J. Thomas Grayston

Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery.

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.

Background and Current Knowledge of Chlamydia pneumoniae and Atherosclerosis

Attributes of Chlamydia pneumoniae of potential importance to a relationship with atherosclerosis are described. Among these are that C. pneumoniae is not new. It is unique. It is a pathogen with which everyone is infected, and it is difficult to treat. It causes immunopathology, myocarditis, and endocarditis and chronicity is a hallmark of Chlamydia infection. Current knowledge of the relation of C. pneumoniae and atherosclerosis comes from observational (e.g., seroepidemiology and tissue studies) and experimental studies. The limitations of the serologic studies of chronic infection are noted as is the conclusive demonstration of an association of C. pneumoniae and atherosclerosis by the repeated and frequent finding of the organism in atherosclerotic tissue. Experimental studies are needed to determine if the association is causal. Such studies should include animal models, basic mechanisms, and secondary prevention antibiotic treatment trials.

Evidence of Systemic Dissemination of Chlamydia pneumoniae via Macrophages in the Mouse

Chlamydia pneumoniae has been postulated to cause systemic disease by infection of monocytes/macrophages and spread via the blood or lymphatics. To investigate how C. pneumoniae disseminates, the ability of the organism to infect murine macrophages in vivo and whether infection can be transferred via macrophages were determined. C. pneumoniae was detected by direct plating, isolation, and polymerase chain reaction in alveolar macrophages from intranasally inoculated mice and peritoneal macrophages from intraperitoneally inoculated mice. C. pneumoniae were also detected in peripheral blood mononuclear cells, but not plasma, of intranasally and intraperitoneally inoculated mice. When alveolar or peritoneal macrophages were adoptively transferred by intraperitoneal injection from infected to uninfected mice, C. pneumoniae DNA was detected by polymerase chain reaction in lung, thymus, spleen, and/or abdominal lymph nodes. These results demonstrate the ability of C. pneumoniae to infect macrophages in vivo and to disseminate systemically via infected macrophages by hematogenous and lymphatic routes.

Murine Models of Chlamydia pneumoniae Infection and Atherosclerosis

Chlamydia pneumoniae have been demonstrated in atherosclerotic lesions but not in normal arteries. An animal model of both C. pneumoniae and atherosclerosis is needed to investigate the role of the organism in atherosclerosis. Apolipoprotein (apo) E-deficient transgenic mice, which spontaneously develop atherosclerosis, and C57BL/6J mice, which only develop atherosclerosis on an atherogenic diet, were evaluated. Following single and multiple intranasal inoculations of apoE-deficient transgenic mice, C. pneumoniae were detected in lung, aorta, and spleen for 20 weeks after inoculation in 25%-100% of mice. In the aorta, C. pneumoniae were detected within the atherosclerotic lesion. In C57BL/6J mice on a nonatherogenic diet, C. pneumoniae were detected in the aorta only 2 weeks after a single intranasal inoculation in 8% of mice. The persistence of C. pneumoniae in atheromas suggests a tropism of C. pneumoniae to the lesion. These mouse models should be useful for studying the pathogenic role of C. pneumoniae in atherosclerosis.

Chlamydia pneumoniae Infection Accelerates the Progression of Atherosclerosis in Apolipoprotein E—Deficient Mice

Accumulating evidence supports an association between Chlamydia pneumoniae infection and atherosclerosis. To determine whether there is a causal relationship, the effects of chronic infection with C. pneumoniae on the development of atherosclerosis in apolipoprotein E (apoE)-deficient mice were evaluated. Eight-week-old male apoE-deficient mice were inoculated intranasally with C. pneumoniae three times, at 8, 9, and 10 weeks of age. The combined area of atherosclerotic lesions in the lesser curvature of the aortic arch was measured en face by computer-assisted morphometry. The lesion area was 2.4-fold greater (P=.05) at 16 weeks of age and 1.6-fold greater (P=.05) at 20 weeks of age in infected mice than in control mice. There were no differences in total plasma cholesterol levels between groups. This study demonstrates that C. pneumoniae infection accelerates the progression of atherosclerosis in the aortic arch of apoE-deficient mice.

Past infection by Chlamydia pneumoniae strain TWAR and asymptomatic carotid atherosclerosis

PURPOSE To determine whether past infection by Chlamydia pneumoniae strain TWAR is associated with asymptomatic atherosclerosis. Previous studies have linked this organism with symptomatic coronary heart disease. SUBJECTS AND METHODS Between 1986 and 1989, 15,800 men and women aged 45 to 64 years were examined as part of the Atherosclerosis Risk in Communities Study, a prospective cohort study of atherosclerosis being conducted in 4 United States communities. The examination included B-mode ultrasonography of the carotid arteries and an assessment of cardiovascular disease risk factors. Carotid wall thickening (blood-intima to medial-adventitial interface) in the absence of clinical cardiovascular disease was considered evidence of asymptomatic atherosclerosis. In 1991, IgG antibody titers to TWAR were assayed by microimmunofluorescence in stored sera from 326 case-control pairs matched by age group, race, sex, examination period, and field center. A titer of 1:8 or higher was considered a positive TWAR antibody response. RESULTS Seventy-three percent of atherosclerosis cases had serologic evidence of past TWAR infection versus 63% of controls (matched odds ratio 1.76; 95% confidence interval, 1.21 to 2.57). After adjustment for age, hypertension, diabetes, cigarette smoking, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and education, the odds ratio for atherosclerosis was essentially unchanged at 2.00 (95% confidence interval, 1.19 to 3.35). The association was stronger for individuals aged 45 to 54 years than for those aged 55 to 64 years. CONCLUSION There was a significant cross-sectional association between past TWAR infection and asymptomatic atherosclerosis. This organism may be a contributor to the pathogenesis of atherosclerosis.

