Identification of metabolically active Chlamydia pneumoniae in symptomatic carotid atherosclerotic plaques.

Objective: Recent reports suggest a causative role of Chlamydophila pneumoniae (CP) in the development of atherosclerosis and its complications. The aim of this study was to investigate the presence of metabolically active C. pneumoniae in human carotid artery plaques retrieved at surgery, by means of tissue culture and PCR and RT-PCR. Methods: Carotid endarterectomy (CE) specimens were obtained at time of surgery from ten patients (six males, four females; mean age: 74 years; five symptomatic) with hemodynamically significant carotid artery stenosis. Under aseptic conditions each CE specimen was divided and cut into three parts, namely: a proximal part(the common carotid artery at the upstream end, that appeared to contain diffuse atherosclerosis or fatty streaks, as well as plaque free segments), a medial part (the carotid bulb, containing raised plaque), and a distal part (the internal carotid artery, at the downstream end, i.e. above the raised plaque). The pieces obtained were immediately homogenized by freezing at -80 °C and grinding with a scalpel. DNA and total RNA extraction from homogenate aliquots was performed. C. pneumoniae 16S rRNA (coding for the small subunit of bacterial ribosomes), MOMP (major outer membrane protein) and Hsp 60 (60 kDa heat shock protein) geneexpression was investigated by means of PCR and RT-PCR. Results: Detection of C. pneumoniae Hsp-60 and 16s rRNA gene expression was positive in only two samples, corresponding to the proximal part of CE specimens retrieved at surgery from two patients with recent symptoms of cerebral ischemia (<8 weeks before surgical intervention). Interestingly PCR and RT-PCR analysis of the specimens of other three patients with a previous, but not recent history of cerebral ischemia, resulted negative. Analysis of C. pneumoniae MOMP gene expression was negative in all samples, regardless of severity and onset of symptomaticity. Conclusions: DNA and RNA amplification from different portions of carotid atherosclerotic plaques proved to be a useful tool to investigate C. pneumoniaelocalization and metabolic activity within carotid atheromatous tissue. The evidence of the gene expression of C. pneumoniae Hsp60 and 16-s rRNA only in the C.E. specimen of two patients with recent ischemic symptoms (i.e. "retinal arteryembolism" and transient hemiparesis) may suggest an implication of metabolically active C. pneumoniae in unstabilization of carotid atheromatous plaque.