Development of a selective and sensitive polymerase chain reaction assay for the detection of Mycoplasma pirum

Abstract A new assay using the polymerase chain reaction to amplify a 173-nucleotide DNA fragment within the 16S ribosomal RNA gene of Mycoplasma pirum has been developed. The assay selectively amplified DNA from all strains of M. pirum tested with a high level of sensitivity, even in a context of human DNA. DNA from other mollicute species, including those closely related to M. pirum , from bacteria phylogenetically close to mollicutes ( Clostridium innocuum, C. ramosum and Bacillus subtilis ), from Escherichia coli and from human peripheral blood mononuclear cells, did not produce the amplified DNA product specific for M. pirum .     

Chronic viral hepatitis C: management update

The management of chronic viral hepatitis C is evolving rapidly. Monotherapy with interferon, the accepted standard of treatment until recently, achieves only a modest sustained virological response rate of 15%. Combination treatment with alpha-2b interferon and ribavirin has been shown to increase sustained response rates to 40% in patients who have never been treated with interferon and to 50% in those who have relapsed following monotherapy with interferon. However, side effects, which have led to the discontinuation of combination treatment in a significant proportion of patients, must be carefully monitored. Treatment with interferon alpha-2b and ribavirin has now been approved in Canada, but the selection and monitoring of patients suitable for combination treatment requires special expertise. Although improvements in current therapeutic options may be possible with more frequent, higher doses or long-acting forms of interferon together with ribavirin, low sustained response rates (i.e., below 30%) for patients with hepatitis C virus genotype 1 emphasize the need for novel antiviral medications that will target the functional sites of the HCV genome.     

Effect of 17days of bed rest on peak isometric force and unloaded shortening velocity of human soleus fibers

The purpose of this study was to examine the effect of prolongedbed rest (BR) on the peak isometric force(P_o) and unloaded shorteningvelocity (V_o)of single Ca²⁺-activated musclefibers. Soleus muscle biopsies were obtained from eight adult malesbefore and after 17 days of 6° head-down BR. Chemicallypermeabilized single fiber segments were mounted between a forcetransducer and position motor, activated with saturating levels ofCa²⁺, and subjected to slacklength steps. V_owas determined by plotting the time for force redevelopment vs. theslack step distance. Gel electrophoresis revealed that 96% of the pre-and 87% of the post-BR fibers studied expressed only the slow type Imyosin heavy chain isoform. Fibers with diameter >100 µm made uponly 14% of this post-BR type I population compared with 33% of thepre-BR type I population. Consequently, the post-BR type I fibers(n = 147) were, on average, 5%smaller in diameter than the pre-BR type I fibers(n = 218) and produced 13% lessabsolute P_o. BR had no overalleffect on P_o per fibercross-sectional area(P_o/CSA), even though halfof the subjects displayed a decline of 9-12% inP_o/CSA after BR. Type Ifiber V_oincreased by an average of 34% with BR. Although the ratio of myosinlight chain 3 to myosin light chain 2 also rose with BR, there was nocorrelation between this ratio andV_o for either thepre- or post-BR fibers. In separate fibers obtained from the originalbiopsies, quantitative electron microscopy revealed a 20-24%decrease in thin filament density, with no change in thick filamentdensity. These results raise the possibility that alterations in thegeometric relationships between thin and thick filaments may be atleast partially responsible for the elevatedV_o of the post-BRtype I fibers.

     

Suppression by Thimerosal of Ex-Vivo CD4+ T Cell Response to Influenza Vaccine and Induction of Apoptosis in Primary Memory T Cells

Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine. Thimerosal was included in the multidose non-adjuvanted pandemic 2009 H1N1 vaccine Panenza. In the context of the analysis of the ex-vivo T cell responses directed against influenza vaccine, we discovered the in vitro toxicity Panenza, due to its content in thimerosal. Because thimerosal may skew the immune response to vaccines, we investigated in detail the ex-vivo effects of thimerosal on the fate and functions of T cells in response to TCR ligation. We report that ex-vivo exposure of quiescent or TCR-activated primary human T cells to thimerosal induced a dose-dependent apoptotic cell death associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, cytochrome c release from the mitochondria and caspase-3 activation. Moreover, exposure to non-toxic concentrations of thimerosal induced cell cycle arrest in G0/G1 phase of TCR-activated T cells, and inhibition of the release of proinflammatory cytokines such as IFN gamma, IL-1 beta, TNF alpha, IL-2, as well as the chemokine MCP1. No shift towards Th2 or Th17 cells was detected. Overall these results underline the proapoptotic effect of thimerosal on primary human lymphocytes at concentrations 100 times less to those contained in the multidose vaccine, and they reveal the inhibitory effect of this preservative on T-cell proliferation and functions at nanomolar concentrations.