Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70%, compared to 64% for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85%, compared to 88% for GWGR. The assessment of RFLP revealed 68% sensitivity and 94% specificity for the B-GPGR strain compared to 84 and 90% for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.

Imbalance of naive and memory T lymphocytes with sustained high cellular activation during the first year of life from uninfected children born to HIV-1-infected mothers on HAART

The immune consequences of in utero HIV exposure to uninfected children whose mothers were submitted to highly active antiretroviral therapy (HAART) during gestation are not well defined. We evaluated 45 HIV-exposed uninfected (ENI) neonates and 45 healthy unexposed control (CT) neonates. All HIV-infected mothers received HAART during pregnancy, and the viral load at delivery was <50 copies/mL for 56.8%. Twenty-three ENI neonates were further evaluated after 12 months and compared to 23 unexposed healthy age-matched infants. Immunophenotyping was performed by flow cytometry in cord and peripheral blood. Cord blood lymphocyte numbers did not differ between groups. However, ENI neonates had a lower percentage of naive T cells than CT neonates (CD4+, 76.6 vs 83.1%, P < 0.001; CD8+, 70.9 vs 79.6%, P = 0.003) and higher percentages of central memory T cells than CT neonates (CD4+, 13.9 vs 8.7%, P < 0.001; CD8+, 8.6 vs 4.8%, P = 0.001). CD38 mean fluorescence intensity of T cells was higher in ENI neonates (CD4+, 62.2 vs 52.1, P = 0.007; CD8+, 47.7 vs 35.3, P < 0.001). At 12 months, ENI infants still had higher mean fluorescence intensity of CD38 on T cells (CD4+, 34.2 vs 23.3, P < 0.001; CD8+, 26.8 vs 19.4, P = 0.035). Despite effective maternal virologic control at delivery, HIV-exposed uninfected children were born with lower levels of naive T cells. Immune activation was present at birth and remained until at least 12 months of age, suggesting that in utero exposure to HIV causes subtle immune abnormalities.

A DNA enzyme targeting Egr-1 inhibits rat vascular smooth muscle cell proliferation by down-regulation of cyclin D1 and TGF-β1

We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-β1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73% VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-β1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-β1. However, ED5 has no effect on VSMC apoptosis.

Effect of aerobic training on EEG alpha asymmetry and depressive symptoms in the elderly: a 1-year follow-up study

The effect of physical exercise on the treatment of depressive elderly adults has not been investigated thus far in terms of changes in cortical hemispheric activity. The objective of the present study was to identify changes in depressive symptoms, quality of life, and cortical asymmetry produced by aerobic activity. Elderly subjects with a diagnosis of major depressive disorder (DSM-IV) were included. Twenty patients (70% females, 71 ± 3 years) were divided into an exercise group (pharmacological treatment plus aerobic training) and a control group (undergoing pharmacological treatment) in a quasi-experimental design. Pharmacological treatment was maintained stable throughout the study (antidepressants and anxiolytics). Subjects were evaluated by depression scales (Beck Depression Inventory, Hamilton Depression Rating Scale, Montgomery-Asberg Depression Rating Scale) and the Short Form Health Survey-36, and electroencephalographic measurements (frontal and parietal alpha asymmetry) before and after 1 year of treatment. After 1 year, the control group showed a decrease in cortical activity on the right hemisphere (increase of alpha power), which was not observed in the exercise group. The exercise group showed a significant decrease of depressive symptoms, which was not observed in the control group. This result was also accompanied by improved treatment response and remission rate after 1 year of aerobic exercise associated with treatment. This study provides support for the effect of aerobic training on alpha activity and on depressive symptoms in elderly patients. Exercise facilitates the treatment of depressive elderly adults, leading to clinical and physical improvement and protecting against a decrease in cortical activity.

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.

Efeito da temperatura de extrusão na absorção de água, solubilidade e dispersibilidade da farinha pré-cozida de milho-soja (70:30)

Com o objetivo de verificar a possibilidade do uso das farinhas pré-cozidas de milho-soja (70:30) por extrusão em produtos alimentícios, foram estudadas suas características tecnológicas. A mistura crua de milho e soja foi extrusada num extrusor Brabender de dupla-rosca com velocidade de alimentação constante de 20,8Kg/h, velocidade da rotação de parafuso de 100rpm e diâmetro de matriz de 5mm. Os perfis da temperatura de barril (TB) foram: zona 1,60ºC (constante); zona 2,90ºC (constante); zona 3, 90, 110, 130 ou 150ºC e zona 4 (matriz), 110, 130 ou 150ºC. Os resultados mostraram que, o aumento da TB nas zonas 3 e 4 causou um aumento na absorção de água, nitrogênio solúvel em água, índice de solubilidade de nitrogênio, proteína dispersível em água e índice de dispersibilidade de proteína. Não foi verificada a atividade do inibidor de tripsina nas farinhas extrusadas estudadas. Sugere-se, portanto, o uso destas nos produtos cárneos e de panificação.