PMS2: a potential prognostic protein marker in oral squamous cell carcinoma.

BACKGROUND: An increase in oral squamous cell carcinoma (OSCC) cases was observed despite the reduction in exposure to classic risk factors. Although the exact cause of this trend remains unknown, epigenetic factors could be contributing to an increased occurrence of these tumors. This study aims to assess the influence of PMS2 protein immunoexpression on the prognosis of patients with OSCC. MATERIAL AND METHODS: This study comprised 76 cases of OSCC treated between 2011 and 2016. Immunohistochemical staining for PMS2 was performed. For evaluation, 10 fields per histological section were photographed at a 400x magnification and positively-stained cells were counted with Image J. Mann-Whitney and Kruskal-Wallis tests were used to compare the immunolabeling pattern with the clinical-pathological and prognostic characteristics. Survival analysis was performed with Chi-square, Long-Rank Mantel-Cox and Cox regression tests (p<0.05). RESULTS: An overexpression of PMS2 was observed in N0/1 tumors and in oral cancers found in unusual locations. In patients ≤60 years of age, high levels of PMS2 (>60%; p=0.041) were associated with low survival (p=0.029). In multivariate analysis, surgery combined with chemotherapy (p=0.030) and high PMS2 immunoexpression (p=0.042) significantly increased the risk of death for ≤60 years old patients. CONCLUSIONS: The findings of this study indicate that PMS2 can be a potential prognostic protein marker in OSCC patients 60 years of age and younger.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Prevalência de tuberculose urogenital no estado do Ceará no ano de 2003 / Prevalence urogenital tuberculosis in Ceará during 2003 year

No período de janeiro à dezembro de 2003 foram enviados ao Laboratório Central do estado do Ceará ( LACEN-Ce), 1.028 espécimes clínicos (urina), coletados de 112 pacientes com suspeita de tuberculose urogenital, onde 79 (7,7) destes espécimes apresentaram baciloscopia e cultura positiva para o complexo Mycobacterium tuberculosis. Dos 112 pacientes apenas 40 (35,7)...

[Evaluation of mutations that confer resistance to nucleoside analogs and protease inhibitors in HIV-1-infected patients] / Evaluación de mutaciones que confieren resistencia a análogos de nucleósidos e inhibidores de la proteasa en pacientes infectados por el VIH-1.

Genotypes that confer drug resistance to reverse transcriptase inhibitors and protease inhibitors were evaluated in HIV-1 proviral DNA obtained from peripheral blood mononuclear cell samples. Fifty-three HIV-1-infected patients were studied, 19 of whom had not received antiretroviral treatment. In the other 34 patients, 9 had been treated with combinations of two reverse transcriptase inhibitors (AZT, ddI, d4T, 3TC) and 25 had been treated with triple antiretroviral therapy including a protease inhibitor (nelfinavir, indinavir, saquinavir, ritonavir). To determine the presence of mutations involved in the development of resistance to reverse transcriptase inhibitors a hybridization Microtiter assay was carried out. Mutations were detected in treated patients as well as in those without previous antiretroviral treatment, with the most frequent mutations being those that confer resistance to AZT, followed by those that develop cross-resistance to ddI/ddC and 3TC, which are the most commonly used drugs to date. No mutations were detected to any nucleoside analog in only 13 cases. To analyze the presence of mutations in the protease gene a dot-blot hybridization was carried out which included the mutations in codons 36, 82 and 90. Mutation 82 was detected in one case. Therefore, with the aim of determining the pattern of genotypic mutations in patients infected with HIV-1 and in order to make the best therapeutic choice, it would be recommended to consider carrying out genotypic resistance assays in clinical practice.

[Evaluation of mutations that confer resistance to nucleoside analogs and protease inhibitors in HIV-1-infected patients. Study Group on Resistance to Antiretroviral Agents]. / Evaluación de mutaciones que confieren resistencia a análogos de nucleósidos e inhibidores de la proteasa en pacientes infectados por el VIH-1. El Grupo de Estudio de Resistencias a Antirretrovirales.

