The association of carotid plaque inflammation and Chlamydia pneumoniae infection with cerebrovascular symptomatology
Article Outline
Objective
Inflammation and infection have been implicated in the pathogenesis of carotid artery atherosclerosis, but their role in cerebrovascular disease symptomatology is not so well defined. We hypothesized that carotid disease symptomatology was associated with specific serologic markers of inflammation and Chlamydia pneumoniae infection and the presence of the pathogen and concentration of tumor necrosis factor-α (TNF-α) on the atheroma.
Methods
In 2004, 78 patients underwent carotid endarterectomy in our department, 46 of whom were symptomatic (group A) and 32 were asymptomatic (group B). A detailed medical history, the presence of atherosclerosis risk factors, ankle-brachial index and boxy mass index were recorded. We measured preoperatively the levels of C-reactive protein, fibrinogen, TNF-α, and the titers of immunoglobulin (Ig) A and IgG antibodies against C pneumoniae in the serum. Finally, the atherosclerotic plaques of all patients were immunohistochemically examined for the presence of C pneumoniae and their TNF-α concentration was determined.
Results
Isolation of the pathogen on the atheromatous lesion was statistically correlated with several risk factors and some of the variables that were tested. After testing independence of association, using the multiple regression analysis, only male gender (P = .024), hypertension (P = .008), hypercholesterolemia (P = .001), and TNF-α plaque values (P = .008) remained significantly associated. Hypertension, serum levels of fibrinogen and anti-C pneumoniae immunoglobulin A, detection of C. pneumoniae on the plaque, and plaque TNF-α values were significantly correlated with carotid disease symptomatology. After multiple analyses, only the presence of the pathogen on the lesion (P = .008) and atheroma TNF-α levels (P = .025) remained significantly associated with cerebrovascular events.
Conclusions
It seems that hypertensive hypercholesterolemic men are more likely to have C. pneumoniae infected carotid plaques and that these plaques have higher TNF-α concentrations. Cerebrovascular disease symptomatology is strongly correlated with both C. pneumoniae infection and TNF-α concentration of the atheroma.
Inflammation is now recognized as an integral part of atherogenesis.1 This low-grade chronic inflammatory process may result from an excessive response to various inflammatory stimuli, including infection. The association of several infectious agents with cardiovascular disease has been investigated in the last two decades.2 The pathogen most strongly associated with atherosclerosis so far is Chlamydia peumoniae.3
Many different kinds of studies have attempted to link this obligate intracellular gram-negative bacterium to the pathogenetic process of atherosclerotic disease. Seroepidemiologic and histopathologic studies, animal models of experimental atherosclerosis and chlamydial infection, and antibiotic intervention studies have all been trying to elucidate the role that this microorganism could play in cardiovascular disease. It is quite interesting to explore whether C pneumoniae is involved in the initiation, progression, or complications of the atheromatous lesion. C pneumoniae serology4 and plaque colonization5 have been correlated with carotid atherosclerosis. Chronic infection from C pneumoniae has also been associated with lesion development and the cerebrovascular symptomatology.6
This sustained dormant infection could be the cause of the increased levels of several inflammatory markers, including C-reactive protein (CRP) and fibrinogen. These markers seem to be equally important for, and indicative of, the clinical manifestations of carotid artery disease. They are probably involved in plaque evolution and stability.7 Cytokines seem to have a central role in atheroma development and rupture.8 Tumor necrosis factor-α (TNF-α), in particular, has been shown to be intimately involved in the atherosclerotic process. It has been known to contribute to the recruitment of inflammatory cells, and vascular smooth muscle and endothelial cells apoptosis, which could lead to cap thinning and plaque destabilization.9 TNF-α receptor levels have been linked to carotid atherosclerosis.10
C pneumoniae induces TNF-α production in infected macrophages, monocytes, smooth muscle or endothelial cells in vitro, and this synergy may be very important for lesion stability.11 To our knowledge, TNF-α lesion concentrations have not yet been correlated with either C pneumoniae detection on the atheroma or with stroke and transient ischemic attacks. The aim of our study was to investigate the relationship of serum and carotid plaque markers of inflammation and C pneumoniae infection with cerebrovascular symptomatology.
