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Institute of Medicine (US) Forum on Microbial Threats; Knobler SL, O'Connor S, Lemon SM, et al., editors. The Infectious Etiology of Chronic Diseases: Defining the Relationship, Enhancing the Research, and Mitigating the Effects: Workshop Summary. Washington (DC): National Academies Press (US); 2004.

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The Infectious Etiology of Chronic Diseases: Defining the Relationship, Enhancing the Research, and Mitigating the Effects: Workshop Summary.

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INFECTIOUS AGENTS AND CARDIOVASCULAR DISEASE

, M.D.

Pfizer Global Research and Development, New London, CT

Atherosclerosis remains the most significant threat to the health of individuals living in the United States and Europe. Myocardial infarctions, strokes, peripheral vascular disease and premature deaths constitute an enormous burden on the healthcare systems of these regions every year. Risk factors for atherosclerosis have been identified and interventions targeting these risks have helped mitigate its impact. The clinical sequelae of atherosclerosis remain significant, however, justifying continued research efforts to enhance the value of available interventions as well as identify presently unappreciated risk factors.

Examination of an atherosclerotic plaque reveals pools of cholesterol under a fibrous cap and the infiltration of monocytes and T cells at its margins. This concentration of white blood cells within the plaque is consistent with an ongoing inflammatory process, influenced by factors not yet fully understood. One such influence may be infection.

That infection may play a role in atherosclerosis was first suggested over one hundred years ago with the finding that acute infection with Bacillus typhosus resulted in fatty sclerotic changes in the arterial wall (Gilbert and Lion, 1889; Nieto, 1998). Interest in the role of infection in atherosclerosis was renewed with the observation that patients with coronary artery disease were more likely than matched controls to have an elevated antibody titer to Chlamydia pneumoniae (Saikku et al., 1988). Since that observation, a number of additional associations have been identified. The chain of events linking infections to the development of atherosclerosis is outlined in Figure 1-5. A local infection may lead to an arterial response through two different routes. First, local infection may trigger the systemic release of various proinflammatory mediators, including cytokines, bacterial lipopolysaccharide, heat shock proteins, immune complexes and, possibly, activated, but uninfected, mononuclear cells. These mediators move through the systemic circulation and incite an immune response in the arterial wall. This response may include the upregulation of receptors on the endothelial cell surface, enhancement of transendothelial migration of inflammatory cells, or activation of white blood cells already existing within the plaque. These activated WBCs may oxidize LDL cholesterol or release proteinases, which then act to destabilize the overlying fibrous cap of the atheroma.

FIGURE 1-5. Pathways through which local infection can lead to atherogenesis.

FIGURE 1-5

Pathways through which local infection can lead to atherogenesis.

The second route by which infection may result in progression or initiation of an atherosclerotic lesion involves the dissemination of organisms from local sites of infection directly to the arterial wall itself. The organisms may traffic to the site within an infected monocyte, attach and then diapedese through the endothelial cell layer, taking advantage of secondary host defense mechanisms to infect distal tissue. Once at the site, the organisms could drive a local inflammatory process or, in addition, infect other cells within the arterial wall.

A number of potential pathogens have been associated with atherosclerosis (Danesh, 1999). The strength of the association varies with the organism but is based on seroepidemiologic studies, histopathologic evidence of disease, animal model data and various pathophysiologic associations. Among possible viral pathogens are cytomegalovirus and herpes simplex (Nieto, 1999; Dunne, 2000). Among bacterial pathogens are various dental organisms, Helicobacter pylori, and Mycoplasma pneumoniae. The most significant amount of preclinical and clinical investigation, however, has focused on C. pneumoniae; as an example of the types of evidence that can implicate a potential infectious pathogen driving some component of the atherosclerotic process, these data will be reviewed in more detail.

