Search

Background: The gut microbiota (GM) is believed to be closely associated with symptomatic carotid atherosclerosis (SCAS), yet more evidence is needed to substantiate the significant role of GM in SCAS. This study, based on the detection of bacterial DNA in carotid plaques, explores the characteristics of GM in SCAS patients with plaque bacterial genetic material positivity, aiming to provide a reference for subsequent research., Methods: We enrolled 27 healthy individuals (NHF group) and 23 SCAS patients (PFBS group). We utilized 16S rDNA V3-V4 region gene sequencing to analyze the microbiota in fecal samples from both groups, as well as in plaque samples from the carotid bifurcation extending to the origin of the internal carotid artery in all patients., Results: Our results indicate significant differences in the gut microbiota (GM) between SCAS patients and healthy individuals. The detection rate of bacterial DNA in plaque samples was approximately 26%. Compared to patients with negative plaques (PRSOPWNP group), those with positive plaques (PRSOPWPP group) exhibited significant alterations in their GM, particularly an upregulation of 11 bacterial genera (such as Klebsiella and Streptococcus) in the gut, which were also present in the plaques. In terms of microbial gene function prediction, pathways such as Fluorobenzoate degradation were significantly upregulated in the GM of patients with positive plaques., Conclusion: In summary, our study is the first to identify significant alterations in the gut microbiota of patients with positive plaques, providing crucial microbial evidence for further exploration of the pathogenesis of SCAS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Lv, Zhang, Fu, Li, Yin, Liu, Xu, Wang, Li, Hao, Zhang and Wang.)

The association of bacteria with arterial plaque lesions in patients with atherosclerosis has been widely reported. However, the role these bacteria play in the progression of atherosclerosis is still unclear. Previous work in our lab has demonstrated that bacteria exist in carotid artery plaques as biofilm deposits. Biofilms are communities of microorganisms enmeshed within a protective, self-produced extracellular matrix and have been shown to contribute to chronic infections in humans. Biofilm communities have the potential to impact surrounding tissues in an infection if they undergo a dispersion response, releasing bacteria into the surrounding environment by enzymatic degradation of the extracellular matrix. One concern relating to these enzymes is that they could cause collateral damage to host tissues. In this study, we present an in vitro multispecies biofilm culturing model used to investigate the potential role of bacterial biofilm dispersion in the progression of atherosclerosis. This work has demonstrated an increase in cell release from mixed-species biofilms formed by bacteria associated with human carotid arterial plaque deposits following treatment with iron or a combination of norepinephrine and transferrin. Greater extracellular lipase, protease, and collagenase/gelatinase activity was also associated with iron-treated biofilms. The results of this work suggest that bacteria in this model undergo iron-induced biofilm dispersion, as evidenced by the increased cell release and higher enzyme activity following treatment. This work demonstrates the potential for multispecies biofilm dispersion to contribute to arterial tissue degradation by bacteria and suggests that in atherosclerotic infections, biofilm dispersion may contribute to thrombogenesis, which can lead to heart attack or stroke. IMPORTANCE Atherosclerosis, or hardening of the arteries, is a leading cause of congestive heart failure, heart attack, and stroke in humans. Mounting evidence, in the literature and from our lab, points to the regular involvement of bacteria within arterial plaque deposits in patients with advanced atherosclerosis. Very little is known about the behavior of these bacteria and whether they may contribute to tissue damage in infected arteries. Tissue damage within the arterial plaque lesion can lead to rupture of the plaque contents into the bloodstream, where a clot may form, resulting in a potential heart attack or stroke. This study shows that plaque-associated bacteria, when cultured as mixed-species biofilms in the laboratory, can release degradative enzymes into their environment as the result of a dispersion response triggered by iron. These degradative enzymes can digest proteins and lipids which are associated with the tissues that separate the plaque lesion from the arterial lumen. Thus, this study demonstrates that if mixed species biofilms are induced to undergo dispersion in an infected atherosclerotic lesion when exposed to an elevated concentration of free iron, they have the potential to contribute to the weakening of arterial tissues, which may contribute to atherosclerotic plaque destabilization.

Increasing attention has been paid to the possible link between periodontal disease and atherosclerosis over the past decade. The aim of this study is to investigate the presence of five periopathogens: Porphyromonas gingivalis ( P.g. ), Aggregatibacter actinomycetemcomitans ( A.a. ), Tannerella forsythia ( T.f. ), Treponema denticola ( T.d. ), and Prevotella intermedia ( P.i. ) in atheromatous plaques obtained from the carotid and coronary arteries in patients who underwent coronary artery bypass graft surgery and carotid endarterectomy. Group I (carotid arteries) consisted of 30 patients (mean age: 54.5 ± 14.8), and group II (coronary arteries) consisted of 28 patients (mean age: 63 ± 12.1). Clinical periodontal examinations consisted of plaque index, gingival index, sulcus bleeding index, and periodontal probing depth and were performed on the day of vascular surgery. The presence of periopathogens in periodontal pockets and atherosclerotic vessels was detected using polymerase chain reaction. In both subgingival plaque and atherosclerotic plaque of carotid arteries, P.g. , A.a. , T.f. , T.d. , and P.i. were detected in 26.7%, 6.7%, 66.7%, 10.0%, and 20.0%, respectively, while for coronary arteries, P.g . was detected in 39.3%, A.a . in 25%, T.f . in 46.4%, T.d. in 7.1%, and P.i. in 35.7%. The presence of five periopathogens in carotid and coronary atherosclerotic vessels showed correlation in regard to the degree of periodontal inflammation. The present study suggests the relationship between periodontal pathogenic bacteria and atherogenesis. Further studies are necessary in relation to the prevention or treatment of periodontal disease that would result in reduced mortality and morbidity associated with atherosclerosis., Competing Interests: No conflict of interest exists., (Copyright © 2021 Verica Pavlic et al.)