Methods
Between January 1 and December 2004, 86 consecutive patients underwent carotid endarterectomy in the Vascular Surgery Unit of the Second Dept of Propedeutic Surgery, Laikon Hospital, Athens University. Three patients failed to provide informed consent and five were excluded according to the exclusion criteria. The study thus included 78 patients: 46 were symptomatic (group A) and 32 were asymptomatic (group B). The study was approved by the hospital Ethics Committee, and informed consent was obtained from all participants.
Indication for surgery was a symptomatic carotid stenosis ≥50% or an asymptomatic carotid stenosis ≥70%. All patients had brain computed tomography (CT) scans and carotid duplex ultrasound scans preoperatively. Symptomatic disease was defined as the presence of clinical manifestations of transient ischemic attack (TIA) or stroke in the last 6 months, irrespective of the brain imaging findings. Of the symptomatic patients, 20 had a recent history of TIA, and 26 had had a stroke. Exclusion criteria for both groups were the presence of chronic or active inflammatory or infectious disease, malignancy; systematic administration (>3 days) of anti-inflammatory drugs, antibiotics, or corticosteroids; and pathologic white blood cell count and erythrocyte sedimentation rate value.
At entry into the study, a complete medical history was taken and risk factors were recorded, including age, gender, coronary artery disease (angina pectoris, myocardial infarction, coronary artery by-pass grafting, percutaneous transluminal coronary angioplasty), diabetes mellitus (controlled with diet, oral hypoglycemic agents or insulin; fasting glucose level >7 mmol/L), hypercholesterolemia (on statin therapy, total cholesterol >5.2 mmol/L), hypertension (on antihypertensive treatment, blood pressure >160 mm Hg systolic or 95 mm Hg diastolic), and smoking history (current or past). The demographic characteristics of the study patients are summarized in Table I.
Table I. Patient demographics and clinical characteristics
| Variables | Group A (n = 46) | Group B (n = 32) | P |
|---|---|---|---|
| Age, years | 70.1 ± 7.5 | 68.1 ± 3.2 | 0.15 |
| Gender: men | 69.6 (32) | 75.0(24) | 0.80 |
| BMI (kg/m2) | 27.7 ± 2.8 | 27.5 ± 2.7 | 0.77 |
| ABI | 0.83 ± 0.17 | 0.89 ± 0.16 | 0.12 |
| Hypertension | 82.6(38) | 53.1(17) | 0.006⁎ |
| Coronary artery disease | 37.0(17) | 56.3(18) | 0.11 |
| Diabetes mellitus | 52.2(24) | 37.5(12) | 0.25 |
| Hypercholesterolemia | 56.5(26) | 62.5(20) | 0.65 |
| Statin therapy | 43.5(20) | 53.1(17) | 0.71 |
| Smoking | 56.5(26) | 65.6(21) | 0.48 |
⁎Significant. |
Height and weight were measured and the body mass index (BMI) of obesity was calculated in all patients. Baseline assessment included measurement of ankle-brachial systolic pressure index (ABI). Systemic blood pressure in both arms and ankles was measured using a hand held 8-MHz Doppler device (Huntleigh Doppler, Cardiff, Wales) and a standard mercury sphygmomanometer.
All our patients, both symptomatic and asymptomatic, were taking low-dose aspirin prophylaxis. Aspirin treatment was interrupted 1 week before the expected day of surgery. Upon admission, venous blood samples were obtained for the determination of CRP, fibrinogen, and TNF-α levels in the serum as well as for the measurement of immunoglobulin (Ig) A and IgG anti-C pneumoniae (CP) titers and the samples were immediately stored at –70°C and analyzed at the end of the study.
CRP levels were measured by the photometric-turbidimetric test for the quantitative determination of human high-sensitivity CRP in serum and plasma (Human, Wiesbaden, Germany). Positive was considered a value >0.5 mg/dL. Fibrinogen was measured using the photometric method. TNF-α levels were calculated by the Titerzyme Enzyme Immunometric Assay of human TNF-α (Assay Designs, Inc, Ann Arbor, Mich). The antibodies used do not show significant cross-reactivity with other known cytokines. All samples were assayed in duplicate by technicians blind to the clinical information. The IgA and IgG anti-CP titers were determined by the quantitative enzyme-linked immunosorbent assay (ELISA) technique (Immunolisa, Orgenics, Israel). They were measured in enzyme immunounits (IEU), and positivity was appointed to values >5 EIU/mL.
