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(Stroke. 1998;29:773-778.)
© 1998 American Heart Association, Inc.

Original Contributions

Distribution of Chlamydia pneumoniae Infection in the Atherosclerotic Carotid Artery

Katsuhiro Yamashita, MD; Kazunobu Ouchi, MD; Mutsunori Shirai, MD; Toshikazu Gondo, MD; Teruko Nakazawa, MD; Haruhide Ito, MD

From the Departments of Neurosurgery (K.Y., H.I.), Microbiology (M.S., T.N.), and Pathology (T.G.), Yamaguchi University School of Medicine, Yamaguchi, Japan, and the Department of Pediatrics, Saiseikai Shimonoseki General Hospital (K.O.), Shimonoseki, Japan.

Correspondence to Katsuhiro Yamashita, Department of Neurosurgery, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, Yamaguchi, 755 Japan. E-mail yamasita-ygc{at}umin.u-tokyo.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose—Chlamydia pneumoniae infection has recently become noteworthy in relation to atherosclerosis. We investigated by immunohistochemistry the distribution of C pneumoniae infection in the atherosclerotic carotid artery.

Methods—Twenty carotid atherosclerotic lesions that were resected during carotid endarterectomy were investigated. Parallel sections were stained immunohistochemically with monoclonal antibodies for a C pneumoniae–specific antigen, macrophages, and smooth muscle cells.

Results—Immunoreactivity for the C pneumoniae–specific antigen was observed in 11 of 20 specimens (55%), and intense immunoreactivity was observed in 7 of 20 (35%). C pneumoniae infection was observed in endothelial cells, macrophages and in smooth muscle cells that had migrated into the atheromatous plaque, as well as in smooth muscle cells and small arteries in the media underlying the atheromatous plaques. C pneumoniae infection was most prominently observed in smooth muscle cells. The severity of the infection as demonstrated by immunohistochemistry was not significantly related to general risk factors for atherosclerosis.

Conclusions—C pneumoniae widely infects endothelial cells, macrophages, and smooth muscle cells in the atherosclerotic carotid artery. The results of the present study can help us to understand how C pneumoniae infection contributes to the progression of carotid atherosclerosis.

Key Words: atherosclerosis • carotid arteries • Chlamydia pneumoniae • infection

	Introduction

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Chlamydia pneumoniae, a gram-negative bacteria, frequently causes community-acquired respiratory infections, such as pharyngitis and pneumonia. There are epidemics of C pneumoniae infection every 5 to 7 years, with 50% to 70% prevalence of seropositivity in adults, and most adults are infected 2 to 3 times in their lifetime.^{1 2}

Recently, C pneumoniae has been linked to an atherosclerotic disease. Since Saikku et al³ first reported that chronic C pneumoniae infection is a risk factor for coronary heart disease, a number of investigations have shown a positive relationship between C pneumoniae infection and atherosclerosis.^{4 5 6 7 8 9 10 11 12 13 14} Shor et al⁹ detected by electron microscopy bodies of C pneumoniae in lipid-rich areas of fibrous plaques and in intimal smooth muscle cells of the coronary arteries. The organism has also been detected in atherosclerotic lesions by immunohistochemistry, the polymerase chain reaction method, and cell culture.^{10 11 12 13 14 15} Moreover, the presence of C pneumoniae in carotid atherosclerotic lesions and its possible contribution to ischemic cerebrovascular diseases have been reported.^{16 17 18} However, the detailed distribution of C pneumoniae infection in the carotid arterial wall and atheromatous plaque remains to be elucidated. The distribution of C pneumoniae infection may indicate how it contributes to the progression of atherosclerotic lesions.

The aim of the present study was to clarify the distribution of C pneumoniae infection in carotid arterial walls and atheromatous plaques, which were resected during carotid endarterectomy (CEA) by immunohistochemistry, with use of monoclonal antibodies for a C pneumoniae–specific antigen, macrophages, and smooth muscle cells. In addition, we investigated the relationship between the severity of the infection, as demonstrated by immunohistochemistry, and general risk factors for atherosclerosis.

