- Email: [email protected]
The Association Between Serological Markers for Chlamydophila pneumoniae and the Development of Abdominal Aortic Aneurysm
The Association Between Serological Markers for Chlamydophila pneumoniae and the Development of Abdominal Aortic Aneurysm Lars Karlsson,1,2 Martin Bj€ orck,1 H akan P€ arsson,1,3 and Anders Wanhainen,1 Uppsala, G€ avle, and Helsingborg, Sweden
Background: To investigate the association between serological markers for Chlamydophila pneumoniae (Cpn) and the development of abdominal aortic aneurysm (AAA) in a populationbased caseecontrol study. Methods: A screening for AAA among 65-75-year-old men and women was performed in a population with high prevalence of disease. Most of the subjects had undergone previous testing at the age of 60, including blood sampling. A total of 42 patients with AAA were compared with 100 age- and gender-matched controls with normal aortas. Cpn immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies present in plasma samples obtained at the time of screening (current) and in the past 5-15 (mean, 12) years (historical) were analyzed. Cpn antibody titers (<1/64, 1/64, 1/264, and 1/1024) were analyzed using the microimmunofluorescence technique. Results: No differences in current Cpn immunoglobulin A and IgG antibodies titers ( p ¼ 0.111 and 0.659), historical titers ( p ¼ 0.449 and 0.228), or titer change (delta) ( p ¼ 0.794 and 0.172) were observed between patients with AAA and controls. In all, 82% of the patients with AAA had a current Cpn IgG titer of 1/1024 as compared with the 70% of the control group. All 11 patients who had an aortic diameter of >40 mm reported having high current Cpn IgG titers. The fact that such a large proportion of the healthy population demonstrated an immune response against Cpn made it difficult to demonstrate possible effects of Cpn infection on AAA formation in a caseecontrol study. Conclusion: No significant associations were found between AAA detected by screening and Cpn antibody titer levels at the time of screening or during past screening at the age of 60.
INTRODUCTION Abdominal aortic aneurysm (AAA) is a chronic degenerative disease with proteolysis of elastic
1 Department of Vascular Surgery, Institution of Surgical Sciences, Uppsala University Hospital, Uppsala, Sweden. 2 Department of Surgery, G€ avle County Hospital, G€ avle, Sweden. 3 Department of Surgery, Helsingborg County Hospital, Helsingborg, Sweden.
Correspondence to: Lars Karlsson, MD, PhD, Department of Vascular Surgery, Institution of Surgical Sciences, Uppsala University Hospital, SE-371 85 Uppsala, Sweden, E-mail: [email protected] Ann Vasc Surg 2011; 25: 322-326 DOI: 10.1016/j.avsg.2010.09.001 Ó Annals of Vascular Surgery Inc. Published online: December 2, 2010
322
lamellae and extracellular matrix proteins brought about mainly by matrix metalloproteinases. Chronic inflammation in the aortic wall suggests that immune responses participate in the pathophysiological process.1,2 Several studies have indicated that infectious agents may be involved in the development of aneurysm disease; both cytomegalovirus and Chlamydophila pneumoniae (Cpn) have been previously identified in the AAA tissue.3-5 As part of the V€asterbotten Intervention Program (VIP), all men and women aged 60 years from the Norsj€ o municipality in northern Sweden donated blood between 1984 and 1994 for future research.6 In 1999, after a median of 12 years, the same population was screened for AAA. The highest prevalence of AAA in a general population until
Vol. 25, No. 3, April 2011
recently was reported.7 In three previous reports, the same population was analyzed for associations between AAA and traditional risk factors for atherosclerosis,8 fibrinolytic factors,9 and antibodies to Streptococcus pyogenes antigens.10 The aim of this study was to investigate the possible association between serological markers for Cpn and the development of AAA in this unique population.
SUBJECTS AND METHODS Between 1984 and 1994, all men and women aged 60 years from the Norsj€ o municipality in northern Sweden were invited to participate in the VIP,6 a program that addressed cardiovascular disease and diabetes. As part of this program, all participants after keeping a fast donated venous blood samples, which were to be stored at the northern Sweden Medical Research Bank for future research purposes. In 1999, a median of 12 years after the initial check-up (60 years), all men and women who by then were aged between 65 and 75 years and residing in the Norsj€ o municipality were screened for AAA and also new blood samples were collected, which were stored at 80 C until analysis. Of the 555 invited subjects, 504 (91%) participated (248 men and 256 women, median age: 70 years) in the study. A maximum infrarenal aortic diameter of 30 mm detected by means of an ultrasonography indicated the presence of an AAA. The study was designed as a caseecontrol study, which included 42 patients with AAA detected by screening, 35 men (83%) and seven women with a mean age of 71.4 years, and 100 age- and gender-matched controls who had participated in both the VIP and the AAA screening but who were otherwise randomly selected from the AAA screening study population. Comparisons between patients with AAA and controls were made on the basis of the following: (1) frozen plasma from the VIP study, when the participants were aged 60 years (‘‘historical data’’), (2) plasma from the AAA screening, when subjects were aged between 65 and 75 years (‘‘current data’’), and (3) the differences between current and historical data (‘‘delta data’’), where the median between assessments of current and historical data was 12 years (mean, 11.6). The study design is presented in Figure 1. Venous blood samples were collected in tubes containing ethylenediaminetetraacetic acid. The plasma samples were snap frozen to 20 C within 1 hour and later stored at 80 C within a week.
