Arindam Maitra, Jayashree Shanker, Debabrata Dash, Prathima Arvind, Vijay V. Kakkar components that serve as their ligands – peptidoglycans for TLR-2, lipopolysaccharides for TLR-4, flagellin for TLR-5 and unmethylated CpG motifs present in bacterial DNA for TLR-9 [6]. The TLRs initiate a cross talk between innate and adaptive immunity [7], resulting in activation of the nuclear factor-κB (NFκB) mediated inflammatory response via the direct regulation of immune responsive genes [8]. The TLR-mediated inflam- mation thus acts as an important pathogenic link between innate immunity and CAD [9]. Gene expression studies on the TLR family have shown that TLR-1, TLR-2 and TLR-4 genes are particularly elevated in human atherosclerotic lesions accompanied by parallel activation of TLR- expressing cells, as shown by the nuclear translocation of NFκB [10]. Recent findings have linked the TLR-mediated signalling cascade to established pathological determinants of plaque development and destabilization [11, 12]. Direct in vivo evidence on the pattern of TLR signalling in LDL receptor and apoE knock-out mice has shown that regulation of TLR expression is critical for maintaining immunological homeostasis [13]. TLRs have sparked great interest in recent times due to their potential for therapeutic manipulation of the innate immune system by way of alternate activation and down-regulation. TLRs can either serve as adjuvants for an immune-based vaccine by the induction of atheroprotective immunity [14] or as antagonists to TLR ligands, thereby arresting disease progression [15-17]. The present investigation is a part of the Indian Atherosclerosis Research Study (IARS), a large genetic epidemiological study on Asian Indians, where the underpinning objectives are to identify a potential vaccine to fight heart disease and to identify a strategic ‘high risk’ population for testing the vaccine. Given the key ability of TLRs to participate both in atheroprotective and pro- atherogenic immune responses, the objective of the present study was to assess the role of TLR in a cohort selected from the IARS by studying the expression levels of TLR-2, TLR-4 and TLR-9 as a pilot exploratory study. Material and methods All participants included in the study were selected from the IARS, an ongoing study, initiated and conducted by the Thrombosis Research Institute (TRI), India, to elucidate the involvement of genetic and non-genetic factors contributing to the aetiopathology of CAD among Asian Indians. The IARS participants were enrolled, based on predefined inclusion/exclusion criteria, from cardiac specialty hospitals in Bangalore and Mumbai situated in the southern and western regions of India. The CAD patients (n = 60) were selected from among the IARS probands who had a strong family history of CVD and age at onset of CAD ≤ 60 years for men and ≤ 65 years for women. All patients had angiographically confirmed presence of CAD. Patients having a history of primary myocardial disease or congenital heart disease were excluded from the study. The asymptomatic controls (n = 79) were matched for age and gender from among volunteers from the general population. The controls were considered healthy based on their ECG result and did not have a personal or family history of CAD. Individuals were considered to be diabetic or hypertensive based on self report of prescription medication. None of the patients were on insulin treatment. Individuals with concomitant infection were not enrolled in the study. Parti- cipation was by informed voluntary written consent. The IARS protocol was approved by the institutional ethics committee and is based on the Indian Council of Medical Research (ICMR) guidelines on Bioethics and the Helsinki Declaration for conducting research on human subjects [18]. RNA isolation Around 22 ml of blood sample was collected from all study participants. The serum, EDTA plasma and citrate plasma were separated into aliquots and preserved for biomarker assays while the blood pellet was used for DNA extraction. Around 3 ml of EDTA whole blood was used for gene expression studies. Total RNA was extracted from fresh whole blood using the QIAamp Blood Mini Kit (Qiagen Inc., Valencia, CA, USA) for RNA extraction as per the recommended protocol. Purified RNA was converted to cDNA using the cDNA archive kit (Applied Biosystems Inc., Foster Coty, CA, USA) and stored at –80°C. TLR assays and data analysis SYBR Green I based cDNA specific real time PCR assays for TLR-2, TLR-4, TLR-9 and GAPDH genes were designed and developed in house. Table I shows the forward and reverse primers as well as the PCR conditions used for the SYBR green assay. Expression analysis of TLR-2, TLR-4 and TLR-9 genes was performed with replicates of the cDNA, using the GAPDH gene as an endogenous control. Relative quantitation of gene expression was performed by a method described previously [19]. The intra-assay coefficient of variation (CV) of the relative quantities obtained for TLR-2, TLR-4 and TLR-9 was 5.9%, 6.4% and 7.6%, respectively, while the inter-assay CV for the same was estimated to be 7.3%, 9.4% and 8.1%, respectively. Statistical analysis Multivariate analysis of fold changes in relative gene expression was performed with and without 782 Arch Med Sci 5, October / 2011