	Materials and Methods

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Twenty specimens of carotid atheromatous plaque were obtained during CEAs performed in 19 patients with symptomatic severe (>70%) carotid artery stenosis. The severity of carotid artery stenosis in these patients was verified by conventional carotid angiography. The symptoms of patients were transient ischemic attacks in 8 of 19 and minor stroke in 11 of 19 patients. The specimens were embedded in paraffin after fixation with 10% buffered formaldehyde and cut serially to expose coronal planes of the carotid artery and atheromatous plaque. Eight serial sections 6 µm thick were prepared from each specimen for the following: hematoxylin-eosin staining, elastica–van Gieson staining to visualize elastic fibers, immunohistochemical staining, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining to detect apoptotic cells.

Immunohistochemical Staining for C pneumoniae Infection and Identification of Cell Types With Infection
Three parallel sections of each specimen were immunohistochemically stained with the following monoclonal antibodies: CF-2, a Chlamydia genus–specific monoclonal antibody that recognizes Chlamydial lipopolysaccharide (Washington Research Foundation) diluted 1:1000; AY-6, a C pneumoniae–specific monoclonal antibody that recognizes a 53-kDa outer membrane protein (Hitachi Chemical), diluted 1:1000; and Virostat 1641, a C trachomatis–specific monoclonal antibody that recognizes a major outer membrane protein (Virostat), diluted 1:100. Another two parallel sections from each specimen were also immunohistochemically stained to identify the cell types infected by C pneumoniae, with two monoclonal antibodies: anti-human -smooth muscle actin (DACO), diluted 1:50, and anti-human macrophage KP-1 (DACO), diluted 1:10. The immunoreactivity was detected by immunoperoxidase staining through use of the streptavidin-biotin-peroxidase method (LSAB kit; DACO) and was visualized with diaminobenzidine tetrahydrochloride as the chromogen. Control slides with HEp-2 cell monolayers infected with C pneumoniae or C trachomatis were processed in parallel with the tissue sections. Finally, sections were counterstained with hematoxylin. Immunoreactivity for C pneumoniae infection was assessed in the whole area of each section according to the following four grades: 3+, intense immunoreactivity in numerous cells; 2+, positive immunoreactivity in a moderate number of cells (10 to 100 cells); 1+, weak immunoreactivity in a few cells; and 0, no immunoreactivity.

In Situ End Labeling of Fragmented DNA (TUNEL Staining)
TUNEL staining was performed with use of an in situ apoptosis detection kit (ApopTag Plus; Oncor Inc) to detect cells with DNA fragmentation. Briefly, deparaffinized sections were incubated with 20 µg/mL proteinase K for 15 minutes at room temperature to digest the proteins in the specimens, washed with water, and treated with 2% vol/vol hydrogen peroxide for 5 minutes to extinguish the endogenous peroxidase activity. Immediately afterward, the sections were washed with distilled water and immersed in equilibration buffer, then in terminal deoxynucleotidyltransferase enzyme solution for 1 hour at 37°C, followed by stop/wash buffer. Subsequently, 35 µL anti-digoxigenin-peroxidase solution was applied to each slide, and peroxidase was detected by staining with 0.025% wt/vol diaminobenzidine. Sections were finally counterstained with hematoxylin. Negative controls were treated with distilled water instead of the terminal deoxynucleotidyltransferase enzyme.

Risk Factors
The general risk factors for atherosclerosis, smoking, hypertension, hypercholesterolemia, diabetes mellitus, and complications of ischemic heart diseases were investigated for each patient. The severity of each risk factor was graded as follows: current smoker (>20 cigarettes/day), former smoker, and never smoked; with hypertension and without hypertension; severe hypercholesterolemia of >250 mg/dL, moderate hypercholesterolemia of 220 to 250 mg/dL, and no hypercholesterolemia; severe diabetes mellitus with systemic complications such as retinal and renal diseases, moderate diabetes mellitus without systemic complications, and no diabetes mellitus.

