Levels of antibodies to microorganisms implicated in atherosclerosis and of C-reactive protein among atomic bomb survivors

Although it has been suggested that cardiovascular disease incidence is increased among atomic bomb survivors, the existence of a causal relationship between radiation exposure and atherosclerosis is unclear. Microbial infections, including those caused by Chlamydia pneumoniae, Helicobacter pylori and cytomegalovirus, have recently been implicated in atherosclerosis. Since immune function is somewhat impaired among atomic bomb survivors, their immune defense against such infections might be diminished. To investigate this possibility, we measured antibody levels to the above microorganisms in the sera of survivors. We found that the levels of IgG and IgA antibodies to Chlamydia pneumoniae decreased significantly with radiation dose, whereas the levels of IgG antibodies to Helicobacter pylori or cytomegalovirus remained unchanged. The inflammation marker C-reactive protein was significantly and positively associated with level of antibodies to Chlamydia pneumoniae only in heavily exposed (>or=1000 mGy) survivors. These results may suggest that among atomic bomb survivors, immune response to Chlamydia pneumoniae is diminished and chronic inflammatory reactions related to Chlamydia pneumoniae infection are present.     

Chlamydia pneumoniae and atherosclerosis

Exposure to Chlamydia pneumoniae is extremely common, and respiratory infections occur repeatedly among most people. Strong associations exist between C. pneumoniae infection and atherosclerosis as demonstrated by: (i) sero-epidemiological studies showing that patients with cardiovascular disease have higher titres of anti-C. pneumoniae antibodies compared with control patients; (ii) detection of the organism within atherosclerotic lesions, but not in adjacent normal tissue by immunohistochemistry, polymerase chain reaction and electron microscopy and by culturing the organism from lesions; and (iii) showing that C. pneumoniae can either initiate lesion development or cause exacerbation of lesions in rabbit and mouse animal models respectively. The association of this organism with atherosclerosis has also provided sufficient impetus to conduct a variety of human secondary prevention antibiotic treatment trials. The results of these studies have been mixed and, thus far, no clear long-lasting benefit has emerged from these types of investigations. Studies of C. pneumoniae pathogenesis have shown that the organism can infect many cell types associated with both respiratory and cardiovascular sites, including lung epithelium and resident alveolar macrophages, circulating monocytes, arterial smooth muscle cells and vascular endothelium. Infected cells have been shown to exhibit characteristics associated with the development of cardiovascular disease (e.g. secretion of proinflammatory cytokines and procoagulants by infected endothelial cells and foam cell formation by infected macrophages). More detailed analysis of C. pneumoniae pathogenesis has been aided by the availability of genomic sequence information. Genomic and proteomic analyses of C. pneumoniae infections in relevant cell types will help to define the pathogenic potential of the organism in both respiratory and cardiovascular disease.     

Chlamydia pneumoniae inhibits activated human T lymphocyte proliferation by the induction of apoptotic and pyroptotic pathways

Chlamydia pneumoniae is an omnipresent obligate intracellular bacterial pathogen that infects numerous host species. C. pneumoniae infections of humans are a common cause of community acquired pneumonia but have also been linked to chronic diseases such as atherosclerosis, Alzheimer's disease, and asthma. Persistent infection and immune avoidance are believed to play important roles in the pathophysiology of C. pneumoniae disease. We found that C. pneumoniae organisms inhibited activated but not nonactivated human T cell proliferation. Inhibition of proliferation was pathogen specific, heat sensitive, and multiplicity of infection dependent and required chlamydial entry but not de novo protein synthesis. Activated CD4(+) and CD8(+) T cells were equally sensitive to C. pneumoniae antiproliferative effectors. The C. pneumoniae antiproliferative effect was linked to T cell death associated with caspase 1, 8, 9, and IL-1β production, indicating that both apoptotic and pyroptotic cellular death pathways were activated after pathogen-T cell interactions. Collectively, these findings are consistent with the conclusion that C. pneumoniae could induce a local T cell immunosuppression and inflammatory response revealing a possible host-pathogen scenario that would support both persistence and inflammation.     

