2014/12/04 by J. Thomas Grayston, Robert J. Belland, Gerald I. Byrne +4 · 2 citations
Immunology and Microbiology · Medicine · Biochemistry, Genetics and Molecular Biology · #Reproductive tract infections research #Urinary Tract Infections Management #Bacterial Identification and Susceptibility Testing
paper · pdf · doi:10.1093/femspd/ftu015
openalex publication_date 2014/12/04 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/30
We are convinced that Chlamydia pneumoniae research has been unfavorably affected by the negative results of antibiotic treatment trials for the secondary prevention of late-stage coronary heart disease (CHD) (O'Connor et al., 2003; Cannon et al., 2005; Grayston et al., 2005). There is a widespread belief that the clinical trials showed that C. pneumoniae has no role in atherosclerotic disease. This flawed causal inference from these trials has contributed to slowing much-needed research on C. pneumoniae. It has also resulted in a nearly complete loss of momentum for research on the infection-based response to injury hypothesis as a key factor in the initiation and progression of CHD. Chlamydia pneumoniae had been considered a possible cause of atherosclerosis. That antibiotics failed to prevent secondary coronary events in patients with established coronary artery disease has been erroneously interpreted as ruling out a causative role for C. pneumoniae. This misinterpretation has had a chilling effect on C. pneumoniae research. Our concern has been confirmed by the sharp drop in published reports (PubMed citations) on C. pneumoniae since 2005. From 1999 to 2005, approximately 375 papers were published each year. There has been a 62% drop from that number with 143 being listed for 2013. The antibiotic treatment trials were not etiologic studies. No inference regarding the role of C. pneumoniae in the cause of atherosclerosis can properly be made from the trials. A prior publication stated that the study design for these trials precluded proving or disproving a role for C. pneumoniae in the initiation or progression of atherosclerosis, and predicted an overreaction to either negative or positive results (Grayston 2000). All subjects of these trials had established coronary artery disease with mostly advanced disease. Most had had a myocardial infarction (MI). The trials studied whether antibiotics could prevent secondary coronary events in patients who previously had had a coronary event. A coronary event was defined as an MI, a specified angina episode, surgical intervention or death. While it was disappointing, it was not surprising that antibiotics given late in the course of atherosclerotic arterial disease did not prevent additional coronary events. The pathogenesis of MI has differences from the pathogenesis of atherosclerosis. In the animal model, antibiotic treatment was effective only when given early after C. pneumoniae inoculation. The animal model treatment studies were published after most of the clinical trials had been initiated. The misinterpretation of the significance of the clinical trial results was already becoming known when the authors first began discussions that eventually led to this manuscript. These discussions occurred at a remote mountainous ranch in Wyoming in 2007. The purpose of this ‘think tank’ was to discuss the state of C. pneumoniae research and to recommend areas for emphasis in future investigations. The stimulus for the retreat was our concern with the lack of progress in C. pneumoniae research. A wide variety of research needing attention was discussed. During the second day of the discussions, the group was coalescing around the idea that while there were many individual technical and conceptual problems in C. pneumoniae research, the most important was the potential role of C. pneumoniae infection in atherosclerotic diseases. The decisions to select this issue as the one for further study and to develop possible approaches for future research were influenced by the enormous importance of atherosclerotic diseases to human health. Atherosclerotic CHD is now the no. 1 killer throughout the industrialized world. This manuscript will present two major topics: first, a description of the evidence for a potential etiologic association of C. pneumoniae and atherosclerosis, and second, a proposed method that could provide data for an etiologic association. Infection has been proposed as a causal factor in heart disease for more than 100 years. In the last two decades, there has been increasing research on the role of infection in atherosclerotic