Statistical Analysis
The relationship between immunoreactivity for C pneumoniae infection in the atherosclerotic carotid artery and general risk factors for atherosclerosis was investigated using multiple regression analysis, and a value of P<.05 was considered to indicate statistical significance.

	Results

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The intensity of CF-2 (a Chlamydia genus–specific monoclonal antibody) immunoreactivity was the same as that of AY-6 (a C pneumoniae–specific monoclonal antibody) in all specimens. Eleven of 20 specimens (55%) were positive for CF-2 and AY-6 immunoreactivity, and intense AY-6 immunoreactivity (grade 3) was observed in 7 of 20 specimens (35%) (Table 1).

View this table: [in this window] [in a new window]   Table 1. Immunoreactivity for CF-2, AY-6, and Virostat Monoclonal Antibodies in the Atherosclerotic Carotid Artery and Risk Factors for Atherosclerosis in 19 Patients

Cells with AY-6 immunoreactivity were observed both in the intima near the atheromatous plaque and in the media, which was partially removed during the CEA procedure. The border between the intima and the media was clearly demonstrated by elastica–van Gieson staining. AY-6 immunoreactivity was observed in endothelial cells of the intima near the atheromatous plaque (Fig 1). Within the atheromatous plaque, macrophages and smooth muscle cells that had migrated from the media into the plaque showed AY-6 immunoreactivity (Figs 2 and 3); cells with AY-6 immunoreactivity were identified by immunohistochemical staining of adjacent sections for KP-1 and -smooth muscle actin. Macrophages with C pneumoniae infection were predominantly located in the subendothelial region. Numerous cells with AY-6 immunoreactivity observed in the media were smooth muscle cells underlying the atheromatous plaques as well as those of small arteries that supply the carotid arterial wall (Figs 4 and 5). These smooth muscle cells were also identified by immunohistochemistry for -smooth muscle actin. The cells with C pneumoniae infection formed clusters that were distributed in a patchy pattern in the atheromatous plaque and the media.

View larger version (142K): [in this window] [in a new window]   Figure 1. Case 2. AY-6 (a C pneumonia–specific monoclonal antibody) immunoreactivity in endothelial cells (arrows). Weak immunoreactivity was also observed in form cells in the intima (below the arrows) (original magnification x100, bar=50 µm).

View larger version (157K): [in this window] [in a new window]   Figure 2. A, AY-6 immunoreactivity in cells within an atheromatous plaque (case 8) (original magnification x56; bar=100 µm). B, Immunohistochemical staining for KP-1 to detect macrophages in the section adjacent to that shown in panel A. Many KP-1 positive cells were scattered throughout the atheromatous plaque with a distribution similar to that for AY-6 (original magnification x56; bar=100 µm).

View larger version (84K): [in this window] [in a new window]   Figure 3. A, Coronal view of the atheromatous specimen of case 12 (hematoxylin-eosin staining; original magnification x10; bar=400 µm). The area in the thickening intima, which is surrounded by arrowheads, is magnified in panels B and C. B, Immunohistochemical staining for -smooth muscle actin to detect smooth muscle cells. Smooth muscle cells that had migrated were located in the atheromatous plaque in the intima (original magnification x50; bar=50 µm). C, AY-6 immunohistochemical staining (original magnification x50; bar=50 µm). The distribution of the AY-6 positive cells is similar to that for -smooth muscle actin as shown in panel B.

View larger version (138K): [in this window] [in a new window]   Figure 4. A, Coronal view of the atheromatous specimen of case 12 (hematoxylin-eosin staining; original magnification x25; bar=200 µm). The area in the media, which is shown below the arrowheads, is magnified in panel B. B, AY-6 immunohistochemical staining. AY-6 immunoreactivity was observed in numerous smooth muscle cells in the media underlying the atheromatous plaques (original magnification x50; bar=75 µm).