Immunoproteomic identification and serological responses to novel Chlamydia pneumoniae antigens that are associated with persistent C. pneumoniae infections

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.     

Analysis of gene expression in penicillin G induced persistence of Chlamydia pneumoniae

Chlamydia pneumoniae is responsible for respiratory tract infections and has been associated to chronic diseases such as atherosclerosis. The involvement of C. pneumoniae in chronic diseases may be correlated to its ability to induce persistent forms in which Chlamydiae remain viable but are not cultivable. The aim of our study is to investigate C. pneumoniae specific gene activities associated with the development of Chlamydial persistence in a cell culture system in the presence of penicillin G. Chlamydia-infected HEp 2 cells were incubated with or without penicillin G for up to 72 hours. The relative mRNA expression levels of early and late genes in treated and untreated cell cultures were determined by Real-time RT-PCR. Our results revealed a consistent down-regulation of Chlamydial hctA and hctB genes (p=0.012 and p=0.003 respectively) in association with up-regulation of htrA gene (p=0.002) during penicillin G-induced persistence suggesting these gene sets as leading candidate for in vivo investigation of the development of persistent Chlamydial infection. In conclusion, the Chlamydial expression pattern of hctA, hctB, and htrA genes may be helpful to identify target molecules to diagnose and treat Chlamydia-associated chronic diseases.     

Genomic screening for Chlamydophila pneumoniae-specific antigens using serum samples from patients with primary infection

Chlamydophila pneumoniae, an obligate intracellular human pathogen, causes respiratory tract infections. The most common techniques used for the serological diagnosis of C.?pneumoniae infections are microimmunofluorescence tests and commercial serological ELISA tests; these are based on the detection of antibodies against whole chlamydial elementary bodies and lipopolysaccharide/outer membrane protein, respectively. Identification of more specific and highly immunodominant antigens is essential for the development of new serodiagnostic assays. To identify novel specific antigens from C.?pneumoniae, we screened 455 genes with unknown function in the genome of C.?pneumoniae J138. Extracts of Saccharomyces cerevisiae cells expressing GFP-tagged C.?pneumoniae proteins were subjected to Western blot analysis using serum samples from C.?pneumoniae-infected patients as the primary antibodies. From this comprehensive analysis, 58 clones expressing C.?pneumoniae open reading frames, including hypothetical proteins, were identified as antigens. These results have provided useful information for the development of new serological tools for the diagnosis for C.?pneumoniae infections and for the development of vaccines in future.     

TLR/MyD88 and liver X receptor alpha signaling pathways reciprocally control Chlamydia pneumoniae-induced acceleration of atherosclerosis

Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and atherothrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in apolipoprotein E (ApoE)(-/-) mice is reciprocally modulated by activation of TLR-mediated innate immune and liver X receptor alpha (LXRalpha) signaling pathways. We infected ApoE(-/-) mice and ApoE(-/-) mice that also lacked TLR2, TLR4, MyD88, or LXRalpha intranasally with C. pneumoniae followed by feeding of a high fat diet for 4 mo. Mock-infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques and the serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE-deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6, and TNF-alpha. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE(-/-) mice that were also deficient in TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE(-/-) mice was further enhanced in ApoE(-/-)LXRalpha(-/-) double knockout mice and was accompanied by higher serum levels of IL-6 and TNF-alpha. We conclude that C. pneumoniae infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism and that LXRalpha appears to reciprocally modulate and reduce the proatherogenic effects of C. pneumoniae infection.     

