(Circulation. 1995;92:3397-3400.)
© 1995 American Heart Association, Inc.
Articles |
Chlamydia pneumoniae (TWAR) in Atherosclerosis of the Carotid Artery
From the Departments of Epidemiology (J.T.G.), Pathobiology (J.T.G., C-c.K., L.A.C., S-p.W.), and Surgery (E.D.S.), University of Washington, Seattle; the Dameron Hospital Heart Institute (A.S.C., R.D.L.), Stockton, Calif; and the Harborview Medical Center (M.J.L.), University of Washington, Seattle.
Correspondence to J. Thomas Grayston, MD, Department of Epidemiology, Box 357236, University of Washington, Seattle, WA 98195-7236.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Chlamydia pneumoniae has been demonstrated in atherosclerotic lesions of coronary arteries and aorta. A seroepidemiological study found C pneumoniae–specific antibody more frequently in persons with significant carotid artery wall thickening than in matched control subjects.
Methods and Results Fresh-frozen or formalin-fixed tissue obtained at carotid endarterectomy was examined by immunocytochemistry (ICC) and the polymerase chain reaction (PCR) for the presence of C pneumoniae. Five of five fresh-frozen and formalin-fixed carotid endarterectomy specimens were positive for C pneumoniae by ICC (three of five by PCR). A total of 56 archival formalin-fixed, paraffin-embedded carotid endarterectomy tissues from three hospitals were examined by ICC. Thirty-two were positive. Thirteen normal carotid artery tissue sections from six patients were negative for C pneumoniae.
Conclusions C pneumoniae organisms are frequently found in the advanced carotid atherosclerotic lesions of persons undergoing endarterectomy. Although these findings do not establish causality for C pneumoniae in carotid artery atherosclerosis, they should stimulate investigation of a possible causal or pathogenic role for the organism in the disease.
Key Words: immunohistochemistry • polymerase chain reaction • chlamydia • immunology
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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The chlamydia isolates called TWAR (after the laboratory designations of the first two isolates, TW-183 and AR-39) were first isolated from respiratory infections a decade ago1 and have subsequently been shown to be a common cause of pneumonia, bronchitis, sinusitis, and pharyngitis in both adults2 and children.3 DNA homology studies and electron microscopy established these organisms as a new third species of chlamydia named Chlamydia pneumoniae.4
The three chlamydia species that infect humans (C trachomatis, C psittaci, and C pneumoniae) are known to cause disease of the heart (myocarditis) and blood vessels (endocarditis).5 Saikku and colleagues6 showed an association of C pneumoniae antibody and coronary artery disease. These seroepidemiological observations were extended by the Finnish group and others.7 8 9 10 In addition, an association of C pneumoniae antibody and carotid artery thickening has been reported.11
The presence of C pneumoniae (TWAR) in atheroma of the coronary artery has been established in several populations by ICC, PCR, and electron microscopy.12 13 14 By ICC techniques, TWAR was demonstrated in macrophages and smooth muscle cells of atheromatous plaques of the aorta.15
This study was undertaken to explore the presence of the organism in other arteries of the body. The finding of C pneumoniae in a high proportion of atheromatous plaque material removed by surgery from carotid arteries led to an expansion of the data by use of archival tissue. As in our other studies, the organism was not found in normal arteries.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Tissue Examined
The tissue studied was obtained at the Dameron Hospital Heart Institute, Stockton, Calif, during surgery. The first group of tissues examined was frozen in dry ice and shipped to Seattle. The specimens were divided and used for culture and PCR. Formalin-fixed tissue in paraffin blocks from the same patients (although a different part of the removed tissue) was available for ICC study. The tissue blocks from carotid endarterectomy typically had several pieces of plaque material embedded together. Serum from a blood sample obtained before surgery was available from the prospectively studied patients. The study was approved by the Institutional Review Committee of the Dameron Hospital, and all patients studied prospectively signed consent forms.
Additional formalin-fixed tissue blocks from carotid endarterectomy were studied. These tissues were from the surgical pathology archives of the Dameron Hospital, the HMC, and the UWMC, Seattle.
The formalin-fixed portion of each carotid artery specimen was paraffin-embedded and processed according to conventional techniques. Serial sections 4 µm thick were cut and placed on glass slides for hematoxylin-eosin and ICC staining.