View larger version (145K): [in this window] [in a new window]   Figure 5. AY-6 immunoreactivity in small arteries in the deep media underlying the atheromatous plaques. The immunoreactivity was observed in endothelial cells and in smooth muscle cells of small arteries (arrows) (original magnification x50; bar=100 µm).

The immunoreactivity for Virostat 1641 (a C trachomatis–specific monoclonal antibody) was not observed in the atheromatous plaque or in the media of any of the specimens. Only a few scattered TUNEL-positive cells were seen in the subendothelial region of the atheromatous plaques in two specimens with intense AY-6 immunoreactivity (data not shown).

The risk factors for atherosclerosis of each patient are shown in Table 1. The positivity of each risk factor for all 19 patients was 31.6% (6 of 19) for smoking, 73.7% (14 of 19) for hypertension, 31.6% (6 of 19) for diabetes, and 10.5% (2 of 19) for hypercholesterolemia. Ischemic heart disease was present in 3 of 19 patients (15.8%). None of these risk factors correlated significantly with AY-6 immunoreactivity, that is, with the severity of C pneumoniae infection.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, C pneumoniae infection of the carotid arterial wall and the atheromatous plaque was observed in 55% of the patients. Although this frequency of infection is very similar to those reported in other studies,^{9 10 11 13 14} C pneumoniae infection was not observed in all our patients. It is more likely that the results of the histopathologic examination underestimate rather than overestimate the frequency of C pneumoniae infection, because only a very small region with a 6-µm thickness of each atherosclerotic lesion was examined.

We demonstrated that C pneumoniae infected both the intima near an atheromatous plaque and the media of the carotid artery. Infection was localized to endothelial cells in the intima and to macrophages and smooth muscle cells in the atheromatous plaque. This finding is similar to those reported for in vivo studies of an aorta and a coronary artery and in vitro studies,^{9 10 11 13 19 20 21} although this is the first report of the infection in endothelial cells in vivo. Macrophage seem to play an important role in the transportation of C pneumoniae bodies and progression of atherosclerosis. It is thought that C pneumoniae is taken up by macrophages in the pharynx or the lung after a chronic infection and transported via the blood to the subendothelial region through the injured endothelium of the artery.^{22 23} According to the hypothesis of Ross,²⁴ macrophages in the intima produce some cytokines and growth factors (such as platelet-derived growth factor) and elicit migration of smooth muscle cells from the media to the intima, as well as an inflammatory response that subsequently leads to the progression of atherosclerosis. Chlamydial organisms survive and multiply in macrophages,^{25 26} and such a chronic infection of C pneumoniae in macrophages is believed to enhance the proliferative and inflammatory processes of atherosclerosis by inducing some cytokines and lipoproteins.^{27 28 29 30}

Infection by C pneumoniae was observed not only in smooth muscle cells that had migrated into the atheromatous plaque but also in smooth muscle cells in the media underlying the atheromatous plaques. The susceptibility of smooth muscle cells to C pneumoniae infection has been demonstrated in previous in vitro studies.^{19 20 21} The outcome of widespread infection of C pneumoniae in smooth muscle cells is unclear compared with that in macrophages. Growth factors such as platelet-derived growth factor in atherosclerotic lesions are also produced and released by smooth muscle cells,^{31 32 33} and this function of smooth muscle cells may be enhanced by chronic C pneumoniae infection.

Infection by C pneumoniae of the wall of small arteries in the media is interesting. It is generally believed that the C pneumoniae body is transported to the carotid arterial wall and the atheromatous plaque predominantly by macrophages that can pass through injured endothelium. However, another route of infection, in which C pneumoniae invades an atheromatous plaque and carotid arterial wall from small arteries in the media (vasa vasorum), is possible.

Recently, many investigations^{34 35 36 37 38} have shown the involvement of apoptosis of both macrophages and smooth muscle cells in atherosclerosis. Although TUNEL staining can cause overestimation of apoptosis, some cells in the atherosclerotic lesions surely undergo apoptotic cell death, and in addition, bacterial infection is a well known cause of apoptosis.³⁹ However, only a few TUNEL-positive cells were observed in the atheromatous plaques in the present study, which is consistent with other reports^{35 38} ; thus, there was a considerable discrepancy between the number of TUNEL-positive cells and the number of cells infected by C pneumoniae. The relationship between atherosclerosis and C pneumoniae infection probably does not involve the process of apoptosis.

