(Circulation. 2000;102:833.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
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
From the Institute for Biomedical Aging Research, Austrian Academy of Sciences (M.M., G.W., Q.X.), the Department of Neurology, University Hospital (S.K., J.W.), and the Institute for General and Experimental Pathology, University of Innsbruck Medical School (G.W.), Innsbruck, Austria.
Correspondence to Dr Qingbo Xu, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail qingbo.xu{at}oeaw.ac.at
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Abstract |
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Background—Atherogenesis involves inflammatory processes in which infections are incriminated as possible contributors.
Methods and Results—We evaluated cardiovascular risk factors as well as seropositivity to Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus in a population-based study. A significant association between prevalence and severity of atherosclerosis in carotid and femoral arteries and IgA antibodies to C pneumoniae was demonstrated that was not substantially altered after adjustment for established risk factors. For anti–H pylori IgG antibodies, significant correlations to vascular disease were restricted to low social status and lesions in carotid arteries. In addition, the study design allowed us to monitor lesion progression over time. In this prospective analysis, C pneumoniae seropositivity emerged as a significant risk predictor. Antibody titers against cytomegalovirus were not a marker for prevalence or incidence of atherosclerosis in this population. Further infection parameters added to the predictive value of chlamydial serology in risk assessment: Mean odds ratios for the prevalence of carotid atherosclerosis were 4.2 and 6.3 for seropositive subjects with elevated C-reactive protein levels and clinical evidence for chronic respiratory infection, respectively. For subjects with all 3 infection parameters, the odds ratio of carotid atherosclerosis reached 10.3 (P<0.0001). Concomitantly, serum antibodies to mycobacterial heat-shock protein 65 (mHSP65) correlated with seropositivity to C pneumoniae and H pylori but not to cytomegalovirus.
Conclusions—This prospective population-based study provides strong evidence for a potential atherogenic role of persistent bacterial infection, especially C pneumoniae, as indicated by serological and clinical data and demonstrates a correlation between immune reactions to mHSP65 and bacterial infections in atherogenesis.
Key Words: aging • infection • antibodies • atherosclerosis • immunology
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Introduction |
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Over the past decade, awareness of possible associations between atherosclerosis and certain persistent bacterial and viral infections has steadily increased. The most compelling evidence derives from seroepidemiological studies, which focused mainly on 3 pathogens: Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus (CMV).1 The hypothesis is strongest for C pneumoniae on the basis of the association between chlamydial seropositivity and risk of cardiovascular and cerebrovascular disease.2 3 Prevalences of IgG antibodies in middle-aged adults exceed 50%, because this common respiratory pathogen causes widespread epidemics and frequent reinfection. These obligate intracellular bacteria are detected frequently within atherosclerotic lesions but rarely in normal vessels.4 They reside mainly in plaque macrophages but also colonize the vessel wall by infecting endothelial and smooth muscle cells.5 Animal models support their possible atherogenic effect,6 and preliminary data from small-scale clinical trials suggest that macrolide antibiotic treatment is beneficial in vascular disease,7 but this remains to be confirmed by larger studies.8
Human heat-shock protein (HSP) production in the arterial wall is essential for cell protection against unfavorable conditions, such as hemodynamic stress, oxidants, and cytokine stimulation. However, HSPs bear the risk of autoimmunity because of their high sequence homology between different species, from prokaryotes to humans.9 Previous epidemiological studies from our laboratory demonstrated that increased serum antibodies to mycobacterial HSP65 (mHSP65), a bacterial homologue of human HSP60, were associated with the prevalence of carotid atherosclerosis independently of other established risk factors,10 and this was subsequently confirmed by other laboratories.11 Furthermore, elevated anti-mHSP65 antibody titers were sustained during a 5-year follow-up and were strongly associated with advanced atherosclerosis and mortality in prospective analyses.12 These serum antibodies cross-react with human HSP60 and mediate cytotoxicity on stressed endothelial cells.13 Therefore, the antimicrobial HSP60/65 immune response may contribute to vascular endothelial injury, which is believed to be a key event in the pathogenesis of atherosclerosis.14
Despite the rapidly growing number of studies about associations between infections and vascular disease,1 consensus on the possible atherogenic effects of infectious agents has not been achieved; pathogenetic mechanisms remain unclear, and the available seroepidemiological studies are subject to certain limitations.1 First, most previous surveys focused on clinical end points, such as coronary heart disease or stroke. The associations obtained do not necessarily prove the existence of atherogenic effects, because infections may provoke ischemia by prothrombotic and other mechanisms. In particular, extrapolation of results from clinical studies to early stages of atherogenesis, which pathologically resembles chronic inflammatory disease, requires caution. Second, the studies that investigated the association of infections with atherosclerosis were, with few exceptions, cross-sectional,15 and those with a prospective design were not conducted in a random population.16 Finally, seropositivity as a marker for infections has the advantage of clinical applicability, but the assessment of infection status based on serology without further clinical or laboratory characterization is subject to diagnostic inaccuracies, especially if seropositivity is common because of the widespread distribution of the incriminated microorganism.
