[Frontiers in Bioscience 8, e36-43, January 1, 2003]

CHLAMYDIAL INFECTIONS OF THE CARDIOVASCULAR SYSTEM

Cho-Chou Kuo and Lee Ann Campbell

Department of Pathobiology, University of Washington, Seattle, WA 98195, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Chlamydial infection and acute cardiac disease
4. C. pneumoniae infection and atherosclerosis
4.1. History
4.2. Seroepidemiology
4.3. Detection of the organism in atheromatous lesions
4.4. Detection of antigen and DNA and organism isolation
4.5. Detection of the organism in peripheral blood mononuclear cells
5. Chlamydial infection of the heart valves
6. Histopathologic findings in atherosclerotic lesions positive for C. pneumoniae
7. Pathogenesis of chlamydial infections
8. Animal models of pneumonia and atherosclerosis
9. Therapy
10. Conclusion
11. References

1. ABSTRACT

This paper presents a review on cardiovascular diseases which can be caused by chlamydial infection with the emphasis in the recent development in association between Chlamydia pneumoniae and cardiovascular disease. The review includes seroepidemiologic observations; the discovery of C. pneumoniae in atheromatous plaques; in vivo studies using animal models indicating that C. pneumoniae is a co-risk factor of hyperlipidemia for atherosclerosis; in vitro studies demonstrating putative mechanisms by which C. pneumoniae could contribute to the immunopathology of atherosclerosis; and early promising antibiotic intervention studies.

2. INTRODUCTION

Chlamydiae are obligate intracellular Gram-negative bacteria (1). These organisms have a unique life cycle and replicate within the membrane-bound phagosomes (inclusions). Two human species, Chlamydia trachomatis (1) and Chlamydia pneumoniae (2), cause distinct disease syndromes. C. trachomatis causes ocular and genital infections, C. pneumoniae the upper and lower respiratory infections. Chlamydia psittaci (1) and

Chlamydia percorum (3) causes infection of avian species and lower mammals. Infection in animals are usually systemic. Humans may be infected from sick animals. However, the virulence of animal species for humans varies. Avian strains are much more virulent to humans than mammalian strains. In humans, infection from exposure to sick birds are usually in the form of severe pneumonia or fever-of-unknown etiology. Infection from mammalian strains are rare. Chlamydiae are susceptible to macrolides, tetracyclines, quinolones, and rifampin groups of antibiotics. However, the antibiotics of choices are macrolides and tetracyclines.

A characteristic of chlamydial infection is chronic or persistent infection (4). The chronic inflammatory response has been considered a major factor in the development of tissue damage leading to functional impairments of the involved organs, such as blindness and fallopian tube obstruction and infertility in C. trachomatis ocular and genital infection, respectively (5). No analogous chronic conditions have been identified with C. pneumoniae infection. However, evidence accumulated during the past decade has indicated that atherosclerosis may be a manifestation of chronic or persistent C.pneumoniae infection of atheromatous plaques (6). These findings prompted the Infectious Disease Society of America to convene a symposium to discuss this topic in 1999 (7). In this chapter, the cardiovascular diseases that can be caused by chlamydial infection will be reviewed. The main focus, however, will be directed to the recent developments in the association of C. pneumoniae and atherosclerosis.

To avoid overwhelming the article with references, only key articles of original and significant work will be provided. Reports on contradictory findings will be quoted for the purpose of discussion. The readers can obtain additional references from review articles provided in this article and from the internet sources.

3. CHLAMYDIAL INFECTION AND ACUTE CARDIAC DISEASE

Although C. trachomatis and the avian species of C. psittaci have been associated with acute cardiac diseases such as myocarditis, endocarditis, and pericarditis in humans--these are sporadic cases. An extensive review on these acute cardiac diseases was reported in 1992 by Odeh and Oliven (8). Only C. pneumoniae has been associated with chronic cardiovascular diseases.

4. C. PNEUMONIAE INFECTION AND ATHEROSCLEROSIS

4.1. History

The association of C. pneumoniae and coronary heart disease (CHD) was first demonstrated by serologic evidence by Saikku et al. in 1988 (9). The presence of the organism in atheromatous tissue of the coronary artery was first demonstrated by electron microscopy (EM) and immunohistochemistry (IHS) by Shor et al. in 1992 (10). The first C. pneumoniae isolate was obtained by Ramirez et

al. in 1996 (11) from the coronary artery of a bypass patient with coronary atherosclerosis. To address whether C. pneumoniae may play a pathogenic role in atherosclerosis, experimental animal models have been employed. For example, using a rabbit model, Muhlestein et al. demonstrated that C. pneumoniae was a co-risk factor with hyperlipidemia for atherosclerosis in 1998 (12). In addition, similar observations were made in a mouse model by Moazed et al. in 1999 (13). Following these publications the research on C. pneumoniae and atherogenesis has increased rapidly over the next decade in various areas. These areas have included seroepidemiology, detection of C. pneumoniae in atherosclerotic lesions in the artery, use of animal models for studying the causative role and pathogenic mechanisms of C. pneumoniae in atherosclerosis (14-16). In addition, in vitro studies have been employed using vascular wall cells to elucidate the cellular and molecular mechanisms of C. pneumoniae infection in atherogenesis (17).