Development of AAA 323
All ethylenediaminetetraacetic acid containing plasma samples were analyzed once using Cpnspecific microimmunofluorescence technique (Ani Labsystems, Vantaa, Finland) for immunoglobulin G (IgG) and immunoglobulin A (IgA).11 The Cpn antibody titers were categorized into the following levels: <1/64, 1/64, 1/264, and 1/1024. The c2 test (Kendall’s tau-b) was used to measure the associations between AAA and Cpn antibodies titers. Statistical evaluations of the data were carried out with a computer software package SPSS PC version 16.0 (SPSS, Chicago, IL). The investigation conformed to the principles outlined in the Declaration of Helsinki, all participants gave their informed consent, and the study was approved by the Committee of Ethics of the Ume a University.
RESULTS Baseline characteristics of the two groups are shown in Table I. Current smoking, cerebrovascular artery disease, and family history of AAA were factors found to be significantly associated with AAA. The distributions of Cpn titers for patients with AAA detected by screening and controls are displayed in Tables II and III. In all, 82% of the patients with AAA had a current Cpn IgG titer of 1/1024 as compared with the 70% of the control group. No significant associations were observed between AAA and historical Cpn IgG and IgA titer levels ( p ¼ 0.449 and 0.228), current titer levels ( p ¼ 0.111 and 0.659), or titer changes (delta) ( p ¼ 0.794 and 0.172). The distribution of aortic diameter for patients with different current Cpn IgG antibody titers is displayed in a box-plot in Figure 2. All patients (11/11) with an aortic diameter of >40 mm had a current Cpn IgG titer of either 1/256 or 1/1024. Both historical and current Cpn antibody titers could be analyzed in 30 of the total 42 patients with AAA.
DISCUSSION Although there are several known causes of aneurysmal dilatation, such as infections and heritable connective tissue disorders, the etiology of the disease is unknown in >90% of the patients with AAA.12 AAA is a disease that involves inflammatory infiltration in the media and adventitia and degeneration of collagen and elastin.1,2,12 However, it is difficult to demonstrate a causative relationship between an infectious agent and a chronic inflammatory process. Many chronic inflammatory diseases have multifactorial etiologies.
324 Karlsson et al.
1984
Annals of Vascular Surgery
1989
1994
1999
60 years of age
42 patients with AAA detected by screening
AAA screening
VIP study mean time 11.6 yr
vs
65 - 75 years of age
100 age- and sex matched controls
Historical data
Δ data
Current data
Fig. 1. Overview of the study design.
Table I. Baseline characteristics of patients with AAA detected by screening and age- and gendermatched controlsa Current data
Controls Mean (95% CI)
AAA Mean (95% CI)
p
Age Women Ever smoked Current smoker Smoke-years Pack-years Hypertension Coronary artery disease Cerebrovascular artery disease Diabetes mellitus First degree relative with AAA
71.4 17 38 1 32 19 26 23 2 9 5
71.4 17 50 18 42 21 45 30 18 10 23
0.95 0.96 0.18 <0.001 0.010 0.59 0.034 0.38 0.002 0.87 0.002
a
(70.9-71.8) (10-24) (27-48) (0-3) (26-37) (12-25) (17-35) (14-32) (0-5) (3-15) (0-9)
(70.6-72.1) (5-28) (34-66) (5-30) (36-49) (15-28) (29-61) (15-45) (5-30) (0-20) (9-36)
Data obtained at AAA screening at the age of 65-75 years.