cardiovascular disease. The accumulated data associating C. pneumoniae with atherosclerosis are particularly compelling. C. pneumoniae is unique among several microbes that have been associated with arterial disease in having been demonstrated with frequency in atherosclerotic lesions but not in normal arterial tissue (Kuo et al., 1995). The organism was found not only in coronary arteries but also in carotid (Jackson et al., 1997), aortic (Kuo et al., 1993), femoral and popliteal arteries (Kuo et al., 1997). These findings have been confirmed by a number of experienced investigative teams using multiple laboratory techniques, including PCR, microscopy with immunocytochemical stain, in situ DNA hybridization and isolation of the organism (Campbell and Kuo 2004; Watson and Alp 2008). In atheromas, C. pneumoniae is found within smooth muscle cells, macrophages and endothelial-derived foam cells (Kuo et al., 1993; Kuo and Campbell 2000). In addition to the human studies, animal studies have contributed to the evidence for C. pneumoniae as an infectious cause of CHD. Chlamydia pneumoniae pulmonary infection has been shown to accelerate atherosclerotic disease in mice and rabbits prone to develop the disease due to genetic manipulation or high-fat diets (Muhlestein et al., 1998; Hu, Pierce and Zhong 1999; Moazed et al., 1999). In rabbits fed a regular diet, C. pneumoniae infection resulted in atherosclerotic changes (Fong et al., 1999). In contrast, although repeated infection did not induce atherosclerosis in normolipidemic mice, inflammatory changes were observed in the heart and aorta (Blessing et al., 2000). If a high-fat diet was initiated concurrently with infection in C57BL/6J mice, atherosclerotic lesion progress was accelerated (Blessing et al., 2002a). However, if infections preceded administration of high-fat diet, no augmentation of lesion development was observed. Taken together, these results suggest that in the mouse model C. pneumoniae infection is a co-risk factor with hyperlipidemia. In human atherosclerotic lesions, although the organism has been cultured only a few times, the organism is frequently detected in mature atherosclerotic lesions by other methods, suggesting persistent infection. Similarly, following repeated pulmonary infection in mice, the organisms can be cultured from the aorta for 1–2 weeks post-infection, but can be detected by other methods in the aorta for 20 weeks post-infection, the endpoint of the experiment (Moazed et al., 1997). In rabbit models of C. pneumoniae accelerated atherosclerosis, azithromycin prevented the accelerated intimal thickening. However, timing of antibiotic treatment was critical in blocking C. pneumoniae accelerated atherosclerosis. Specifically, initiation of treatment with either clarithromycin or azithromycin within a week after the first of three inoculations with C. pneumoniae was efficacious, while delayed treatment initiated 6 weeks after the first inoculation was not (Fong 2000). Studies done in apoE knockout mice using different treatment regimens of azithromycin (a dose after each of two inoculations or a 6-week course initiated after the third inoculation) did not demonstrate any reduction in C. pneumoniae accelerated atherosclerosis (Rothstein et al., 2001; Blessing et al., 2005). Importantly, following antibiotic treatment in both rabbit and mouse models, C. pneumoniae DNA or antigen was detected in the aorta, suggesting that infection was refractory to treatment (Muhlestein et al., 1998; Rothstein et al., 2001; Fong et al., 2002). Other biological effects consistent with a role of C. pneumoniae in atherosclerotic processes have been demonstrated in animal models including increased T-cell influx into the atherosclerotic lesion and earlier formation of complex lesions (Ezzahiri et al., 2002), enhanced endothelial dysfunction (Liuba et al., 2000, 2003), apoptosis of endothelial cells and degenerative changes associated with necrosis (Birck et al., 2013), and plaque destabilization as suggested by increased production of matrix metalloproteinases and reduced area of the fibrous cap (Ezzahiri et al., 2003) as well as increased intra-plaque hemorrhage in older mice (Campbell et al., 2010). The human and animal studies have been used to demonstrate the role of C. pneumoniae infection in atherosclerosis. The question of how C. pneumoniae infection exacerbates atherosclerosis has been addressed in cell culture systems. In the presence of LDL, C. pneumoniae induces foam cell formation and simulates LDL oxidation, through chlamydial LPS and Hsp60, respectively (Kalayoglu and Byrne 1998; Kalayoglu et al., 1999a,b). Scavenger receptors mediate oxidized LDL (oxLDL) uptake (Ross 1993). For endothelial cells, the lectin-like oxLDL receptor (LOX-1) is the major receptor for uptake of oxLDL (Sawamura et al., 1997; Kume et al., 1998). This scavenger receptor is also found on macrophages and smooth muscle cells (Moriwaki et al., 1998; Aoyama et al., 2000). Expression of LOX-1 is increased in hyperlipidemia and atherosclerotic lesions and activation of LOX-1 results in the up-regulation of pro-atherogenic factors (Kataoka et al., 1999; Chen et al., 2000; Li and Mehta 2000; Li et al., 2003; Zhu et al., 2005). Chlamydia pneumoniae has been shown to bind to the LOX-1 receptor, up-regulate LOX-1 expression, induce the expression of adhesion molecules and matrix metalloproteinases through LOX-1 activation, and promote uptake of ox-LDL (Yoshida et al., 2006; Campbell et al., 2012, 2013). Chlamydia pneumoniae infection of macrophages inhibits the expression of the cholesterol transporters ABCA1 and ABCG1 which play critical roles in cholesterol efflux and homeostasis (Liu et al., 2010; Korhonen et al., 2013; Zhao et al., 2014). Chlamydia pneumoniae infection of vascular cells also induces the expression of pro-inflammatory cytokines, chemokines and growth factors, all of which could contribute to the chronic inflammatory processes of atherosclerosis (Kaukoranta-Tolvanen et al., 1996; Hu, Pierce and Zhong 1999; Gaydos 2000; Kothe et al., 2000; Netea et al., 2000, 2002; Summersgill et al., 2000; Coombes and Mahony 2001; Blessing et al., 2002b; Mamata et al., 2007; Eitel et al., 2012). The findings of C. pneumoniae in atherosclerotic lesions provided the impetus for a number of investigators to attempt treatment of CHD with antibiotics known to be effective against C. pneumoniae. Results from three large randomized double blind studies of the effect of antibiotic treatment for secondary prevention of coronary events have been reported (O'Connor et al., 2003; Cannon et al., 2005; Grayston et al., 2005). Two of these studies had prolonged antibiotic treatment for one year (Cannon et al., 2005; Grayston et al., 2005). There were no differences in outcome between the groups given antibiotics or placebos. The overall results from a number of smaller trials were similarly negative (Andraws, Berger and Brown 2005). The significance of the failure of antibiotics to reduce coronary events in patients with established CHD has been discussed above. It is now well established that both atherosclerosis and C. pneumoniae infection are first seen in early childhood and that the prevalence of both increase with age. A variety of studies in different populations using different techniques have shown that atherosclerosis begins in childhood. In autopsy studies in Japanese children, evidence of early atherosclerosis, fatty streaks, was found in 29% of aortas in those <1 year old and in 3.1% of coronary arteries of children 1–9 years old (Tanaka et al., 1988). In a US autopsy study of coronary arteries, the prevalence of fatty streaks in the coronary arteries increased with age from 50% at 2–15 years of age to 85% at 21–39 years of age. Raised fibrous-plaque lesions were seen in 8% of children 2–15 years of age and in 69% of adults 26–39 years of age (Berenson et al., 1998). Another study showed lipid-laden macrophages in the intima of the aorta and coronary arteries of young American children killed in motor accidents, with over 50% aged 10–14 years having some evidence of early atherosclerosis (Stary 1989). In the Pathobiological Determinants of Atherosclerosis in Youth study (McGill et al., 2000), in those 15–19 years old at time of death, raised fatty streaks were found in 20% of aortas and 10% of right coronary arteries. The prevalence increased to 40% of aortas and 30% of right coronary arteries showing the lesions by age 30–34. Studies on the etiology of atherosclerosis must begin early in life: Atherosclerotic lesions have been found in very young children and the prevalence increases with age. Chlamydia pneumoniae infection first appears in early childhood and accelerates with school attendance. In animals, C. pneumoniae pulmonary infection accelerates development of atherosclerotic disease. Utilizing an intravascular ultrasound technique, it was found that 17% of otherwise healthy heart donors <20 years of age, 37% of those aged 20–29 years, 60% of those aged 30–39 years, 71% of those aged 40–49 years and 85% of those ≥50 years of age had evidence of coronary intimal thickening (Tuzcu et al., 