Questioning the utility of pooling samples in microarray experiments with cell lines

We describe a microarray experiment using the MCF-7 breast cancer cell line in two different experimental conditions for which the same number of independent pools as the number of individual samples was hybridized on Affymetrix GeneChips. Unexpectedly, when using individual samples, the number of probe sets found to be differentially expressed between treated and untreated cells was about three times greater than that found using pools. These findings indicate that pooling samples in microarray experiments where the biological variability is expected to be small might not be helpful and could even decrease one's ability to identify differentially expressed genes.     

Nitric oxide activation of p38 mitogen-activated protein kinase in 293T fibroblasts requires cGMP-dependent protein kinase

An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated protein kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent protein kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent protein kinase-1alpha caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMP-dependent protein kinase.     

Preclinical development of an in vivo BCG challenge model for testing candidate TB vaccine efficacy

There is an urgent need for an immunological correlate of protection against tuberculosis (TB) with which to evaluate candidate TB vaccines in clinical trials. Development of a human challenge model of Mycobacterium tuberculosis (M.tb) could facilitate the detection of such correlate(s). Here we propose a novel in vivo Bacille Calmette-Guérin (BCG) challenge model using BCG immunization as a surrogate for M.tb infection. Culture and quantitative PCR methods have been developed to quantify BCG in the skin, using the mouse ear as a surrogate for human skin. Candidate TB vaccines have been evaluated for their ability to protect against a BCG skin challenge, using this model, and the results indicate that protection against a BCG skin challenge is predictive of BCG vaccine efficacy against aerosol M.tb challenge. Translation of these findings to a human BCG challenge model could enable more rapid assessment and down selection of candidate TB vaccines and ultimately the identification of an immune correlate of protection.     

Two Doses of Candidate TB Vaccine MVA85A in Antiretroviral Therapy (ART) Na?ve Subjects Gives Comparable Immunogenicity to One Dose in ART+ Subjects

Tuberculosis (TB) is a global public health problem exacerbated by the HIV epidemic. Here we evaluate a candidate TB vaccine, MVA85A, in a Phase I study in HIV-infected adults in Senegal. 24 patients were enrolled: Group 1∶12, antiretroviral therapy (ART) naïve, adults, with CD4 counts >300 and HIV RNA load <100 000 copies/ml. Group 2∶12 adults, stable on ART, with CD4 counts >300, and an undetectable HIV RNA load. Safety was evaluated by occurrence of local and systemic adverse events (AEs) and by monitoring of CD4 count, HIV RNA load, haematology and biochemistry. Immunogenicity was evaluated by ex-vivo interferon-gamma ELISpot assay. 87.7% of AEs were mild; 11.6% were moderate; and 0.7% were severe. 29.2% of AEs were systemic; 70.8% were expected local AEs. There were no vaccine-related Serious Adverse Events (SAEs) or clinically significant effects on HIV RNA load or CD4 count. In ART naive subjects, the first MVA85A immunisation induced a significant immune response at 1 and 4 weeks post-immunisation, which contracted to baseline by 12 weeks. Durability of immunogenicity in subjects on ART persisted out to 24 weeks post-vaccination. A second dose of MVA85A at 12 months enhanced immunogenicity in ART naïve subjects. Subjects on ART had higher responses after the first vaccination compared with ART naïve subjects; responses were comparable after 2 immunisations. In conclusion, MVA85A is well-tolerated and immunogenic in HIV-infected subjects in Senegal. A two dose regimen in ART naïve subjects is comparable in immunogenicity to a single dose in subjects on ART.Clinicaltrials.gov trial identifier {"type":"clinical-trial","attrs":{"text":"NCT00731471","term_id":"NCT00731471"}}NCT00731471.     

Alternative communication systems for people with severe motor disabilities: a survey

We have now sufficient evidence that using electrical biosignals in the field of Alternative and Augmented Communication is feasible. Additionally, they are particularly suitable in the case of people with severe motor impairment, e.g. people with high-level spinal cord injury or with locked-up syndrome. Developing solutions for them implies that we find ways to use sensors that fit the user's needs and limitations, which in turn impacts the specifications of the system translating the user's intentions into commands. After devising solutions for a given user or profile, the system should be evaluated with an appropriate method, allowing a comparison with other solutions. This paper submits a review of the way three bioelectrical signals - electromyographic, electrooculographic and electroencephalographic - have been utilised in alternative communication with patients suffering severe motor restrictions. It also offers a comparative study of the various methods applied to measure the performance of AAC systems.