Finally, the carotid plaque surgical specimens were collected immediately after surgery and divided in half longitudinally. One half was sliced and four plates for each patient were kept at –70°C until the time of the assay. The presence of C pneumoniae on the specimen was detected with immunohistochemistry (IHC) techniques, using the alkaline phosphatase staining method of DynaChrome (ThermoShandon, Pittsburgh, Pa) and the monoclonal antibody to C pneumoniae (Biogenex, San Ramon, Calif). The technique was considered positive when cell groups and not isolated cells were stained positively. The other half was homogenized in an ice-cold homogenization buffer. The homogenates were centrifuged and the supernatants were stored at –70°C until analysis. Tissue content of TNF-α was determined by the use of an ELISA kit (Biosource International, Inc, Camarillo, Calif). The minimum detectable dose of TNF-α is 3.0 pg/mL.
In the statistical analysis, continuous variables are expressed as mean ± SD, and categoric variables are presented as percentages. Comparisons of continuous variables among the groups were made using the Pearson parametric or the Spearman nonparametric test, as appropriate. The t test or the Mann-Whitney U test were used, as appropriate, for pair-wise comparisons between the groups of interest. Associations between two categoric variables were tested by the χ2 test or the Fisher’s exact test, as appropriate. After determining univariate associations, multivariate analysis (multiple linear regression analysis) was used to determine independence of associations. All tests were two-tailed and P < .05 was considered significant. All the analyses were done with the SPSS 9.0 statistical software (SPSS Inc, Chicago, Ill).
Results
As can be seen in Table I, there were no statistically significant differences in most of the traditional cardiovascular risk factors of coronary artery disease, diabetes mellitus, hypercholesterolemia, smoking, age, gender, BMI, ABI, or statin therapy between the symptomatic and the asymptomatic patients. Only hypertension was significantly more common in group A compared with group B patients (P = .006).
Serology revealed no difference between the two groups in CRP and TNF-α values or in the levels of anti-CP IgG (Table II). Fibrinogen serum values were significantly higher in the symptomatic group (P = .044). The mean anti-CP IgA levels in group A patients were almost double those in group B (P = .006). One in four symptomatic patients had anti-CP IgA values <8.2 EIU/mL, whereas in asymptomatic patients, the highest quartile started at 7.0 EIU/mL. IgA seropositivity, according to the standards set by the manufacturer (>5EIU/mL), was also significantly correlated with the symptomatic form of the disease (P < .001). Four of five subjects were positive in group A. Only a third of the second group was positive (Fig 1). For symptomatic patients with carotid artery disease, the odds ratio of being IgA seropositive was 9.2 (95% confidence interval [CI], 3.2 to 26.1) times greater than that of being seronegative. This is quite a significant odds ratio in favor of IgA seropositivity being a risk factor of cerebrovascular events, since the lower confidence limit is well above 1.
Table II. Inflammatory serum markers, Chlamydia pneumoniae serology, immunohistochemical detection of the pathogen on the atheroma, and tumor necrosis factor-α plaque values in symptomatic and asymptomatic patients
| Variables | Group A (n = 46) | Group B (n = 32) | P |
|---|---|---|---|
| CRP (mg/dL) | 1.93±3.36 | 1.79±2.75 | 0.83 |
| Fibrinogen (mg/dL) | 240.1±71.7 | 206.8±68.3 | 0.044⁎ |
| TNF-α (pg/mL) | 21.8±13.5 | 17.1±6.0 | 0.070 |
| Anti-CP† | |||
| IgA (EIU/mL) | 11.06±8.6 | 5.53±8.32 | 0.006⁎ |
| IgG (EIU/mL) | 36.2±29.0 | 28.6±24.2 | 0.23 |
| IgA+ | 78.3 (36) | 31.3 (10) | <0.001⁎ |
| IgG+ | 84.8 (39) | 87.5 (28) | 1.00 |
| IHC+ | 80.4 (37) | 25.0 (8) | <0.001⁎ |
| Plaque TNF-α (pg/mL) | 227.7±130.9 | 18.7±16.5 | <0.001⁎ |
⁎Significant value. |
†Anti-CP IgA+ when >5 EIU/mL; Anti-CP IgG+ when >5 EIU/mL. |
Fig 1.