4.2. Seroepidemiology

Since the original report by Saikku in 1988 showing an association of antibodies against C. pneumoniae and chronic CHD (9,18,19), which was later confirmed by Thom et al. (20,21), over 50 papers have appeared in the literature that have investigated the association of anti-C. pneumoniae antibody and CHD. Except for a few negative studies (22), the vast majority of the studies demonstrated a positive association. The average odds-ratio in these studies is in general 2.0 or greater and the risk is independent of other risk factors of atherosclerosis, i.e. hypercholesterolemia, cigarette smoking, hypertension, diabetes, and family history (20,21,23-25) (Table 1). The association of C. pneumoniae antibody and CHD was made despite the relatively high prevalence of antibody in the general population (26). Fifty percent of middle-age and 70% to 80% of older adults have antibody against C. pneumoniae. The seroepidemiologic studies have also demonstrated an association of antibody against C. pneumoniae and carotid artery stenosis (27), aortic aneurysm (28,29), and cerebrovascular disease (30,31). The early reports on the positive association of C. pneumoniae antibodies and CHD raised the question as to whether the organism is present in the atheromatous lesions. This lead to the next stage of studies to determine if the organism could be detected in atheromatous lesions.

4.3. Detection of the organism in atheromatous lesions

There have been over 45 publications demonstrating C. pneumoniae organisms in atheromatous tissues by IHC, PCR, EM and isolation following the original reports by Shor et al. in 1992 (10) and Kuo et al. in 1993 (32).

4.4. Detection of Antigen and DNA and organism isolation

Overall, C. pneumoniae was detected by IHC, PCR, or both in atheromatous tissues from 50% of subjects but was rarely found (1%) in normal vascular tissues (33,34). The most commonly used antibodies for IHC staining are monoclonal antibodies against C. pneumoniae species-specific antigens and Chlamydia-genus specific lipopolysaccharide antigen (33). Only a few papers reported failure in detecting C. pneumoniae in atheromatous tissues (35-37). Finding the organism in lesions but not in normal arteries may suggest that the organism is involved in the disease process. The lesions in which C. pneumoniae were detected were obtained from the coronary artery, including coronary bypass of the saphenous vein (38), carotid, pulmonary, iliac, femoral, and popliteal arteries and the aorta (33) from patients with coronary artery stenosis (angina and myocardial infarction) (10,32) and carotid artery stenosis (39), stenosis of the arteries of the lower extremities (claudication) (40) and aortic aneurysm (29,41-43). C. pneumoniae was also detected in nonrheumatotic aortic stenotic valves (44-46). However, this finding has been disputed (47).

The youngest age at which C. pneumoniae was detected in the artery was 15 years (48). In the Pathological Determination of Atherosclerosis in Youth study, a higher detection rate was found in subjects 25-34 years old (7 (26%) of 27 subjects) than in those 15-24 years old (1 (5%) of 22 subjects), suggesting a pathogenic role of C. pneumoniae in the atherosclerotic process (49).

Variability in detection rates has been noted among investigators, arteries, and methods used for detection (33). These rates have been reported to range from 0% to 100%. One reason for this variability is the localized nature of C. pneumoniae in lesions and the fact that most studies examined only one or a few 4 um sections with IHC and 8 um sections with PCR. In general, detection rates were higher with IHC than PCR whether the same tissues or different tissues were tested. This discrepancy has been attributed to the presence of tissue inhibitors for PCR. A lack of correlation between IHC and PCR has also been noted. In our studies in which specimens were positive by both methods, only 25%-50% of specimens were positive by both tests (33). The lack of correlation has also been noted between detection and serology (32,50-53). Finally, there has been no correlation shown between the presence of the organism and the severity of atherosclerosis or clinical diagnosis, such as acute myocardial infarction, thrombosis or ulceration of atheroma, and rupture of aneurysm (33).

The organism has been isolated from atheromatous tissue of the coronary and carotid arteries by several independent investigators (11,39,52). However, isolation has been the most difficult to achieve and the most important step toward determining a causal relationship between C. pneumoniae and atherogenesis and fulfillment of Koch's postulate.

4.5. Detection of the organism in peripheral blood mononuclear cells

In attempting to identify a marker for laboratory diagnosis of C. pneumoniae infection in coronary disease patients and for assessment of the outcome of antibiotic therapy, PCR has been used for direct detection of C. pneumoniae in circulating monocytes. The detection rates ranged from 8.8% to 59% in patients with coronary artery disease including unstable angina and myocardial infarction (54-56) and abdominal aortic aneurysm (57) patients. Similar detection rates ranging from 8.9% to 46% (54,58,59) have also been shown in healthy blood donors. The presence of the organism in circulating mononuclear cells may indicate a mechanism by which C. pneumoniae disseminate systemically from the lungs to other organs (60).