Genotypes that confer drug resistance to reverse transcriptase inhibitors and protease inhibitors were evaluated in HIV-1 proviral DNA obtained from peripheral blood mononuclear cell samples. Fifty-three HIV-1-infected patients were studied, 19 of whom had not received antiretroviral treatment. In the other 34 patients, 9 had been treated with combinations of two reverse transcriptase inhibitors (AZT, ddI, d4T, 3TC) and 25 had been treated with triple antiretroviral therapy including a protease inhibitor (nelfinavir, indinavir, saquinavir, ritonavir). To determine the presence of mutations involved in the development of resistance to reverse transcriptase inhibitors a hybridization Microtiter assay was carried out. Mutations were detected in treated patients as well as in those without previous antiretroviral treatment, with the most frequent mutations being those that confer resistance to AZT, followed by those that develop cross-resistance to ddI/ddC and 3TC, which are the most commonly used drugs to date. No mutations were detected to any nucleoside analog in only 13 cases. To analyze the presence of mutations in the protease gene a dot-blot hybridization was carried out which included the mutations in codons 36, 82 and 90. Mutation 82 was detected in one case. Therefore, with the aim of determining the pattern of genotypic mutations in patients infected with HIV-1 and in order to make the best therapeutic choice, it would be recommended to consider carrying out genotypic resistance assays in clinical practice.

Association between HIV and other DNA viruses in vitro.

To investigate the association of human immunodeficiency virus (HIV) with various DNA viruses, including hepatitis B virus (HBV), cytomegalovirus (CMV) and Epstein-Barr virus, (EBV), simultaneous detection of HIV p24 antigen, HBV surface antigen and DNA, CMV-DNA and EBV-DNA expression was performed in phytohemagglutinin-stimulated peripheral blood mononuclear (PBMC) culture supernatants obtained from 54 individuals at risk for HIV infection. HIV expression in PBMC culture supernatants never occurred alone; expression of other viruses was always detected in the 24 samples expressing HIV antigen in vitro. Furthermore, in 16 patients expression of other viruses was detected without HIV expression, and in 14 patients none of the tested viruses were detected. These results indicate a strong association between the presence of HIV antibody and expression of DNA viruses in vitro (p = 0.0001). The coexpression of these viruses could be related to the evolution of HIV infection and AIDS.

Detection of hepatitis B virus DNA and determination of surface antigen expression in peripheral blood mononuclear cells from patients with AIDS.

The polymerase chain reaction (PCR) was used to analyze the presence of hepatitis B virus (HBV) DNA in serum and peripheral blood mononuclear cells (PBMCs) from 20 patients with AIDS with and without conventional HBV serological markers. DNA sequences of HBV were detected in PBMCs from 13 patients, nine of whom were positive for anti-HBc only and four of whom were also positive for anti-HBs. When PBMCs from patients were activated in culture with phytohemagglutinin, the presence of HBsAg could be detected in the culture supernatants from four of 13 patients with HBV DNA in their PBMCs; for two of the four, HBV DNA could also be detected in the culture supernatant after DNA amplification. It was observed that HBV DNA sequences found in PBMCs can be reactivated by mitogen stimulation in some HIV-1 infected patients.

Detection of human immunodeficiency virus type 1 by PCR before seroconversion in high-risk individuals who remain seronegative for prolonged periods.

HIV-1 seronegative patients at high risk for HIV infection were followed up. In 1990 PCR was positive for HIV DNA sequences in samples of 17 seronegative patients who continued to report for surveillance of HIV infection. There was clear evidence of seroconversion in four of these 17 seronegative patients, while in one patient an indeterminate result for HIV was repeatedly obtained in different samples. The other 12 patients continue to be seronegative without any evidence of HIV infection except the presence of provirus in peripheral blood mononuclear cells. It is important to apply the PCR technique together with tests to detect other virological and immunological markers, in order to identify seronegative carriers and thus avoid HIV transmission by them.

[Detection of the HIV p24 antigen on lymphocyte membranes using flow cytometry]. / Detección de antígeno p24 del VIH en las membranas linfocitarias por citometría de flujo.

The evaluation of the presence of p24 antigen on the membrane of peripheral blood mononuclear cells from 31 HIV infected individuals is presented. The study was performed by indirect immunofluorescence and flow cytometry and the data were analyzed by the Kolmogorov-Smirnov test. Values obtained [D/s(n)] result from the comparison of the fluorescence histograms of each sample with a control one. Cases showing p24 Ag on peripheral blood mononuclear cells also presented percentages of CD3, HLA-DR positive cells significantly higher than p24 negative ones. In addition, D/s(n) values were superior in symptomatic patients than in asymptomatic ones, which indicate the existence of a correlation between flow cytometry results, viral replication and clinical course. Nevertheless in this study, as well and in previous ones, a high degree of cross reactivity between the anti-p24 monoclonal antibody employed and normal lymphocytes has been observed. This reactivity is localized preferentially in the CD4 positive subset.