In this prospective survey, we investigated the association of immune reactions to C pneumoniae, H pylori, and CMV with carotid and femoral atherosclerosis as quantified by ultrasound in the population-based Bruneck study. In addition, we scrutinized this association by laboratory and clinical markers for persistent infection and by correlating bacterial infections with serum antibodies to mHSP65.
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Methods |
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Subjects
Human sera were derived from the Bruneck Study, a large population-based study on the epidemiology and etiology of atherosclerosis and arterial disease. The study population is an age- and sex-stratified random sample of all inhabitants of Bruneck 40 to 79 years old. In each of the fifth to eighth decades of age (40 to 49, 50 to 59, 60 to 69, and 70 to 79 years), 125 men and 125 women were selected for inclusion (n=1000). A total of 93.6% participated, with data assessment completed in 919 subjects. Between 1990 and 1995, a subgroup of 63 individuals died or moved away. In the remaining population, follow-up was 96.5% complete, which left 826 subjects for the present analysis. All participants gave informed consent before entering the study.
Clinical Examination and Laboratory Methods
All participants underwent a clinical examination with
cardiological and neurological priority and completed standardized
questionnaires on current and past exposure to candidate vascular risk
factors as described previously.17 18 The term "chronic
respiratory infections" encompassed chronic bronchitis, emphysema,
and asthma, which were defined according to standard criteria. Blood
samples were taken from the antecubital vein after subjects had fasted
and abstained from smoking for 
12 hours.17 Laboratory
parameters were examined by standard
methods.17 18
Scanning Protocol and Definition of Ultrasound End Points
The ultrasound protocol involves scanning of the internal
(bulbous and distal segments) and common (proximal and distal segments)
carotid arteries on either side with a 10-MHz imaging probe and 5-MHz
Doppler.19 20 Atherosclerotic lesions were defined by
2 ultrasound criteria: (1) wall surface (protrusion into the lumen or
roughness of the arterial boundary) and (2) wall texture
(echogenicity). The maximum axial diameter of plaques was assessed in
each of the additional 16 vessel segments, and a sensitive and
reproducible atherosclerosis score was calculated by
addition of all diameters. The accuracy of this procedure had been
established previously.17 Scanning was performed twice, in
1990 and 1995, by the same experienced sonographer, who was unaware of
the subjects’ clinical and laboratory characteristics. Incident
(early) atherosclerosis was defined by the occurrence
of new plaques in previously normal segments (1990 to 1995). Advanced
atherogenesis shown to originate primarily from
atherothrombosis18 was assumed whenever the progression
criterion (increase in plaque diameter exceeding twice the measurement
error) was met and a narrowing of the lumen >40% was
achieved.19 20 The cutoff of 40% was adopted from
previous epidemiological analyses in this
cohort.19 20 In 1995, the ultrasound examination was
extended to the femoral arteries (40 mm proximal and 10 mm
distal to the bifurcation into the superficial and deep branches). The
intima-media thickness was measured in 1995 at the far wall of the
femoral arteries and common carotid arteries with the ultrasound beam
directed through the axis of the vessel. It was defined as the distance
between the lumen-intima interface and the leading edge of the
media-adventitia interface.19
Specific Tests for Infections and Measurement of Anti-mHSP65
Antibodies
Serum antibodies against C pneumoniae, H
pylori, and CMV were determined by 3 commercial test systems as
part of the 1995 evaluation: SeroCP-IgA (Savyon Diagnostics
Ltd), Helicobacter-IgG (Medac), and CMV-IgG ELISA (Medac). The
analyses were performed and calculated according to the
manufacturer’s instructions. Antibodies against mHSP65 were determined
by ELISAs according to an established protocol.12 In
brief, microtiter plates were coated with 1 µg/mL PBS with
recombinant mHSP65 overnight at 4°C and incubated with 100 µL human
serum diluted 1 in 10 to 1 in 5160 for 1 hour. A serum dilution was
considered positive for antibodies to mHSP65 if the optical density at
410 nm exceeded 0.400.