C. Pneumoniae and Atherosclerosis Seroepidemiologic Studies

Since the initial study that identified an association between elevated C. pneumoniae antibody titers and the prevalence of coronary artery disease, over thirty additional studies have been performed and multiple review articles published. These studies used different antibody detection assays with different titer cutoffs, different case definitions of coronary artery disease, and were performed in different geographic regions. Overall, it appears that elevated antibody titers to C. pneumoniae are associated with a three-fold increase in the likelihood of having coronary artery disease. The association identified in seroepidemiologic studies using titers to predict the incidence, distinct from the prevalence, of heart disease, however, only variably detect an association and, when positive, only in the range of a 20–40 percent increased risk (Dunne, 2000). While the implications of these different findings are being evaluated, the main value of these seroepidemiologic studies may be the attention they have brought to the potential for any association at all.

Histopathology

The next series of studies involve histopathologic examinations of the atheromatous plaque. In the first 15 studies reported in the literature which were conducted in the United States and Europe, approximately 45 percent of the total of 574 samples examined were found to contain evidence of C. pneumoniae by either immunohistochemistry, electron microscopy, in situ polymerase chain reaction (PCR) or, rarely, culture. The primary criticism of these studies has focused on the lack of standardization of the assay techniques but, given the bulk of the observations from these and subsequent studies, it seems likely that this pathogen can be found in the plaque.

Because antibody titers merely suggest historical exposure to the pathogen, there has been recent interest in the use of PCR to identify individuals that may have an active infection with C. pneumoniae. PCR has been used to assess both histopathologic specimens and circulating white blood cells. In four published papers, patients with a history of coronary artery disease were more likely than controls to have C. pneumoniae identified in circulating monocytes by PCR (Dunne, 2000). In a fifth paper, the incidence was not significantly different but the C. pneumoniae rRNA copy number was higher in patients with heart disease (Berger et al., 2000). Of interest, the proportion of individuals with PCR positive cells in these studies ranged from 9 to 60 percent in the patients with heart disease and 2 to 46 percent in the controls. While this range of exposure may be explained by epidemiologic influences, technical concerns about assay methodologies remain and efforts at standardization have been initiated (Dowell et al., 2001). When the technical concerns have been addressed, it will also be important to understand why otherwise normal individuals have evidence of this pathogen circulating in what should be a sterile space.

Animal Models

In addition to serologic and histologic evidence associating C. pneumoniae and atherosclerosis, a number of animal models have been established. Evidence that C. pneumoniae can either initiate or accelerate the atherosclerotic lesion has come from work with both mice (NIH/s, ApoE-deficient, and LDL-receptor knock-out strains) and New Zealand White rabbits. These animals generally need to consume a high cholesterol diet in order to develop observable changes, though it is possible, in one of the rabbit models, to observe effects without an atherogenic diet (Fong et al., 1999). In the LDL receptor knockout mouse, intranasal inoculation with the C. pneumoniae AR39 strain twice monthly for six months was performed prior to sacrifice of the animals. Uninfected mice fed a high cholesterol diet had a lesion area index (defined as the size of a digitized image of the lesion divided by the aorta luminal surface and multiplied by one hundred) of 18, while infected animals given a high cholesterol diet had an index of 42. This 130 percent increase in lesion size suggests that infection with chlamydia can accelerate the growth of an atherosclerotic plaque (Hu et al., 1999).

There are limitations to the interpretation of animal models of atherosclerosis. In some of these models the atherosclerotic lesions observed are consistent with a very early pathologic process that does not mirror the lesions responsible for causing human disease. The atherosclerotic lesions in these models generally do not rupture or lead to clinical disease in the animal. While these data do support the potential for a contribution of chlamydia to lipid accumulation at the site, they do not provide conclusive evidence that infection will lead to plaque rupture.

Chlamydia Pathogenesis and Atherogenesis

A fourth line of persuasive evidence comes from similarities in the pathophysiology of C. pneumoniae infection and atherogenesis. The generation of an atherosclerotic plaque is generally felt to be a chronic process. To the extent that a chlamydia infection, in addition to any acute effects, has a chronic component to its pathophysiology, an association with atherosclerosis can be more easily defended. The demonstration that chlamydia may exist in a persistent state may serve to explain the latent nature of a chlamydia infection.