The use of serologic tests for the diagnosis of chlamydial infections

Serology is commonly used for the diagnosis of acute Chlamydia pneumoniae infections and also for the diagnosis of complicated Chlamydia trachomatis infections. Furthermore, recent sero-epidemiological studies have linked C. pneumoniae infection with several diseases traditionally considered non-infectious. The objectives of this mini-review are to critically review and discuss some selected analytical and methodological aspects, controversies and current problems in chlamydial serodiagnosis. To illustrate our views we present some original data of the comparison of current technologies. The review of the literature revealed high variability in methodologies applied to different studies. This observation was supported by our own data, which explains occasional conflicting clinical interpretation. Although the microimmunofluorescence (MIF) technique is generally considered as the gold standard for serodiagnosis of chlamydial infections, assay conditions are highly variable and hence pose a major problem in the interpretation of the results. For instance, many recent studies linking C. pneumoniae and atherosclerosis have utilized MIF techniques with variable threshold criteria for the positivity, in combination with selection bias of cases and controls possibly leading to conflicting results. Variability of assay conditions is also a common problem with Western blots, and interpretation is problematic when both anti-C. pneumoniae and anti-C. trachomatis antibodies are present. Furthermore, there is a lot of disagreement in serological criteria applied to recently emerged enzyme immunoassay (EIA) techniques when these assays are used for acute and non-acute clinical conditions and their association with Chlamydiae. In conclusion, standardization of serological techniques and the development of uniform criteria for interpretation of serologic findings is necessary to increase our knowledge of the biology of Chlamydiae, pathogenesis of any chlamydial infection and chronic infections in particular.     

Chlamydophila pneumoniae and human cytomegalovirus in atherosclerotic carotid plaques--combined presence and possible interactions

The aim of our study was to investigate the combination of Chlamydophila pneumoniae and human cytomegalovirus (HCMV) as a pathogenic factor in atherosclerosis. Accordingly, we tested by means of PCR and immunohistochemistry the presence of these pathogens in the same atherosclerotic carotid specimen. The histology of the samples and the patients' antibodies against these pathogens were evaluated. Further, we examined the impact of C. pneumoniae and HCMV infection on the gene expression of the human monocytic cell line U937. Six of the 22 samples contained only C. pneumoniae, 4 contained only HCMV, 7 contained both C. pneumoniae DNA and/or antigens of both pathogens, and 5 samples were negative. No correlation was found between the presence of these microbes and either the cellular structure of the plaques, or the serostatus of the patients. The infection of U937 cells with HCMV and especially C. pneumoniae induced inflammation and atherosclerosis-related genes. Furthermore, the doubly-infected cells produced higher levels of the mRNA of pro-platelet basic protein and fatty acid binding protein 4. In conclusion, C. pneumoniae is often present in combination with HCMV in atherosclerotic carotid lesions. The in vitro coinfection model reveals that the doubly-infected monocytes are potent expressors of proatherosclerotic genes, suggesting that this coinfected population may accelerate the process of atherosclerosis.     

Is infection with Chlamydia pneumoniae a causative agent in atherosclerosis?

There is mounting evidence to suggest that Chlamydia pneumoniae might play a role in atherosclerosis. Serological studies and detection of the microorganism in atheromatous lesions were the first indications of an association between C. pneumoniae and the disease. Studies suggest that anti-chlamydial chemotherapy has a favorable effect on cardiovascular disease in humans. Moreover, infection of animals with C. pneumoniae induces inflammatory changes in the aorta that are suggestive of atherosclerosis and accelerates the progression of existing atherosclerotic lesions. If the pathogenic role of C. pneumoniae in atherosclerosis is defined more conclusively by future studies, the development of preventive or therapeutic measures against infection might provide an effective strategy to reduce the risk of atherosclerosis.     