Statistical Analyses
The association between immune reaction to bacterial/viral
pathogens and anti-mHSP65 antibodies was investigated by comparing
antibody levels across categories of increasing titers of antibodies to
C pneumoniae, H pylori, and CMV. Logistic
regression models were built to analyze strength and type of
association between antibody titers and prevalent or incident
atherosclerosis (incident nonstenotic
atherosclerosis or incident stenosis). The test
procedure was based on maximum-likelihood estimators, and the goodness
of fit of each model was assessed by the test of Hosmer and
Lemeshow.21 Effect modification was tested by the
inclusion of interaction terms. Base models were adjusted for age and
sex only (plus baseline atherosclerosis score in the
prospective analysis). Multivariate equations
were controlled for potential effects of age, sex, smoking, alcohol
intake, diabetes, impaired glucose tolerance, lipoprotein (a),
apolipoprotein B, ferritin, hypertension, fibrinogen, and social
status.18 The forced entry of all these variables
yielded results virtually identical to those of a forward stepwise
selection procedure (data not presented). In the main
analysis (Tables 1 to 3
), log-transformed
antibody titers were treated as a continuous variable, and odds
ratios were calculated for a 1-titer increase in given serological
parameters. This procedure, contrary to most previous
evaluations in this field, avoided arbitrary categorizations of
subjects according to their antibody titer. Because there is no
consensus on how to define seropositivity, categorizations may give
rise to spurious relations, especially if guided by a post hoc
decision. In a second step, antibody titers were modeled as a set of
indicator variables, and odds ratios of
atherosclerosis were computed for each category to
investigate the scale of the association (Figure 1). Finally, the relation between
atherosclerosis and immune reaction to C
pneumoniae was reanalyzed with respect to clinical
(manifest chronic respiratory diseases) and/or laboratory (high
C-reactive protein [CRP]) evidence of persistent infection status
(Figure 2). For ease of
presentation, we used a categorization of IgA antibody
titers to C pneumoniae (negative <16, positive 16) in
this analysis. Logistic regression models were supplemented by
linear regression analyses using intima-media thickness as a
continuous outcome variable. Multivariate
regression models were built as described above.
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Results |
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Association of Antibody Titers With Carotid and Femoral Atherosclerosis
Seroprevalence was assessed by commercial ELISA tests measuring IgA antibodies to C pneumoniae and IgG antibodies to H pylori and CMV. Chlamydial IgA antibodies are expected to be a more reliable marker for chronicity of chlamydial infections than seroprevalences of IgG antibodies.22 Antibody titers to C pneumoniae were strongly associated with male sex and age (P<0.001); a moderate association was observed with smoking (P<0.1). Anti–H pylori antibodies were more frequent in subjects of low social status and patients with ulcer disease (P<0.001 and P<0.1, respectively). Serum antibodies to CMV were significantly higher in women (P<0.001) and increased with age and low social status (P<0.05).
Anti–C pneumoniae IgA antibodies were associated with the
prevalence of atherosclerosis in carotid and femoral
arteries (P=0.011 and P=0.035, Table 1
). Results remained significant
after adjustment for multiple risk factors, including age, sex,
hypertension, smoking, apolipoprotein B, lipoprotein (a), diabetes,
impaired glucose tolerance, fibrinogen and ferritin levels, alcohol
consumption, and social status. Anti–H pylori IgG
antibodies correlated significantly with carotid
atherosclerosis when the statistical analysis
was restricted to low social status (P=0.027). No
correlation between immune responses to H pylori and
atherosclerosis in femoral arteries could be obtained.