A few pediatric cases with brain vasculitis most frequently affecting the middle cerebral artery have been reported in association with Mycoplasma pneumoniae infection, but involvement of the common carotid artery (CCA) before the bifurcation has not been reported to date. We report herein a case of 10-year-old boy with common carotid arteritis and polymyalgia associated with Mycoplasma pneumoniae infection. His fever and cough began 2 weeks before, and his right upper and lower extremity pains began 2 days before admission. He had initially been treated with clarithromycin followed by tosufloxacin, but his symptoms persisted. His M. pneumonia-specific antibody titer was high on admission (1:10240 by particle agglutination method) and the gene of M. pneumoniae was detected in a throat swab specimen by the loop-mediated isothermal amplification method with initial high levels of serum interleukin-8, tumor necrosis factor-α, and interleukin-18 along with elevated blood levels of complements. On the 5th day of hospitalization, vascular echograms of the extremities and neck showed increasing intima-media thickness of bilateral CCAs without stenosis and/or thrombosis and T2-weighted with lipid suppression magnetic resonance imaging of the neck showed high signal intensity of bilateral CCA walls. Coagulation studies were unremarkable and no autoantibodies were detected as far as tested. He was successfully treated by intravenous administration of prednisolone and was stable without any neurological sequelae 17 months after the onset without medication. His particle agglutination titer decreased to 1:5120, and serum interleukin-8, tumor necrosis factor-α, interleukin-18, and complement levels returned to normal., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: To establish SD rat model with type 2 diabetes mellitus (DM) and concomitant chronic periodontitis (CP) and to evaluate the influence of periodontitis on the vascular lesions of type 2 diabetes rats. Methods: Totally 241 clean level SD rats were randomly divided into four groups, group A (normal control, NC, n= 27), group B (DM, n= 34), group C (CP, n= 90) and group D (DM+CP, n= 90). The rats of DM group were fed with high-fat and high-sugar diet for 8 to 10 weeks, and then were multiply injected with small dose streptozotocin under the condition of ice bath. Blood sugar levels after the injection were dynamically monitored at 72 h, 1 week, 2 weeks and 4 weeks, respectively. The CP model was established by means of ligation. Bilateral maxillary first and second molars were selected and ligated using 0.2 mm orthodontic wires binding with 4-0 surgical suture soaked with Porphyromonas gingivalis (Pg) suspension. After a period of 14 weeks, all the rats were put to death. Maxillary samples were subjected to methylene blue staining to observe alveolar bone loss. Bilateral carotid artery specimens were collected. The left carotid artery specimens were used to detect the prevalence of Pg using quantitative real-time PCR. The right carotid artery specimens were used to observe pathological changes. Results: Blood sugar levels of rats in group B and D increased and changed sharply after Streptozotocin injection with in 1 week. Symptoms of 'more drink, more food and body weight loss' appeared. The fasting blood glucose (FBG) was more than 7.8 mmol/L and (or) the random blood glucose (RBG) was more than 17.8 mmol/L. Both FBG and RBG became stable after 2 to 3 weeks. Levels of HbA1C in group B and D ([7.32±0.45]%, [9.41±0.45]%) were significantly higher than that of group A ([4.02±0.45]%) ( P< 0.01). Rats of group D were observed the most severe bone loss showing wider interdental space and furcation involvement. Pathological results of carotid artery tissues of group D showed the worst lesions including thinning and calcification of vessel walls, and breaking down or disappearance of elastic fibers. The prevalences of DNA of Pg in groups of A, B, C and D were 3/7, 3/7, 6/7 and 7/7, respectively. The bacteria numbers detected by quantitative real-time PCR in groups C and D were significantly higher than that of groups A and B ( P< 0.01). Conclusions: Rat model of type 2 DM with periodontitis was successfully established in the present study. Carotid artery specimens from DM+CP model rats showed typical vascular lesions such as calcification and fiber disorders. Pg was found in all carotid specimens and the highest bacteria numbers were detected in the composite model rats. The Pg might play a role in the progress of diabetes vascular lesions.