Microbiologic examination of 500 aneurysms showed that 37% were positive in bacterial culture, and of these, 80% were identified as normal skin flora.13 A wide variety of bacteria can be isolated from aortic aneurysms.14 Of these, Cpn infection might be regarded as one potential etiological factor in the pathogenesis of AAA. Cpn has a tendency to persist and cause chronic infections. Recently, the detection of chlamydial lipopolysaccharide in AAA tissue was reported,15 suggesting the presence of Cpn in diseased aortic tissue and its possible role in triggering inflammation. Cpn can escape the immune response within the host by using different mechanisms. First, several studies indicate that when Cpn comes into contact with agents, such as tobacco smoke, antibiotics, or cytokines, it enters a persistent phase characterized by the inhibition of productive infection, reduced bacterial cell division, and formation of aberrant bodies.16 Second, Cpn can survive, be metabolically active, and multiply within phagocytes through modification of metabolic pathways within
eukaryotic cells.17 The innate immune system may not be sufficient for the elimination of Cpn. Third, evasion of T-cell recognition includes suppression of major histocompatibility complex expression on human monocytes.18 Fourth, apoptosis of the infected cell is a common response to microbial activity. Chlamydia possesses antiapoptotic activity for evasion from cytotoxic T-cells.19 It is unlikely that Cpn is an innocent bystander because metabolically inactive or killed organisms are eliminated from the system, as opposed to actively-growing Cpn. Cpn is commonly diagnosed in AAA by using techniques such as polymerase chain reaction, immunohistochemistry, electron microscope, and culture.4,5,20,21 Serologic studies conducted on patients with AAA have demonstrated an increased prevalence of specific Cpn antibodies in these patients when compared with controls.5,22,23 We have previously reported higher antibody titers against Cpn in patients operated for AAA as compared with age- and gender-matched controls;5 in addition, Lindholt et al. reported that the
Vol. 25, No. 3, April 2011
Development of AAA 325
Table II. Distribution of historical IgG titers, IgA titers, current IgG titers, and IgA titers for patients with AAA detected by screening and controls <1/64
Historical IgG titers AAA 3 Control 8 Historical IgA titers AAA 9 Control 20 Current IgG titers AAA 3 Control 8 Current IgA titers AAA 12 Control 30
1/64
1/256
1/1024
Total
2 11
7 27
21 54
33 100
10 29
9 34
4 16
32 99
0 7
4 15
33 70
40 100
5 19
13 29
10 22
40 100
Table III. Distribution of delta (currenthistorical titers) IgG titers and IgA titers for patients with AAA detected by screening and controls
Delta IgG titers AAA Control Delta IgA titers AAA Control
0
+
Total
1 8
24 69
5 23
30 100
5 25
15 52
10 22
30 99
expansion of AAA was related to Cpn antibody titer, suggesting that a chronic infection may contribute to pathogenesis and development of AAA.24 However, most previous studies on AAA are based on the final stage of the disease, when degeneration has already occurred. We wanted to investigate an earlier stage of the pathogenesis of the disease and the unique study design permitted us to analyze plasma drawn from individuals 12 years before the detection of a small AAA by screening. Some important limitations with the study design need to be addressed. Respiratory tract infections with Cpn are common, with about 70% of an adult population having antibodies against Cpn.25 The limited number of patients in this study and the fact that only 30 of the total 42 cases could be analyzed made the caseecontrol study design susceptible to type-II statistical error. Moreover, the etiology of AAA is thought to be multifactorial, which makes it difficult to find evidence of one specific cause. Different infectious agents may participate in the pathogenesis of AAA, and we have previously found that an immune response against S pyogenes protein G-related alpha 2M-binding protein may be of
Fig. 2. Box-plot of the distribution of aortic diameters for patients with different current Cpn IgG antibody titers, showing the 2.5%, 25%, 50%, 75%, and 97.5% cumulative relative frequencies. Open circles represent cases with values 1.5-3 box lengths from the upper or lower edge of the box and asterisks represent cases with values >3 box lengths from the upper or lower edge of the box.
importance in the early pathogenesis of AAA.10 Although the present study found no significant association between AAA detected by screening and Cpn antibody titer levels in blood samples obtained 12 years before the screening or at screening, there was an interesting tendency toward high IgG titers among subjects with larger AAAs. On the basis of the present study, it is therefore difficult to rule out that an immune response against Cpn participates in the development of AAAs, especially among a subset of genetically-vulnerable subjects.
CONCLUSIONS In this population-based caseecontrol study, no significant association was found between AAA detected by screening and Cpn antibody titer levels in blood obtained 12 years before the screening or at screening. However, because of the methodological limitations it is difficult to conclusively rule out on the basis of the findings of the present study that an immune response against Cpn participates in the development of AAAs. The authors thank professor G€ oran Hallmans and his staff at the northern Sweden Medical Research Bank, Ume a University Hospital, for their expertise and help with the blood samples. This work was supported by the County of G€ avleborgs L€ an Research and Development (FoU) Center, Zoega medical research fund, the Schyberg foundation for medical research, and the Swedish Research Council (grant K2007-64X-2040601-3).