Although antibodies to C pneumoniae and H
pylori predicted the risk of carotid
atherosclerosis (Figure 1), elevated antibody
titers to CMV were not associated with atherosclerotic lesions in
carotid or femoral arteries. The power of our statistical
analysis was >0.9 to detect a difference of 1 titer between
outcome categories at a level of 
=0.05.
Intima-media thickness is a well-established surrogate and precursor of
definite atherosclerosis. When this outcome
variable was substituted for the atherosclerosis
categories, C pneumoniae remained a significant risk
factor (Table 2). Significance for
anti–H pylori IgG antibodies was again limited to subjects
of low social status and lesions in carotid arteries. Finally,
IgG titers to CMV were definitely unrelated to vessel wall thickness.
In this analysis, the power of our study would allow detection
of correlates as low as r=0.09 (power=0.8, =0.05).
The study design permitted us to study changes in carotid
atherosclerosis during a 5-year follow-up and to define
distinct stages in the atherogenesis process. Immune reactions to
C pneumoniae were associated with early and advanced stages
of atherosclerosis, whereas seroprevalences of
antibodies to H pylori and CMV were not (Table 3).
Notably, none of these findings were substantially altered when the analysis was restricted to nonsmokers. When subjects seropositive to C pneumoniae had additional serological evidence for infections with either H pylori or CMV or both, predictive significance for atherosclerosis did not change.
Respiratory Infections, CRP Levels, and Anti-mHSP65
Antibodies
The presence of IgA antibodies to C pneumoniae is
supposed to be indicative for ongoing chronic
infection,22 but substantial fluctuations in antibody
responses during chlamydial disease may weaken statistical
associations. Hence, evaluation of infection activity should rely on
further clinical and laboratory parameters. In our study,
high CRP levels (Figure 2A) and clinical signs of chronic
respiratory infections (Figure 2B) were positively correlated
with carotid atherosclerosis and substantially improved
the predictive accuracy of chlamydial serology in risk assessment. Odds
ratios increased to 4.2 when seropositivity to C pneumoniae
was combined with elevated CRP levels and to 6.3 when clinical criteria
for chronic respiratory infections were present (P<0.05
for effect modification). Overall, the better the status of persistent
infection was established by combination of seroepidemiological,
clinical, and laboratory data, the higher the predictive significance
for atherosclerosis development was. Odds ratios were
1.5 (P=0.097), 4.3, and 10.3 (P<0.0001) for
those subjects with 1, 2, and 3 risk factors, respectively (Figure 2C).
Previously, we have provided evidence that increased concentrations of
anti-mHSP65 antibodies are sustained in subjects with carotid
atherosclerosis in a 5-year follow-up
study.12 We now attempt to clarify relationships of
anti-mHSP65 antibodies with infectious agents. Antibody levels to
C pneumoniae and antibody titers to H pylori
correlated significantly with anti-mHSP65 antibodies (P=0.03
and P=0.001). Conversely, levels of anti-CMV antibody titers
were not associated with either atherosclerosis or
anti-mHSP65 antibodies (P=0.17). Subjects seronegative to
CMV had detectable anti-mHSP65 antibodies, as did all individuals in
this population. But in contrast to C pneumoniae and H
pylori, seropositivity to CMV did not alter these titers
significantly (Table 4). Altogether, our
findings suggest that immune reactions to mHSP65 are linked with
bacterial but not viral infections.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Consensus about the role of infections in atherosclerosis has not been achieved despite several seroepidemiological studies. Available data derive almost exclusively from cross-sectional and retrospective analyses using such clinical end points as coronary heart disease or stroke as outcome variables.1 To the best of our knowledge, the present study is the first prospective survey about atherosclerosis and immune reactions to C pneumoniae, H pylori, and CMV conducted in a large random population. Among the 3 microorganisms, C pneumoniae was the most reliably associated with atherosclerosis. The general consistency of the data in all analysis, ie, different stages of atherosclerosis (precursor to stenosis), and several vascular territories as well as in the cross-sectional and prospective analysis, supports the existence of a real association. Seropositivity for H pylori yielded some association with carotid atherosclerosis, but data were less consistent. The inconclusive results may be partly explained by the strong association of H pylori infection with confounding factors, such as low social class.23 In addition, the genetic polymorphism of various strains concerning virulence factors like the cytotoxin-associated gene A seems to be decisive for their pathogenic contribution.24 Finally, our study could not furnish any evidence for a contribution of CMV to atherogenesis in the general community. At this point, it should be noted that only carotid and femoral arteries were examined in this study, and extrapolation of our results to other vascular territories, eg, coronary arteries, requires caution.
