Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro. Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

Complementary and alternative medicine (CAM) in obstetrics and gynaecology: a survey of office-based obstetricians and gynaecologists regarding attitudes towards CAM, its provision and cooperation with other CAM providers in the state of Hesse, Germany.

PURPOSE: Whereas we have some information on complementary medicine in the field of oncology, little is known about complementary medicine in the field of obstetrics and gynaecology especially outside of hospitals. METHODS: All office-based obstetricians and gynaecologists in the state of Hesse, Germany, were contacted and asked to fill in an assessment form regarding cooperation in the field of complementary and alternative medicine (CAM), as well as the perceived efficacy of various CAM methods for a number of pathological conditions in the field of obstetrics and gynaecology. RESULTS: It was found that more than half of Hessian office-based obstetricians and gynaecologists had existing cooperation regarding CAM, especially with colleagues, but also midwives, pharmacists, physiotherapists, and health practitioners. The probability of cooperation was significantly inversely associated with age. It was found that the probability for advising CAM differed between various health problems. The following CAM methods were considered reasonable for the treatment of different conditions: phytotherapy for climacteric complaints and premenstrual syndrome; homoeopathy for puerperal problems; acupuncture and traditional Chinese medicine for complaints during pregnancy; and dietary supplements for the side effects of cancer therapy. CONCLUSIONS: The analysis shows that there is much cooperation in the field of CAM. Comparison between physicians' perceived efficacy of CAM methods and objective findings shows that there is a need for the provision of valid information in the field.

Caffeine inhibits cytokine expression in lymphocytes.

Caffeine alters intracellular calcium signalling patterns in lymphocytes which are important for the specific regulation of activation and effector function in lymphocytes. The effect of caffeine on calcium signalling is probably mediated via a ryanodine receptor type 3 dependent intracellular calcium store which releases calcium after exposure to caffeine. Also, caffeine decreases lymphocyte cytotoxicity against allogenic myocyte. Which cytotoxic mechanisms are actually altered by caffeine is unknown. In mouse splenocyte cultures containing about 87% lymphocytes we show that concanavalin A (ConA, 5 microg/ml) stimulated cells increase the expression of TNF-alpha, IL-2 and IFN-gamma (ELISA) significantly. Caffeine (3.75 mM) inhibits cytokine expression of ConA stimulated cells almost completely. Ryanodine (1 microM) specifically blocks ryanodine receptors and thereby prevents caffeine induced calcium release. In our experiments, however, ryanodine has no effect on ConA stimulated IL-2 and IFN-gamma expression and only suppresses TNF-alpha expression by 20%. Furthermore, ryanodine does not prevent the inhibitory effect of caffeine on TNF-alpha, IL-2 and IFN-gamma expression in stimulated effector cells. We postulate that caffeine suppresses cytokine expression and thereby contributes to decreased cytotoxicity of lymphocytes against allogenic myocytes. The ryanodine receptor dependent intracellular calcium store does not seem to play a significant role in this process. Possibly, the blockade of IP3 receptors by caffeine is more important for cytokine suppression.