326 Karlsson et al.
REFERENCES 1. Koch AE, Haines GK, Rizzo RJ, et al. Human abdominal aortic aneurysms: immunophenotypic analysis suggesting an immune mediated response. Am J Pathol 1990;137: 1199-1213. 2. Satta J, Laurila A, P€a€akk€ o P, et al. Chronic inflammation and elastin degradation in abdominal aortic aneurysm disease: an immunohistochemical and electron microscopic study. Eur J Vasc Endovasc Surg 1998;15:313-319. 3. Yonemitsu Y, Nakagawa K, Tanaka S, Mori R, Suqimachi K, Sueishi K. In situ detection of frequent and active infection of human cytomegalovirus in inflammatory abdominal aortic aneurysm: possible pathogenic role in sustained chronic inflammatory reaction. Lab Invest 1996;74:723-736. 4. Juvonen J, Juvonen T, Laurila A, et al. Demonstration of Chlamydia pneumoniae in the walls of the abdominal aortic aneurysm. J Vasc Surg 1997;25:499-505. 5. Karlsson L, Gnarpe J, N€a€as J, et al. Detection of viable Chlamydia pneumoniae in abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2000;19:630-635. 6. V€ asterbottens L€ans Landsting. Protokoll Fr an F€ orvaltningsutskottets Sammantr€ade 1984-12-17 [Protocol from the County Council Board Meeting, December 17, 1984]. Ume a, Sweden: V€asterbottens L€ans Landsting, 1984. 470. 7. Wanhainen A, Bj€ orck M, Boman K, Ruteg ard J, Bergqvist D. The influence of diagnostic criteria on the prevalence of abdominal aortic aneurysm. J Vasc Surg 2001;34:229-235. 8. Wanhainen A, Bergqvist D, Boman K, Nilsson T, Ruteg ard J, Bj€ orck M. Factors associated with the development of abdominal aortic aneurysm. A study based on historicaland cross-sectional screening data. J Vasc Surg 2005;41: 390-396. 9. Wanhainen A, Nilsson TK, Bergqvist D, Boman K, Bj€ orck M. Elevated tissue plasminogen activator in patients with screening-detected abdominal aortic aneurysm. J Vasc Surg 2007;45:1109-1113. 10. Wanhainen A, Rasmussen M, Bj€ orck L, Bj€ orck M. Immuneresponse against Streptococcus pyogenes in the development of abdominal aortic aneurysmda population-based casecontrol study. VASA 2008;37:143-149. 11. Gnarpe J, N€ a€ as J, Lundb€ack A. Comparison of a new commercial EIA kit and the microimmunofluorescence technique for determination of IgG and IgA antibodies to Chlamydia pneumoniae. AMPIS 2000;108:819-824. 12. Sakalihasan N, Limet R, Defawe OD. Abdominal aortic aneurysm (seminar). Lancet 2005;365:1577-1589.
Annals of Vascular Surgery
13. Farkas JC, Fichelle JM, Laurian C, et al. Long-term followup of positive cultures in 500 abdominal aortic aneurysms. Arch Surg 1993;128:284-288. 14. Marques da Silva R, Caugant DA, Eribe ER, et al. Bacterial diversity in aortic aneurysms determined by 16S ribosomal RNA gene analysis. J Vasc Surg 2006;44:1055-1060. 15. Vikatmaa P, Lajunen T, Ikonen TS, et al. Chlamydial lipopolysaccharide (cLPS) is present in atherosclerotic and aneurysmal arterial wall-cLPS levels depend on disease manifestation. Cardiovasc Pathol 2010;19:48-54. 16. Wiedeman JA, Kaul R, Heuer LS, Thao NN, Pinkerton KE, Wenman WM. Tobacco smoke induces persistent infection of Chlamydia pneumoniae in Hep-2 cells. Microb Pathog 2004;37:141-148. 17. Gaydos C, Summersgill JT, Sahney NN, Ramirez JA, Quinn TC. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells and aortis artery smooth muscle cells. Infect Immun 1996;64:1614-1620. 18. Zhong G, Fan T, Liu L. Chlamydia inhibits interferon gamma-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1. J Exp Med 1999;189:1931-1938. 19. Fan T, Lu H, Hu H, et al. Inhibition of apoptosis in Chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation. J Exp Med 1998;187:487-496. 20. Petersen E, Boman J, Persson K, et al. Chlamydia pneumoniae in abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 1998;15:138-142. 21. Meijer A, van der Vliet JA, Roholl PJM, Gielis-Proper SK, de Vries A, Ossewarde JM. Chlamydia pneumoniae in abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol 1999;19: 2680-2686. 22. Wolski A, Korobowicz E, Siezieniewska Z, et al. Immunofluorescence in situ and the serologic indices of Chlamydia pneumoniae infection in patients with an abdominal aortic aneurysm. Pol J Pathol 2002;53:223-228. 23. Schillinger M, Domanovits H, Mlekusch W, et al. Anti Chlamydia antibodies in patients with thoracic and abdominal aortic aneurysms. Wien Klin Wochenschr 2002;114: 972-977. 24. Lindholt JS, Ashton HA, Scott RAP. Indicators of infection with C. pneumoniae (IgA antibodies) are associated with expansion of abdominal aortic aneurysms. J Vasc Surg 2001;34:212-215. 25. Kuo CC, Grayston JT, Campbell YA, Goo R, Wissler W, 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.
