Background-Recovery of the intracellular bacterium Chlamydia pneumoniae from atherosclerotic plaques has initiated large studies on antimicrobial therapy in coronary artery disease. The basic concept that antibiotic therapy may eliminate and prevent vascular infection was evaluated in vitro and in vivo by examining the antibiotic susceptibility of C pneumoniae in circulating human monocytes, which are thought to transport chlamydiae from the respiratory tract to the vascular wall. Methods and Results-Blood monocytes (CD14ϩ) from 2 healthy volunteers were obtained before and after oral treatment with azithromycin or rifampin and then inoculated with a vascular C pneumoniae strain and continuously cultured in the presence of the respective antibiotic. Progress of infection and chlamydial viability was assessed by immunogoldlabeling and detection of C pneumoniae-specific mRNA transcripts. Circulating monocytes from patients undergoing treatment with experimental azithromycin for coronary artery disease were examined for C pneumoniae infection by cell culture. Antibiotics did not inhibit chlamydial growth within monocytes. Electron microscopy showed development of chlamydial inclusion bodies. Reverse transcription-polymerase chain reaction demonstrated continuous synthesis of chlamydial mRNA for 10 days without lysis of the monocytes. The in vivo presence of viable pathogen not eliminated by azithromycin was shown by cultural recovery of C pneumoniae from the circulating monocytes of 2 patients with coronary artery disease. Key Words: Chlamydia pneumoniae Ⅲ atherosclerosis Ⅲ infection Ⅲ azithromycin Ⅲ rifampin T he obligate intracellular pathogen Chlamydia pneumoniae has been related to atherosclerosis by seroepidemiology, detection of chlamydial structures and, most recently, recovery of viable chlamydiae from atherosclerotic plaques. 1,2 Chlamydiae may thus have a role in the multifactorial pathogenesis of atherosclerosis, a chronic inflammatory condition of the vascular wall. 3,4 On the basis of these findings, antimicrobial treatment studies are currently underway, with the intention to improve the clinical condition of patients with coronary artery disease (CAD) by chlamydial eradication from atheromatous plaques. 5 Endothelial and smooth muscle cells can acquire a productive chlamydial infection that can be inhibited in vitro by common antichlamydial antibiotics. 6 However, chlamydiae are notorious for causing chronic infections, 7 and treatment failure is common. These infections and failures may be due to the establishment of a nonreplicating but viable state of chlamydiae in the host cells. 8 Systemic dissemination of C pneumoniae from the respiratory tract to the cells of the vascular wall requires a cellular transport system, because chlamydiae replicate exclusively within their host cells. The elementary body, the metabolically inactive extracellular life stage of chlamydiae, has never been found circulating free within the bloodstream. Circulating monocytes, which are pivotal to the development of ather...

P-selectin and its ligand, PSGL-1, are cell adhesion molecules that facilitate interaction of platelets, leukocytes and endothelial cells. Polymorphisms of these genes have been reported to be associated with coronary heart disease (CHD). In the present study, we characterized the entire coding regions of P-selectin and PSGL-1 genes in CHD patients and healthy controls. The 17 exons of the P-selectin gene and exon 2 of the PSGL-1 gene were screened for single nucleotide polymorphisms (SNPs) by exon re-sequencing in 88 CHD patients and 96 controls. For rapid genotyping of the SNPs we developed PCR techniques with sequence-specific primers (PCR-SSP). By using PCR-SSPs we genotyped 261 CHD patients and 214 controls for 5 SNPs in P-selectin and 2 SNPs in PSGL-1. In addition to the already described SNPs in P-selectin (S290N, N562D, V599L and T715P), we identified a novel SNP in exon 5 (V168M). The P-selectin 715P allele was more frequent among CHD patients with hypercholesterolemia compared to patients with normal cholesterol levels. A SNP (M621) in the PSGL-1 gene was found close to the P-selectin binding site and the 621 allele revealed a higher prevalence in the control group indicating a protective effect of the mutation. The molecular characterization of P-selectin and PSGL-1 in a case-control study including CHD patients and healthy controls revealed evidence for association of the genes with development of the disease. However, the functional role of the gene variants should be elucidated by further experimental data.

Because individual diagnoses of vascular infection with Chlamydia pneumoniae depend entirely on surgically removed tissues, a better assay to predict vascular infection is needed. Polymerase chain reaction detection of chlamydial DNA was applied to CD14-positive cells collected from 238 patients with angiographically identified unstable angina or acute myocardial infarction. C. pneumoniae was detected in 52 (28%) of 188 persons with unstable angina and in 13 (26%) of 50 persons with myocardial infarction. Differences between groups were not significant. C. pneumoniae is present in monocytes/macrophages of a significant proportion of persons with progressive coronary artery disease. Infarction is not accompanied by a rise in chlamydial detection rates. The potential role of chlamydiae in coronary atherosclerosis may therefore be more related to acceleration of disease or systemic effects by persistent infection than to sudden initiation of infarction by acute infection.

Chlamydia pneumoniae uses peripheral blood monocytes (PBMC) for systemic dissemination and has been linked to atherogenesis by inflammation mediated via TLR2/4 and CD14. We found 12.8% of 610 coronary artery disease (CAD) patients of Central European background to be chronically infected with C. pneumoniae based on the repeated detection of chlamydial DNA in PBMC. Among those the À159C4T CD14 promoter polymorphism was more frequent (OR 1.7, 95% CI 1.08-2.65, P ¼ 0.0224) than among C. pneumoniae-negative subjects matched for age and gender. The Arg753Gln TLR2 and Asp299Gly TLR4 polymorphisms were not related to chlamydial infection. Susceptibility for chronic chlamydial infection of PBMC in CAD patients appears associated with the CD14-159C4T promoter polymorphism encoding for enhanced CD14 expression.