David Stirling

A short history of the polymerase chain reaction.

A Short History of the Polymerase Chain Reaction. In the 1950s, the discovery of PCR is the subject of claim and counterclaim that has yet to be.Methods Mol Biol. A short history of the polymerase chain reaction. Author information: 1Division of Cancer and.A Short History of the Polymerase Chain Reaction. Full Text, Download PDF 103K. And although this does risk overstating the impact of PCR outside the scientific community, the comparison.

Interleukin 6 and Haemostasis

Interleukin 6 (IL-6) is a 26 kDa protein produced by many cell types including activated monocytes and macrophages, endothelial cells, adipose cells and the Th-2 subset of Thelper cells (Aarden et al, 1987; Jirik et al, 1989; MohamedAli et al, 1997; Laharrague et al, 2000). After interaction with a specific saturable receptor, present on a great variety of responsive cells, it promotes a range of activities (Fig 1 and Table I) including antiviral effects (Sehgal & Sagar, 1980), enhancement of proliferation of haemopoietic progenitors (Wong et al, 1988) and induction of acutephase reactant release from hepatocytes (Gauldie et al, 1990) ± a feature shared with other cytokines, collectively known as the IL-6 cytokine family. It is also this last action that leads to the production of C-reactive protein (CRP), a useful surrogate marker of IL-6 (Heinrich et al, 1990). IL-6 may also be involved in the pathogenesis of disease as has been demonstrated in myeloma (Kawano et al, 1988). The subject of this review, however, is the role of IL-6 in haemostasis, which is mediated through a series of differing effects on endothelial cells, leucocytes and hepatocytes with promotion of synthesis of coagulation factors such as fibrinogen, tissue factor and factor VIII (Amrani, 1990; Cermak et al, 1993; Stirling et al, 1998), and also through stimulation of platelet production (Burstein, 1997).

JAK2 V617F and CALR mutations are not mutually exclusive; findings from retrospective analysis of a small patient cohort

Lopes, R.D., Hylek, E.M., Hanna, M., Al-Khalidi, H.R., Ansell, J., Atar, D., Avezum, A., Bahit, M.C., Diaz, R., Easton, J.D., Ezekowitz, J.A., Flaker, G., Garcia, D., Geraldes, M., Gersh, B.J., Golitsyn, S., Goto, S., Hermosillo, A.G., Hohnloser, S.H., Horowitz, J., Mohan, P., Jansky, P., Lewis, B.S., Lopez-Sendon, J.L., Pais, P., Parkhomenko, A., Verheugt, F.W., Zhu, J. & Wallentin, L. (2011) Apixaban versus warfarin in patients with atrial fibrillation. New England Journal of Medicine, 365, 981–992. Keeling, D., Baglin, T., Tait, C., Watson, H., Perry, D., Baglin, C., Kitchen, S. & Makris, M. (2011) Guidelines on oral anticoagulation with warfarin fourth edition. British Journal of Haematology, 154, 311–324. Linkins, L.A., Choi, P.T. & Douketis, J.D. (2003) Clinical impact of bleeding in patients taking oral anticoagulant therapy for venous thromboembolism: a meta-analysis. Annals of Internal Medicine, 139, 893–900. NHS Improvement (2009) Heart and Stroke Improvement. Commissioning for Stroke Prevention in Primary Care. The Role of Atrial Fibrillation. www.improvement.nhs.uk/heart/portals/docu ments2009. Patel, M.R., Mahaffey, K.W., Garg, J., Pan, G., Singer, D.E., Hacke, W., Breithardt, G., Halperin, J.L., Hankey, G.J., Piccini, J.P., Becker, R.C., Nessel, C.C., Paolini, J.F., Berkowitz, S.D., Fox, K.A. & Califf, R.M. (2011) Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. New England Journal of Medicine, 365, 883–891.

Four factor deficiency.

Four factor deficiency is variably associated with mild to fatal bleeding. We describe a 3-month-old boy, born of consanguineous parents, who presented with a right subdural haematoma and a clotting screen showing a prothrombin time (PT) > 100 s, an activated partial thromboplastin time (aPTT) > 150 s, a fibrinogen of 0.4 g/l, and fibrinogen degradation products < 1 microg/ml. He was given 300 U of factor IX concentrate (containing factors II and X) and 1 mg of vitamin K intravenously. Forty-five minutes later, clotting tests showed a PT of 24 s, an aPTT of 31 s and a fibrinogen of 2.6 g/l. The patient was found to be deficient in all the vitamin K-dependent factors: factors II, VII, IX and X, protein C and protein S. A 14-base deletion was found in intron 1 (bases 1056-1069) of the gamma-carboxylase gene. The patient and his elder sister were homozygous for this deletion, whereas both parents were heterozygous. The deletion destroys a reverse palindromic sequence (TTGAGGCAA) of the type often associated with cis-acting elements. Our results suggest that this element may be involved in the regulation of gamma-carboxylase expression. Expression studies are being completed so that this region can be definitively ascribed as a cis-acting element involved in gene regulation.

TGF‐β is not the principal immunosuppressive component in coagulation factor concentrates

Coagulation factor concentrates are known to inhibit a variety of immune reactions when assessed in vitro. This study assessed the immunomodulatory activity of a wide range of coagulation factor concentrates by measuring their inhibition of PHA‐stimulated lymphocyte proliferation and reduction in IL‐2 secretion. The hypothesis that TGF‐β is responsible for most of these effects was tested by measuring biologically active TGF‐β and immunoreactive TGF‐β1 in the concentrates and comparing the levels recorded with immunosuppressive activity. In addition, the coagulation factors were compared directly with a standard preparation of TGF‐β in a TGF‐β‐specific bioassay and in lymphocyte proliferation assays. Although there was a broad correlation between levels of total or active TGF‐β and immunosuppressive activity across all of the coagulation factors tested, individual data sets showed clear discrepancies. Implying that TGF‐β probably serves as a surrogate marker for other immunomodulatory contaminants and that neither TGF‐β nor any other single substance could account for all of the immunosuppressive activity observed. Furthermore, there was a difference of more than 100‐fold in the relative potencies of coagulation factors and pure TGF‐β, when compared in immunosuppression assays, indicating that the different assays did not measure the same substance. Whereas anti‐TGF‐β antibody almost completely blocked the activity of coagulation factor concentrates (TGF‐β‐specific bioassay) and abrogated the effect of authentic TGF‐β (immunosuppression assays) at high concentrations it achieved <50% reversal of the immunosuppressive effects of coagulation factors in immunosuppression assays. These findings indicated that TGF‐β accounted for only a minor proportion of the immunosuppressive activity in most coagulation factor concentrates.

A novel method for quantitation of human von Willebrand factor messenger RNA

We have developed a novel assay for quantitation of human von Willebrand factor (vWF) messenger RNA (mRNA) using the ABI 7700 quantitative PCR system (Applied Biosystems, Warrington, Cheshire, UK). To allow accurate measurement of the vWF mRNA concentration in samples, we have used 18S ribosomal RNA as an internal control. The specificity of the assay was demonstrated by polyacrylamide gel electrophoresis and ABI Prism BigDye terminator cycle sequencing of the PCR product. We have also assessed the assay by using RNA from both a hepatocyte cell line which does not express vWF mRNA, and an endothelial cell line which does express vWF mRNA. By mixing varying amounts of these RNAs together, we were able to adjust the vWF quantity in known increments while keeping the 18S ribosomal RNA internal control constant. The quantitation of vWF mRNA correlated with the proportion of endothelial cell RNA (r(2)=0.912, p<0.001).