[Detection of HBV-DNA in peripheral blood mononuclear cells of anti-HIV-positive patients and its relation to other serological markers of HBV]. / Detección del ADN-VHB en células mononucleares de sangre periférica de pacientes anti-VIH-positivos, y su relación con otros marcadores serológicos del VHB.

HBV infection has been investigated in 47 anti-human HIV positive patients in relation to a similar group of 33 anti-HIV negative patients. Serological HBV markers were found in 87% of anti-HIV positive patients. The difference in markers of viral replication (HBeAg, HBV-DNA) was not statistically significant between the two groups. It has been suggested that HBV infection of peripheral blood mononuclear cells could be a cofactor implicated in the development of immunodeficiency due to HIV. For this reason we have investigated the presence of HBV-DNA in peripheral blood mononuclear cells by in situ hybridization. Although its detection was more frequent in anti-HIV positive patients than in anti-HIV negative ones (p < 0.05), it was not related to clinical state of immunodeficiency. With regard to serological HBV markers, HBV-DNA was detected in peripheral blood mononuclear cells from antiHBc w/o antiHBs patients. This fact means the virus may persist in this cells after recovery and suggest they could serve as additional reservoirs of HBV. These cells, that contain the HBV genome, could be implicated in the perpetuation, reactivation of the infection and in its transmission.

Detection of hepatitis B virus DNA in chronic carriers by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect hepatitis B virus DNA (HBV-DNA) in serum samples of 104 chronic HBV carriers. Of 22 patients positive for both HBV surface (HBsAg) and HBVe (HBeAg) antigens, seven were positive for HBV-DNA on dot blot hybridisation, and all 22 positive in the PCR. Of 41 HBsAg positive patients who had antibodies against HBeAg (anti-HBe), only three were positive for DNA-HBV on dot blot hybridisation, however DNA was detected in 30 of them with the PCR. Similarly, of 41 individuals with antibodies against HBsAg (anti-HBs), 23 yielded positive results in the PCR technique, although dot blot hybridisation detected HBV-DNA in only one patient. These results indicate that while serological and conventional DNA hybridisation assays are not sensitive enough to determine the infectivity of HBV chronic carriers, PCR is an accurate method for establishing the status and progression of disease in these patients.

Evidence of an in vitro association between human immunodeficiency virus antigen P24 and Epstein-Barr virus DNA.

To investigate the association between human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV), simultaneous determinations of HIV antigen (HIV Ag) p24 and EBV DNA were performed in lymphocyte culture supernatants from 63 individuals at risk of HIV infection. In vitro data, together with HIV immune status results, were subjected to a statistical analysis. HIV infection was identified in 49 patients (78%); of these, in vitro EBV DNA was found in 44 individuals (90%), while in only 3 of the 14 non-infected ones (21%). Statistical analysis demonstrated a close relationship between evidence of HIV infection and in vitro detection of EBV DNA (87.3% concordant with 95% confidence interval: 76.5%-94.5%). Furthermore, a strong dependence was revealed between the presence of EBV DNA and HIV Ag in culture (p less than 0.00001). These results indicate the existence of in vitro viral interactions, with likely in vivo implications in the pathogenesis and evolution of HIV infection.

[Detection of hepatitis B virus DNA in peripheral blood mononuclear cells from patients with various hepatopathies using in situ hybridization]. / Detección del DNA del virus de la hepatitis B en células mononucleares de sangre periférica de pacientes con distintas hepatopatías mediante hibridación "in situ".

