Prognostication of prostate cancer based on TOP2A protein and gene assessment: TOP2A in prostate cancer.

BACKGROUND: TOP2A encodes for topoisomerase IIα, a nuclear enzyme that controls DNA topological structure and cell cycle progression. This enzyme is a marker of cell proliferation in normal and neoplastic tissues; however, little information is available about its expression in prostate cancer (PCa). METHODS: Immunohistochemistry (IHC) was automated using mouse monoclonal antibody against TOP2A (clone SWT3D1; DAKO, Carpenteria, CA, USA) at dilution 1:800 and Flex Plus detection system in autostainer 48Ultra (DAKO). FISH was performed using TOP2A (17q21)/ CEP17 probe kit (Kreateck Biotechnology, San Diego, CA, USA). Biochemical and pathological data from 193 patients with PCa were retrieved for the analysis, whose significance was considered when p < 0.05. Also, fractal analysis was performed in a subset of 20 randomly selected cases. RESULTS: TOP2A protein expression correlated with higher Gleason scores and higher levels of preoperative PSA (p = 0.018 and p = 0.011). Patients with higher levels of TOP2A presented shorter biochemical recurrence-free survival (BRFS) (p = 0.001). In multivariate analysis, we found that TOP2A remained an independent prognostic factor of BRFS, with a relative risk of 1.98 (p = 0.001; 95% CI, 1.338-2.93); thus, cases that expressed high levels of this enzyme had a shorter BRFS compared with TOP2A-negative or TOP2A-low cases. No alterations in TOP2A gene status nor correlation between FISH and IHC results were observed. Concerning fractal analysis, patients who expressed higher levels of TOP2A have angiolymphatic invasion and presented higher Gleason scores (p = 0.033 and p = 0.025, respectively). Also, patients with higher expression of TOP2A presented shorter BRFS (p = 0.001). CONCLUSIONS: This is the first study to perform TOP2A protein and gene digital assessment and fractal analysis in association with BRFS in a large series of PCa. Also, we show that TOP2A gene copy number alterations are not observed in this type of tumor. So, higher protein expression of TOP2A is not related to gene amplification in PCa. Furthermore, TOP2A protein assessment has prognostic importance and, due to its relation with poor outcome, TOP2A IHC evaluation in the biopsy can represent an important tool for selecting the most suitable surgical and clinical approach for patients with PCa.

A macrolactone from benzo[a]phenazine with potent activity against Mycobacterium tuberculosis.

We report here an alternative to the MCPBA or ozonolysis-based oxidation methods of quinoxaline-featuring compounds prepared from beta-lapachones. The use of peracetic acid allowed a simple preparation of the corresponding macrolactones by cleavage of the ring system. These lactones were evaluated for their antimycobacterial potential and compound 4 turned out to have an MIC of 0.62 microg per mL on Mycocabteriumtuberculosis H37Rv. These results justify further research into its value as a potential lead for an original treatment of tuberculosis.

Flow cytometry evaluation of the T-cell receptor Vbeta repertoire among HIV-1 infected individuals before and after antiretroviral therapy.

HIV-1 infection leads to serious impairment of the immune system and perturbations in the T cell receptor Vbeta repertoire are also described. Immune reconstitution can be potentially achieved in response to HAART. In the present study 10 patients were investigated for the Vbeta pattern expression before and after six months of HAART. TCR were analyzed for T CD4+ and CD8+ subsets, separately, by flow cytometry, using a monoclonal antibody set of 24 different Vbeta chains. Compared to eight Brazilian healthy controls, no differences in Vbeta pattern of expression was observed for patients before or on antiretroviral therapy. Some chains such as Vbeta 3, 14, 16, 20 and 21.3 were over utilized by both T subsets, independently of HIV infection and/or antiretroviral treatment, differing from the ones described for individuals of other nationalities. However, when each patient was taken individually, particular alterations were detected for the Vbeta gene usage, compared to controls, for all individuals. After treatment, significant Vbeta usage changes were observed for seven patients. One or more chains on both T subsets were engaged in this process, defining a preferential oligoclonal profile for TCR repertoire distribution, after HAART. Although no pattern of specific Vbeta changes was detected in the circulating T cells, we cannot exclude that differential immune responses to HIV or other important antigens are being focused by these cells.

Comparison of flow cytometric and Alamar Blue tests with the proportional method for testing susceptibility of Mycobacterium tuberculosis to rifampin and isoniazid.

The performance of flow cytometry and the microplate Alamar Blue assay in determining susceptibility of Mycobacterium tuberculosis was assessed by testing 150 Brazilian isolates. The overall agreement was 97.3 and 98% for isoniazid and 94.7 and 100% for rifampin by flow cytometry and MABA, respectively. This study was entirely done in a developing country.

Cryopreservation of peripheral blood mononuclear cells does not significantly affect the levels of spontaneous apoptosis after 24-h culture.

Studies performed with malaria patients living in endemic areas are frequently conducted in laboratories located hundreds of kilometer away from research centers, due to the difficulties in performing the assays in field conditions. Thus, we considered the potential indication of cryopreservation of peripheral blood mononuclear cells (PBMC), in most fieldwork, and decided to evaluate the effect of cryopreservation of PBMC on spontaneous apoptosis. The membrane integrity of PBMC was tested using three previously described protocols of cryopreservation. Cell samples were obtained from 19 healthy volunteers. Percentage of apoptotic nuclei in short-term PBMC cultures was determined by a sensitive method using 7-aminoactinomycin D followed by flow cytometry. Our results indicate that although cryopreservation can to some extent affect lymphocyte membrane integrity rates, flow cytometry analysis showed that frequencies of spontaneous apoptosis in cryopreserved cells were not significantly modified after 24-h culture. It is concluded that cryopreserved PBMC could be used for measuring spontaneous apoptosis and therefore, could be employed for the study of populations living in areas distant from research centers, allowing the comparative evaluation of samples obtained at different time.