2001). Seroepidemiologic studies with the microimmunofluorescence (MIF) test have provided information on C. pneumoniae infections in populations. Using several different serum banks, data on the prevalence and incidence of C. pneumoniae infections at different ages have been obtained (Aldous et al., 1992; Grayston 1994). The age-specific prevalence of C. pneumoniae-specific IgG antibody has shown a similar curve in several countries. Fig. 1 shows age prevalence in over 1000 Seattle residents below 20 years of age. The greatest increase in prevalence is seen from 5 to 15 years of age. While the prevalence rate is low in children under 5, many of the mild infections in these very young children may not produce long-lasting IgG antibody. This phenomenon has been demonstrated in very young children infected in the eye with C. trachomatis. Often two or three reinfections occur before antibody persists (Grayston et al., 1985). Prevalence of C. pneumoniae MIF IgG antibody by age in Seattle children (Grayston 1994). Sera from a family study in Seattle from 1963 to 1979 (Aldous et al., 1992) were used to determine incidence rates of C. pneumoniae infection based on 4-fold antibody titer rises. High annual rates of antibody rises were seen in the 5–9 year age group (9.2%) and the 10–14 year group (6.2%). These rates are consistent with the increase in prevalence rates from 5–14 years. The available clinical data suggest that many of the antibody conversions occurred in children who were asymptomatic. The symptomatic children usually had cough, a hallmark of C. pneumoniae disease. These incidence rates were confirmed by a study of sera collected longitudinally from children in Sweden. The reported annual MIF antibody conversion rates were 8.0% in 8 to 12 year olds and 5.9% in 12 to 16 year olds (Haidl, Sveger and Persson 1994). Chlamydia pneumoniae antibody prevalence rates continue to rise throughout adult life reaching 70–80% in the elderly. The rates are approximately the same in both sexes until age 15. Thereafter, the rates are higher in males, Fig. 2. The C. pneumoniae gender prevalence is different from other organisms causing respiratory infections where the prevalence rates are higher in women, presumably because they are in closer contact with children who are often the source of family infection. There is no known reason for C. pneumoniae to be more common in but it is that atherosclerotic diseases are more common in Prevalence of MIF antibody to C. pneumoniae by age in among in IgG from 8 to (Grayston We of no to C. pneumoniae etiology of CHD and have proposed an study by laboratory studies. could be the and and the studies The for a study to a causal between C. pneumoniae and This is made by in for both early atherosclerosis and C. pneumoniae infection. The of of to study the of chronic by the first of in the at the of data to study the of both and chronic in the and that of for to that cause chronic as by are similar to those for that cause The same three are These are data for the first of family present and of However, was found to be more for chronic infections since is not in the after but causal and may occur at any time in the life of the infected found that two major of studies for the of of infectious and the to the of The to be for chronic infections where most The method that was by who that most infected will be and the emphasis of is on the of infection than the development of disease. The for this of study is the out by and in from to where it was established that infection progression to disease et al., et al., The which multiple for isolation in family antibody and of disease in each was in a number of other studies to into and infections The of the was in for a number of and chronic and diseases. were used over the course of years to between and heart to an infectious to this very large in children a now that is by treatment of et al., The of atherosclerotic cardiovascular disease has early in artery in children and young with other factors LDL atherosclerosis in et al., 2010). the known of infectious in chronic inflammatory and the between C. pneumoniae respiratory infections and atherosclerotic it be possible to the to to our on the role of C. pneumoniae infections in cardiovascular disease. cardiovascular research has led to a of processes atherosclerosis, from initiation to progression of arterial techniques have also advanced in and may now research to and in the causal of atherosclerosis and plaque The of the carotid artery intima by may in early vascular changes of atherosclerosis et al., The