Background: To investigate the association between serological markers for Chlamydophila pneumoniae (Cpn) and the development of abdominal aortic aneurysm (AAA) in a populationbased caseecontrol study. Methods: A screening for AAA among 65-75-year-old men and women was performed in a population with high prevalence of disease. Most of the subjects had undergone previous testing at the age of 60, including blood sampling. A total of 42 patients with AAA were compared with 100 age- and gender-matched controls with normal aortas. Cpn immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies present in plasma samples obtained at the time of screening (current) and in the past 5-15 (mean, 12) years (historical) were analyzed. Cpn antibody titers (<1/64, 1/64, 1/264, and 1/1024) were analyzed using the microimmunofluorescence technique. Results: No differences in current Cpn immunoglobulin A and IgG antibodies titers ( p ¼ 0.111 and 0.659), historical titers ( p ¼ 0.449 and 0.228), or titer change (delta) ( p ¼ 0.794 and 0.172) were observed between patients with AAA and controls. In all, 82% of the patients with AAA had a current Cpn IgG titer of 1/1024 as compared with the 70% of the control group. All 11 patients who had an aortic diameter of >40 mm reported having high current Cpn IgG titers. The fact that such a large proportion of the healthy population demonstrated an immune response against Cpn made it difficult to demonstrate possible effects of Cpn infection on AAA formation in a caseecontrol study. Conclusion: No significant associations were found between AAA detected by screening and Cpn antibody titer levels at the time of screening or during past screening at the age of 60.
INTRODUCTION Abdominal aortic aneurysm (AAA) is a chronic degenerative disease with proteolysis of elastic
1 Department of Vascular Surgery, Institution of Surgical Sciences, Uppsala University Hospital, Uppsala, Sweden. 2 Department of Surgery, G€ avle County Hospital, G€ avle, Sweden. 3 Department of Surgery, Helsingborg County Hospital, Helsingborg, Sweden.
Correspondence to: Lars Karlsson, MD, PhD, Department of Vascular Surgery, Institution of Surgical Sciences, Uppsala University Hospital, SE-371 85 Uppsala, Sweden, E-mail: [email protected] Ann Vasc Surg 2011; 25: 322-326 DOI: 10.1016/j.avsg.2010.09.001 Ó Annals of Vascular Surgery Inc. Published online: December 2, 2010
322
lamellae and extracellular matrix proteins brought about mainly by matrix metalloproteinases. Chronic inflammation in the aortic wall suggests that immune responses participate in the pathophysiological process.1,2 Several studies have indicated that infectious agents may be involved in the development of aneurysm disease; both cytomegalovirus and Chlamydophila pneumoniae (Cpn) have been previously identified in the AAA tissue.3-5 As part of the V€asterbotten Intervention Program (VIP), all men and women aged 60 years from the Norsj€ o municipality in northern Sweden donated blood between 1984 and 1994 for future research.6 In 1999, after a median of 12 years, the same population was screened for AAA. The highest prevalence of AAA in a general population until
Vol. 25, No. 3, April 2011
recently was reported.7 In three previous reports, the same population was analyzed for associations between AAA and traditional risk factors for atherosclerosis,8 fibrinolytic factors,9 and antibodies to Streptococcus pyogenes antigens.10 The aim of this study was to investigate the possible association between serological markers for Cpn and the development of AAA in this unique population.
SUBJECTS AND METHODS Between 1984 and 1994, all men and women aged 60 years from the Norsj€ o municipality in northern Sweden were invited to participate in the VIP,6 a program that addressed cardiovascular disease and diabetes. As part of this program, all participants after keeping a fast donated venous blood samples, which were to be stored at the northern Sweden Medical Research Bank for future research purposes. In 1999, a median of 12 years after the initial check-up (60 years), all men and women who by then were aged between 65 and 75 years and residing in the Norsj€ o municipality were screened for AAA and also new blood samples were collected, which were stored at 80 C until analysis. Of the 555 invited subjects, 504 (91%) participated (248 men and 256 women, median age: 70 years) in the study. A maximum infrarenal aortic diameter of 30 mm detected by means of an ultrasonography indicated the presence of an AAA. The study was designed as a caseecontrol study, which included 42 patients with AAA detected by screening, 35 men (83%) and seven women with a mean age of 71.4 years, and 100 age- and gender-matched controls who had participated in both the VIP and the AAA screening but who were otherwise randomly selected from the AAA screening study population. Comparisons between patients with AAA and controls were made on the basis of the following: (1) frozen plasma from the VIP study, when the participants were aged 60 years (‘‘historical data’’), (2) plasma from the AAA screening, when subjects were aged between 65 and 75 years (‘‘current data’’), and (3) the differences between current and historical data (‘‘delta data’’), where the median between assessments of current and historical data was 12 years (mean, 11.6). The study design is presented in Figure 1. Venous blood samples were collected in tubes containing ethylenediaminetetraacetic acid. The plasma samples were snap frozen to 20 C within 1 hour and later stored at 80 C within a week.