5. CHLAMYDIAL INFECTION OF THE HEART VALVES

As mentioned previously, C. pneumoniae has been detected in the sclerotic aortic valves of non-rheumatic origin (44-46). Finding of the organism in the aortic valves raised the possibility that C. pneumoniae could also infect other cardiac valves. The fact that the organism has been found only in the aortic valves may indicate that it is an extension of the infection of the aorta.

6. HISTOPATHOLOGIC FINDINGS IN ATHEROSCLEROTIC LESIONS POSITIVE FOR C. PNEUMONIAE

No distinguishable differences in histopathology were observed between C. pneumoniae-positive and C. pneumoniae-negative atheromatous lesions under light microscopy in tissue sections stained with hematoxylin and eosin (6). Utilizing IHC, the organism has been detected in early (fatty streaks) and advanced (atheromatous plaques) lesions, but rarely in histologically normal-looking arteries. The organism is often located in foam cells within the plaque and in macrophages infiltrating the intima, media and adventitia. By double-labeling or co-localization staining, the organism has been located in macrophages and the smooth muscle cells (61), but is rarely found in endothelial cells (62). EM demonstrates that the organisms are found within cytoplasmic vacuoles of foam cells (32) (Figure 1).

7. PATHOGENESIS OF CHLAMYDIA INFECTIONS

Infection with C. trachomatis and C. pneumoniae induces chronic inflammatory reactions, consisting of macrophages and lymphocytes, at the site of infection (6). Atherosclerosis has been regarded as a chronic inflammatory reaction with lipid accumulation. Both in vivo and in vitro experiments have shown that C. pneumoniae infection induces pro-inflammatory cytokines. Infection of macrophages results in the production of TNF-alpha, interleukin-1beta (IL-1beta), IL-8 (for review see (17)). In the atheromatous lesion, C. pneumoniae infection of macrophages may induce foam cell formation by chlamydial LPS (63) and oxidation of low density lipoprotein (a key lipid in foam cells) by chlamydial heat shock protein-60 (hsp-60) (64). Chlamydial hsp-60 can activate macrophage TNF-alpha and expression of matrix metaloproteinase (65), which may cause plaque destabilization.

How C. pneumoniae establishes persistent infection in arterial cells has been investigated in vitro. It has been found that the interaction of monocytes with endothelial (66) and smooth muscle (67) cells promotes the growth of C. pneumoniae in these cells. However, different mechanisms seem to be involved in the growth promotion in different cell types. In endothelial cells, the growth is enhanced by the insulin-like growth factor-2 (IGF-2) secreted by monocytes (68), while a direct cell-to-cell contact is required for the growth promotion in smooth muscle cells (67).

8. ANIMAL MODELS OF PNEUMONIA AND ATHEROSCLEROSIS

The most commonly used animals for studying the pathogenesis of C. pneumoniae induced pneumonitis and atherosclerosis are mice. Rabbits have also been used for studying atherosclerosis. Using the genetically induced hyperlipidemic animals (apoE-knockout mice) (13,69) and diet-induced hyperlipidemic animals (LDL receptor knockout mice (70), C57BL/6J mice (71), and New Zealand white rabbits (12,72)) in these studies have shown that intranasal inoculation with C. pneumoniae accelerate plaque development in these hyperlipidemic animals. However, others have not observed an exacerbation (73). In the absence of hyperlipidemia infection induces inflammatory reactions in the artery, but no definitive atherosclerotic lesions have been induced (74,75). In general, the findings from these animal experiments indicate that C. pneumoniae is a co-risk factor of hyperlipidemia for atherosclerosis.

9. THERAPY

The most commonly used antibiotics for treating chlamydial infections are tetracyclines, macrolides, and azalides (26). These drugs have been used to determine whether treatment would ameliorate the effect of C. pneumoniae infection on atherosclerosis in animals and decrease clinical events in patients with coronary artery disease.

Treatment studies in animal models have yielded variable results. Muhlestein et al. showed that a 7-week course of azithromycin started immediately following the inoculation was effective in preventing the enhancing effects of C. pneumoniae on atherosclerotic lesion development in diet-induced hyperlipidemic rabbits (12). A similar effect was obtained by Fong et al. (76). However, in Fong's study the beneficial effects were observed with the early, but not late treatment with azithromycin. In the apoE-deficient mouse model, a two-week treatment with azithromycin had no effect on C. pneumoniae accelerated lesion development (77).