Enzymology and molecular biology of prokaryotic sulfite oxidation

The intracellular bacterial pathogen Chlamydia pneumoniae causes respiratory tract infection and has been associated with atherosclerosis and coronary artery disease. Since atherosclerosis is a progressive disease and is considered to be a chronic inflammation of the artery vessel wall, the interaction of C. pneumoniae with cells of the vasculature that can result in a local inflammatory response is of paramount importance. In this essay we review the pathophysiology of atherosclerosis in the context of C. pneumoniae infection and present an integrated model that includes the involvement of C. pneumoniae in all stages of atherogenesis including initiation, inflammation, fibrous plaque formation, plaque rupture and thrombosis. We hypothesize that acute and persistent infection of professional immune cells (T-cells, monocytes and macrophages) and non-immune cells (endothelial cells and smooth muscle cells) contributes to a sustained inflammatory response mediated by extensive cellular 'crosstalk' and numerous cytokines/chemokines. This cascade of inflammatory mediators may contribute to cellular dysfunction and tissue remodelling of the arterial intima. An improved understanding of the precise mechanism(s) of C. pneumoniae involvement in atherogenesis may help resolve the question of causality however, at the present time, we interpret the data as favoring a contributory rather than a causal role. Future research directed at the discovery of chlamydial virulence factors necessary for intracellular survival and subsequent alterations in host cell gene expression including signalling pathways may be important for the design of future clinical trials.     

Chlamydia pneumoniae infection alters the junctional complex proteins of human brain microvascular endothelial cells

Chlamydia pneumoniae has been identified and associated with multiple sclerosis (MS) and Alzheimer's disease (AD) pathogenesis, although the relationship of this organism in these diseases remains controversial. We have hypothesized that one potential avenue of infection is through the junctional complexes between the blood-brain barrier (BBB) endothelia. C. pneumoniae is characteristically a respiratory pathogen, but has been implicated in atherosclerosis, coronary artery disease, and neuroinflammatory conditions. C. pneumoniae infection may lead to endothelial damage, junctional alterations, and BBB breakdown. Therefore, in this study, C. pneumoniae infection of human brain microvascular endothelial cells (HBMECs) resulted in increased expression of the zonula adherens proteins beta-catenin, N-cadherin, and VE-cadherin, and decreased expression of the tight junctional protein occludin, as determined by immunocytochemistry and Western blot analyses. These events may underlie a mechanism for the regulation of paracellular permeability while maintaining barrier integrity during C. pneumoniae infection associated with neuropathologies such as MS and AD.     

Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-cell responses to nasopharyngeal colonisation

The nasopharyngeal commensal bacteria Streptococcus pneumoniae is also a frequent cause of serious infections. Nasopharyngeal colonisation with S. pneumoniae inhibits subsequent re-colonisation by inducing Th17-cell adaptive responses, whereas vaccination prevents invasive infections by inducing antibodies to S. pneumoniae capsular polysaccharides. In contrast, protection against invasive infection after nasopharyngeal colonisation with mutant S. pneumoniae strains was associated with antibody responses to protein antigens. The role of colonisation-induced Th17-cell responses during subsequent invasive infections is unknown. Using mouse models, we show that previous colonisation with S. pneumoniae protects against subsequent lethal pneumonia mainly by preventing bacteraemia with a more modest effect on local control of infection within the lung. Previous colonisation resulted in CD4-dependent increased levels of Th17-cell cytokines during subsequent infectious challenge. However, mice depleted of CD4 cells prior to challenge remained protected against bacteraemia, whereas no protection was seen in antibody deficient mice and similar protection could be achieved through passive transfer of serum. Serum from colonised mice but not antibody deficient mice promoted phagocytosis of S. pneumoniae, and previously colonised mice were able to rapidly clear S. pneumoniae from the blood after intravenous inoculation. Thus, despite priming for a Th17-cell response during subsequent infection, the protective effects of prior colonisation in this model was not dependent on CD4 cells but on rapid clearance of bacteria from the blood by antibody-mediated phagocytosis. These data suggest that whilst nasopharyngeal colonisation induces a range of immune responses, the effective protective responses depend upon the site of subsequent infection.     

Epidemiologic aspects of M. pneumoniae disease complications: a review

As early as the 1940s, erythema multiforme exudativum (Stevens-Johnson syndrome) and hemolytic anemia were associated with outbreaks of atypical pneumonia, a disease later found to be caused by Mycoplasma pneumoniae. Epidemiologic evidence has also associated neurological complications, especially aseptic meningitis and meningoencephalitis, with M. pneumoniae infections. Urticarial and morbilliform skin rashes often appear late in the course of M. pneumoniae pneumonia. A multitude of other complications have been ascribed to M. pneumoniae infections, often reported as case reports diagnosed by serologic antibody titers only. More systematic investigations are needed to assess the frequency of complications to M. pneumoniae infections. Isolation of the agent, not only serologic titer rises, should be required before a syndrome is attributed to M. pneumoniae infection.     