Multiple reaction monitoring-MS analysis of lipid extracts from human carotid endarterectomy and carotid artery samples from young individuals consistently demonstrated the presence of bacterial serine dipeptide lipid classes, including Lipid 654, an agonist for human and mouse Toll-like receptor (TLR)2, and Lipid 430, the deacylated product of Lipid 654. The relative levels of Lipid 654 and Lipid 430 were also determined in common oral and intestinal bacteria from the phylum Bacteroidetes and human serum and brain samples from healthy adults. The median Lipid 430/Lipid 654 ratio observed in carotid endarterectomy samples was significantly higher than the median ratio in lipid extracts of common oral and intestinal Bacteroidetes bacteria, and serum and brain samples from healthy subjects. More importantly, the median Lipid 430/Lipid 654 ratio was significantly elevated in carotid endarterectomies when compared with control artery samples. Our results indicate that deacylation of Lipid 654 to Lipid 430 likely occurs in diseased artery walls due to phospholipase A2 enzyme activity. These results suggest that commensal Bacteriodetes bacteria of the gut and the oral cavity may contribute to the pathogenesis of TLR2-dependent atherosclerosis through serine dipeptide lipid deposition and metabolism in artery walls., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Background and Aims: Several studies have confirmed the presence of bacterial DNA in atherosclerotic plaques, but its contribution to plaque stability and vulnerability is unclear. In this study, we investigated whether the bacterial plaque-profile differed between patients that were asymptomatic or symptomatic and whether there were local differences in the microbial composition within the plaque., Methods: Plaques were removed by endarterectomy from asymptomatic and symptomatic patients and divided into three different regions known to show different histological vulnerability: A, upstream of the maximum stenosis; B, site for maximum stenosis; C, downstream of the maximum stenosis. Bacterial DNA composition in the plaques was determined by performing 454 pyrosequencing of the 16S rRNA genes, and total bacterial load was determined by qPCR., Results: We confirmed the presence of bacterial DNA in the atherosclerotic plaque by qPCR analysis of the 16S rRNA gene but observed no difference (n.s.) in the amount between either asymptomatic and symptomatic patients or different plaque regions A, B and C. Unweighted UniFrac distance metric analysis revealed no distinct clustering of samples by patient group or plaque region. Operational taxonomic units (OTUs) from 5 different phyla were identified, with the majority of the OTUs belonging to Proteobacteria (48.3%) and Actinobacteria (40.2%). There was no difference between asymptomatic and symptomatic patients, or plaque regions, when analyzing the origin of DNA at phylum, family or OTU level (n.s.)., Conclusions: There were no major differences in bacterial DNA amount or microbial composition between plaques from asymptomatic and symptomatic patients or between different plaque regions, suggesting that other factors are more important in determining plaque vulnerability., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Ischaemia of the lower limbs is frequently followed by inflammation and, in advanced cases, necrosis of peripheral tissues. Whether this is caused by arterial hypoperfusion only or by the presence of bacteria in the arterial walI as well remains unclear. The aim of the study was to prove the presence and source of bacteria in arterial specimens and evaluate their chemotactic properties resulting in the formation of periarterial cellular infiltrates., Materials and Methods: Bacterial culture and testing for 16sRNA were performed in fragments of popliteal artery harvested from amputated limbs. Carotid artery plaques served as controls. Fragments of arteries were transplanted into scid mice to evaluate their chemotactic activity for macrophages., Results: a) higher prevalence of isolates and 16sRNA in atherosclerotic popliteal than carotid arteries, b) high density of plaque and periarterial infiltrates and mRNA level for pro-inflammatory cytokines in popliteal arteries, c) prevalent microbes were Staphylococcus aureus, S. epidermidis and Enterococci, d) foot skin and arterial bacterial phenotypes and DNA revealed evident similarities, and e) more intensive mouse macrophage accumulation in popliteal than carotid implants into scid mice., Conclusions: The presence of bacteria in the lower limb arterial wall was documented. They may predispose to inflammation secondary to ischaemic changes.

In the present study, human atherosclerotic carotid arteries were examined following endarterectomy for the presence of the Gram-positive bacterium Propionibacterium acnes and its potential association with biofilm structures within the arterial wall. The P. acnes 16S rRNA gene was detectable in 4 of 15 carotid artery samples, and viable P. acnes was one among 10 different bacterial species recoverable in culture. Fluorescence in situ hybridization analysis of 5 additional atherosclerotic carotid arteries demonstrated biofilm bacteria within all samples, with P. acnes detectable in 4 samples. We also demonstrated that laboratory-grown cultures of P. acnes biofilms were susceptible to induction of a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of iron-bound transferrin or with free iron. The production and release of lipolytic and proteolytic extracellular enzymes by P. acnes were shown to increase in iron-induced dispersed biofilms, and these dispersion-induced P. acnes VP1 biofilms showed increased expression of mRNAs for the triacylglycerol lipases PPA2105 and PPA1796 and the hyaluronate lyase PPA380 compared to that in untreated biofilms. These results demonstrate that P. acnes can infect the carotid arteries of humans with atherosclerosis as a component of multispecies biofilms and that dispersion is inducible for this organism, at least in vitro, with physiologically relevant levels of norepinephrine resulting in the production and release of degradative enzymes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Over the last few years the role of microorganisms in the pathogenesis of atherosclerosis has been widely discussed. Chlamydia pneumoniae activates immune cells to produce cytokines that are responsible for the formation of atheromatous carotid lesions., Material and Methods: The study was carried out at the Department of Vascular, General and Transplantation Surgery, Wroclaw Medical University, in 2002-2003, on 100 consecutive symptomatic patients with internal carotid stenosis, who underwent an endarterectomy procedure. Each patient had their carotid artery sampled in order to find C. pneumoniae DNA using the nested PCR method and some cytokines (TGF-β, VEGF, FGF, TNF-α) using immunohistochemical examination. The control group consisted of 20 young organ donors who had been diagnosed with brain death and who had their healthy carotid artery harvested. Analogous genetic and immunohistochemical tests were performed., Results: We did not confirm the presence of either cytokines or C. pneumoniae in the healthy carotid arteries. The presence of FGF was probably due to intima fibroblast activity, which is responsible for elastin and collagen synthesis for the extracellular matrix. C. pneumoniae was discovered in 68% of patients with carotid plaques. Three cytokines (TGF-β, FGF, TNF-α) were detected in atherosclerotic internal carotid arteries as well., Conclusion: Chronic infection by C. pneumoniae may exacerbate carotid plaque development and may lead to its destabilization.