We investigated well-characterized general risk factors for atherosclerosis (hypertension, hypercholesterolemia, diabetes, and cigarette smoking) for each patient, and none of these risk factors was related to the severity of C pneumoniae infection as demonstrated by immunohistochemistry, even though hypertension was observed in 74% of patients. This result is similar to those reported in previous studies of C pneumoniae infection of the coronary artery^{7 14} and does not support a positive relationship between C pneumoniae infection and atherosclerosis. However, some serological investigations of C pneumoniae infection have provided evidence for a significant relationship between infection and atherosclerosis based on the correction of general risk factors for atherosclerosis.^{4 5 7 16} Therefore, C pneumoniae infection can be related to carotid atherosclerosis independently of the general risk factors and will become the next risk factor for atherosclerosis.

In conclusion, we investigated the distribution of C pneumoniae infection in atherosclerotic carotid lesions resected during CEA. Infection by C pneumoniae was widespread in endothelial cells, macrophages, and smooth muscle cells that had migrated into the atheromatous plaque. In addition, smooth muscle cells and small arteries in the media of the carotid artery were also infected. This investigation has not necessarily established the etiologic role of C pneumoniae infection in the development of atherosclerosis, and further studies are required to confirm all of Koch's postulates, which establish the etiologic role of C pneumoniae infection in atherosclerosis.⁴⁰ However, clinical evidence that C pneumoniae infection contributes to atherosclerosis is accumulating,^{41 42} and an animal model of C pneumoniae infection in the atherosclerotic lesion has been developed.²³ The demonstration of the distribution of C pneumoniae infection in the present study, together with the animal model of C pneumoniae infection, will help to clarify how C pneumoniae infection contributes to the progression of carotid atherosclerotic lesions.

	Acknowledgments

 
We thank Dr Shiro Kashiwagi and Dr Shoichi Kato for helpful discussions in preparation of the manuscript and Fumio Iwasaki for his technical assistance in immunohistochemistry. We also thank the doctors in the departments of neurosurgery of Ube Kosan Central Hospital, Saiseikai Yamaguchi General Hospital, and National Yamaguchi Hospital for providing the carotid atheromatous plaque used in our study.

Received November 18, 1997; revision received January 12, 1998; accepted January 12, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Leinonen M. Pathogenetic mechanisms and epidemiology of Chlamydia pneumoniae. Eur Heart J. 1993;14(suppl K):57–61.

2. Kanamoto Y, Ouchi K, Mizui M, Ushio M, Usui T. Prevalence of antibody to Chlamydia pneumoniae TWAR in Japan. J Clin Microbiol. 1991;29:816–818.[Abstract/Free Full Text]

3. Saikku P, Leinonen M, Mattila K, Ekman MR, Nieminen MS, Makela PH, Huttonen JK, Valtonen V. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet. 1988;2:983–986.[Medline] [Order article via Infotrieve]

4. Thom DH, Grayston JT, Siscovick DS, Wang SP, Weiss NS, Daling JR. Association of prior infection with Chlamydia pneumoniae and angiographically demonstrated coronary artery disease. JAMA. 1992;268:68–72.[Abstract/Free Full Text]

5. Saikku P, Leinonen M, Tenkanen L, Linnanmaki E, Ekman MR, Manninen V, Manttari M, Frick MH, Huttunen JK. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Intern Med. 1992;116:273–278.

