11-20, 2000

Authors: Leonardi, Elena | Dalla Palma, Paolo | Reich, Alessandro | Caffo, Orazio | Luciani, Lucio

Article Type: Research Article

Abstract: A total of 238 cases of bladder carcinoma stages Ta, Tis, T1 were submitted prospectively to multiparameter flow cytometry and immunohistochemical study in order to determine the biological aggressiveness of the tumour. DNA index (DI), S‐phase fraction (SPF) obtained by bivariate cytokeratin 7/DNA analyses, and the immunohistochemical evaluation of p53 and MIB‐1 were studied in relation to the traditional prognostic factors in bladder cancer (stage and grade). The variance analysis results showed that DNA aneuploidy was significantly associated with high stage (p=0.0001), high grade (p=0.0001), high SPF value ≥5.5% (p=0.0001), MIB‐1 positivity ≥31% (p=0.0001) and high expression of p53 (staining …involving >50% of cells, p=0.0001). Even if there was no statistical significance the hypotetraploid class (1.70 < DI< 1.89) showed poor prognostic biomarkers more frequently than the other aneuploid classes. Out of 238 cases, 101 were also submitted to flow cytometric measurement of MIB‐1 (fMIB‐1) to study the correlation between cell proliferation and DNA content. Data obtained from fresh, 3 :1 methanol/acetone fixed samples were compared with values obtained from both cell cycle analysis methods and routine application of the MIB‐1 immunostaining in histological sections. fMIB‐1 values were positively correlated with SPF values (r=0.801, p<0.01) and S+G2M fraction (percentage of cells in S and in G2M phases) (r=0.763, p<0.01) but no correlation with paraffin sections was found. A fMIB‐1 value >7% was strongly associated with aneuploidy (p=0.0001). The determination of DNA content coupled with the study of the epithelial (cytokeratin 7) and proliferative (MIB‐1) markers could be useful in providing important information on the biological behaviour of superficial bladder tumours. Show more

Keywords: DNA content, bladder cancer, p53, cell proliferation, MIB‐1

Citation: Analytical Cellular Pathology, vol. 21, no. 1, pp. 21-33, 2000

Authors: Neri, Monica | Geido, Elio | Filiberti, Rosangela | Orecchia, Roberto | Di Vinci, Angela | Cafferata, Mara | Tassara, Elisabetta | Puntoni, Riccardo | Giaretti, Walter

Article Type: Research Article

Abstract: The glycophoryn A (GPA) assay evaluates somatic in vivo mutations. It is considered a cumulative biodosimeter for genotoxic exposures and is under evaluation in cancer risk assessment. GPA, a polymorphic membrane protein of the erythrocytes, determines the MN blood groups. The N0 and NN variant frequencies (VF) may be detected in MN subjects (about 50% of the population) by flow cytometry using two differently labelled antibodies. We explored if GPA N0 and NN VF might be relevant to the assessment of individual lung cancer risk and susceptibility, in a small population with a high prevalence of heavy tobacco …smokers: 8 lung cancer patients and 16 subjects with non‐malignant lung diseases associated with increased risk of lung cancer. There was a wide interindividual variability and complete overlap between non‐neoplastic and neoplastic patients. A significant positive correlation was seen with smoking duration in N0 VF (p=0.04, age‐adjusted). Current smokers (n=12) displayed higher N0 values than never (n=1) or ex‐smokers (n=11), 36.3±18.2 and 21.0±13.2, respectively (p<0.01). No association was shown with occupational exposure. The present exploratory study suggests that assessment of individual lung cancer risk and susceptibility by the GPA assay does not seem to be feasible. The assay appears to provide a biomarker of longterm exposure to tobacco smoke. Show more

Citation: Analytical Cellular Pathology, vol. 21, no. 1, pp. 35-40, 2000

Authors: Joshi, Mandar S. | Crouser, Elliott D. | Julian, Mark W. | Schanbacher, Brandon L. | Bauer, John Anthony

Article Type: Research Article

Abstract: Primary defects in mitochondrial function have been implicated in over 100 diverse diseases. In situ, mitochondria possess unique and well‐defined morphology in normal healthy cells, but diseases linked to defective mitochondrial function are characterized by the presence of morphologically abnormal and swollen mitochondria with distorted cristae. In situ study of mitochondrial morphology is established as an indicator of mitochondrial health but thus far assessments have been via subjective evaluations by trained observers using discontinuous scoring systems. Here we investigated the value of digital imaging analysis to provide for unbiased, reproducible, and convenient evaluations of mitochondrial ultrastructure. Electron photomicrographs of ileal …mucosal mitochondria were investigated using a scoring system previously described by us, and also analyzed digitally by using six digital parameters which define size, shape, and electron density characteristics of over 700 individual mitochondria. Statistically significant changes in mitochondrial morphology were detected in LPS treated animals relative to vehicle control using both the subjective scoring system and digital imaging parameters (p<0.05). However, the imaging approach provided convenient and high throughput capabilities and was easily automated to remove investigator influences. These results illustrate significant changes in ileal mucosal mitochondrial ultrastructure during sepsis and demonstrate the value of digital imaging technology for routine assessments in this setting. Show more

Keywords: Digital image analysis, endotoxin, mitochondria, sepsis, ultrastructure

Citation: Analytical Cellular Pathology, vol. 21, no. 1, pp. 41-48, 2000