The present study adds further dimensions to the relation between infections and atherogenesis in that the predictive significance of chlamydial IgA antibodies for carotid atherosclerosis substantially increased (OR=10.3) when elevated CRP levels and clinical evidence for chronic respiratory infections, as well as the fact that chronic respiratory infections amplified atherosclerosis risk in the absence of chlamydial antibodies, were taken into account. The finding that laboratory and clinical markers for persistent infection added significantly to the predictive value of chlamydial serology in risk assessment underscores the importance of chlamydial infections in atherogenesis and demonstrates the limited accuracy of chlamydial antibodies as a single criterion of infection.25 Discrepancies in the results of the few previous studies in this field15 may be explained by this methodological shortcoming. The observation that chronic respiratory infections, as defined by clinical criteria, predict the prevalence of carotid atherosclerosis even without seropositivity to C pneumoniae implies that the total infectious load might be even more important than a single infectious agent and that elevated CRP levels in patients with cardiovascular disease might be partly due to infection.
How infections are involved in vascular disease remains to be elucidated. Increased blood viscosity, hypercoagulability, and alterations of the serum lipid profile are postulated mechanisms.1 As in the case of C pneumoniae or CMV, vessel wall colonization may contribute to local inflammation by cytokine induction or antigen stimulation.26 In contrast, H pylori infections might stimulate atherogenesis via indirect effects, such as systemic inflammation or (auto) immune reactions,11 because these bacteria are rapidly eliminated in the circulation before they reach the vessel wall. Immune reactions to HSP60 are a possible link between the various incriminated microorganisms and atherogenesis.26 Abundant bacterial HSP60 may evoke an anti–self-immune response in susceptible individuals because of its high sequence homology with the human homologue.27 Indeed, circulating HSP60 antibodies are absent in specific pathogen–free animals, suggesting that induction of HSP antibodies depends primarily on infections. Likewise, the age-dependent increase of anti-HSP60 titers in humans might be due to lifetime exposure to environmental microorganisms.28 The prospective Bruneck study yielded evidence for a sustained elevation of anti-mHSP65 antibody titers in subjects with atherosclerosis.12 These serum antibodies cross-react with bacterial and human HSP60 and mediate vascular cytotoxicity on stressed endothelial cells.13 26 Cells of the arterial wall overexpress HSP60 in response to various harmful stimuli, including hemodynamic stress, infections, oxidants, and cytokines, to protect themselves against these unfavorable conditions.29 Interestingly, persistent chlamydial infections are associated with an abundance of HSP60.30 The frequent colocalization of bacterial and human HSP60 within atherosclerotic lesions provides a possible explanation of how C pneumoniae might contribute to the pathophysiology of atheroma.31 32 In addition to molecular mimicry, HSP60 activates vascular adhesion molecule expression, induces the production of proinflammatory cytokines, and regulates the expression of matrix metalloproteinases in macrophages.32 33 In the present study, we demonstrated for the first time that anti-mHSP65 correlates significantly with antibodies against C pneumoniae and H pylori in the general community, suggesting that immune reactions to HSP60 in atherogenesis are at least partially due to bacterial infections.
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Acknowledgments |
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This work was supported by grants P-12213-MED (to Dr Wick) and P-13099-BIO (to Dr Xu) from the Austrian Science Fund and P7919 (to Dr Xu) from the Austrian National Bank. We are grateful to the companies Medac (Hamburg) and Savyon Diagnostics Ltd (Israel) for providing their commercial ELISA kits and to A. Jenewein for technical assistance.