6. Grayston JT. Chlamydia in atherosclerosis. Circulation. 1993;87:1408–1409.[Free Full Text]

7. Linnanmäki E, Leinonen M, Mattila K, Nieminen MS, Valtonen V, Saikku P. Chlamydia pneumoniae–specific circulating immune complexes in patients with chronic coronary heart disease. Circulation. 1993;87:1130–1134.[Abstract/Free Full Text]

8. Mendall MA, Carrington D, Strachan D, Patel P, Molineaux N, Levi J, Toosey T, Camm AJ, Northfield TC. Chlamydia pneumoniae: risk factors for seropositivity and association with coronary heart disease. J Infect. 1995;30:121–128.[Medline] [Order article via Infotrieve]

9. Shor A, Kuo CC, Patton DL. Detection of Chlamydia pneumoniae in the coronary artery atheroma plaque. S Afr Med J. 1992;82:158–161.[Medline] [Order article via Infotrieve]

10. Kuo CC, Shor A, Campbell LA, Fukushi H, Patton DL, Grayston JT. Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J Infect Dis. 1993;167:841–849.[Medline] [Order article via Infotrieve]

11. Kuo CC, Gown AM, Benditt EP, Grayston JT. Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain. Arterioscler Thromb. 1993;13:1501–1504.[Abstract/Free Full Text]

12. Kuo CC, Grayston JT, Campbell LA, Goo YA, Wissler RW, Benditt EP. Chlamydia pneumoniae (TWAR) in coronary arteries of young adults (15–34 years old). Proc Natl Acad Sci U S A. 1995;92:6911–6914.[Abstract/Free Full Text]

13. Campbell LA, O'Brien ER, Cappuccino AL, Kuo CC, Wang SP, Stewart D, Patton DL, Cummings PK, Grayston JT. Detection of Chlamydia pneumoniae TWAR in human coronary atherectomy tissues. J Infect Dis. 1995;172:585–588.[Medline] [Order article via Infotrieve]

14. Muhlestein JB, Hammond EH, Carlquist JF, Radicke E, Thomson MJ, Karagounis LA, Woods ML, Anderson JL. Increased incidence of Chlamydia species within the coronary arteries of patients with symptomatic atherosclerosis versus other forms of cardiovascular disease. J Am Coll Cardiol. 1996;27:1555–1561.[Abstract]

15. Jackson LA, Campbell LA, Kuo CC, Rodriguez DI, Lee A, Grayston JT. Isolation of Chlamydia pneumoniae from a carotid endarterectomy specimen. J Infect Dis. 1997;176:292–295.[Medline] [Order article via Infotrieve]

16. Melnick SL, Shahar E, Folsom AR, Grayston JT, Sorlie PD, Wang SP, Szxlo M. Past infection by Chlamydia pneumoniae strain TWAR and asymptomatic carotid atherosclerosis. Am J Med. 1993;95:499–504.[Medline] [Order article via Infotrieve]

17. Grayston JT, Kuo CC, Coulson AS, Campbell LA, Lawrence RD, Lee MJ, Strandness ED, Wang SP. Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery. Circulation. 1995;92:3397–3400.[Abstract/Free Full Text]

18. Wimmer MLJ, Sandmann-Strupp R, Saikku P, Haberl RL. Association of chlamydial infection with cerebrovascular disease. Stroke. 1996;27:2207–2210.[Abstract/Free Full Text]

19. Godzik KL, O'Brien ER, Wang SK, Kuo CC. In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae. J Clin Microbiol. 1995;33:2411–2414.[Abstract]

20. Gaydos CA, Summersgill JT, Sahney NN, Ramirez JA, Quinn TC. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells. Infect Immun. 1996;64:1614–1620.[Abstract]

21. Knoebel E, Vijayagopal P, Figueroa JE II, Martin DH. In vitro infection of smooth muscle cells by Chlamydia pneumoniae. Infect Immun. 1997;65:503–506.[Abstract]

22. Hammerschlag MR, Chirgwin K, Roblin PM, Gelling M, Dumornay W, Mandel L, Smith P, Schachter J. Persistent infection with Chlamydia pneumoniae following acute respiratory illness. Clin Infect Dis. 1992;14:178–182.[Medline] [Order article via Infotrieve]

