ABSTRACT: To date there is no clear explanation as to how Chlamydia pneumoniae heat shock protein 60 (cHSP60) gets activated either through TLR-2/4, MAPKinase (p38/JNK/ERK), apoptotic/antiapoptotic, chemokines and inflammatory cytokines pathways leading to coronary artery disease (CAD). Hence to better understanding towards cHSP60 signaling in CAD patients, we performed experiments at RNA levels in cHSP60 positive and negative groups of CAD patients. For the determination of positivity for C. pneumoniae, Helicobacter pylori, Cytomegalovirus and Herpes Simplex Virus in atheromatous plaque multiplex Real Time PCR was performed. Monoplex Real Time PCR was also performed with 16S rRNA and HSP60 gene Chlamydia pneumoniae. Further study was performed only on cHSP60 positive (negative for H. pylori, CMV & HSV-1) and cHSP60 negative (also negative for H. pylori, CMV & HSV-1) CAD patients. 1. Patients Forty patients (28 males, 12 females) in age group (51±13 year) attending Department of Cardio Thoracic & Vascular Surgery, Safdarjung Hospital, New Delhi from September 2007 to April 2008 were enrolled in the study. These patients had manifestations of CAD and were undergoing open by-pass heart surgery. Prior written consent was obtained from each patient. The study received clearance from Ethical committee, Safdarjung hospital. Patients who had a recent acute coronary syndrome or transient ischemic attack were not included in our study. 2. Atheroma collection and handling Atheromatous tissue were collected in aseptic conditions and placed in 30 ml transport medium immediately upon resection. Three types of transport medium were used for tissue: viz. Dulbecco?s phosphate-buffered saline (PBS; Gibco Life Technologies, Paisley, Scotland) for DNA, RNA later (Ambion Inc., Woodward st. Austin, Texas, USA) for RNA and optimum cutting temperature compound (Sigma, St. Louis, MO, USA) for protein. 3. Atheroma DNA & RNA isolation and testing Total DNA was isolated for checking the positivity for C. pneumoniae from atheromatous plaques as described earlier [1]. Total RNA from the atheroma samples were isolated using RNeasy fibrous tissue mini kit (Qiagen Sciences, Maryland, USA) and the concentration was determined by a RNA dye-binding assay (Pico-Green, Molecular Probes, Eugene, OR). For cDNA synthesis, RETRO script (Ambion Inc., Woodward st. Austin, Texas, USA) was used as per manufacturer instructions. For detecting positivity for C. pneumoniae, Helicobacter pylori, Cytomegalovirus and Herpes Simplex Virus in atheromatous plaques, multiplex RT-PCR was performed [as describe in reference 1]. Monoplex RT PCR amplification primers and probes were custom designed by Applied Biosystems (Foster City, CA, USA). Briefly, sequences for the 16S rRNA and HSP60 gene of the organisms of interest were aligned and inspected for regions of conserved and variable sequences. 4. Microarray experiment Further study was performed only on cHSP60 positive (negative for H. pylori, CMV & HSV-1) and cHSP60 negative (also negative for H. pylori, CMV & HSV-1) CAD patients. cHSP60 positive samples represented the test samples and cHSP60 negative samples represented the control samples. 3 samples were analysed: samples 17 and 23 were biological replicates of test samples and also performed in duplicate as technical replicates, and sample 26 was a control sample and was also performed in duplicate as a technical replicate. Reference 1. Hem C Jha, Pragya Srivastava, Aabha Divya , Jagdish Prasad, Aruna Mittal. Prevalence of Chlamydophila pneumoniae is higher in aorta and coronary artery than in carotid artery of coronary artery disease patients. APMIS, 2009: 117 (12): 905-911.