Chlamydia pneumoniae infection results in generalized bone loss in mice

Osteoporosis is associated with a general bone loss. Whether infections could contribute to osteoporosis is not known. Chlamydia pneumoniae causes chronic infections and produces potentially bone resorptive cytokines. The effect of C. pneumoniae infection was investigated in vivo in 10-week old mice (c57BL/6) and in vitro in the human osteoblast-like cell line hFOB 1.19 (hFOB). Bone mineral density (BMD) was measured before and 16 days after infection. C. pneumoniae-infected mice had decreased (p<0.05) total and subcortical BMD at the distal femur and proximal tibia compared with controls, but no body-weight gain differences. IL-6 (56 vs. 39pg/mL, p=0.02) and IL-1beta (11 vs. 0pg/mL, p=0.003) levels in sera, and CD3(+) T-cells (p=0.04) were higher in infected mice compared with controls. In vitro, hFOB infected with C. pneumoniae was associated with increased IL-6 (p=0.01) and RANKL (p<0.05) mRNA expression; additionally, IL-6 secretion increased in a dose-dependent manner (p<0.05). In summary, mice infected with C. pneumoniae had generalized bone loss associated with increased IL-6 and IL-1. In addition, C. pneumoniae established an infection in an osteoblast cell line in vitro with similar cytokine profiles as those in vivo, supporting a causal linkage.     

Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry.

Chlamydia pneumoniae is a common cause of community-acquired pneumonia and it has been associated with atherosclerosis. C. pneumoniae has usually been diagnosed by serology using a microimmunofluorescence test, but more recently polymerase chain reaction (PCR) has been viewed as an advantageous alternative. We developed a quantitative real-time PCR for detection of C. pneumoniae. Primers were targeted for the pmp4 gene, and the PCR fragment was detected real-time with a fluorescence resonance energy transfer probe set using a LightCycler instrument. The PCR was used on DNA released from 50 microm sections of paraffin-embedded formalin-fixed lung tissue from experimentally infected mice. Thereby, the number of C. pneumoniae genomes was determined. To our knowledge this is the first time quantification of C. pneumoniae DNA has been attempted on paraffin-embedded formalin-fixed tissue. C. pneumoniae-specific immunohistochemistry (IHC) was done on 5 microm sections adjacent to the sections used in PCR, and the number of inclusions were counted in each section. Good correlation was found when comparing results from PCR and IHC, which is in contrast to many previous studies.     

Vaccination with DNA vector expressing chlamydial low calcium response protein E (LcrE) against Chlamydophila pneumoniae infection

Chlamydophila pneumoniae is an obligate intracellular human pathogen, which causes acute respiratory tract infections and can also cause chronic infections. C. pneumoniae possess type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and the host cell cytosol. Low calcium response protein E (LcrE) is a part of TTSS. The gene of LcrE in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin, this gene was also cloned into a eukaryotic expression vector (pΔRC). One group of BALB/c mice received an intramuscular pΔRC inoculation then the mice were immunized with purified LcrE protein; the second group of mice was immunized two times with the recombinant plasmid (pΔRCLcrE), and the third group was primed with pΔRCLcrE inoculation then boosted with LcrE protein. LcrE-specific antibody response was induced by DNA immunization with a shift towards Th1 isotype pattern compared to protein-immunization, this shifting pattern was observed in plasmid primed then protein-boosted animals. DNA immunization given as a priming and followed by a protein booster significantly reduced the number of viable bacteria in the lungs after challenge with C. pneumoniae. These results confirm that immunization with pΔRCLcrE can be an effective part of a vaccination schedule against C. pneumoniae.