Anti-Chlamydia pneumoniae (Cp) immunoglobulin A (IgA) seropositivity in patients with symptomatic and asymptomatic carotid artery disease.
Immunohistochemical plaque positivity was far more common in the first group (P < .001). Almost four of five symptomatic atheromas stained positive for the presence of C pneumoniae, whereas only one quarter of the group B patients’ plaques displayed the characteristic positive stain (Fig 2). Mean TNF-α symptomatic lesion level was >10 times higher than the corresponding asymptomatic lesion value (P < .001). Fig 3 clearly shows the significant difference in plaque TNF-α concentrations between symptomatic and asymptomatic patients. It is also evident that symptomatic seropositive subjects for anti-CP IgA have higher plaque TNF-α levels than those who are symptomatic seronegative, but this association was just over the limit of statistical significance (P = .056). TNF-α serum values were strongly associated with the concentration of TNF-α on the atheroma (P < .001). The five variables that were significantly correlated with carotid symptomatology were entered into a multivariate analysis model (multiple linear regression) with symptomatic disease as the dependent variable. Table III shows that the presence of C pneumoniae on the plaque and lesion TNF-α values remained risk factors for cerebrovascular symptomatology.
Fig 2.
Chlamydia pneumoniae was detected much more often in the carotid plaques of symptomatic patients. IHC, Immunohistochemistry.
Fig 3.
Tumor necrosis factor-α (TNF-α) plaque levels were significantly higher in symptomatic patients compared with asymptomatic patients. Especially in symptomatic anti-Chlamydia pneumoniae (Cp) immunoglobulin A (IgA) seropositive subjects, the levels of TNF-α on the atheroma were higher than in seronegative subjects, but the difference was not statistically significant (P = .056). Range bars indicate median and interquartile range and whiskers indicate outliers.
Table III. Multiple linear regression analysis⁎
| Standardized coefficient (B) | SE (SEB) | t | P | |
|---|---|---|---|---|
| Hypertension | .128 | .118 | 1.182 | .241 |
| Fibrinogen | .150 | .001 | 1.622 | .109 |
| Anti-CP IgA | .086 | .006 | .868 | .388 |
| IHC+ | .317 | .115 | 2.744 | .008† |
| Plaque TNF-α | .248 | .001 | 2.297 | .025† |
⁎Symptomatic carotid disease is the dependent variable and hypertension, fibrinogen and anti-CP IgA serum values, IHC plaque positivity, and TNF-α plaque levels are the independent variables. |
†Significant. |
Immunohistochemical detection of the pathogen on the atheroma was found to have many significant associations (Fig 4). Men’s plaques were found infected in >80% of the cases, while only half of women’s lesions stained positive for C pneumoniae (P = .041). Among the traditional cardiovascular risk factors, hypertension (P < .001) hypercholesterolemia (P = .039), and smoking (P = .005) were associated with IHC atheroma positivity. Only 10% of IHC-positive patients were normotensive, and about 45% of IHC-negative subjects were hypertensive. Almost three of four hypercholesterolemic patients, but less than half of normocholesterolemic ones had C pneumoniae infected plaques. Among smokers, the lesions of >75% stained positively, whereas in the nonsmoking population, this was the case in <45%. IHC-positive subjects were also older, with higher BMI and higher TNF-α serum values. Mean anti-CP IgA serum level (Fig 5) was almost double in patients with infected plaques compared with IHC-negative ones (P = .001). Finally, TNF-α atheroma concentration was many times higher in patients with C pneumoniae plaque infection compared with those that stained negatively for the pathogen (P < .001). All the independent variables associated with IHC were introduced in the multivariate regression analysis. Only male gender (P = .024), hypertension (P = .008), hypercholesterolemia (P = .001), and high TNF-α plaque values (P = .008) retained their significance.
Fig 4.