Development of AAA 323
All ethylenediaminetetraacetic acid containing plasma samples were analyzed once using Cpnspecific microimmunofluorescence technique (Ani Labsystems, Vantaa, Finland) for immunoglobulin G (IgG) and immunoglobulin A (IgA).11 The Cpn antibody titers were categorized into the following levels: <1/64, 1/64, 1/264, and 1/1024. The c2 test (Kendall’s tau-b) was used to measure the associations between AAA and Cpn antibodies titers. Statistical evaluations of the data were carried out with a computer software package SPSS PC version 16.0 (SPSS, Chicago, IL). The investigation conformed to the principles outlined in the Declaration of Helsinki, all participants gave their informed consent, and the study was approved by the Committee of Ethics of the Ume a University.
RESULTS Baseline characteristics of the two groups are shown in Table I. Current smoking, cerebrovascular artery disease, and family history of AAA were factors found to be significantly associated with AAA. The distributions of Cpn titers for patients with AAA detected by screening and controls are displayed in Tables II and III. In all, 82% of the patients with AAA had a current Cpn IgG titer of 1/1024 as compared with the 70% of the control group. No significant associations were observed between AAA and historical Cpn IgG and IgA titer levels ( p ¼ 0.449 and 0.228), current titer levels ( p ¼ 0.111 and 0.659), or titer changes (delta) ( p ¼ 0.794 and 0.172). The distribution of aortic diameter for patients with different current Cpn IgG antibody titers is displayed in a box-plot in Figure 2. All patients (11/11) with an aortic diameter of >40 mm had a current Cpn IgG titer of either 1/256 or 1/1024. Both historical and current Cpn antibody titers could be analyzed in 30 of the total 42 patients with AAA.
DISCUSSION Although there are several known causes of aneurysmal dilatation, such as infections and heritable connective tissue disorders, the etiology of the disease is unknown in >90% of the patients with AAA.12 AAA is a disease that involves inflammatory infiltration in the media and adventitia and degeneration of collagen and elastin.1,2,12 However, it is difficult to demonstrate a causative relationship between an infectious agent and a chronic inflammatory process. Many chronic inflammatory diseases have multifactorial etiologies.
324 Karlsson et al.
1984
Annals of Vascular Surgery
1989
1994
1999
60 years of age
42 patients with AAA detected by screening
AAA screening
VIP study mean time 11.6 yr
vs
65 - 75 years of age
100 age- and sex matched controls
Historical data
Δ data
Current data
Fig. 1. Overview of the study design.
Table I. Baseline characteristics of patients with AAA detected by screening and age- and gendermatched controlsa Current data
Controls Mean (95% CI)
AAA Mean (95% CI)
p
Age Women Ever smoked Current smoker Smoke-years Pack-years Hypertension Coronary artery disease Cerebrovascular artery disease Diabetes mellitus First degree relative with AAA
71.4 17 38 1 32 19 26 23 2 9 5
71.4 17 50 18 42 21 45 30 18 10 23
0.95 0.96 0.18 <0.001 0.010 0.59 0.034 0.38 0.002 0.87 0.002
a
(70.9-71.8) (10-24) (27-48) (0-3) (26-37) (12-25) (17-35) (14-32) (0-5) (3-15) (0-9)
(70.6-72.1) (5-28) (34-66) (5-30) (36-49) (15-28) (29-61) (15-45) (5-30) (0-20) (9-36)
Data obtained at AAA screening at the age of 65-75 years.