23. Moazed TC, Kuo CC, Grayston JT, Campbell LA. Murine models of Chlamydia pneumoniae infection and atherosclerosis. J Infect Dis. 1997;175:883–890.[Medline] [Order article via Infotrieve]

24. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med. 1986;314:488–500.[Medline] [Order article via Infotrieve]

25. Kuo CC. Cultures of Chlamydia trachomatis in mouse peritoneal macrophages: factors affecting organism growth. Infect Immun. 1978;20:439–445.[Abstract/Free Full Text]

26. Chen WJ, Kuo CC. A mouse model of pneumonitis induced by Chlamydia trachomatis: morphologic, microbiologic, and immunologic studies. Am J Pathol. 1980;100:365–377.[Abstract]

27. Saikku P. Chlamydia pneumoniae infection as a risk factor in acute myocardial infarction. Eur Heart J. 1993;14(suppl K):62–65.

28. Rothermel CD, Schachter J, Lavrich P, Lipsitz EC, Francus T. Chlamydia trachomatis-induced production of interleukin-1 by human monocytes. Infect Immun. 1989;57:2705–2711.[Abstract/Free Full Text]

29. Kaukoranta-Tolvanen SS, Teppo AM, Leinonen M, Saikku P, Laitinen K. Chlamydia pneumoniae induces the production of TNF-, IL-1ß, and IL-6 by human monocytes. Proc Eur Soc Chlamydia Res. 1992;2:85.

30. Dinarello CA. Interleukin-1 and its biologically related cytokines. In: Cohen S, ed. Lymphokines and the Immune Response. Boca Raton, Fla: CRC Press; 1990: 145–179.

31. Sterpetti AV, Cucina A, Fragale A, Lepidi S, Cavallaro A, Santoro-D'Angelo L. Shear stress influences the release of platelet derived growth factor and basic fibroblast growth factor by arterial smooth muscle cells. Eur J Vasc Surg. 1994;8:138–142.[Medline] [Order article via Infotrieve]

32. Köster R, Windstetter Überfuhr P, Baumann G, Nikol S, Höfling B. Enhanced migratory activity of vascular smooth muscle cells with high expression of platelet-derived growth factor A and B. Angiology. 1995;46:99–106.

33. Murry CE, Bartosek T, Giachelli CM, Alpers CE, Schwartz SM. Platelet-derived growth factor-A mRNA expression in fetal, normal adult, and atherosclerotic human aortas: analysis by competitive polymerase chain reaction. Circulation. 1996;93:1095–1106.[Abstract/Free Full Text]

34. Geng YJ, Libby P. Evidence for apoptosis in advanced human atheroma: colocalization with interleukin-1ß-converting enzyme. Am J Pathol. 1995;147:251–266.[Abstract]

35. Isner JM, Kearney M, Bortman S, Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation. 1995;91:2703–2711.[Abstract/Free Full Text]

36. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995;95:2266–2274.

37. Björkerud S, Björkerud B. Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol. 1996;149:367–380.[Abstract]

38. Hegyi L, Skepper JN, Cary NRB, Mitchinson MJ. Form cell apoptosis and the development of the lipid core of human atherosclerosis. J Pathol. 1996;180:423–429.[Medline] [Order article via Infotrieve]

39. Müller A, Hacker J, Brand BC. Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection. Infect Immun. 1996;64:4900–4906.[Abstract]

40. Moxon ER. Microbes, molecules and man: The Mitchell Lecture, 1992. J R Coll Physicians Lond.. 1993;27:169–174.[Medline] [Order article via Infotrieve]

41. Gupta S, Leatham EW, Carrington D, Mendall MA, Kaski JC, Camm AJ. Elevated Chlamydia pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of myocardial infarction. Circulation. 1997;96:404–407.[Abstract/Free Full Text]

42. Gurfinkel E, Bozovich G, Daroca A, Beck E, Mautner B, for the ROXIS Study group. Randomised trial of roxithromycin in non-Q-wave coronary syndromes: ROXIS Pilot Study. Lancet. 1997;350:404–407.[Medline] [Order article via Infotrieve]