There have been four small scale therapeutic trials using roxithromycin, clarithromycin and azithromycin for secondary prevention of acute coronary events (78-81). The sample sizes range from 80 to 300 patients with the duration of treatment ranging from one or two courses of a 3 day treatment (78) to the longest of 3 months (81). Follow-ups ranged from 30 days to 1 year and 6 months. Three of these four studies showed statistically significant beneficial effects (p=0.03). The one study which did not show a reduction in the outcome of acute cardiac events involved 302 patients who were given azithromycin (500 mg per day for 3 days followed by 500 mg per week for 3 months) and examined at 3 and 6 months (81).

Two large scale multi-center double-blind clinical trials of azithromycin on CHD are being conducted in North America. These two studies were the WIZARD (Weekly Intervention with Zithromax for Atherosclerosis and its Related Disorders (82) and ACES (Zithromax and Coronary Events Study) (83). The enrollment criteria were post myocardial infarction for both studies. Additional criteria were C. pneumoniae antibody titers of 1:16 or greater in the WIZARD study and 50% stenosis in the ACES study. The dosage was 600 mg azithromycin once a week for 3 months in the WIZARD study and for 12 months in the ACES study. Of the 3,300 patients in the WIZARD study, half were treated with azithromycin and half with placebo. Patients were followed for 1 year and 6 months. In the ACES study, half of the 4,000 patients were treated with azithromycin and half with placebo. The endpoints in both studies were CHD events. The WIZARD study has been completed in the year 2001 and the data are being analyzed. The expected date of completion for the ACES study is December 2003.

10. CONCLUSION

The most significant development in chlamydial infection and cardiovascular biology has been the observation of an association between chlamydial infection and cardiovascular disease, the discovery of C. pneumoniae in atheromatous plaques, in vivo experimental studies in animals indicating that C. pneumoniae is a co-risk factor of hyperlipidemia for atherogenesis, and in vitro studies demonstrating putative mechanisms by which C. pneumoniae could contribute to the immunopathology of atherosclerosis. Early promising pilot intervention studies have paved the way for large scale controlled clinical trials. Future studies in molecular and cellular mechanisms of the pathogenic role of C. pneumoniae-related atherosclerosis will define the role of C. pneumoniae in atherosclerosis. This will lead to the development of new preventive or intervention measures.

11. REFERENCES

1. Moulder J.W., T.P. Hatch, C-C Kuo, J. Schachter, & J. Storz: Genus Chlamydia, Jones, Rake, and Stearns 1945,55. In N.R. Krieg (ed.), Bergey's Manual of Systematic Bacteriology. Baltimore, MD: Williams and Wilkins. vol 1, 729-739 (1984)

2. Grayston J.T, C-C Kuo, L.A. Campbell & S-P Wang: Chlamydia pneumoniae sp. nov. for Chlamydia strain TWAR. Int J Syst Bacteriol 39, 88-90 (1988)

3. Fukushi H & K. Hirai: Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants. Int J Syst Bacteriol 42, 306.308 (1992)

4. Beatty W, R.P. Morrison & G.I. Byrne: Persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis. Microb Rev 58, 686-699 (1994)

5. Grayston J.T, S-P Wang, L-J Yeh & C-C Kuo: Importance of reinfection in the pathogenesis of trachoma. Rev Infect Dis 7, 717-725 (1985)

6. Kuo C-C & L.A. Campbell: Pathologic manifestation of chlamydial infection. Am Heart J 138, S496-S499 (1999)

7. Gilbert D.N. & J.T. Grayston (eds): The potential etiologic role of Chlamydia pneumoniae in atherosclerosis. A multidisciplinary meeting to promote collaborative research. J Infect Dis 181(suppl 3), S393-S586 (2000)

8. Odeh M & A. Oliven: Chlamydial infection of the heart. Eur J Clin Microbiol Infect Dis 11, 885-893 (1992)

9. Saikku P, K. Mattila, M.S. Nieminen, J.K. Huttunen, M. Leinonen, M-R Ekman, P.H. Makela, & V. Valtonen: Serological evidence of an association of a novel Chlamydia TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 2, 983-986 (1988)

10. Shor A, C-C Kuo & D.L. Patton: Detection of Chlamydia pneumoniae in coronary artery fatty streaks and atheromatous plaques. S Afr Med J 82, 158-161 (1992)

11. Ramirez J.A. and the Chlamydia pneumoniae/Atherosclerosis study group. Isolation of Chlamydia pneumoniae from the coronary artery of a patient with coronary atherosclerosis. Ann Intern Med 125, 979-982 (1996)

12. Muhlestein J.B, J.L. Anderson, E.H. Hammond, L. Zhao, S. Treharn, E.P. Schwobe & J.F. Carlquist: Infection with Chlamydia pneumoniae accelerates the development of atherosclerosis and treatment with azithromycin prevents it in a rabbit model. Circulation 97, 633-636 (1998)