Confirmed Previous Infection With Chlamydia pneumoniae (TWAR) and Its Presence in Early Coronary Atherosclerosis

BACKGROUND Chlamydia pneumoniae has been identified in coronary atheroma, but concomitant serum antibody titers have been inconsistently positive and unavailable before the detection of early or advanced atherosclerotic lesions. METHODS AND RESULTS This retrospective investigation was performed on premortem serum specimens and autopsy tissue from 60 indigenous Alaska Natives at low risk for coronary heart disease, selected by the potential availability of their stored specimens. Serum specimens were drawn a mean of 8.8 years (range, 0.7 to 26.2 years) before death, which occurred at a mean age of 34.1 years (range, 15 to 57 years), primarily from noncardiovascular causes (97%). Coronary artery tissues were independently examined histologically and, for C pneumoniae organism and DNA, by immunocytochemistry (ICC) and polymerase chain reaction (PCR) with species-specific monoclonal antibody and primers. Microimmunofluorescence detected species-specific IgG, IgA, and IgM antibody in stored serum. C pneumoniae, frequently within macrophage foam cells, was identified in coronary fibrolipid atheroma (raised lesions, Stary types II through V) in 15 subjects (25%) and early flat lesions in 7 (11%) either by PCR (14, 23%) or ICC (20, 33%). The OR for C pneumoniae in raised atheroma after a level of IgG antibody > or =1:256 >8 years earlier was 6.1 (95% CI, 1.1 to 36.6) and for all coronary tissues after adjustment for multiple potential confounding variables, including tobacco exposure, was 9.4 (95% CI, 2.6 to 33.8). CONCLUSIONS Serological evidence for C pneumoniae infection frequently precedes both the earliest and more advanced lesions of coronary atherosclerosis that harbor this intracellular pathogen, suggesting a chronic infection and developmental role in coronary heart disease.

INCREASED FREQUENCY OF SERUM ANTIBODIES TO CHLAMYDIA TRACHOMATIS IN INFERTILITY DUE TO DISTAL TUBAL DISEASE

186 infertile women underwent standard infertility investigations (including hysterosalpingography and, in 87 women, laparoscopy) and tests for the presence of antibody to Chlamydia trachomatis. 73% of the women with distal occlusion of the fallopian tubes and 21% with peritubal adhesions alone had antibodies to C. trachomatis, but none of those with normal tubes did (p less than 0.001 and less than 0.005, respectively). No other infertility factors were associated with an increased frequency of antibodies to C. trachomatis. Since the presence or absence of antibodies to C. trachomatis was as discriminatory in the detection of tubal disease in infertile women as was the hysterosalpingogram, the serological test for C. trachomatis should become part of a routine infertility investigation.

Chlamydia pneumoniae, Herpes Simplex Virus Type 1, and Cytomegalovirus and Incident Myocardial Infarction and Coronary Heart Disease Death in Older Adults The Cardiovascular Health Study

BackgroundWhether serological evidence of prior infection with Chlamydia pneumoniae, herpes simplex virus type 1 (HSV-1), and cytomegalovirus (CMV) is associated with myocardial infarction (MI) and coronary heart disease (CHD) death remains a source of controversy. Methods and ResultsWe conducted a nested case-control study among participants in the Cardiovascular Health Study, a cohort study of persons aged ≥65 years. Cases experienced an incident MI and CHD death (n=213). Control subjects were matched to cases by age, sex, clinic, year of enrollment, and month of blood draw (n=405). Serum was analyzed for IgG antibodies to C pneumoniae, HSV-1, and CMV. After adjustment for other risk factors, the risk of MI and CHD death was associated with the presence of IgG antibodies to HSV-1 (odds ratio [OR] 2.0, 95% CI 1.1 to 3.6) but was not associated with the presence of IgG antibodies to either C pneumoniae (OR 1.1, 95% CI 0.7 to 1.8) or CMV (OR 1.2, 95% CI 0.7 to 1.9). Although there was little association with low to moderate C pneumoniae antibody titers (≤1:512), high-titer (1:1024) C pneumoniae antibody was associated with an increased risk (OR 2.2, 95% CI 1.1 to 4.4). ConclusionsAmong older adults, the presence of IgG antibodies to HSV-1 was associated with a 2-fold increase in the risk of incident MI and CHD death. For C pneumoniae, only high-titer IgG antibodies were associated with an increased risk of MI and CHD death. The presence of IgG antibodies to CMV was not associated with risk among the elderly.

Chlamydia pneumoniae Serology: Interlaboratory Variation in Microimmunofluorescence Assay Results

The lack of standardization in chlamydia serology has made interpretation of published data difficult. This study was initiated to determine the extent of interlaboratory variation of microimmunofluorescence (MIF) test results for the serodiagnosis of Chlamydia pneumoniae infections. Identical panels of 22 sera were sent to 14 laboratories in eight countries for the determination of IgG and IgM antibodies by MIF. Although there was extensive variation in the numeric titer values, the overall percentage agreement with the reference standard titers from the University of Washington was 80%. For results by serodiagnostic category, the best agreement was for four-fold rise in IgG titers, while the lowest agreement was for negative or low IgG titers. Agreement for IgM titers was 50%-95%. Four laboratories failed to discern false-positive IgM titers possibly because of the presence of rheumatoid factor. Further studies are underway to determine the source of interlaboratory variation for the MIF test.