Changes in antibody titers against Chlamydia pneumoniae after coronary angioplasty

International Journal of Cardiology 95 (2004) 293 – 297 www.elsevier.com/locate/ijcard

Changes in antibody titers against Chlamydia pneumoniae after coronary angioplasty Gu¨lay Yetkin a, Ertan Yetkin b,*, Yu¨ksel Aksoy b, Oguz Alp Gurbuz a, Ali Mert c a

Department of Microbiology, Inonu University Faculty of Medicine, Malatya, Turkey Department of Cardiology, Inonu University Faculty of Medicine, Malatya, Turkey c Department of Microbiology, SSK Education Hospital, Ankara, Turkey

b

Received 18 December 2002; accepted 2 April 2003

Abstract Objectives: The potential role of common infectious agents in the pathogenesis and progression of atherosclerosis has been studied increasingly over the last decade. The evidence for Chlamydia pneumoniae as a potential causative agent is strong and is based on the findings of numerous sero-epidemiological studies, examination of atheromatous plaque specimens, in vitro animal models. We performed a prospective study in percutaneous transluminal coronary angioplasty (PTCA) patients to investigate whether the angioplasty procedure influenced the specific humoral immune response reaction against C. pneumoniae antigens. Methods: We studied 76 patients who successfully underwent PTCA for de novo lesions. Blood samples were drawn immediately before PTCA and 1 month after PTCA. IgG and IgA antibodies against C. pneumoniae (strain CDC/CWL-029) were determined by an in-house developed enzyme immunoassay. Results: At the time of angioplasty 75% and 34% of the patients had seropositive antibodies to elementary bodies (EBs) of classes IgG and IgA, respectively. Mean titers of IgG antibodies before and 1 month after PTCA were 46 F 31 and 50 F 28 relative units (RU/ml) ( P>0.05). One month after PTCA, 97% and 34% of the patients had seropositive antibodies to EBs of classes IgG and IgA, respectively. We divided our patients into two groups on the basis of IgG seropositivity (group I: Chlamydia antibody IgG seronegative patients, group II: Chlamydia antibody IgG seropositive) before PTCA. Significant increase in the antibody titers of IgG (12 F 5 vs. 40 F 18, P < 0.001) and IgA (0.6 F 0.33 vs. 1.15 F 0.83, P = 0.007) was observed in group I patients 1 month after PTCA and 88% of them gained IgG seropositivity. There were no significant changes in IgG and IgA antibody levels in group II after PTCA. Conclusion: We have demonstrated a statistically significant rise in C. pneumoniae antibodies (especially IgG) induced by PTCA in patients previously seronegative. D 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Chlamydia pneumoniae; Coronary angioplasty

1. Introduction Atherosclerosis and related diseases, in particular myocardial infarction and coronary artery disease (CAD) are major causes of morbidity and mortality worldwide. Differences in the prevalence of conventional cardiovascular risk factors such as smoking, hypertension and dyslipidemia do not account fully temporal and geographical variations in the prevalence and severity of CAD [1,2]. The potential role of common infectious agents in the pathogenesis and progression of atherosclerosis has been studied increasingly over the last decade.

* Corresponding author. Tel.: +90-422-341-0660. E-mail address: [email protected] (E. Yetkin). 0167-5273/$ - see front matter D 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2003.04.045

Chlamydia pneumoniae is an important respiratory pathogen that accounts for up to 10% of cases of community acquired pneumonia [3,4]. The evidence for C. pneumoniae as a potential causative agent for CAD is strong and is based on the findings of numerous seroepidemiological studies, examination of atheromatous plaque specimens and in vitro animal models. However, several studies do not support a link between C. pneumoniae and CAD [5 –7] and Danesh et al. have reported that neither IgA and IgG titers are strongly predictive of CAD in general population [8,9]. We performed a prospective study in percutaneous transluminal coronary angioplasty (PTCA) patients to investigate whether the angioplasty procedure would have an influence on the humoral immune response reaction against C. pneumoniae antigens.

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G. Yetkin et al. / International Journal of Cardiology 95 (2004) 293–297 Table 2 Antibody titers of IgG and IgA class before and after PTCA in group I and group II

2. Materials and methods 2.1. Patient population Between December 1999 and March 2000, 86 patients who were consecutive candidates for elective PTCA of a de novo lesion were enrolled in the study. Patients who had previous myocardial infarction, previous PTCA or coronary artery by-pass grafting or unstable angina were not included in the study. One patient died shortly after the PTCA and was excluded from the study. From nine patients, we were unable to obtain control blood samples at follow-up for various reasons. Study analysis was performed on the remaining 76 patients. All patients had given written informed consent to participate in the study prior to PTCA. 2.2. Blood sampling and serological analysis Blood samples were drawn immediately before PTCA and 1 month after PTCA. Serum samples were centrifuged and stored at 80 jC within 2 h after venasection. Serological analysis was carried out without prior knowledge of clinical data. IgG and IgA antibodies against C. pneumoniae (strain CDC/CWL-029) were determined as described for C. trachomatis [10] (EUROIMMUN Laboratorium fu¨r Experimentelle Immunologie GmbH, Germany). Chlamydial elementary bodies (EBs) purified from cell lysates were treated with sodium dodecyl sulphate. The used solution contains all relevant antigens localised in the outer membrane of the EBs. The main portion was provided by the major outer membrane protein antigen (MOMP). C. pneumoniae was propagated in 12-well microtiter plates using optimal conditions [11]. The test kit contained eight break-off reagent wells coated with C. pneumoniae antigens. The serum samples for analysis were diluted 1:101 with sample buffer. In the first reaction step, diluted patient