Objectives: Interest in periodontitis as a potential risk factor for atherosclerosis and its complications resulted from the fact that the global prevalence of periodontal diseases is significant and periodontitis may induce a chronic inflammatory response. Many studies have analyzed the potential impact of the Porphyromonas gingivalis, major pathogen of periodontitis, on general health. The purpose of this study was to find the presence of the Porphyromonas gingivalis DNA in the atherosclerotic plaques of coronary and carotid arteries and in the periodontal pockets in patients with chronic periodontitis, who underwent surgery because of vascular diseases., Methods and Results: The study population consisted of 91 patients with coronary artery disease or scheduled for carotid endarterectomy. The presence of Porphyromonas gingivalis DNA in atheromatous plaques and in subgingival samples was determined by PCR. Bacterial DNA was found in 21 of 91 (23%) samples taken from vessels and in 47 of 63 (74.6%) samples from periodontal pockets., Conclusions: Porphyromonas gingivalis DNA is frequently found in atheromatous plaques of patients with periodontitis. That is why more research should be conducted to prove if this periopathogen may have an impact on endothelium of patients at risk of atherosclerosis.

Unlabelled: Atherosclerosis, a disease condition resulting from the buildup of fatty plaque deposits within arterial walls, is the major underlying cause of ischemia (restriction of the blood), leading to obstruction of peripheral arteries, congestive heart failure, heart attack, and stroke in humans. Emerging research indicates that factors including inflammation and infection may play a key role in the progression of atherosclerosis. In the current work, atherosclerotic carotid artery explants from 15 patients were all shown to test positive for the presence of eubacterial 16S rRNA genes. Density gradient gel electrophoresis of 5 of these samples revealed that each contained 10 or more distinct 16S rRNA gene sequences. Direct microscopic observation of transverse sections from 5 diseased carotid arteries analyzed with a eubacterium-specific peptide nucleic acid probe revealed these to have formed biofilm deposits, with from 1 to 6 deposits per thin section of plaque analyzed. A majority, 93%, of deposits was located proximal to the internal elastic lamina and associated with fibrous tissue. In 6 of the 15 plaques analyzed, 16S rRNA genes from Pseudomonas spp. were detected. Pseudomonas aeruginosa biofilms have been shown in our lab to undergo a dispersion response when challenged with free iron in vitro. Iron is known to be released into the blood by transferrin following interaction with catecholamine hormones, such as norepinephrine. Experiments performed in vitro showed that addition of physiologically relevant levels of norepinephrine induced dispersion of P. aeruginosa biofilms when grown under low iron conditions in the presence but not in the absence of physiological levels of transferrin., Importance: The association of bacteria with atherosclerosis has been only superficially studied, with little attention focused on the potential of bacteria to form biofilms within arterial plaques. In the current work, we show that bacteria form biofilm deposits within carotid arterial plaques, and we demonstrate that one species we have identified in plaques can be stimulated in vitro to undergo a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of transferrin. Biofilm dispersion is characterized by the release of bacterial enzymes into the surroundings of biofilm microcolonies, allowing bacteria to escape the biofilm matrix. We believe these enzymes may have the potential to damage surrounding tissues and facilitate plaque rupture if norepinephrine is able to stimulate biofilm dispersion in vivo. This research, therefore, suggests a potential mechanistic link between hormonal state and the potential for heart attack and stroke., (Copyright © 2014 Lanter et al.)

Chlamydia pneumoniae (Cpn) could play an important role in the development of atherosclerosis. Cpn interferes with HIF-1alpha regulation in infected host cells during intracellular replication in hypoxia. We obtained carotid artery specimens with low (n=38), high (n=25) levels of stenosis and 10 middle cerebral arteries. Fifty eight percent of the carotids with low levels of stenosis showed evidence of the viable organism. Ninety one percent of the positive results were derived from pre-atheromatous lesions. Only 12 percent of plaques removed at endarterectomy showed the presence of Cpn DNA. All middle cerebral arteries failed to show evidence of live Chlamydia. Ninety one percent of sera from 22 endarterectomy patients failed to show the presence of Cpn antibodies. Immunohistology of carotid arteries with low levels of stenosis was used to confirm the presence of HIF-1alpha in infected specimens and showed a correlation between the over-expression of HIF-1alpha and Cpn in the plaque (p less than 0.05). Cpn might play an important role in activation and development of the initial stages of atherosclerotic lesions.