13. Moazed C, L.A. Campbell, M.E. Rosenfeld, J.T. Grayston & C-C Kuo: Chlamydia pneumoniae infection accelerates the progression of atherosclerosis in ApoE-deficient mice. J Infect Dis 180, 238-241 (1999)

14. Campbell L.A, C-C Kuo & J.T. Grayston: Chlamydia pneumoniae and cardiovascular disease. Emerg Infect Dis 4, 571-579 (1998)

15. Camm A.J. & K.M. Fox: Chlamydia pneumoniae (and other infective agents) in atherosclerosis and acute coronary syndromes. How good is the evidence? Eur Heart J 21, 1046-1051 (2000)

16. Wong Y-K, P.J. Gallagher & M.E. Ward: Chlamydia pneumoniae and atherosclerosis. Heart 81, 232-238 (1999)

17. Mahoney J, & B. Coombs: Chlamydia pneumoniae and atherosclerosis: does the evidence support a causal or confirmatory role? FEMS Microbiol Lett 197, 1-9 (2001)

18. Leinonen M, E. Linnanmaki, K. Mattila, M.S. Nieminen, V. Valtonen, M. Leirisalo-Repo & P. Saikku: Circulating immune complexes containing chlamydial lipopolysaccharide in acute myocardial infarction. Microb Pathog 9, 67-73 (1990)

19. Linnanmaki E, M. Leinonen, K. Mattila, M.S. Nieminen, V. Valtonen V & P. Saikku: Chlamydia pneumoniae-specific circulating immune complexes in patients with chronic coronary heart disease. Circulation 87, 1130-1134 (1993)

20. Thom D.H, S-P Wang, J.T. Grayston, D.S. Siscovick, D.K. Stewart, R.A. Kronmal & N.S. Weiss: Chlamydia pneumoniae strain TWAR antibody and angiographically demonstrated coronary artery disease. Arterioscler Thromb 11, 547-551 (1991)

21. Thom D.H, J.T. Grayston JT, D.S. Siscovick, S-P Wang, N.S. Weiss & J.R.Daling: Association of prior infection with Chlamydia pneumoniae and angiographically demonstrated coronary artery disease. JAMA 268, 68-72 (1992)

22. Ridka P.M, R.B. Kunsin, M.J. Stampfer, S. Poulin & C.H. Hennekens: Prospective study of Chlamydia pneumoniae IgG seropositivity and risks of future myocardial infarction. Circulation 99, 1161-1164 (1999)

23. Saikku P, M. Leinonen, L. Tenkanen, E. Linnanmaki, M-R Ekman,V. Manninen, M. Manntari, H. Frick & J.K. Huttunen: Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Intern Med 116, 273-278 (1992)

24. Mendall M.A, D. Carrington, D. Strachan, P. Patel, N. Molineaux, J. Levi, T. Toosey, A.J. Camm & T.C. Northfield: Chlamydia pneumoniae: Risk factors for seropositivity and association with coronary heart disease. J Infect 30, 121-128 (1995)

25. Sessa R, M.D. Pietro, I. Santino, M. del Piano, A. Varveri, A. Dagianti & M. Penco: Chlamydia pneumoniae infection and atherosclerotic coronary disease. Am Heart J 137, 1116-1119 (1999)

26. Kuo C-C, L.A. Jackson, L.A. Campbell & J.T. Grayston: Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 8, 451-461 (1995)

27. Melnick S.L, E.E. Shahar, A.R. Folson, J.T. Grayston, P.D. Sorlie, S-P Wang & M. Szklo for the Atherosclerosis Risk in Communities (ARIC) Study Investigators. Am J Med 95, 499-504 (1993)

28. Lindholt J.S, S. Juul, S. Vammen, I. Lind, H. Fasting & E.W. Henneberg: Immunoglobulin A antibodies against Chlamydia pneumoniae are associated with expansion of abdominal aortic aneurysm. British J Surg 86, 634-638 (1999)

29. Blanchard J.F, H.K. Armenian, R. Peeling, P.P. Friesen, C. Shen & R.C. Brunham: The relationship between Chlamydia pneumoniae infection and abdominal aortic aneurysm: Case-control study. Clin Infect Dis 30, 946-947 (2000)

30. Wimmer M.L.J, R. Sandmann-Strupp, P. Saikku & R.L. Haberl: Association of chlamydial infection with cerebrovascular disease. Stroke 227, 2207-2210 (1996)

31. Fagerberg B, J. Gnarpe, H. Gnarpe, S. Agewall & J. Wikstrand: Chlamydia pneumoniae but not cytomegalovirus antibodies are associated with future risk of stroke and cardiovascular disease. A prospective study in middle-aged to elderly men with treated hypertension. Stroke 30, 299-305 (1999)

32. Kuo C-C, A. Shor, L.A. Campbell, H. Fukushi, D.L. Patton & J.T. Grayston: Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J Infect Dis 167, 841-849 (1993)