ICC Staining for Detection of Chlamydia-Specific
Antigens
To determine the presence of chlamydia, one slide from each
specimen was stained with CF-2, a genus-specific monoclonal
antibody directed against chlamydial lipopolysaccharide.
Immunoperoxidase staining was done by the
avidin-biotin-peroxidase method as described
previously.13 Briefly, it involved deparaffinizing
sections, blocking endogenous peroxidase activity with
H2O2, and applying the primary CF-2
antibody (1:6000 dilution) for 60 minutes. A biotinylated
anti-mouse secondary antibody was then applied for 30 minutes,
followed by an avidin-biotin-peroxidase conjugate (ABC Elite,
Vector Laboratories) for 30 minutes at room temperature. Standard
peroxidase enzyme substrate, 3,3'-diaminobenzidine with nickel
chloride, was then added to yield a black reaction product. The
slides were counterstained with methyl green. For each experiment, a
slide with C pneumoniae–infected HL cells was used as a
positive control, and a tissue section stained with mouse ascitic fluid
from mice inoculated with NS-O myeloma cells was used as a negative
control. Ten of the carotid artery tissue sections that stained
positive with CF-2 were stained with TT-401 (1:2000 dilution), a
C pneumoniae species–specific monoclonal antibody, and
KK-12 (1:6000 dilution), a C trachomatis
species–specific monoclonal antibody.15
Detection of C pneumoniae–Specific DNA by
PCR
PCR was done with the HL-1, HR-1 primer set as described by
Campbell et al.16 DNA was purified from tissues either as
described previously13 or by Qiagen columns according to
the directions of the manufacturer (Qiagen). Mock extractions of buffer
were done and amplified to ensure that no contamination occurred during
DNA isolation. Controls consisting of PCR reagents without any specimen
and with various dilutions of purified C pneumoniae DNA were
done in each PCR run as the negative and positive controls,
respectively. Confirmation of presumptive positive specimens and
detection of products below the sensitivity of agarose gels were
done by immunochemiluminescence using the 474-bp Pst I
C pneumoniae–specific fragment as a probe of Southern
transfers as described by Kuo et al.13 If the specimen was
negative, aliquots were seeded with 100 fg of C pneumoniae
DNA and reamplified to rule out that the negative results were due to
inhibition of PCR. If inhibition was observed, drop dialysis against
sterile water was performed and PCR was repeated.
To minimize the risk of contamination, tissue homogenization, DNA extraction, sample preparation, PCR amplification, and product analysis were done in separate rooms. For tissue homogenization, separate pairs of forceps, scissors, and mortar and pestle were used for each tissue. The rooms used for tissue homogenization, DNA extraction, and sample preparation were equipped with germicidal lamps. Before and after use, the laboratory benches were wiped with Absolve (DuPont NEN) and the UV lights were turned on for 30 minutes.
Cell Culture for C pneumoniae
The HL cell line was
used for isolation of C
pneumoniae.17
Microimmunofluorescence
Serology
The chlamydia microimmunofluorescence
technique18 with whole elementary bodies of TWAR strain
AR-39 as antigen was used for serology. Antibodies in the IgG and IgA
serum fractions were measured.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Surgical specimens from five patients undergoing carotid artery surgery were studied prospectively. The results are shown in Table 1
. All had advanced atherosclerotic plaques, as shown on
hematoxylin-eosin–stained sections; four were calcified. All
had chlamydia genus–specific antigen demonstrated by ICC, although
the number of positive fragments varied among the patients. The density
of antigen staining in two of these patients was greater than had been
seen in our previous studies of coronary arteries and
aorta.12 13 14 15 PCR for
TWAR-specific DNA was positive in
three patients (two of three fresh-frozen and one of two
formalin-fixed specimens). All the patients had some TWAR antibody,
although the titers varied. None of the four isolation attempts by
culture were successful.
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To obtain more information on carotid artery tissue and C
pneumoniae, we studied formalin-fixed blocks of tissue from
carotid endarterectomies from the archives of the Department of
Pathology of the Dameron Hospital, HMC, and UWMC. Table 2
summarizes the results of the ICC studies of these
tissues. Tissue from 56 patients was examined, and 31 were positive for
chlamydia antigen.