Microbiologic examination of 500 aneurysms showed that 37% were positive in bacterial culture, and of these, 80% were identified as normal skin flora.13 A wide variety of bacteria can be isolated from aortic aneurysms.14 Of these, Cpn infection might be regarded as one potential etiological factor in the pathogenesis of AAA. Cpn has a tendency to persist and cause chronic infections. Recently, the detection of chlamydial lipopolysaccharide in AAA tissue was reported,15 suggesting the presence of Cpn in diseased aortic tissue and its possible role in triggering inflammation. Cpn can escape the immune response within the host by using different mechanisms. First, several studies indicate that when Cpn comes into contact with agents, such as tobacco smoke, antibiotics, or cytokines, it enters a persistent phase characterized by the inhibition of productive infection, reduced bacterial cell division, and formation of aberrant bodies.16 Second, Cpn can survive, be metabolically active, and multiply within phagocytes through modification of metabolic pathways within
eukaryotic cells.17 The innate immune system may not be sufficient for the elimination of Cpn. Third, evasion of T-cell recognition includes suppression of major histocompatibility complex expression on human monocytes.18 Fourth, apoptosis of the infected cell is a common response to microbial activity. Chlamydia possesses antiapoptotic activity for evasion from cytotoxic T-cells.19 It is unlikely that Cpn is an innocent bystander because metabolically inactive or killed organisms are eliminated from the system, as opposed to actively-growing Cpn. Cpn is commonly diagnosed in AAA by using techniques such as polymerase chain reaction, immunohistochemistry, electron microscope, and culture.4,5,20,21 Serologic studies conducted on patients with AAA have demonstrated an increased prevalence of specific Cpn antibodies in these patients when compared with controls.5,22,23 We have previously reported higher antibody titers against Cpn in patients operated for AAA as compared with age- and gender-matched controls;5 in addition, Lindholt et al. reported that the
Vol. 25, No. 3, April 2011
Development of AAA 325
Table II. Distribution of historical IgG titers, IgA titers, current IgG titers, and IgA titers for patients with AAA detected by screening and controls <1/64
Historical IgG titers AAA 3 Control 8 Historical IgA titers AAA 9 Control 20 Current IgG titers AAA 3 Control 8 Current IgA titers AAA 12 Control 30
1/64
1/256
1/1024
Total
2 11
7 27
21 54
33 100
10 29
9 34
4 16
32 99
0 7
4 15
33 70
40 100
5 19
13 29
10 22
40 100
Table III. Distribution of delta (currenthistorical titers) IgG titers and IgA titers for patients with AAA detected by screening and controls
Delta IgG titers AAA Control Delta IgA titers AAA Control
0
+
Total
1 8
24 69
5 23
30 100
5 25
15 52
10 22
30 99
expansion of AAA was related to Cpn antibody titer, suggesting that a chronic infection may contribute to pathogenesis and development of AAA.24 However, most previous studies on AAA are based on the final stage of the disease, when degeneration has already occurred. We wanted to investigate an earlier stage of the pathogenesis of the disease and the unique study design permitted us to analyze plasma drawn from individuals 12 years before the detection of a small AAA by screening. Some important limitations with the study design need to be addressed. Respiratory tract infections with Cpn are common, with about 70% of an adult population having antibodies against Cpn.25 The limited number of patients in this study and the fact that only 30 of the total 42 cases could be analyzed made the caseecontrol study design susceptible to type-II statistical error. Moreover, the etiology of AAA is thought to be multifactorial, which makes it difficult to find evidence of one specific cause. Different infectious agents may participate in the pathogenesis of AAA, and we have previously found that an immune response against S pyogenes protein G-related alpha 2M-binding protein may be of
Fig. 2. Box-plot of the distribution of aortic diameters for patients with different current Cpn IgG antibody titers, showing the 2.5%, 25%, 50%, 75%, and 97.5% cumulative relative frequencies. Open circles represent cases with values 1.5-3 box lengths from the upper or lower edge of the box and asterisks represent cases with values >3 box lengths from the upper or lower edge of the box.
importance in the early pathogenesis of AAA.10 Although the present study found no significant association between AAA detected by screening and Cpn antibody titer levels in blood samples obtained 12 years before the screening or at screening, there was an interesting tendency toward high IgG titers among subjects with larger AAAs. On the basis of the present study, it is therefore difficult to rule out that an immune response against Cpn participates in the development of AAAs, especially among a subset of genetically-vulnerable subjects.
CONCLUSIONS In this population-based caseecontrol study, no significant association was found between AAA detected by screening and Cpn antibody titer levels in blood obtained 12 years before the screening or at screening. However, because of the methodological limitations it is difficult to conclusively rule out on the basis of the findings of the present study that an immune response against Cpn participates in the development of AAAs. The authors thank professor G€ oran Hallmans and his staff at the northern Sweden Medical Research Bank, Ume a University Hospital, for their expertise and help with the blood samples. This work was supported by the County of G€ avleborgs L€ an Research and Development (FoU) Center, Zoega medical research fund, the Schyberg foundation for medical research, and the Swedish Research Council (grant K2007-64X-2040601-3).