of has in children because of the is used to early atherosclerosis, since it of cardiovascular and to It has been used to and in the and are being with and to plaque in more and can be by a known as the between two major arteries in the carotid or and the femoral or the time for the to a given the arteries produce a higher and may have an to other techniques due to of and time for a A et al., the published on advanced techniques that have potential for in clinical Studies will be to further these However, one can that methods will be for in studies of atherosclerosis and have been made to the of C. pneumoniae infection et al., there is lack of on methods for infection with C. pneumoniae. research have provided information to and approaches for C. pneumoniae infection for a family A of approaches that can be considered for C. pneumoniae infection is provided of C. DNA this is both and et al., et al., and 2012). It can also be which will infection to be with clinical C. pneumoniae organism although it is and can also provide for further studies, the rate is low the as a et al., 2010). of isolation methods is of C. pneumoniae the of and of the can also both the and they are these will results and not be for the proposed of C. including cytokines, and although C. pneumoniae is known to induce inflammatory of these be used for C. pneumoniae infection due to the lack of of the receptors for C. pneumoniae. the by C. pneumoniae receptors can be used for C. pneumoniae infection. However, due to the of molecules and the response can only be used to whether the subjects have been infected with C. pneumoniae. T-cell are more to and also more T-cell are not for for C. pneumoniae infection. However, C. pneumoniae T-cell can be for the roles of T-cell in C. pneumoniae et al., can be used for and in the et al., 2014). can be used to either the of et al., or the and for both and the of each in a given For and cells can be used for C. pneumoniae infection by C. pneumoniae DNA and for the and by using the and in the the of C. it is important to the in the and the same the from the of will be used for C. pneumoniae the overall and antigen of C. pneumoniae-specific will be The can be out as for C. antibody et al., 2010). These will a for each study During the same will be collected for clinical for both C. pneumoniae infection and the and antibody These will be with clinical observed in the same study of C. pneumoniae infection rate and with clinical diseases will of disease with C. pneumoniae infection. of and with clinical diseases will to associated with clinical diseases or lack of diseases. of pneumoniae antibody and with clinical diseases will to both and as has been done for C. et al., 2012). between the three will into the of the and on C. pneumoniae infection and and on to C. pneumoniae infection. A family study could be that provide information atherosclerotic disease among family and the prevalence of C. pneumoniae in and as well as of and to C. pneumoniae in Chlamydia and the of and to infection et al., 2008). and methods may be to on expression associated with infection and C. pneumoniae These associated with in vascular may further those who from vascular disease. the in vascular it also be possible to information on the initiation and progression of atherosclerotic lesions in and arteries. data on and infection provide into a possible etiologic association et al., 2008). The of many other potential etiologic factors and other For C. pneumoniae research, a number of and be from a These the to develop and the of of C. pneumoniae for studies, of the between and disease C. pneumoniae associated with and of genetic associated with infection and cardiovascular health. The of the American of to test cholesterol and in some to cholesterol treatment of young children the early age for prevention of heart disease et al., The proposed family study could in design studies that to the CHD is the cause of in the industrialized world. including changes has reduced CHD and of life for but there is no for the disease. It is critical that the of early atherosclerotic changes be to provide an for prevention or atherosclerosis and evidence of prior C. pneumoniae infection are found in most If C. pneumoniae infection or other microbes are found to be associated with the initiation and development of a antibiotic treatment or a could be the of the research to the role of infection in heart disease be a Studies as a the be This is to the of our and The authors and Campbell for with this manuscript. The retreat was in by research from the of of