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The difference in percent positive between the archival specimens and the fresh specimens was not statistically significant. The majority of tissue pieces (lesion fragments) examined from the positive patients of the Dameron Hospital and HMC were positive (61 of 100). Two of 25 positive patients had only one piece positive.
The Figure shows photomicrographs of the ICC stain of a
positive section with control.
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Five of the Dameron Hospital ICC positive carotid tissues and five of the HMC positive tissues were stained with C pneumoniae and C trachomatis species–specific monoclonal antibodies to demonstrate the species specificity of the ICC positive tissues with the genus-specific antibody. All 10 stained with the C pneumoniae–specific antibody but not with the C trachomatis antibody.
PCR was attempted with these fixed carotid tissues with limited success. Most of the HMC specimens had been decalcified with formic acid. Of the five that were not decalcified, two were ICC positive, and one of the two was PCR positive. No other HMC specimen was PCR positive. Four of the 20 Dameron Hospital archival tissues were PCR positive; three of these were ICC positive, and one was not. EDTA was used for decalcification, and each of the positive tissues had been treated. The PCR-positive, ICC-negative patient brings the number of Dameron Hospital archival specimens positive for TWAR to 14 and the total for the archival specimens to 32. PCR was not attempted with the UWMC specimens, all of which had been decalcified with formic acid.
Thirteen specimens of normal carotid arteries (8 specimens from one person) were obtained at autopsy from six patients, 21 to 46 years old, and examined for TWAR antigen by ICC. All were negative.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The major finding of this investigation is that C pneumoniae can frequently be demonstrated in atherosclerotic plaques removed from carotid arteries. This extends the finding of C pneumoniae in atherosclerotic lesions in coronary arteries and aorta to another major artery in which atherosclerotic disease causes an important health hazard. Finding the organism in the arterial lesion does not prove that C pneumoniae is the cause of or plays a role in the progression of atherosclerosis. However, having a known pathogen replicating in the viable cells of atheromata raises the possibility that C pneumoniae contributes to the pathogenesis of atherosclerosis.
Most of the persons studied were consecutive patients undergoing endarterectomy in the three hospitals during the years indicated. We have no information on how representative they are of persons with carotid atherosclerosis in the population at large.
The ICC findings in our series of studies of coronary artery and aorta atheromata12 13 14 15 were confirmed and supported in a number of ways. Two monoclonal antibodies, a genus- and a species-specific antibody, were used. Whenever the genus antibody, which is more avid, was positive, the C pneumoniae–specific antibody was positive. Multiple observers, including pathologists with extensive experience with ICC, have confirmed the findings.12 13 14 15 Double immunocytochemical staining showed that the C pneumoniae antigen is associated with macrophages and smooth muscle cells.15 Because of the very small (4-µm) area of an atheroma represented on a microscopic slide, it is more likely that the ICC results are an underestimation than an overestimation of the frequency of C pneumoniae organisms in atheromata.
Because all previous ICC-positive results for chlamydia in arteries have proved to be C pneumoniae, in this study not all ICC genus–positive specimens were stained with C pneumoniae–specific antibody. Five carotid artery positive specimens each from Dameron Hospital and HMC were confirmed as C pneumoniae with the species-specific antibody.
Our PCR technique for identifying C pneumoniae–specific DNA has been successful with throat swab specimens. PCR has been more sensitive than culture and been closely associated with serological evidence of infection.19 20 Although the PCR results with arterial tissue have confirmed that C pneumoniae is present in atheromata, PCR has been less sensitive than ICC in identifying the organism.13 14 In fixed tissues, formalin may degrade DNA.21 With fresh tissue, there is still some lack of sensitivity that may be due to inhibitors but remains to be explained.