Chlamydia exists as elementary bodies in the environment. Upon entry into a host cell the elementary body undergoes a series of transformations that allow it ultimately to replicate. At this stage it is referred to as a reticulate body. After cell division, it again reverts to an elementary body and is released from the host cell. If, however, host cell conditions are not favorable, chlamydia will not progress through cell division and instead moves into what has been referred to as a persistent state, appearing morphologically as a large, aberrant form (Beatty et al., 1994). The organism has been found to persist in cell culture in this state for prolonged periods of time and, in vitro, to be relatively refractory to antibiotic therapy.

While evidence for a persistent state has not been established in clinical specimens, it remains possible that chlamydia could contribute to a chronic condition by remaining relatively dormant, while still influencing the condition of the host cell. A series of experiments (Zhong et al., 1999; 2000), has offered some insights as to why a chronically infected host cell is not destroyed by the immune system. It appears that chlamydia can selectively inhibit IFN-gamma-inducible MHC class I and II expression and thereby evade antigen presentation on the cell surface. Inhibition of this process by bacterial protein synthesis inhibitors such as chloramphenicol suggests that it is dependent on chlamydial protein synthesis.

Clinically latent infections have been demonstrated with a number of chlamydia species. The blinding eye disease trachoma has occurred decades after exposure to either C. trachomatis or C. pneumoniae. Infertility can result from chronic infection of the upper genital tract with C. trachomatis, a process that can take place over years. C. pneumoniae has also been isolated from the respiratory tract long after resolution of an acute infection.

Atherosclerosis is now considered to be an inflammatory disease (Ross, 1999). The association of C. pneumoniae with atherogenesis is supported by the possibility that C. pneumoniae contributes to this inflammation. Based on data from animal models, and supported by the PCR examinations of circulating white blood cells and histologic examinations of atherosclerotic tissue, a respiratory tract infection could lead to dissemination of C. pneumoniae in monocytes. These monocytes release factors that enhance the likelihood of endothelial infection with chlamydia (Lin et al., 2000). Once infected, the endothelial cells could affect the local arterial environment in three ways. Transendothelial migration of the monocytes is enhanced (Molestina et al., 1999). The infected endothelial cells release tissue factor and platelet aggregation inhibitor, which leads to enhanced coagulability at the site. And thirdly, mitogenic factors are released through an NF-Kβ related mechanism, leading to smooth muscle cell proliferation (Miller et al., 2000). This triad, subendothelial monocyte accumulation, hypercoagulability at the site of the atheroma and smooth muscle cell proliferation, is the hallmark of an atherosclerotic plaque and, as such, provides further support for a contribution of local C. pneumoniae infection to this inflammatory state.

Clinical Trials with Antibiotics

Even with continued gaps in our understanding of the association between infection and atherosclerosis, the significance of coronary artery disease as an unmet medical need has driven interest in conducting antibiotic intervention studies. Based on the various supportive data discussed thus far, a number of clinical trials designed to investigate the role of antibiotic intervention in reducing the incidence of atherosclerotic disease have been initiated. There is certainly more work that needs to be done preclinically, including additional studies outlining the role of C. pneumoniae in atherogenesis, improving the capabilities around diagnostic testing, understanding the influence of antibiotics, alone or in combination, on chlamydia replication, further exploring animal models of in vivo pathogenesis, and better defining the lifecycle of chlamydia, and specifically the persistent state.

There are a number of challenges to studying the use of antibiotics in clinical coronary artery disease. While several risk factors for coronary artery disease are already well established, the relationship between these risk factors and C. pneumoniae infection has not been fully examined. As such associations become better known, the use of these risk factors as selection criteria may become useful. Clinical studies will need to address this problem of multiple competing risks even while the appropriateness of controlling for these factors in any statistical analyses, or selecting the target group of patients to treat, remains open to debate.