We have investigated, by "in situ" hibridisation, the presence of hepatitis B virus (HBV) DNA in peripheral blood mononuclear cells (PBMC) from 45 patients with acute and chronic hepatic disorders directly related with HBV or with some seric HBV marker. Results has been related with serological markers and the different types of hepatopaties. The HBV-DNA was detected in PBMC more frequently in patients with hepatic alterations more prolongated (chronic active hepatitis, chronic persistent hepatitis and cirrhosis) than in acute hepatitis patients. It was not detected in any asymptomatic patient with HBV serological markers. As regards HBV serological markers, HBV-DNA was detected in PBMC in 8/11 HBsAg positive patients and in 11/34 HBsAg negative patients: 3 antiHBc positive, 5 antiHBc and antiHBs positive and 3 without conventional seric markers. The detection of HBV-DNA in antiHBc and/or antiHBs positive subjects means the virus may persist after recovery of infection and suggests PMBC could serve as additional reservoirs for reinfection of hepatocytes leading to a reactivation of the liver disease. Our results suggest that HBV infection of PBMC is a frequent event during HBV infection and can have important consequences fundamentally with respect to pathogenic mechanisms of HBV induced liver disease and to the transmission of the virus.

[Detection and antigenic distribution of hepatitis B virus in liver tissue and its relation to other serological markers of viral replication]. / Detección y distribución antigénica del virus de la hepatitis B en tejido hepático y su relación con otros marcadores serológicos de replicación viral.

The aim of this work has been the production of specific monoclonal antibodies against HBV-antigens and their utilisation in order to study their distribution on liver tissue. The monoclonal antibodies anti-HBc and anti-HBs were obtained by the modified hybridoma technique. This study was performed on 50 patients affected by several chronic hepatopathies. For the detection of the antigens, avidin-biotin-peroxidase complex immunostaining was used. Both cytoplasmic and membranous HBsAg were detected in 15 out of 16 HBsAg+ patients; 8 of 12 HBsAg-/anti-HBc+ patients and 1 HBsAg-/antiHBc- patient. Cytoplasmic and nuclear HBcAg was observed in 12 of 16 HBsAg+ patients and 4 of 20 HBsAg- patients. Although the presence of serum HBsAg is an index of liver infection, in some HBsAg-/antiHB+ patients (20%) with undetectable levels of HBsAg, hepatic injury may be disclosed by the detection of other markers of active viral replication.

[Hepatitis B virus (HBV) replication in patients with various types of chronic hepatopathy]. / Estudio de la replicación del virus de la hepatitis B (VHB) en pacientes con distintas formas de hepatopatía crónica.

We study retrospectively the viral replication state (HBV) of 50 patients with chronic hepatic alterations. The seric DNA-HBV and/or intrahepatic (molecular hybridization), the intrahepatic distribution of HBV antigens (specific monoclonal antibodies labelled with immunoperoxidase), conventional seric HBV markers (commercial enzymoimmunoessay) and the different histopathologic features. We found a correlation between DNA-HBV "in situ" and HBcAg intrahepatic and the seric DNA-HBV production. 81% of the patients with HBsAg (+) had intrahepatic HBcAg and 85% (11/13) of them showed the antigen in their cytoplasms. Patients with HBcAg also had seric and liver DNA-HBV (+). The lack of seric HBsAg did not mean that non-active replication of HBV did not exist because 20% of the patients with HBsAg (-) showed seric and "in situ" DNA-HBV and cytoplasmic HBcAg. The detection of DNA-HBV in endothelial cells and vascular elements in hepatic tissue show that the rate of the HBV host cells is greater.

[The detection of the DNA of the hepatitis B virus in patients with probable non-A, non-B hepatitis]. / Detección del DNA del virus de la hepatitis B en pacientes con probable hepatitis noA-noB.

We found the presence of hepatitis B virus in 17 cases of non-A-non-B hepatitis using the DNA detection technique in serum of patients with a type of chronic hepatopathy. This finding supports the needs to determine this seric marker in all patients afflicted with chronic hepatopathy before the diagnosis of hepatitis B is excluded.

[Presence of HBsAg in lymphocyte cultured from patients infected with human immunodeficiency virus (HIV)]. / Presencia de HBsAg en cultivos de linfocitos de pacientes infectados por el virus de la inmunodeficiencia humana (VIH).

The behavior of the hepatitis B virus was investigated in mononuclear cell cultures in nine patients infected by the human immunodeficiency virus. Although only one of them was a carrier of HBsAg and four had anti-HBs in their sera, HBsAg was detected in the supernatant of the cultures from all patients. These results suggest that mononuclear cells might act as a reservoir for hepatitis B virus, and that the concomitant infection by this virus and human immunodeficiency virus may alter the natural evolution of any of both conditions.