Mycotic aneurysms of the extracranial carotid artery are rare and warrant surgical intervention. Management involves open and endovascular approaches. We report the case of a 67-year-old woman with an Escherichia coli soft-tissue infection of the right retropharyngeal space and subsequent mycotic carotid aneurysm and thrombosis of the internal jugular vein. The patient presented with a pulsatile mass and right middle cerebral artery stroke. Our surgical management involved coil embolization of the aneurysm to provide for vascular control, with resection of the common carotid artery, internal carotid artery, and extracranial carotid artery branches, along with the internal jugular vein., (Copyright (c) 2010 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Background and Purpose: The overall burden of prior infections may contribute to atherosclerosis and stroke risk. We hypothesized that serological evidence of common infections would be associated with carotid plaque thickness in a multiethnic cohort., Methods: Antibody titers to 5 common infectious microorganisms (ie, Chlamydia pneumoniae, Helicobacter pylori, cytomegalovirus, and herpesvirus 1 and 2) were measured among stroke-free community participants and a weighted index of infectious burden was calculated based on Cox models previously derived for the association of each infection with stroke risk. High-resolution carotid duplex Doppler studies were used to assess maximum carotid plaque thickness. Weighted least squares regression was used to measure the association between infectious burden and maximum carotid plaque thickness after adjusting for other risk factors., Results: Serological results for all 5 infectious organisms were available in 861 participants with maximum carotid plaque thickness measurements available (mean age, 67.2+/-9.6 years). Each individual infection was associated with stroke risk after adjusting for other risk factors. The infectious burden index (n=861) had a mean of 1.00+/-0.35 SD and a median of 1.08. Plaque was present in 52% of participants (mean, 0.90+/-1.04 mm). Infectious burden was associated with maximum carotid plaque thickness (adjusted increase in maximum carotid plaque thickness 0.09 mm; 95% CI, 0.03 to 0.15 mm per SD increase of infectious burden)., Conclusions: A quantitative weighted index of infectious burden, derived from the magnitude of association of individual infections with stroke, was associated with carotid plaque thickness in this multiethnic cohort. These results lend support to the notion that past or chronic exposure to common infections, perhaps by exacerbating inflammation, contributes to atherosclerosis. Future studies are needed to confirm this hypothesis and to define optimal measures of infectious burden as a vascular risk factor.

The aim of our studies was to create diagnostically informative criteria of hematologic and biochemical analyses and their combinations as well as to establish relation between Chlamidia Pneumoniae A and atherosclerosis. This is to be done for the purpose of establishing the entire pathogenesis carotid and coronary atherosclerosis and inflammation. The investigation of Chlamidia Pneumoniae infection in proved to be a possible risk factor for the carotid atherosclerosis developed in adults that makes the course of atherosclerosis heavier. As a result of the atherosclerotic process, changes in the blood laboratory parameters were expressed more cases of pneumonia in the children.

Coronary artery disease (CAD) is a public health problem accounting for an estimated one-third of deaths overall. A potential link between infectious agents and atherosclerosis has been suggested. Data obtained from several seroepidemiological studies have suggested that infection with Chlamydophila pneumoniae, Helicobacter pylori, cytomegalovirus and herpes simplex virus-1 can initiate or maintain the atherosclerotic process. However, there is no single study in which multiple infectious agents have been detected together in different vascular locations in the same population. This would help in determining if there is any leading pathogen in atheromatous plaques of CAD patients. Hence, we screened for C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations of CAD patients using quantitative real-time (RT) PCR. We performed multiplex RT-PCR for detecting pathogens, viz. C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations of CAD patients. Percent positivity scores for C. pneumoniae, H. pylori, CMV and HSV-1 in different vascular locations were as follows: aorta (64.7, 35.3, 11.7 and 11.7 respectively); carotid (27.2, 27.2, 9 and 0 respectively); coronary artery (58.3, 33.3, 16.6 and 8.3 respectively). Combined positivity for C. pneumoniae (C. pneumoniae IgA and RT-PCR for C. pneumoniae) was the highest compared with all other groups. Aorta and coronary artery were more susceptible to these pathogens as compared with carotid artery. Moreover, CAD patients' characteristics were associated with C. pneumoniae positivity (C. pneumoniae IgA and RT-PCR), suggesting thereby that C. pneumoniae may have caused chronic persistent infection in CAD.

Chlamydophila pneumoniae has been implicated in atherosclerosis, but the role of this obligate intracellular pathogen in the development of the above pathology is still unclear. In particular, its presence and quantitative distribution within lesional areas has not yet been defined. We studied 18 carotid biopsies obtained from patients undergoing endoartherectomy. By laser microdissection (LCM), two different sites (intra-plaque and plaque-adjacent areas) were taken from each lesion, and the presence and quantity of the pathogen DNA were determined by real-time polymerase chain reaction (Real-time PCR). A total of 8 plaques, exclusively from patients with unstable angina, were positive in real-time PCR. The bacterial DNA was detected in both lesional areas of 3 plaques which contained the highest number of DNA copies (1,900 to 2,200 copy numbers), while C. pneumoniae DNA was detected only in the intra-plaque area of the other 5 positive (500 to 1,600 copy numbers). No C. pneumoniae DNA was found in the other 10 plaques of which 6 were from patients with unstable angina and 4 from stable angina patients. No DNA from Helicobacter pylori or Cytomegalovirus was found in any plaque. This is the first report where both the target lesion and an adjacent reference site were evaluated for the presence of C. pneumoniae DNA by the combination of LCM and Real-time PCR assays. The integration of these two methodologies offer an excellent tool for in situ studies and may help to elucidate the putative role of C. pneumoniae in atherosclerosis.