Immunohistochemistry of plaque specimens. A, Characteristically (red fast stain) stained plaque for the detection of Chlamydia pneumoniae by use of a specific monoclonal antibody for Chlamydia, an avidin-biotin complex, and alkaline phosphatase (magnification ×400), B, Atheroma negative for C pneumoniae (magnification ×200).
Fig 5.
Immunoglobulin A (IgA) serum values in patients with Chlamydia pneumoniae (Cp) infected (IHC+) and non-infected (IHC–) plaques (P = .001, Mann-Whitney test). IHC, Immunohistochemistry. Range bars indicate median and interquartile range and whiskers indicate outliers.
Discussion
Chronic inflammation and infection seem to play an important role in the development of atherosclerotic lesions. Melnick et al12 were the first to associate C pneumoniae seropositivity with carotid atherosclerosis. C pneumoniae seropositivity was found to have a significant association with acute stroke and TIAs.13 Elevated anti-CP IgA and IgG antibodies were associated with an increase of intima–media thickness or progression of carotid stenosis.14 A recent study has shown an increased aortic intima–media thickness in healthy children with persistent C pneumoniae seropositivity.15 In a large prospective study, C pneumoniae IgG seropositivity seems to influence significantly the future occurrence of cerebrovascular events.7 Other studies have failed to show the association of C pneumoniae serology with cerebrovascular disease and symptomatology.16, 17 In our study, IgA seropositivity was associated with the symptomatic form of carotid disease. After multiple analyses, the association was no longer statistically significant.
Microimmunofluorescence (MIF) is the established serologic method for diagnosing C pneumoniae infection. We used ELISA, which has been shown to have an overall correlation of about 90% with the MIF test in terms of sensitivity and specificity and is considered less subjective and less operator dependent.18 In a significant comparative study, there was a good concordance of MIF and ELISA results.19 IgA antibodies against C pneumoniae have shown little correlation with the detection of the microorganism in atherosclerotic plaques.5 Others, supporting our findings, still consider Chlamydia serology a marker of atheroma infection.20
According to the Centers for Disease Control and Prevention USA guidelines,21 IHC, polymerase chain reaction (PCR), and culture are the three methods approved for C pneumoniae detection on the lesion. The validity of our IHC method had been recently questioned,22 but a comparative study that we have traced in the literature shows a good correlation between PCR and IHC results.23
C pneumoniae was detected in 57.7% of the plaques, which is well within similar figures in the literature, although some using PCR have failed to detect the pathogen in carotid atheromas.24 Plaque infection was much more common among men, which was probably due to their overwhelming percentage of smokers. It is generally accepted that smoking favors the development of respiratory infections, including chlamydial ones.25 Hypertension and hypercholesterolemia have also been associated with C pneumoniae detection on the atheroma. From the literature, hypertension has been linked to C pneumoniae serology,26 and hyperlipidemia is considered a co-risk factor of the pathogen for atherosclerosis.27
The association of plaque infection and plaque TNF-α concentration is extremely important. TNF-α is produced in large quantities by almost all cells of the atherosclerotic plaque when infected by C pneumoniae.28 It has been correlated with the serologic diagnosis of C pneumoniae infection,29 but it has not yet been associated with plaque infection. When monocytes are exposed to the action of specific antichlamydial antibiotics (eg, the quinolones) the levels of production of TNF-α are significantly reduced.30 Others have found that specifically in carotid lesions, chlamydial lipopolysaccharide increases cytokine expression, including a 50-fold increase in TNF-α expression.31
IgA seropositivity and C pneumoniae PCR-positive peripheral blood mononuclear cells have already been linked to cerebrovascular symptomatology.6, 32 Others have failed to associate plaque detection of the pathogen with symptomatic stenosis.33 In another arterial bed, isolation of the microorganism in femoral plaques has been correlated with the severity of claudication.34 Experimental data show that carotid C pneumoniae infection leads to progression of atherosclerotic lesions with a more vulnerable morphology in LDLr–/– mice.35
Although the large antibiotic intervention trials in coronary patients had no positive effect, antibiotic treatment of C pneumoniae seropositive patients resulted in reduced progression of early carotid atherosclerosis.36 The issue is still unsettled, but published reports seem to support the association of C pneumoniae plaque infection with cerebrovascular symptomatology.