Table 1 Baseline characteristics of patients who underwent PTCA Variables

Group I* (n = 19) Group II (n = 57) Total (n = 76)

Age (year) Female patients

52 F 10 5 (26%)

Risk factors Hypertension DM Smoking Hypercholesterolemia

7 2 6 6

Coronary artery lesions LAD 3 RCA 11 LCx 8

(36%) (10%) (31%) (31%)

55 F 8 14 (24%)

54 F 9 19 (25%)

16 (29%) 5 (9%) 15 (27%) 16 (28%)

23 (30%) 7 (10%) 21 (27%) 22 (29%)

8 28 24

11 39 32

PTCA, percutaneous transluminal coronary angioplasty; LAD, left anterior descending coronary artery; RCA, right coronary artery; LCx, left circumflex coronary artery; DM, diabetes mellitus, *, vs. group II P>0.05.

Group I (n = 19)

Group II (n = 57)

Before PTCA IgG (RU/ml) IgA (RE/ml)

12 F 5* 0.60 F 0.33**

57 F 28 1.17 F 0.87

After PTCA IgG (RU/ml) IgA (RE/ml)

40 F 18 1.15 F 0.83

53 F 30 0.95 F 0.66

PTCA, percutaneous transluminal coronary angioplasty. *, vs. after PTCA P < 0.001; **, vs. after PTCA P = 0.007.

samples were incubated with the wells. To detect the bound antibodies, a second incubation was carried out using enzyme-labelled anti-human IgG (also IgA), which was capable of promoting a colour reaction. Photometric measurement of the colour intensity were made at a wavelength of 450 nm and a reference wavelength of >620 nm within 30 min of the adding the stop solution. The upper limit of the reference range of non-infected persons (cut-off value) for IgG recommended by EUROIMMUN is 20 relative units (RU/ml). After calculation of mean value of calibration for IgG, extinction values of serum samples exceeding those of the calibration serum 2 (20 RU/ml) were considered as positive, those below as negative. The cut-off ratio for IgA antibody was accepted as 1; recommended by EUROIMMUN. Ratios z 1 were considered as positive, ratios < 1 as negative for IgA seropositivity. These ratios were calculated according to formula: extinction of serum samples (RE/ml)/ extinction of the calibrator (cut-off). 2.3. Statistical analysis Statistical analysis was performed on a personal computer using the SPSS for Windows Release 7.5 Standard version. Data in the text were given as mean F S.D. and percentage. Numerical and categorical variables were compared with paired and unpaired Student’s t-test and Chisquare (v2) test. The changes in seroprevalence were statistically tested with the v2-test and changes in antibody titers in seronegative patients were compared with Wilcoxon matched pairs test. Statistical significance was defined as P < 0.05.

3. Results Baseline characteristics of the patients are shown in Table 1. In a total of 86 lesions, the percentage of diameter stenosis decreased from 78% to 22%. At the time of angioplasty 75% and 34% of the 76 patients had seropositive antibodies to EBs of classes IgG and IgA, respectively. Mean titers of IgG antibodies before and 1 month after PTCA were 46 F 31 and 50 F 28 RU/ml ( P>0.05). Mean

G. Yetkin et al. / International Journal of Cardiology 95 (2004) 293–297

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Fig. 1. Changes in antibody levels of IgG after PTCA in IgG seronegative patients. n, after PTCA; x, before PTCA.

titers of IgA antibodies at the same time points were 1.03 F 0.81 and 0.99 F 0.70 RE/ml ( P>0.05). One month after PTCA, 97% and 34% of the patients had seropositive antibodies to EBs of classes IgG and IgA, respectively. We grouped our patients into Chlamydia antibody IgG seronegative (group I) and IgG seropositive (group II) before PTCA. Characteristics of group I and II patients were presented in Table 1. There were no statistically significant differences between two groups regarding these variables. Mean IgG and IgA titers of the seronegative patients before PTCA (group I) were 12 F 5 RU/ml and 0.60 F 0.33 RE/ml, respectively. Mean IgG and IgA titers of the seronegative patients (group I) after PTCA were 40 F 18 RU/ml and 1.15 F 0.83 RE/ml (Table 2). A significant increase in IgG antibody titers was observed in group I patients 1 month after PTCA ( P < 0.001) (Fig. 1) and 88% of them became seropositive. Although there was a significant rise in IgA titers in group I patients ( P = 0.007), this increase was not

observed in IgA seropositivity after PTCA ( P>0.05). There were no significant changes in IgG (Fig. 2) and IgA antibody levels in group II after PTCA.