Background: Chlamydia pneumoniae has been repeatedly associated with atherosclerotic cardiovascular diseases. We investigated the pattern of distribution of C. pneumoniae among patients with carotid atherosclerotic disease evaluating chlamydial load in carotid plaque, peripheral blood mononuclear cells (PBMC) and lymph node from same patient., Methods and Results: Thirty carotid plaques, 30 PBMC and 30 lymph nodes were examined by real-time PCR assay. C. pneumoniae DNA was detected, in carotid plaques, PBMC and lymph nodes in 11 patients; in carotid plaques and PBMC in five patients; in PBMC and lymph nodes in four patients; in lymph nodes in two patients; and in PBMC only in one patient. C. pneumoniae DNA in PBMC significantly coincided with the presence of the respective DNA in carotid plaque (p=0.0001) and lymph node (p=0.02). A higher chlamydial load was detected in PBMC than in lymph nodes and carotid plaques. More than 90% of patients with carotid plaques, PBMC and lymph nodes positive to C. pneumoniae were symptomatic, smokers, hypertensives, dyslipidemics and showed carotid plaques with rupture on the surface, hemorrhage and thrombosis., Conclusion: The measurement of chlamydial load in PBMC may be helpful in the future to assess the state of C. pneumoniae infection and the risk of developing sequelae.

Objective: To investigate the possible link between symptomatic carotid atherosclerotic plaque and Chlamydia pneumoniae., Background: Recently, several studies have demonstrated that there may be a possible link between Chlamydia pneumonia and carotid atherosclerosis, however the real role of Chlamydia pneumoniae is not completely understood., Method: This is a prospective study with a total of 52 patients analyzed. All patients had been submitted to endarterectomy, and had suffered thrombotic ischemic stroke or transient ischemic attack up to 60 days prior to the surgery. Every patient presented carotid stenosis over 70%. The plaque was removed during the surgery and the laboratory exams were immediately done. Evaluation of Chlamydia pneumoniae DNA was done using polymerase chain reaction (PCR)., Results: The PCR analyses of all 52 patients were negative for Chlamydia pneumoniae., Conclusion: These initial results do not show a relationship between Chlamydia pneumoniae and symptomatic carotid atherosclerotic plaque.

Objective: Inflammation and infection have been implicated in the pathogenesis of carotid artery atherosclerosis, but their role in cerebrovascular disease symptomatology is not so well defined. We hypothesized that carotid disease symptomatology was associated with specific serologic markers of inflammation and Chlamydia pneumoniae infection and the presence of the pathogen and concentration of tumor necrosis factor-alpha (TNF-alpha) on the atheroma., Methods: In 2004, 78 patients underwent carotid endarterectomy in our department, 46 of whom were symptomatic (group A) and 32 were asymptomatic (group B). A detailed medical history, the presence of atherosclerosis risk factors, ankle-brachial index and boxy mass index were recorded. We measured preoperatively the levels of C-reactive protein, fibrinogen, TNF-alpha, and the titers of immunoglobulin (Ig) A and IgG antibodies against C pneumoniae in the serum. Finally, the atherosclerotic plaques of all patients were immunohistochemically examined for the presence of C pneumoniae and their TNF-alpha concentration was determined., Results: Isolation of the pathogen on the atheromatous lesion was statistically correlated with several risk factors and some of the variables that were tested. After testing independence of association, using the multiple regression analysis, only male gender (P = .024), hypertension (P = .008), hypercholesterolemia (P = .001), and TNF-alpha plaque values (P = .008) remained significantly associated. Hypertension, serum levels of fibrinogen and anti-C pneumoniae immunoglobulin A, detection of C. pneumoniae on the plaque, and plaque TNF-alpha values were significantly correlated with carotid disease symptomatology. After multiple analyses, only the presence of the pathogen on the lesion (P = .008) and atheroma TNF-alpha levels (P = .025) remained significantly associated with cerebrovascular events., Conclusions: It seems that hypertensive hypercholesterolemic men are more likely to have C. pneumoniae infected carotid plaques and that these plaques have higher TNF-alpha concentrations. Cerebrovascular disease symptomatology is strongly correlated with both C. pneumoniae infection and TNF-alpha concentration of the atheroma.

A polymerase chain reaction (PCR) method for the detection of the glmM gene, selected as Helicobacter pylori target sequence, was improved. While performing pathogenicity island cagA gene detection to discriminate pathogenic strains in atherosclerotic carotid samples, several cagA-positive but glmM-negative samples were found. Polymorphisms present in the region amplified in the nested PCR reaction could explain this result; primers were therefore designed to perform a seminested reaction; this modification optimized sensitivity while maintaining specificity. A real-time PCR for Helicobacter DNA detection was also setup. The combination of all 4 PCR reactions detected 83% of H. pylori DNA-positive samples in atherosclerotic carotid tissue, 64% of which were cagA gene positive.

Objectives: A possible role of some microorganisms has been proposed in the pathogenesis of atherosclerosis, but it is still an unresolved issue. We investigated the presence of Chlamydia pneumoniae and Helicobacter pylori DNA in carotid artery atherosclerotic plaques by using PCR., Methods: One hundred and four patients with atherosclerotic diseases were included. The study group consisted of 52 atherosclerotic plaque specimens obtained from the carotid arteries of patients who had carotid endarterectomy and the control group consisted of 52 specimens obtained from the macroscopically healthy regions of ascending aorta in patients who had undergone coronary artery bypass grafting. The presence of C. pneumoniae and H. pylori DNA in endarterectomy specimens were demonstrated by PCR., Results: C. pneumoniae DNA was detected in 16 of 52 (30.8%) atherosclerotic plaques and 1 of 52 (1.9%) macroscopically healthy ascending aorta wall specimens (P < 0.001). H. pylori DNA was detected in 9 of 52 (17.3%) atherosclerotic plaques and none of the controls (P = 0.003)., Conclusions: The higher incidence of C. pneumoniae and H. pylori DNA in atherosclerotic plaques suggests that these microorganisms may play a role in the pathogenesis of atherogenesis.