326 Karlsson et al.
REFERENCES 1. Koch AE, Haines GK, Rizzo RJ, et al. Human abdominal aortic aneurysms: immunophenotypic analysis suggesting an immune mediated response. Am J Pathol 1990;137: 1199-1213. 2. Satta J, Laurila A, P€a€akk€ o P, et al. Chronic inflammation and elastin degradation in abdominal aortic aneurysm disease: an immunohistochemical and electron microscopic study. Eur J Vasc Endovasc Surg 1998;15:313-319. 3. Yonemitsu Y, Nakagawa K, Tanaka S, Mori R, Suqimachi K, Sueishi K. In situ detection of frequent and active infection of human cytomegalovirus in inflammatory abdominal aortic aneurysm: possible pathogenic role in sustained chronic inflammatory reaction. Lab Invest 1996;74:723-736. 4. Juvonen J, Juvonen T, Laurila A, et al. Demonstration of Chlamydia pneumoniae in the walls of the abdominal aortic aneurysm. J Vasc Surg 1997;25:499-505. 5. Karlsson L, Gnarpe J, N€a€as J, et al. Detection of viable Chlamydia pneumoniae in abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2000;19:630-635. 6. V€ asterbottens L€ans Landsting. Protokoll Fr an F€ orvaltningsutskottets Sammantr€ade 1984-12-17 [Protocol from the County Council Board Meeting, December 17, 1984]. Ume a, Sweden: V€asterbottens L€ans Landsting, 1984. 470. 7. Wanhainen A, Bj€ orck M, Boman K, Ruteg ard J, Bergqvist D. The influence of diagnostic criteria on the prevalence of abdominal aortic aneurysm. J Vasc Surg 2001;34:229-235. 8. Wanhainen A, Bergqvist D, Boman K, Nilsson T, Ruteg ard J, Bj€ orck M. Factors associated with the development of abdominal aortic aneurysm. A study based on historicaland cross-sectional screening data. J Vasc Surg 2005;41: 390-396. 9. Wanhainen A, Nilsson TK, Bergqvist D, Boman K, Bj€ orck M. Elevated tissue plasminogen activator in patients with screening-detected abdominal aortic aneurysm. J Vasc Surg 2007;45:1109-1113. 10. Wanhainen A, Rasmussen M, Bj€ orck L, Bj€ orck M. Immuneresponse against Streptococcus pyogenes in the development of abdominal aortic aneurysmda population-based casecontrol study. VASA 2008;37:143-149. 11. Gnarpe J, N€ a€ as J, Lundb€ack A. Comparison of a new commercial EIA kit and the microimmunofluorescence technique for determination of IgG and IgA antibodies to Chlamydia pneumoniae. AMPIS 2000;108:819-824. 12. Sakalihasan N, Limet R, Defawe OD. Abdominal aortic aneurysm (seminar). Lancet 2005;365:1577-1589.
Annals of Vascular Surgery
13. Farkas JC, Fichelle JM, Laurian C, et al. Long-term followup of positive cultures in 500 abdominal aortic aneurysms. Arch Surg 1993;128:284-288. 14. Marques da Silva R, Caugant DA, Eribe ER, et al. Bacterial diversity in aortic aneurysms determined by 16S ribosomal RNA gene analysis. J Vasc Surg 2006;44:1055-1060. 15. Vikatmaa P, Lajunen T, Ikonen TS, et al. Chlamydial lipopolysaccharide (cLPS) is present in atherosclerotic and aneurysmal arterial wall-cLPS levels depend on disease manifestation. Cardiovasc Pathol 2010;19:48-54. 16. Wiedeman JA, Kaul R, Heuer LS, Thao NN, Pinkerton KE, Wenman WM. Tobacco smoke induces persistent infection of Chlamydia pneumoniae in Hep-2 cells. Microb Pathog 2004;37:141-148. 17. Gaydos C, Summersgill JT, Sahney NN, Ramirez JA, Quinn TC. Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells and aortis artery smooth muscle cells. Infect Immun 1996;64:1614-1620. 18. Zhong G, Fan T, Liu L. Chlamydia inhibits interferon gamma-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1. J Exp Med 1999;189:1931-1938. 19. Fan T, Lu H, Hu H, et al. Inhibition of apoptosis in Chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation. J Exp Med 1998;187:487-496. 20. Petersen E, Boman J, Persson K, et al. Chlamydia pneumoniae in abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 1998;15:138-142. 21. Meijer A, van der Vliet JA, Roholl PJM, Gielis-Proper SK, de Vries A, Ossewarde JM. Chlamydia pneumoniae in abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol 1999;19: 2680-2686. 22. Wolski A, Korobowicz E, Siezieniewska Z, et al. Immunofluorescence in situ and the serologic indices of Chlamydia pneumoniae infection in patients with an abdominal aortic aneurysm. Pol J Pathol 2002;53:223-228. 23. Schillinger M, Domanovits H, Mlekusch W, et al. Anti Chlamydia antibodies in patients with thoracic and abdominal aortic aneurysms. Wien Klin Wochenschr 2002;114: 972-977. 24. Lindholt JS, Ashton HA, Scott RAP. Indicators of infection with C. pneumoniae (IgA antibodies) are associated with expansion of abdominal aortic aneurysms. J Vasc Surg 2001;34:212-215. 25. Kuo CC, Grayston JT, Campbell YA, Goo R, Wissler W, 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.