33. Kuo C-C & L.A. Campbell: Detection of Chlamydia pneumoniae in arterial tissues. J Infect Dis 181 (suppl 3), S432-436 (2000)

34. Taylor-Robinson D. Chlamydia pneumoniae in vascular tissue. Atherosclerosis 1998 140 (suppl 1), S21-S24 (1998)

35. Weiss S.M, P.M. Roblin, C.A. Gaydos, P. Cummings, D.L. Patton, N. Schulhoff, J. Shani, R. Frankel, K. Penney, T.C. Quinn, M.R. Hammerschlag & J. Schachter: Failure to detect Chlamydia pneumoniae in coronary atheromas of patients undergoing atherectomy. J Infect Dis 173, 957-962 (1996)

36. Patterson D.L, J. Hall J, S.J. Rasmussen & P. Timms: Failure to detect Chlamydia pneumoniae in atherosclerotic plaques of Australian patients. Pathology 30, 167-172 (1998)

37. Lindholt J.S, L. Ostergard, E.W. Henneberg, H. Fasting & P. Andersen: Failure to demonstrate Chlamydia pneumoniae in symptomatic abdominal aortic aneurysms by a nested polymerase chain reaction (PCR). Eur J Vasc Endovasc Surg 15, 161-164 (1998)

38. Bartels C, M. Maass, G. Bein, R. Malisius, N. Brill, J.F.M. Bechtel, F. Sayk, A.C. Feller & H-H Sievers: Detection of Chlamydia pneumoniae but not cytomegalovirus in occluded saphenous vein coronary artery bypass grafts. Circulation 99, 879-882 (1999)

39. Jackson L.A, L.A. Campbell, C-C Kuo, D.I. Rodriguez, A. Lee & J.T. Grayston: Isolation of Chlamydia pneumoniae from a carotid endarterectomy specimen. J Infect Dis 176, 292-295 (1997)

40. Kuo C-C, A.S. Coulson, L.A. Campbell, A.L. Cappuccio, R.D. Lawrence, S-P Wang & J.T. Grayston: Detection of Chlamydia pneumoniae in atherosclerotic plaques in the walls of arteries of lower extremities from patients undergoing bypass operation for arterial obstruction. J Vasc Surg 26, 29-31 (1997)

41. Blasi F, F. Denti, M. Ebra, R. Cosentini, R. Raccanelli, A. Rinaldi, L. Fagetti, G. Esposito, U. Ruberti & L. Allergra: Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. J Clin Microbiol 34, 2766-2769 (1996)

42. Juvonen J, T. Juvonen T, A. Laurila, H. Alakarppa, K. Lounatmaa, H-M Surcel, M. Leinonen, M.I. Kairaluoma & P. Saikku: Demonstration of Chlamydia pneumoniae in the walls of abdominal aortic aneurysms. J Vas Surg 25, 499-505 (1997)

43. Meijer A, A. van der Vliet, P.J.M. Roholl, S.K. Gielis-Proper, A. de Vries & 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 19, 2680-2686 (1999)

44. Nystrom-Rosander C, S. Thelin, E. Hjelm, O. Lindquist, C. Pahlson & G. Friman: High incidence of Chlamydia pneumoniae in sclerotic heart valves of patients undergoing aortic valve replacement. Scand J Infect Dise 29, 361-365 (1997)

45. Juvonen J, A. Laurila, T. Juvonen, H. Alakarppa, H-M Surcel, K. Lounatmaa, J. Kuusisto & P. Saikku : Detection of Chlamydia pneumoniae in human nonrheumatic stenotic aortic valves. J Am Coll Cardiol 29, 1054-1059 (1997)

46. Juvonen J, T. Juvonen, A. Laurila, J. Kuusisto, E. Alarakkola, T. Sarkioja, C.A. Bodian, M.I. Kairaluoma & P. Saikku: Can degenerative aortic valve stenosis be related to persistent Chlamydia pneumoniae infection? Ann Intern Med 128, 741-744 (1998)

47. Andersen J.J, S. Farholt & J.S. Jensen: Failure to detect Chlamydia pneumoniae in calcific and degenerative arteriosclerotic aortic valves excised during open heart surgery. APMIS 106, 717-720 (1998)

48. Taylor-Robinson D, G. Ong, B.J. Thomas, M.L. Rose & M.H. Yacoub: Chlamydia pneumoniae in vascular tissues from heart-transplant donors. Lancet 351, 1255 (1998)

49. Kuo C-C, J.T. Grayston, L.A. Campbell, Y.A. Goo, R.W. Wissler & E.P. Benditt: Chlamydia pneumoniae (TWAR) in coronary arteries of young adults (15-34 years old). Proc Natl Acad Sci USA 92, 6911-6914 (1995)