TNF-α atheroma concentrations have never before, to our knowledge, been related to stroke and TIAs. The only hint of information we have had so far is that in symptomatic patients with carotid stenosis of >70%, the levels of messenger RNA for TNF-α are increased in the plaque area.37 TNF-α, probably through its apoptotic properties, may be responsible for plaque rupture.38 It also contributes to the acceleration of atherosclerosis by C pneumoniae in mice.39 C pneumoniae in synergy with TNF-α stimulates matrix metalloproteinase production, which could be very important for plaque stability.40 Chlamydial heat shock protein 60 induces TNF-α and matrix metalloproteinase production by macrophages.41
A significant limitation of our study is that C pneumoniae detection and TNF-α concentration of the lesion are postoperative findings. Therefore, either the patient is already symptomatic or the probable carotid embolic source has been treated, which makes these findings of rather theoretic clinical utility. However, IgA seropositivity could prove clinically useful.
Conclusion
Our data suggest that cerebrovascular symptomatology is associated both with plaque inflammation, expressed by plaque TNF-α values, and plaque C pneumoniae infection. Plaque TNF-α production is strongly related to the presence of the pathogen. Serum TNF-α and anti-CP IgA levels, which have been correlated with atheroma TNF-α concentration and C pneumoniae infection, respectively, could in the future serve as serologic predictors of symptomatic lesions, if prospective studies confirm these findings. Clinical and therapeutic implications of these results remain to be evaluated.
Author contributions
We thank Dr Dimitrios Dimitroulis for his valuable advice in the analysis and interpretation of the data and Dr Maria Poulakou for her help in data collection.
References
- . Inflammation as a cardiovascular risk factor. Circulation. 2004;109(Suppl II):2–10
- . The multiple mechanisms by which infection may contribute to atherosclerosis development and course. Circ Res. 2002;90:2–4
- . Chlamydia pneumoniae—an infectious risk factor for atherosclerosis?. Nat Rev Microbiol. 2004;2:23–32
- . Infections, immunity, and atherosclerosis: associations of antibodies to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus with immune reactions to heat-shock protein 60 and carotid or femoral atherosclerosis. Circulation. 2000;102:833–839
- Chlamydia pneumoniae in carotid artery atherosclerosis (A comparison of its presence in atherosclerotic plaques, healthy vessels and circulating leucocytes from the same individuals). Stroke. 2002;33:2756–2761
- Chlamydia pneumoniae DNA in patients with symptomatic carotid atherosclerotic disease. J Vasc Surg. 2003;37:1027–1031
- Markers of inflammation and infection influence the outcome of patients with baseline asymptomatic carotid lesions (A 5-year follow-up study). Stroke. 2006;37:482–486
- . Novel clinical markers of vascular wall inflammation. Circ Res. 2001;89:763–771
- . Tumor necrosis factor-α in cardiovascular biology and the potential role for anti-tumor necrosis factor-α therapy in heart disease. Pharmacol Ther. 2002;94:123–135
- Tumor necrosis factor receptor levels are associated with carotid atherosclerosis. Stroke. 2002;33:31–38
- Foam cell formation inhibits growth of C. pneumoniae but does not attenuate Chlamydia pneumoniae-induced secretion of proinflammatory cytokines. Circulation. 2002;105:1976–1982
- Past infection by Chlamydia pneumoniae strain TWAR and asymptomatic carotid atherosclerosis. Am J Med. 1993;95:499–504
- . Chlamydia pneumoniae antibody titers are significantly associated with acute stroke and transient cerebral ischemia (The West Birmingham Stroke Project). Stroke. 1998;29:404–410
- Impact of infectious burden on progression of carotid atherosclerosis. Stroke. 2002;33:2581–2586
- Increased aortic intima-media thickness in 11-year old healthy children with persistent Chlamydia pneumoniae seropositivity. Arterioscler Thromb Vasc Biol. 2006;26:649–655
- . Chlamydia pneumoniae infection is not involved in carotid artery stenosis. Atherosclerosis. 2002;163:165–168