Received December 30, 1999; revision received March 20, 2000; accepted March 27, 2000.
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B. Ludewig, P. Krebs, and E. Scandella Immunopathogenesis of atherosclerosis J. Leukoc. Biol., August 1, 2004; 76(2): 300 - 306. [Abstract] [Full Text] [PDF]
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A. Mazzone, M. C. Epistolato, R. De Caterina, S. Storti, S. Vittorini, S. Sbrana, J. Gianetti, S. Bevilacqua, M. Glauber, A. Biagini, et al. Neoangiogenesis, T-lymphocyte infiltration, and heat shock protein-60 are biological hallmarks of an immunomediated inflammatory process in end-stage calcified aortic valve stenosis J. Am. Coll. Cardiol., May 5, 2004; 43(9): 1670 - 1676. [Abstract] [Full Text] [PDF]
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B. Zal, J. C. Kaski, G. Arno, J. P. Akiyu, Q. Xu, D. Cole, M. Whelan, N. Russell, J. A. Madrigal, I. A. Dodi, et al. Heat-Shock Protein 60-Reactive CD4+CD28null T Cells in Patients With Acute Coronary Syndromes Circulation, March 16, 2004; 109(10): 1230 - 1235. [Abstract] [Full Text] [PDF]
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J. Zhu, R. J. Katz, A. A. Quyyumi, D. A. Canos, D. Rott, G. Csako, A. Zalles-Ganley, J. Ogunmakinwa, A. G. Wasserman, and S. E. Epstein Association of Serum Antibodies to Heat-Shock Protein 65 With Coronary Calcification Levels: Suggestion of Pathogen-Triggered Autoimmunity in Early Atherosclerosis Circulation, January 6, 2004; 109(1): 36 - 41. [Abstract] [Full Text] [PDF]
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B. OSTERUD and E. BJORKLID Role of Monocytes in Atherogenesis Physiol Rev, October 1, 2003; 83(4): 1069 - 1112. [Abstract] [Full Text] [PDF]
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P. J. Lindsberg and A. J. Grau Inflammation and Infections as Risk Factors for Ischemic Stroke Stroke, October 1, 2003; 34(10): 2518 - 2532. [Abstract] [Full Text] [PDF]
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Y. Agmon, B. K. Khandheria, I. Meissner, T. M. Petterson, W. M. O'Fallon, T. J. H. Christianson, D. O. Wiebers, T. F. Smith, J. M. Steckelberg, and A. J. Tajik Lack of association between Chlamydia pneumoniae seropositivity and aortic atherosclerotic plaques: A Population-Based transesophageal echocardiographic study J. Am. Coll. Cardiol., May 7, 2003; 41(9): 1482 - 1487. [Abstract] [Full Text] [PDF]
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P. Khairy, S. Rinfret, J.-C. Tardif, R. Marchand, S. Shapiro, J. Brophy, and J. Dupuis Absence of Association Between Infectious Agents and Endothelial Function in Healthy Young Men Circulation, April 22, 2003; 107(15): 1966 - 1971. [Abstract] [Full Text] [PDF]
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M. Mayr, S. Kiechl, M. A. Mendall, J. Willeit, G. Wick, and Q. Xu Increased Risk of Atherosclerosis Is Confined to CagA-Positive Helicobacter pylori Strains: Prospective Results From the Bruneck Study Stroke, March 1, 2003; 34(3): 610 - 615. [Abstract] [Full Text] [PDF]
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T. Huittinen, M. Leinonen, L. Tenkanen, M. Manttari, H. Virkkunen, T. Pitkanen, E. Wahlstrom, T. Palosuo, V. Manninen, and P. Saikku Autoimmunity to Human Heat Shock Protein 60, Chlamydia pneumoniae Infection, and Inflammation in Predicting Coronary Risk Arterioscler Thromb Vasc Biol, March 1, 2002; 22(3): 431 - 437. [Abstract] [Full Text] [PDF]
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