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				H. Yoneda, K. Miura, H. Matsushima, K. Sugi, T. Murakami, K. Ouchi, K. Yamashita, H. Itoh, T. Nakazawa, M. Suzuki, et al. Aspirin inhibits Chlamydia pneumoniae-induced NF-{kappa}B activation, cyclo-oxygenase-2 expression and prostaglandin E2 synthesis and attenuates chlamydial growth J. Med. Microbiol., May 1, 2003; 52(5): 409 - 415. [Abstract] [Full Text] [PDF]

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				C. Stollberger and J. Finsterer Role of Infectious and Immune Factors in Coronary and Cerebrovascular Arteriosclerosis Clin. Vaccine Immunol., March 1, 2002; 9(2): 207 - 215. [Full Text] [PDF]

				P. B. Gorelick Stroke Prevention Therapy Beyond Antithrombotics: Unifying Mechanisms in Ischemic Stroke Pathogenesis and Implications for Therapy: An Invited Review Stroke, March 1, 2002; 33(3): 862 - 875. [Abstract] [Full Text] [PDF]

				P. Lavallee, V. Perchaud, M. Gautier-Bertrand, D. Grabli, and P. Amarenco Association Between Influenza Vaccination and Reduced Risk of Brain Infarction Stroke, February 1, 2002; 33(2): 513 - 518. [Abstract] [Full Text] [PDF]

				J. Boman and M. R. Hammerschlag Chlamydia pneumoniae and Atherosclerosis: Critical Assessment of Diagnostic Methods and Relevance to Treatment Studies Clin. Microbiol. Rev., January 1, 2002; 15(1): 1 - 20. [Abstract] [Full Text]

				Z. G. Nadareishvili, D. E. Koziol, B. Szekely, C. Ruetzler, R. LaBiche, R. McCarron, T. J. DeGraba, and S. Jander Increased CD8+ T Cells Associated With Chlamydia pneumoniae in Symptomatic Carotid Plaque Editorial Comment Stroke, September 1, 2001; 32(9): 1966 - 1972. [Abstract] [Full Text] [PDF]

				L. B. Goldstein, R. Adams, K. Becker, C. D. Furberg, P. B. Gorelick, G. Hademenos, M. Hill, G. Howard, V. J. Howard, B. Jacobs, et al. Primary Prevention of Ischemic Stroke : A Statement for Healthcare Professionals From the Stroke Council of the American Heart Association Circulation, January 2, 2001; 103(1): 163 - 182. [Full Text] [PDF]

				L. B. Goldstein, R. Adams, K. Becker, C. D. Furberg, P. B. Gorelick, G. Hademenos, M. Hill, G. Howard, V. J. Howard, B. Jacobs, et al. Primary Prevention of Ischemic Stroke : A Statement for Healthcare Professionals From the Stroke Council of the American Heart Association Stroke, January 1, 2001; 32(1): 280 - 299. [Full Text] [PDF]

				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]

				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]

				J. Rodel, M. Woytas, A. Groh, K.-H. Schmidt, M. Hartmann, M. Lehmann, and E. Straube Production of Basic Fibroblast Growth Factor and Interleukin 6 by Human Smooth Muscle Cells following Infection with Chlamydia pneumoniae Infect. Immun., June 1, 2000; 68(6): 3635 - 3641. [Abstract] [Full Text] [PDF]

				A. Shor and J. I. Phillips Chlamydia pneumoniae and Atherosclerosis JAMA, December 1, 1999; 282(21): 2071 - 2073. [Full Text] [PDF]

				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]

				S. Sasu, D. LaVerda, N. Qureshi, D. T. Golenbock, and D. Beasley Chlamydia pneumoniae and Chlamydial Heat Shock Protein 60 Stimulate Proliferation of Human Vascular Smooth Muscle Cells via Toll-Like Receptor 4 and p44/p42 Mitogen-Activated Protein Kinase Activation Circ. Res., August 3, 2001; 89(3): 244 - 250. [Abstract] [Full Text] [PDF]

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