50. Davidson M, C-C Kuo, J.P. Middaugh, L.A. Campbell, S-PWang S-P, W.P. III. Newman, J.C. Finley & J.T. Grayston: Confirmed previous infection with Chlamydia pneumoniae (TWAR) and its presence in early coronary atherosclerosis. Circulation 98, 628-633 (1998)

51. Maass M, Gieffers J, Krause E, Engel PM, Bartels C, Solbach W. Poor correlation between microimmunofluorescence serology and polymerase chain reaction for detection of vascular Chlamydia pneumoniae infection in coronary artery disease patients. Med Microbiol Immunol 187, 103-106 (1998)

52. Maass M, C. Bartels, P.M. Engel, U. Mamat & H-H Sievers: Endovascular presence of viable Chlamydia pneumoniae is a common phenomenon in coronary artery disease. J Am Coll Cardiol 31, 827-832 (1998)

53. Bartels C, M. Maass, G. Bein, N. Brill, J.F.M. Bechtel, R. Leyh & H-H Sievers: Association of serology with the endovascular presence of Chlamydia pneumoniae and cytomegalovirus in coronary artery and vein graft disease. Circulation 101, 137-141 (2000)

54. Boman J, S. Soderberg, J. Forsberg, L.S. Birgander, A. Allard, K. Persson, E. Jidell, U. Kumlin, P. Juto, A. Waldenstrom & G. Wadell: High prevalence of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells in patients with cardiovascular disease and in middle-aged blood donors. J Infect Dis 178, 274-277 (1998)

55. Wong Y-k, K.D. Dawkins & M.E. Ward: Circulating Chlamydia pneumoniae DNA as a predictor of coronary artery disease. J Am Coll Cardiol 34, 1435-1439 (1999)

56. Giefers J, H. Fullgraf, J. Jahn, M. Kinger, K. Dalhoff, H.A. Katus, W. Solbach & M. Maass: Chlamydia pneumoniae infection in circulating human monocytes is refractory to antibiotic treatment. Circulation 103, 351-356 (2001)

57. Blasi F, J. Boaman, G. Esposito, G. Mellsano, R. Chiesa, R. Cosentini, P. Tarsia, Y. Tshomba, M. Betti, M. Alessi, N. Morelli & L. Allegra: Chlamydia pneumoniae DNA detection in peripheral blood mononuclear cells is predictive of vascular infection. J Infect Dis 180, 2074-2076 (1999)

58. Bodetti T.J & P. Timms: Detection of Chlamydia pneumoniae DNA and antigen in the circulating mononuclear cell fractions of human and koalas. Infect Immun 68, 2744-2747 (2000)

59. Haranaga S, H. Yamaguchi, G.F. Leparc, H. Fiedman & Y. Yamamoto: Detection of Chlamydia pneumoniae antigen in PBMCs of healthy blood donors. Transfusion 41, 1114-1119 (2001)

60. Kuo C-C & L.A. Campbell: Is infection with Chlamydia pneumoniae a causative agent in atherosclerosis? Mol Med Today 4, 426-430 (1998)

61. Kuo C-C, A.M. Gown, E.P. Benditt & J.T. Grayston: Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain. Arterioscler Thromb 13, 1501-1504 (1993)

62. Yamashita K, K. Ouchi, M. Shirai, T. Gondo, T. Nakazawa & H. Ito: Distribution of Chlamydia pneumoniae infection in the athrosclerotic carotid artery. Stroke 29, 773-778 (1998)

63. Kalayoglu M.V & G.S. Bryne: Induction of macrophage foam cell formation by Chlamydia pneumoniae. J Infect Dis 177, 725-729 (1998)

64. Kalayoglu M.V, Indrawati, R.P. Morrison, S.G. Morrison, Y. Yuan & G.I. Bryne: Chlamydial virulence determinants in atherogenesis: the role of chlamydial lipopolysaccharide and heat shock protein 60 in macrophages-interactions. J Infect Dis 181 (suppl 3), S483-S489 (2000)

65. Kol A, G.K. Sukhova, A.H. Lichtman & P. Libby: Chlamydial heat shock protein 60 localized-human atheroma and regulates macrophage tumor necrosis factor-alpha and matrix metalloproteinase expression. Circulation 98, 300-307 (1998)

66. Lin T-M, L.A. Campbell, M.E. Rosenfeld & C-C Kuo: Monocyte-endothelial cell coculture enhances infection of endothelial cells with Chlamydia pneumoniae. J Infect Dis 181, 1096-1000 (2000)

67. Puolakkainen M, L.A. Campbell, T-M Lin, T. Richards & C-C Kuo: Coculture of monocytes with arterial smooth muscle cells enhances growth of Chlamydia pneumoniae. p. 57-60. In J. Schachter (ed.), the Proceedings of the 10th International Symposium on Human Chlamydial Infections, held in Antalya, Turkey, June 16-21, 2002.