- Chlamydia pneumoniae antibodies and high lipoprotein (a) levels do not predict ischemic cerebral infarctions: results from a nested case-control study in Northern Sweden. Stroke. 1999;30:2013–2018
- . Chlamydia pneumoniae and atherosclerosis—what we know and what we don’t. Clin Microbiol Infect. 2002;8:2–13
- Importance of methodology in determination of Chlamydia pneumoniae seropositivity in healthy subjects and in patients with coronary atherosclerosis. J Clin Microbiol. 2003;41:4049–4053
- Detection of Chlamydia pneumoniae but not of Helicobacter pylori in symptomatic atherosclerotic carotids, associated with enhanced serum antibodies, inflammation and apoptosis rate. Atherosclerosis. 2003;168:153–162
- Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada). Clin Infect Dis. 2001;33:492–503
- Immunohistostaining assays for detection of Chlamydia pneumoniae in atherosclerotic arteries indicate cross-reactions with non-chlamydial plaque constituents. J Clin Microbiol. 2004;42:3219–3224
- . Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry. J Mic Meth. 2001;46:241–251
- . No evidence of involvement of Chlamydia pneumoniae in severe cerebrovascular atherosclerosis by means of quantitative real-time polymerase chain reaction. Stroke. 2004;35:2024–2028
- Smoking, season, and detection of Chlamydia pneumoniae DNA in clinically stable COPD patients. BMC Inf Dis. 2002;2:12–18
- Increased prevalence of Chlamydophila pneumoniae but not Epstein-Barr antibodies in essential hypertensives. J Hum Hypertens. 2003;17:21–27
- . Chlamydia pneumoniae and hyperlipidemia are co-risk factors for atherosclerosis: infection prior to induction of hyperlipidemia does not accelerate development of atherosclerotic lesions in C57BL/6J mice. Inf Imm. 2002;70:5332–5334
- Hydroxymethylglutaryl coenzyme A reductase inhibitors modify the inflammatory response of human macrophages and endothelial cells infected with Chlamydia pneumoniae. Circulation. 2000;101:1760–1763
- Chlamydia pneumoniae seropositivity and cardiovascular risk factors: The InCHIANTI Study. J Am Geriatr Soc. 2004;52(10):1626–1631
- Effect of levofloxacin on the viability of intracellular Chlamydia pneumoniae and modulation of proinflammatory cytokine production by human monocytes. Diagn Mirobiol Infect Dis. 2004;50:205–212
- . Lipopolysaccharide-induced cytokine and chemokine expression in human carotid lesions. J Vasc Res. 2005;42:266–271
- Chlamydia pneumoniae serology is associated with thrombosis-related but not with plaque-related microembolization during carotid endarterectomy. Stroke. 2002;33:1249–1254
- Chlamydia pneumoniae, herpes simplex virus and cytomegalovirus in symptomatic and asymptomatic high-grade internal carotid artery stenosis (Does infection influence plaque stability?). Vasa. 2005;34:163–169
- Inflammation and Chlamydia pneumoniae infection correlate with the severity of peripheral arterial disease. Eur J Vasc Endovasc Surg. 2006;31:509–515
- Delivery of Chlamydia pneumoniae to the vessel wall aggravates atherosclerosis in LDLr-/- mice. Cardiovasc Res. 2006;69:280–288
- . Reduced progression of early carotid atherosclerosis after antibiotic treatment and Chlamydia pneumoniae seropositivity. Circulation. 2002;106:2428–2433
- . Expression of inflammatory mediators and adhesion molecules in human atherosclerotic plaque. Neurology. 1997;49(5 Suppl 4):S15–S19
- . Tumor necrosis factor alpha mediated apoptosis in vascular smooth muscle cells requires p73. Am J Physiol Cell Physiol. 2005;289:C199–C206
- . TNF-α plays a role in the acceleration of atherosclerosis by Chlamydia pneumoniae in mice. Infect Immun. 2005;73:3164–3165
- . Chlamydia pneumoniae enhances cytokine-stimulated human monocyte matrix metalloproteinases through a prostaglandin E2-dependent mechanism. Infect Immun. 2005;73:632–634
- . Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-alpha and matrix metalloproteinase expression. Circulation. 1998;98:300–307
Competition of interest: none.
PII: S0741-5214(06)01493-5
doi:10.1016/j.jvs.2006.08.029
© 2006 The Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.