The normal carotid artery tissue specimens examined were not age-matched to the case specimens. Obtaining appropriate comparison of normal artery tissue has been a problem throughout our studies.13 14 15 It is virtually impossible to find older adults without atherosclerosis. Data suggesting that the organism is not found in normal-appearing arteries have been restricted to studies of arteries of young persons or normal parts of arteries with atherosclerosis elsewhere. We also failed to demonstrate C pneumoniae in arteries not expected to develop atherosclerosis, such as the internal mammary artery. Recently we studied age- and sex-matched normal coronary artery segments with material provided from the multicenter study entitled "Pathobiological Determinants of Atherosclerosis in Youth,"22 in which forensic autopsies on persons 15 to 34 years old were studied. Although most of the segments with atheromatous plaques were positive for C pneumoniae, none of 31 normal control coronary artery segments were positive.23
Although C pneumoniae infection can be effectively treated with tetracyline and macrolide drugs, it is not known whether the organisms in atheromata, in which there is probably a low level of replication, could be eradicated by treatment or whether eradication of the organisms would have a favorable effect on the lesions. Nevertheless, because of the lack of effective medical treatment of carotid artery narrowing, a controlled treatment trial with an effective antibiotic might be considered for patients ineligible for surgery or before the need for surgery.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a grant (RO1-AI-21885) from the National Institutes of Health.
Received July 5, 1995; revision received August 23, 1995; accepted September 11, 1995.
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F. Chierichetti, E. Arbustini, V. Arici, S. P. Moghadam, B. Conti, and A. Bagliani Identification of Chlamydia pneumoniae DNA in Carotid Plaques Angiology, October 1, 2000; 51(10): 827 - 830. [Abstract] [PDF]
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S A Morre, W Stooker, W K Lagrand, A J C van den Brule, and H W M Niessen Microorganisms in the aetiology of atherosclerosis J. Clin. Pathol., September 1, 2000; 53(9): 647 - 654. [Abstract] [Full Text] [PDF]
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C. Espinola-Klein, H.-J. Rupprecht, S. Blankenberg, C. Bickel, H. Kopp, G. Rippin, G. Hafner, U. Pfeifer, and J. Meyer Are Morphological or Functional Changes in the Carotid Artery Wall Associated With Chlamydia pneumoniae, Helicobacter pylori, Cytomegalovirus, or Herpes Simplex Virus Infection? Stroke, September 1, 2000; 31(9): 2127 - 2133. [Abstract] [Full Text] [PDF]
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M. Mayr, S. Kiechl, J. Willeit, G. Wick, and Q. Xu 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, August 22, 2000; 102(8): 833 - 839. [Abstract] [Full Text] [PDF]
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C. Schmidt, J. Hulthe, J. Wikstrand, H. Gnarpe, J. Gnarpe, S. Agewall, and B. Fagerberg Chlamydia pneumoniae Seropositivity Is Associated With Carotid Artery Intima-Media Thickness Stroke, July 1, 2000; 31(7): 1526 - 1531. [Abstract] [Full Text] [PDF]
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C. J. Wiedermann, S. Kiechl, S. Dunzendorfer, P. Schratzberger, G. Egger, F. Oberhollenzer, and J. Willeit Association of endotoxemia with carotid atherosclerosis and cardiovascular disease: Prospective results from the bruneck study J. Am. Coll. Cardiol., December 1, 1999; 34(7): 1975 - 1981. [Abstract] [Full Text] [PDF]
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J. Boman, C. A. Gaydos, and T. C. Quinn Molecular Diagnosis of Chlamydia pneumoniae Infection J. Clin. Microbiol., December 1, 1999; 37(12): 3791 - 3799. [Full Text]
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Y. Fan, S. Wang, and X. Yang Chlamydia trachomatis (Mouse Pneumonitis Strain) Induces Cardiovascular Pathology following Respiratory Tract Infection Infect. Immun., November 1, 1999; 67(11): 6145 - 6151. [Abstract] [Full Text] [PDF]
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A. Meijer, J. A. van der Vliet, P. J. M. Roholl, S. K. Gielis-Proper, A. de Vries, and J. M. Ossewaarde Chlamydia pneumoniae in Abdominal Aortic Aneurysms : Abundance of Membrane Components in the Absence of Heat Shock Protein 60 and DNA Arterioscler Thromb Vasc Biol, November 1, 1999; 19(11): 2680 - 2686. [Abstract] [Full Text] [PDF]