4. Discussion Infectious causes for disease with clear inflammatory components, such as cardiovascular disease [12 – 14] have been proposed for more than 100 years but interest in this topic has recently re-emerged because of a body of evidence implicating specific infectious agents in the development and progression of atherosclerosis and related cardiovascular disease [15 – 17]. One agent that has attracted particular attention is the obligate intracellular prokaryotic pathogen, C. pneumoniae. In 1988, Saikku et al. [17] first suggested a role for C. pneumoniae in CAD. Investigators in different institutions studying patients at various stages of CAD have

Fig. 2. Changes in antibody levels of IgG after PTCA in IgG seropositive patients. n, after PTCA; x, before PTCA.

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identified connections between positive anti-C. pneumoniae antibody serology and atherosclerotic disease. In this study, we had several main findings: (1) PTCA induced a significant rise in IgG anti-Chlamydia antibodies in group I patients (initially seronegative). (2) 88% of the group I patients become seropositive 1 month after the PTCA procedure. (3) IgA antibody levels also showed a significant increase after PTCA in IgG seronegative patients. A rise in antibody titers is generally considered evidence of an acute Chlamydial infection or reinfection. A 2-fold or more rise in titers has been proposed as a diagnostic criterion. Balloon angioplasty causes serious damage to the target artery, including plaque disruption, fissuring and endothelial damage [18]. As a consequence of this injury and the consequent healing process, antigens from the microorganism already present within the plaque may gain access to the cells of the immune system and in this way trigger a rise in antibody levels. One might expect that antibodies against C. pneumoniae would have risen in patients who were seropositive before PTCA. But only patients with low antibody titers and seronegativity showed a 3 – 4-fold increase in the antibody levels of IgG class and became seropositive. A significant increase in IgA antibodies was also observed in IgG seronegative patients. We believe that the observed rise of IgG antibody levels in group I patients reflects an immunologic reaction induced by angioplasty. The pattern of changes in IgG antibody titers suggests that there are differences in respect to immune response between group I and group II. We observed a paradoxical rise of IgG antibody titers in initially seronegative patients. The rise of antibodies in seronegative patients may explained by the induction of immune response stimulated by the antigens from the lesions. However, explanation of non-increased or divergent response of the seropositive patients is quite difficult. But the adverse finding can be attributable to boostering or immune complex forming effects of liberated antigens from the lesion in initially seropositive patients. Tiran et al. [19] has shown a significant increase in C. pneumoniae IgG antibody titers after PTCA. An increase in seropositive patients was also observed after PTCA. However, seronegative and positive patients were compared using antibodies to lipopolisaccharide (LPS) of classes IgG, IgA and IgM. C. pneumoniae antibodies showed a much higher rate of seropositive patients and there was no correlation to antiLPS antibody titers. In addition, our inclusion criteria differed from those of Tiran et al.’s study in which 57% of the patients included in the study had either previous myocardial infarction and/or PTCA and/or coronary artery by-pass grafting (CABG). These events could also alter the immune response to C. pneumoniae, so we only included the patients who had no previous myocardial infarction, PTCA, unstable angina and CABG. Recently it has been demonstrated that coronary angioplasty has resulted in increased detection of C. pneumoniae deoxyribonucleic acid

in blood samples taken from coronary sinus immediately after the procedure [20]. Adult infections are thought to be mainly reinfections characterised by specific IgG and IgA antibody responses without changes in IgM [21]. Thus, accurate detection of acute infection requires paired samples taken 4– 6 weeks apart and the demonstration of a 2-fold or more rising titer. Serological criteria for chronic infection are more controversial but a persistent elevation of specific IgA and IgG is generally accepted as an indication of chronic infection. Specific IgG antibodies against C. pneumoniae are thought to disappear about 3 years following primary C. pneumoniae infection [22]. The finding of IgA seropositivity lower than that of IgG is possibly the result of a more rapid disappearance of IgA. IgA is thought to have much shorter half life than IgG. The observation of a significant change in C. pneumoniae titres following arterial manipulation highlights the weaknesses of serological testing for the diagnosis of infection in CAD. It is possible that many seronegative patients with CAD do have localised infection within plaques which will not be recognised by serological tests. The incidence of seropositivity 1 month after PTCA was high in this study (97%), but was in accordance with the studies examining plaque tissue (as opposed to serological testing). Ouchi et al. [23] detected C. pneumoniae in 75% of coronary atherectomy specimens and detection of C. pneumoniae in atherosclerotic tissue with the polymerase chain reaction has been reported to be as high as 75– 84% [24 – 27]. Juvonen et al. [28] detected C. pneumoniae in all specimens (100%) taken from the aorta by the techniques of immuncytochemistry, polymerase chain reaction, electron microscopy and culture method.

5. Conclusion We have shown a statistically significant rise in C. pneumoniae antibodies induced by PTCA in previously seronegative patients. Mechanical disruption of plaque tissue appears to affect the serological response to C. pneumoniae. This finding highlights the difficulties of relying on serological diagnosis of C. pneumoniae infection in CAD.

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