68. Lin T-M, L.A. Campbell, M.E. Rosenfeld & C-C Kuo: Human monocyte-derived insulin-like growth factor-2 enhances the infection of human arterial endothelial cells by Chlamydia pneumoniae. J Infect Dis 183, 1368-1372 (2001)

69. Burnet M.S, C.A. Gaydos, G.E. Madico, S.M. Glad, B. Paigen & T.C. Quinn & S.E. Epstein: Atherosclerosis in apoE knockout mice infected with multiple pathogens. J Infect Dis 183, 226-231 (2001)

70. Hu H, G.N. Pierce & G. Zhong: The atherogenic effects of chlamydia are dependent on serum cholesterol and specific to Chlamydia pneumoniae. J Clin Invest 103, 747-753 (1999)

71. Blessing E, L.A. Campbell, M.E. Rosenfeld, N. Cough & C-C Kuo: Chlamydia pneumoniae infection accelerates hyperlipidemia induced atherosclerotic lesion development in C57BL/6J mice. Atherosclerosis 158, 13-17 (2001)

72. Fong I.W, B. Chiu, E. Viira, D. Jang & J.B. Mahony: De novo induction of atherosclerosis by Chlamydia pneumoniae in a rabbit model. Infect Immun 11, 6048-6055 (1999)

73. Caliguri G, M. Rottenberg, A. Nicoletti, H. Wigzell & G. Hansson: Chlamydia pneumoniae infection does not induce or modify atherosclerosis in mice. Circulation 103, 2834-2838 (2001)

74. Blessing E, T-M Lin, L.A. Campbell, M.E. Rosenfeld, D. Lloyd & C-C Kuo: Chlamydia pneumoniae induces inflammatory changes in the heart and aorta of normolipidemic C57BL/6J mice. Infect Immun 68, 4765-4768 (2000)

75. Laitinen K, A. Laurila, L. Pyhala, M. Leinonen & P. Saikku: Chlamydia pneumoniae infection induces inflammatory changes in the aortas of rabbits. Infect Immun 65, 4832-4835 (1997)

76. Fong I.W, B. Chiu, E. Viira, D. Jang, M.W. Fond, R. Peeling & J.B. Mahoney: Can an antibiotic (macrolide) prevent Chlamydia pneumoniae-induced atherosclerosis in rabbit model? Clin Diag Lab Immunol 6, 851-894 (1999)

77. Rothstein N.M, T.C. Quinn, G. Madico, C.A. Gaydos & C.J. Lowenstein: Effect of azithromycin on murine arteriosclerosis exacerbated by Chlamydia pneumoniae. J Infect Dis 183, 232-238 (2001)

78. Gupta S, E.W. Leatham, D. Carrington, M.A. Mendall, J.C. Kaski & A.J. Camm: Elevated Chlamydia pneumoniae antibodies, cardiovascular events and azithromycin in male survivors of myocardial infarction. Circulation 46, 404-407 (1997)

79. Gurfinkel E, G. Bozovich, E. Beck, E. Testa, B. Livellara & B. Mautner for the ROXIS Study Group. Treatment with the antibiotic roxithromcyin in patients with acute non-Q-wave coronary syndromes. The final report of the ROXIS Study. Eur Heart J 20, 121-127 (1999)

80. Muhlestein J.B, J.L. Anderson, J.F. Carlquist, K. Salunke, B.D. Horne, R.R. Pearson, T.J. Bunch, A. Allen, S. Trehan & C. Nelson: Randomized secondary prevention trial of azithromycin with coronary artery disease. Primary clinical results of the ACADEMIC study. Circulation 102, 1755-1760 (2000)

81. Sinisalo J, K. Mattila, V. Valtonen, O. Anttonen, J. Juvonen, J. Melin, H. Vuorinen-Markkola & M.S. Nieminen for the Clarithromycin in acute coronary syndrome patients in Finland (CLARIFY) Study Group. Effect of 3 months of antimicrobial treatment with Clarithromycin in acute non-Q-wave coronary syndrome. Circulation 105, 1555-1560 (2002)

82. Dunne M.W. Rationale and design of a secondary prevention trial of antibiotic use in patients after myocardial infarction: The WIZARD (Weekly Intervention with Zithromax (Azithromycin) for atherosclerosis and its related disorders) trial. J Infect Dis 181 (suppl 3), S572-578 (2000)

83. Jackson L.A. Description and status of the azithromycin and coronary event study (ACES). J Infect Dis 181 (suppl 3), S579-581 (2000)

Key Words: Chlamydial Infection, Chlamydia Pneumoniae, Cardiovascular Disease, Atherosclerosis, Review

Send correspondence to: Cho-Chou Kuo, M.D., Ph.D., Department of Pathobiology, Box 357238, University of Washington, Seattle, Washington U.S.A., Tel: 206-543-8689, Fax: 206-543-3873, E-mail: cckuo@u.washington.edu