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 P. J. Cook Antimicrobial therapy for Chlamydia pneumoniae: its potential role in atherosclerosis and asthma J. Antimicrob. Chemother., August 1, 1999; 44(2): 145 - 148. [Full Text] [PDF]
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 P.-Y. Lovey, A. Morabia, D Bleed, O Péter, G Dupuis, and J Petite Long term vascular complications of Coxiella burnetii infection in Switzerland: cohort study BMJ, July 31, 1999; 319(7205): 284 - 286. [Abstract] [Full Text]
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M. Thomas, Y. Wong, D. Thomas, M. Ajaz, V. Tsang, P. J. Gallagher, and M. E. Ward Relation Between Direct Detection of Chlamydia pneumoniae DNA in Human Coronary Arteries at Postmortem Examination and Histological Severity (Stary Grading) of Associated Atherosclerotic Plaque Circulation, June 1, 1999; 99(21): 2733 - 2736. [Abstract] [Full Text] [PDF]
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Y-K Wong, P J Gallagher, and M E Ward Chlamydia pneumoniae and atherosclerosis Heart, March 1, 1999; 81(3): 232 - 238. [Abstract] [Full Text]
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K. Knudsen, A. S. Madsen, P. Mygind, G. Christiansen, and S. Birkelund Identification of Two Novel Genes Encoding 97- to 99-Kilodalton Outer Membrane Proteins of Chlamydia pneumoniae Infect. Immun., January 1, 1999; 67(1): 375 - 383. [Abstract] [Full Text] [PDF]
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M. Davidson, C.-C. Kuo, J. P. Middaugh, L. A. Campbell, S.-P. Wang, W. P. Newman III, J. C. Finley, and J. T. Grayston Confirmed Previous Infection With Chlamydia pneumoniae (TWAR) and Its Presence in Early Coronary Atherosclerosis Circulation, August 18, 1998; 98(7): 628 - 633. [Abstract] [Full Text] [PDF]
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P. M. Ridker Inflammation, Infection, and Cardiovascular Risk : How Good Is the Clinical Evidence? Circulation, May 5, 1998; 97(17): 1671 - 1674. [Full Text] [PDF]
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K. Yamashita, K. Ouchi, M. Shirai, T. Gondo, T. Nakazawa, and H. Ito Distribution of Chlamydia pneumoniae Infection in the Atherosclerotic Carotid Artery Stroke, April 1, 1998; 29(4): 773 - 778. [Abstract] [Full Text] [PDF]
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J. B. Muhlestein, J. L. Anderson, E. H. Hammond, L. Zhao, S. Trehan, E. P. Schwobe, and J. F. Carlquist Infection With Chlamydia pneumoniae Accelerates the Development of Atherosclerosis and Treatment With Azithromycin Prevents It in a Rabbit Model Circulation, February 24, 1998; 97(7): 633 - 636. [Abstract] [Full Text] [PDF]
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P. J. Cook, D. Honeybourne, G. Y. H. Lip, D. G. Beevers, R. Wise, and P. Davies Chlamydia pneumoniae Antibody Titers Are Significantly Associated With Acute Stroke and Transient Cerebral Ischemia : The West Birmingham Stroke Project Stroke, February 1, 1998; 29(2): 404 - 410. [Abstract] [Full Text] [PDF]
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P. Libby, D. Egan, and S. Skarlatos Roles of Infectious Agents in Atherosclerosis and Restenosis: An Assessment of the Evidence and Need for Future Research Circulation, December 2, 1997; 96(11): 4095 - 4103. [Full Text]
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A. Laurila, A. Bloigu, S. Nayha, J. Hassi, M. Leinonen, and P. Saikku Chronic Chlamydia pneumoniae Infection Is Associated With a Serum Lipid Profile Known to Be a Risk Factor for Atherosclerosis Arterioscler Thromb Vasc Biol, November 1, 1997; 17(11): 2910 - 2913. [Abstract] [Full Text]
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B. Chiu, E. Viira, W. Tucker, and I.W. Fong Chlamydia Pneumoniae, Cytomegalovirus, and Herpes Simplex Virus in Atherosclerosis of the Carotid Artery Circulation, October 7, 1997; 96(7): 2144 - 2148. [Abstract] [Full Text]
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M. Maass, E. Krause, P.M. Engel, S. Kruger, and M. Maass Endovascular Presence of Chlamydia pneumoniae in Patients with Hemodynamically Effective Carotid Artery Stenosis Angiology, August 1, 1997; 48(8): 699 - 706. [Abstract] [PDF]
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 MORE EVIDENCE LINKS CHLAMYDIA TO ATHEROSCLEROSIS Journal Watch (General), December 29, 1995; 1995(1229): 3 - 3. [Full Text]
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