It has been suggested that some microorganisms may play a role in the etiology or progression of atherosclerotic plaques. The purpose of this study was to assess for the presence of Helicobacter pylori and cytomegalovirus (CMV) DNA using polymerase chain reaction (PCR) technique in vascular-wall specimens obtained during autopsy. Four to 5 mm long samples from 3 different vascular wall specimens (coronary, carotid and abdominal aortas) of 30 patients (23 male, 7 female) were taken for pathologic and microbiologic investigations during autopsy. H. pylori DNA was found in 48.2% atherosclerotic and 19.6% non-atherosclerotic vascular wall specimens, whereas CMV DNA was found in 37.9% atherosclerotic and 32.7% non-atherosclerotic vascular wall specimens. In terms of CMV DNA detection, no statistically significant differences between the atherosclerotic and non-atherosclerotic groups were present (P > 0.05). However, there was a statistically significant difference between the atherosclerosis and non-atherosclerotic groups in terms of H. pylori DNA in coronary and abdominal aorta arteries (p = 0.016 and p = 0.0029 respectively) but not in carotid arteries (p = 1.00). In conclusion, the correlation between H. pylori and atherosclerosis could be suggested. These finding warrant further investigation regarding the role of H. pylori in atherosclerosis.

Objective: The role of Chlamydia pneumoniae in atherosclerosis is still debated. In this study a novel mouse model was applied to determine the direct impact of C. pneumoniae on the arterial wall and the development of atherosclerosis., Methods: Direct effects of C. pneumoniae on collar-induced atherosclerosis were studied after local delivery of C. pneumoniae to carotid arteries of LDL receptor-deficient (LDLr-/-) mice., Results: The presence of C. pneumoniae in the vessel wall was quantified by RT-PCR (6.2 x 10(4) copies/artery) and resulted in a 2.0-fold increase in intima/media ratios (p<0.05) and a 1.7-fold increase in stenosis (p<0.05). Immunostaining revealed a 2.98-fold (p<0.01) increased macrophage content and a tendency towards lower numbers of smooth muscle cells and collagen in lesions of infected carotid arteries. Direct delivery of another respiratory pathogen, Mycoplasma pneumoniae, to the carotids did not affect size or composition of the atherosclerotic lesions. Presence of C. pneumoniae in the carotid arteries resulted within 7 days in a marked upregulation of the expression of MCP-1 (p<0.01) and ICAM-1 as determined on mRNA and protein levels. These in vivo data were in line with data obtained with in vitro infections of macrophages and endothelial cells with C. pneumoniae., Conclusions: We conclude that C. pneumoniae in carotid arteries leads to more pronounced atherosclerotic lesions with a more vulnerable morphology and that this model is suitable to monitor direct effects of C. pneumoniae on atherogenesis.

Objective: Chlamydia pneumoniae (Cpn) infection of vascular smooth muscle cells increases interleukin-6 (IL-6) secretion in vitro. In vivo, IL-6 stimulates liver C-reactive protein (CRP) production. Because serum levels of IL-6 and CRP are independent risk factors for stroke and myocardial infarction (MI), we investigated whether Cpn burden in carotid plaques might provide a link between plaque IL-6 expression and elevated serum levels of IL-6 and CRP., Methods and Results: Consecutive patients undergoing elective carotid endarterectomy were studied. Serum levels of CRP and IL-6 were measured before surgery. Immunohistochemistry and real-time quantitative (k)RT-PCR were used to detect Cpn and the expression of IL-6 within carotid plaques. Cpn mRNA was present in 19 (37%) of 51 patients, suggesting viable infections. These patients had evidence for infection by PCR and immunohistochemistry. The Cpn burden, measured by real-time quantitative (k)-PCR using the number of organisms normalized against the number of eukaryotic cells in the tissue, was associated with plaque expression of IL-6 (Spearman R=0.55; P<0.0001), which was associated with serum levels of IL-6 (R=0.56; P<0.0001) and CRP (R=0.80; P<0.0001)., Conclusions: IL-6 secretion in atherosclerotic plaques infected with Cpn could explain elevated serum inflammatory markers in individuals at risk for stroke and MI.

Background and Purpose: It is not well established what are the features, if any, that distinguish symptomatic from asymptomatic carotid atherosclerotic plaques. Inducible heme oxygenase-1 (HO-1) is a component of cellular defense mechanisms against oxidative stress. We aimed to assess the presence of Helicobacter pylori (H pylori) and the expression of HO-1 in carotid atherosclerotic plaques of patients with and without prior neurologic symptoms attributable to the operated artery., Methods: We examined 25 symptomatic and 23 asymptomatic carotid atherosclerotic plaques removed during endarterectomy and 7 normal carotid arteries obtained at autopsy. We investigated the presence of H pylori DNA in the vessel wall and performed immunohistochemical detection of HO-1., Results: H pylori DNA was present in 28 plaques and HO-1 was expressed in 30 plaques. HO-1 was found in 27 H pylori-positive specimens but in only 3 H pylori-negative specimens (P<0.001). All 7 normal carotid arteries were negative for both H pylori and HO-1. Although 82% of asymptomatic specimens were positive for H pylori and 87% for HO-1, only 36% of symptomatic specimens were positive for both H pylori and HO-1 (P<0.01)., Conclusions: This study suggests a strong association between H pylori infection and expression of HO-1 in carotid atherosclerotic plaques. There was a substantial prevalence of these features in specimens obtained from asymptomatic subjects.

