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In order to elucidate tumoral progression and drug resistance, cultured cell lines are valuable tools applied on tumor related assays provided they are well established and characterized. Our laboratory settled the NG97 cell line derived from a human astrocytoma grade III, which started to develop and express important phenotypical characteristics of an astrocytoma grade IV after injection in the flank of nude mice. Astrocytomas are extremely aggressive malignancies of the Central Nervous System (CNS) and account for 46% of all primary malignant brain tumors. Progression to worse prognosis occurs in 85% of the cases possibly due to changes in cell tumor microenvironment and through biological pathways that are still unclear.~Background~Background~This work focused on characterizing the NG97 cell line specifically after being recovered from the xenotransplant, who maintained their undifferentiated characteristics along the following 60th passages in vitro. These cells were subcultivated to evaluate the possible contribution of these undifferentiated characteristics to the malignant progression phenotype. These characteristics were the expression of molecules involved in the processes of migration, dedifferentiation and chromosomal instability.~Methods~Methods~Results showed that NG97(ht) had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP) and vimentin. In addition, beta1 integrins were present in intermediate levels while alpha5 integrins had a high expression profile as well as fibronectin and laminin. Cytogenetic analysis of NG97(ht) revealed several chromosomal abnormalities, 89% of the cells showed to be hyperdiploid and the modal number was assigned to be 63. Several acrocentric chromosomes were visualized and at least 30 figures were attributed to be murine. These findings suggest a possible fusion between the original NG97 cells with stromal murine cells in the xenotransplant.~Results~Results~In this study the NG97(ht) cells were characterized to embryonic recovery patterns of intermediate filaments, adhesion molecules expression, chromosomal imbalances and murine chromosomes. In the latter case, these presumably chromosomes were originated as fusions between murine stroma cells and NG97 cell lineage in the xenotransplant. Our results emphasize important queries about astrocytomas tumor progression.~Conclusion~Conclusions [ABSTRACT FROM AUTHOR]

Objective: To evaluate cell-mediated immune response to Bacillus Calmette-Guérin (BCG) vaccination in uninfected, HIV-1-exposed infants, comparing it with unexposed children., Design: It is designed as a cross-sectional study., Methods: BCG-specific lymphoproliferation and T-cell subsets (CD4(+), CD8(+) and TCR γδ(+)) by flow cytometry and interleukin-10, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) concentration by ELISA were analyzed in HIV-exposed and unexposed infants. Whole blood lymphocyte immunophenotyping and blood counts were performed in exposed children. Nonparametric tests were used (P < 0.05)., Results: Given the ontogeny of the immune system, exposed infants were separated into three groups according to age: exposed 1 (E1, aged 6.1-8.8 months), E2 (aged 9.1-17.1 months) and E3 (aged 18.1-26.3 months). Unexposed infants (UE group) and E1 were matched for age. Cell proliferation was not different among the three exposed groups, neither for BCG nor for phytohemagglutinin (PHA)-stimulated cultures. Furthermore, BCG-stimulated lymphoproliferation was reduced in the E1 group in comparison with the UE group. T-lymphocyte subpopulations also showed differences, with the youngest HIV-exposed groups (E1 and E2) showing a predominant proliferation of CD4(+) T cells in cultures with BCG, whereas E3 and UE groups had a robust γδ(+) T-cell expansion. There was lower IFN-γ concentration in the samples from E1 group in comparison with all of the other groups. The unexposed infants showed higher TNF-α concentration in cultures with BCG and PHA in comparison with E1 group., Conclusion: BCG-specific T-cell proliferation was reduced in HIV-exposed uninfected infants and IFN-γ concentration was lower in younger exposed infants, showing a delay in immune system maturation of HIV-exposed infants.

Little is known about the vaccine protective response for infants born from HIV-infected mothers. We evaluated the antibody response to hepatitis B, tetanus, and diphtheria vaccine in vertically HIV-exposed uninfected infants and compared them to those of control infants not exposed to the virus. The quantitative determination of specific neutralizing antibodies against hepatitis B, diphtheria, and tetanus were performed blindly on serum samples. The results showed that 6.7% of the HIV-exposed uninfected individuals were nonresponders to hepatitis B vaccine (anti-HBs titer, <10 mIU/ml), and 64.4% were very good responders (anti-HBs titer, ≥1,000 mIU/ml), whereas only 3.6% of the nonexposed infants were nonresponders (χ(2)=10.93; 1 df). The HIV-exposed uninfected infants showed protective titers for diphtheria and tetanus but lower geometric mean anti-tetanus titers compared to those of the HIV-unexposed infants. Our data point to the necessity of evaluating vaccine immune responses in these children and reinforced that alterations in lymphocyte numbers and functions reported for newborns from HIV-infected mothers interfere with the vaccine response.

On the basis of transmission electron microscopy observations in tumor cell lines, oncologists have made innumerous diagnostic and therapeutical progresses. Following this path, the UNICAMP immunopathologies laboratory established the NG97 cell line derived from a human astrocytoma grade III, which when injected to the athymic nude mouse flank developed a grade IV astrocytoma. In this study, we focused on ultrastructural characterization of the NG97 cells after being recovered from xenotransplant (NG97ht). These cells in culture were assayed by two different electron microscopy procedures to characterize ultrastructures related to grade IV astrocytomas and to observe their structures through cell subcultivation. Additionally, comparative morphological descriptions of different cell passages in these technical procedures could be a useful tool for improving electron microscopy cell lineage protocols. Results from many cell passage observations showed ultrastructural similarities, which suggest malignant and glioblastoma phenotypes. In the first procedure, NG97ht cells were harvested and then incorporated into agarose before subjecting them to electron microscopy protocols, whereas in the second one, monolayer cells grew first on cover slides. Comparison among protocols revealed that organelles, cytoplasmatic extensions, spatial conformation of filopodia, and cell attachment to substrate were more preserved in the second procedure. Furthermore, in this latter procedure, a unique ellipsoidal structure was observed, which was already described when dealing with gliosarcoma cell line elsewhere. Therefore, these analyses demonstrated a morphological characterization of a new NG97ht cell line using electron transmission microscopy. Moreover, it has been shown that the second procedure provides more detailed information compared with the first.

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gammadelta-positive (gammadelta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gammadelta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gammadelta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gammadelta(+) cell expansions were similar with the two vaccines.