Many questions remain regarding antimicrobial activity within the plaque. While there is clinical evidence that patients with either genitourinary tract or respiratory tract infections due to chlamydia can have the clinical course of their disease positively impacted by antibiotic intervention, it remains unknown whether antibiotic treatment will affect either the replication or pathogenicity of chlamydia infections in the atherosclerotic plaque. It may not be possible to either document infection at the arterial site or substantiate a positive microbiologic outcome. There remain concerns that to the extent that cells contain chlamydia in the persistent state, it may not be possible to fully eradicate the organism. Standard in vitro testing may be inadequate to fully address this issue, given that the contribution of the immune system to clearance of infected cells is not measured.

Specific concerns about the design of clinical trials also exist. The appropriate patient population to treat is not clear. If C. pneumoniae is the target organism, patient selection criteria specific to the organism could be useful. Antibody titers are a crude estimate of previous exposure but may not be adequate to select those patients actively infected. As identification of infection within the atheroma is not presently possible, surrogates of active infection are needed. Perhaps, in the future, there will be a role for the measurement of C. pneumoniae DNA in circulating white blood cells. As is typical with cardiovascular studies of coronary artery disease, the event rates are typically low. Selection of patients likely to have a primary event is critical to ensuring that any treatment effect can be observed. Setting the sample size is made difficult by not having any estimate of the potential treatment effects; in order to avoid missing a potential effect, efficacy rates may need to be assumed to be low. These two issues require that definitive studies be large in order to have sufficient statistical power to determine treatment effects. Interpretation of the results from smaller studies is consequently more problematic.

The results of ongoing clinical trials will be best able to answer questions that are focused on the merits of the antibiotic intervention in the specific population of patients enrolled, and focused on the prespecified endpoints. The results will be compelling to the extent that the studies are adequately powered and the chosen endpoints are clinically relevant. The ongoing trials are less likely to be able to define the mechanism of action underlying any observed treatment effect. Pre- and post-treatment measurement of such inflammatory indices as C-reactive protein, cytokines, fibrinogen and tissue factor could be performed, but determining whether any changes are a direct result of immune specific activity or an indirect result of reducing the burden of organisms may be problematic.

A number of antibiotic intervention studies have been initiated. The antibiotics used have been either macrolides, doxycycline or a fluoroquinolone, given the in vitro activity of classes of drugs against C. pneumoniae. Of the completed studies, the two earliest reported promising results, both with short-term therapies, but the small sample size of these trials precludes any definitive conclusions (see Table 1-2). In general, the trial design has varied such that no two are the same. They have focused on primary or secondary prevention, different antibiotic interventions, different durations of therapy, different cardiovascular endpoints and different manifestations of atherosclerosis at baseline. This variability in study design is a consequence of many of the issues noted above and is to be expected at this early stage of the development of a potential new intervention strategy. Results of future study designs that incorporate data-driven refinements in patient selection, duration of dosing and choice of antibiotic will be required before a complete assessment of the value of antibiotic intervention can be made.

TABLE 1-2. Clinical Trials with Antibiotics for Primary and Secondary Prevention of Atherosclerosis Diseases.

TABLE 1-2

Clinical Trials with Antibiotics for Primary and Secondary Prevention of Atherosclerosis Diseases.

Conclusion

Atherosclerosis is an inflammatory disease. That infection may serve as a root cause of this inflammation is supported by a number of different lines of evidence. At present, the most compelling data support the role of C. pneumoniae through pathogens, such as cytomegalovirus and dental organisms, should not be discounted. The macrophage is a critical component in the pathway to atherosclerotic inflammation. To the extent that an infectious process activates a macrophage, either in the local arterial milieu or at a distant site, there is the potential for that macrophage to stimulate both local lipid accumulation and the instability that presages plaque rupture. Given the burden that coronary artery disease imparts on the healthcare system and on society in general, efforts to both understand the role of infection in atherogenesis, and to develop targeted intervention strategies, should continue apace.

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Copyright © 2004, National Academy of Sciences.
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