Innovative electrochemical approach for an early detection of microRNAs

The recent findings of circulating cell-free tissue specific microRNAs in the systemic circulation and the potential of their use as specific markers of disease highlight the need to make microRNAs testing a routine part of medical care. At the present time, microRNAs are detected by long and laborious techniques such as Northern blot, RT-PCR, and microarrays. The originality of our work consists in performing microRNAs detection through an electrochemical genosensor using a label-free method. We were able to directly detect microRNAs without the need of PCR and a labeling reaction. The test is simple, very fast and ultrasensitive, with a detection limit of 0.1 pmol. Particularly feasible for a routine microRNAs detection in serum and other biological samples, our technical approach would be of great scientific value and become a common method for simple miRNAs routine detection in both clinical and research settings

A shift to Th0 cytokine production by CD4(+) cells in human longevity: Studies on two healthy centenarians

Centenarians, particularly healthy centenarians, constitute the example of successful aging and the study of their immune status can help to define the endpoint of the changes occurring throughout life. We characterized T cell clones (TCC) of two healthy centenarians, studying their phenotypes and production of representative Th1 and Th2 cytokines (IFN-gamma and IL-4) and compared them with TCC obtained by three young normal subjects; in all 180 TCC were analyzed, In young donors, 35 TCC were CD4(+), 56 CD8(+) and 2 were alpha beta(+)CD4(-)CD8(-) (double negative). In centenarians, we obtained 46 CD4(+) TCC, 38 CD8(+), 2 CD4(+)CD8(+) (double positive) and 1 gamma delta(+) double negative. Of the young subjects' TCC, 71% produced IFN-gamma but no IL-4 (Th1 pattern) and this prevalence decreased to 39% in TCC from the centenarians. The number of clones showing the opposite Th2 pattern was similar in young and aged donors (3 out of 93 TCC and 2 out of 87 TCC, respectively). The intermediate profile of TCC producing both IL-4 and IFN-gamma (Th0) was found in 25.8% of clones from young people, but it almost doubled to 58.6% in centenarians. The analysis shows that the Th profiles of CD8(+) TCC is nearly superimposable in the two groups, whereas a major shift from a Th1 to a Th0 pattern is presented by CD4(+) TCC. The balance provided by a majority of CD4(+) TCC showing a Th0 pattern may ensure both humoral and cell-mediated defences. In CD8(+) TCC, however, a Th1 pattern still is present, possibly for efficient generation of cytotoxic responses. These findings should be extended by studying other centenarians and elderly subjects

T cell responses to highly active antiretroviral therapy defined by chemokine receptors expression, cytokine production, T cell receptor repertoire and anti-HIV T-lymphocyte activity

The immunological correlates of highly active antiretroviral therapy (HAART)-induced suppression of human immunodeficiency virus type 1 (HIV-1) replication have been investigated. 20 HIV-1-infected patients with mean CD4(+) T cell count of 298/mul, plasma viral load of 4.7 log(10) copies/ml and naive for protease inhibitors (PI) were studied during 12 months of HAART. An increased number of both CD4(+) and CD8(+) naive T cells and a normalization of the frequency of CCR5- and CXCR4-expressing CD4(+) T cells were readily observed after starting therapy. Single cell analysis of cytokine production after 12 months of HAART showed an increased number of interleukin (IL)-2-, but not IIL-4- and (IFN)-gamma-, producing T cells and a decreased percentage of CD8+ TFN-gamma + cells. A correlation between the frequency of IFN-gamma-producing T cells and that of memory, CCR5(+) and CD95(+) T cells was demonstrated in both CD4(+) and CD8(+) subsets. The diversity of T cell receptor (TCR) variable beta (BV) chain repertoire significantly increased after 12 months of HAART within the CD4(+) but not the CD8(+) T cell subset. However, the level of perturbation of the third complementarity-determining region (CDR3), was not significantly modified by effective therapy. The number of anti-HIV Gag and Pol cytotoxic T lymphocytes precursors (CTLp) decreased during HAART and highly correlated with the CD8 IFN-gamma response. Ameliorated clinical conditions were observed in all patients in absence of any opportunistic infections during all the study period. These observations indicate that a better restoration of immunity may be obtained in patients starting HAART at less advanced stages of the disease