Browsing by Author "Chakrewarthy, S."

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    Optimization of reverse transcriptase PCR for selected hepatic cytokines in Wistar Rats
    (Faculty of Graduate Studies, University of Kelaniya, 2015) Samaranayake, H.A.E.; Thammitiyagodage, M.G.; Galhena, B.P.; Chakrewarthy, S.; Wickremasinghe, A.R.
    Expression patterns of hepatic cytokines elucidates the immune and pathological pathways involved in inflammatory responses. Cytokine mRNA quantification is widely used approach in this regard that involves RNA extraction, cDNA synthesis and real-time polymerase chain reaction of selected targets. In the present study, we optimized the reverse transcriptase PCR conditions for selected hepatic cytokines; TNF alpha and IL 6 in Wistar Rats. Liver tissues obtained from Wistar rats were washed with diethylpyrocarbonate (DEPC) treated water and frozen immediately in liquid nitrogen. Samples were stored at -800C. Total RNA was extracted from 0.1 g of liver tissue using Trizol@ according to the manufacturer‘s instructions. Subsequently, cDNA was synthesized from 2000ng of RNA using random primers and M-MLV reverse transcriptase enzyme. PCR for target cytokines was carried out using newly synthesized cDNA based on following PCR conditions. For TNF alpha, 5'-TTC TGT CTA CTG AAC TTG GGG GTG ATC GGT CC-3' and 5'-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG -3' were used as primers. PCR was optimized with initial denaturation at 940C for 1 and 30 sec followed by 35 cycles of 30 sec denaturation at 940C, 1 min annealing and 1 min extension at 720C. A temperature gradient of 530C, 550C and 570C was used for annealing step. Final extension was done at 720C for 3 min. For IL 6, 5‘-TCC TAC CCC AAC TTC CAA TGC TC-3‘ and 5‘-TTG GAT GGT CTT GGT CCT TAG CC-3‘were used as primers. PCR was optimized with initial denaturation at 940C for 1 and 30 sec followed by 35 cycles of 30 sec denaturation at 940C, 1 min annealing and 1 min extension at 720C. A temperature gradient of 570C, 590C, and 610C was used for annealing step. Final extension was done at 720C 3 min. Based on PCR products of TNF alpha and IL-6 separated by agarose gel electrophoresis, annealing temperatures for both genes were decided as 550C and 590C respectively.
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    Validation of the World Health Organization/ International Society of Hypertension (WHO/ISH) cardiovascular risk predictions in Sri Lankans based on findings from a prospective cohort study
    (Public Library of Science, 2021) Thulani, U.B.; Mettananda, K.C.D.; Warnakulasuriya, D.T.D.; Peiris, T.S.G.; Kasturiratne, K.T.A.A.; Ranawaka, U.K.; Chakrewarthy, S.; Dassanayake, A.S.; Kurukulasooriya, S.A.F.; Niriella, M.A.; de Silva, S.T.; Pathmeswaran, A.; Kato, N.; de Silva, H.J.; Wickremasinghe, A.R.
    INTRODUCTION AND OBJECTIVES: There are no cardiovascular (CV) risk prediction models for Sri Lankans. Different risk prediction models not validated for Sri Lankans are being used to predict CV risk of Sri Lankans. We validated the WHO/ISH (SEAR-B) risk prediction charts prospectively in a population-based cohort of Sri Lankans. METHOD: We selected 40-64 year-old participants from the Ragama Medical Officer of Health (MOH) area in 2007 by stratified random sampling and followed them up for 10 years. Ten-year risk predictions of a fatal/non-fatal cardiovascular event (CVE) in 2007 were calculated using WHO/ISH (SEAR-B) charts with and without cholesterol. The CVEs that occurred from 2007-2017 were ascertained. Risk predictions in 2007 were validated against observed CVEs in 2017. RESULTS: Of 2517 participants, the mean age was 53.7 year (SD: 6.7) and 1132 (45%) were males. Using WHO/ISH chart with cholesterol, the percentages of subjects with a 10-year CV risk <10%, 10-19%, 20%-29%, 30-39%, ≥40% were 80.7%, 9.9%, 3.8%, 2.5% and 3.1%, respectively. 142 non-fatal and 73 fatal CVEs were observed during follow-up. Among the cohort, 9.4% were predicted of having a CV risk ≥20% and 8.6% CVEs were observed in the risk category. CVEs were within the predictions of WHO/ISH charts with and without cholesterol in both high (≥20%) and low(<20%) risk males, but only in low(<20%) risk females. The predictions of WHO/ISH charts, with-and without-cholesterol were in agreement in 81% of subjects (ĸ = 0.429; p<0.001). CONCLUSIONS: WHO/ISH (SEAR B) risk prediction charts with-and without-cholesterol may be used in Sri Lanka. Risk charts are more predictive in males than in females and for lower-risk categories. The predictions when stratifying into 2 categories, low risk (<20%) and high risk (≥20%), are more appropriate in clinical practice.