Data published over the past decade show that Chlamydia pneumoniae is likely associated with the development of atherosclerosis. The aim of this study was to ascertain whether C. pneumoniae infections occur more frequently in patients with atherosclerosis than in healthy subjects. A total of 517 persons were studied. Serum samples, leukocytes, and tissue samples were assayed for the presence of C. pneumoniae-specific IgG and IgA antibodies and C. pneumoniae DNA. C. pneumoniae DNA was found in renal, iliac, and brachial vessels, but it was not detected in radial arteries. C. pneumoniae DNA was found most often in directional coronary atherectomy tissue specimens (11/41, 26.8%), but it was also found in the leukocytes of 14.9% (28/188) of patients with atherosclerosis and 24.6% (28/114) of patients without atheroma changes in vessels. Specific IgG and IgA antibodies were present in 63.8 and 49.9% of atheroma patients, respectively. The prevalence of C. pneumoniae antibodies differs significantly in patients with and without atherosclerosis (for IgG, p=0.002, and for IgA, p=0.006). The identification of persons with chlamydial infection of atherosclerotic arteries necessitates the examination of vascular tissues obtained during revascularization procedures. Serological investigation alone cannot identify individuals with vascular chlamydial infections. Detection of C. pneumoniae DNA in peripheral blood mononuclear cells does not seem to be the exclusive marker of persistent vascular infection. A more easily accessible parameter that allows prediction of chlamydial vascular infection is required.

Background: Epidemiological studies have shown that periodontitis may be associated with presence of atherosclerosis. DNA from periodontal pathogens has been detected in atherosclerotic lesions, but viable oral bacteria have not yet been isolated from atherosclerotic plaques. The purpose of the present study was to determine if viable oral bacteria could be isolated from atherosclerotic lesions and if DNA from periodontal pathogens could be detected by use of polymerase chain reaction (PCR) techniques., Methods: Seventy-nine specimens of atherosclerotic plaque removed from carotid or femoral arteries during surgery were immediately transferred to reduced transport fluid and brought to the laboratory. The calcified tissue was meticulously cut into fine pieces and used for cultivation of Porphyromonas gingivalis, Prevotella intermedia, P. nigrescens, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, and oral streptococci. The material from 24 of the specimens was homogenized, DNA was extracted, and PCR amplification of 16S rDNA with universal and specific primers was carried out. Finally, the PCR products were sequenced., Results: None of the samples yielded growth of the oral bacteria under investigation. In all the 24 specimens bacterial DNA was detected and likewise DNA of P. intermedia was found in the samples. P. nigrescens and P. gingivalis were found sporadically., Conclusions: Viable oral bacteria could not be isolated from the atheromas, but the data confirm that DNA of periodontal pathogens can be detected in atherosclerotic plaques. However, the finding that DNA from P. intermedia constantly occured in the examined samples was new. Further studies may focus on the simultaneous occurrence of identical clones of this species in subgingival plaque and atherosclerotic plaques.

An association of the obligatory intracellular gram-negative pathogen Chlamydia pneumoniae with coronary artery disease, myocardial infarction, and atherosclerosis was suggested. The presence of C pneumoniae was determined in different arteries (n = 165) from 23 control cases and 10 patients with stroke including coronary arteries, carotid arteries, basilar artery, and middle cerebral arteries of normal controls and patients with stroke using nested polymerase chain reaction (PCR) and immunohistochemistry (IHC). Atherosclerosis was detected in 51.5% of all investigated arteries. No significant differences were detected between controls (59.1% by IHC, 45.5% by nested PCR) and patients with stroke (40% by IHC, 40% by nested PCR). This is the first investigation demonstrating C pneumoniae by IHC and nested PCR in different intracerebral arteries in control persons and patients with stroke. No significant correlation between the presence of chlamydial DNA or antigens in arteries and stroke could be demonstrated. The presence of the C pneumoniae is indicative of a correlation between infection and atherosclerosis, but not of a specific vascular neuropathology such as stroke.

Recent and historical evidence is consistent with the view that atherosclerosis is an infectious disease or microbial toxicosis impacted by genetics and behavior. Because small bacterial-like particles, also known as nanobacteria have been detected in kidney stones, kidney and liver cyst fluids, and can form a calcium apatite coat we posited that this agent is present in calcified human atherosclerotic plaques. Carotid and aortic atherosclerotic plaques and blood samples collected at autopsy were examined for nanobacteria-like structures by light microscopy (hematoxylin-eosin and a calcium-specific von Kossa staining), immuno-gold labeling for transmission electron microscopy (TEM) for specific nanobacterial antigens, and propagation from homogenized, filtered specimens in culture medium. Nanobacterial antigens were identified in situ by immuno-TEM in 9 of 14 plaque specimens, but none of the normal carotid or aortic tissue (5 specimens). Nanobacteria-like particles were propagated from 26 of 42 sclerotic aorta and carotid samples and were confirmed by dot immunoblot, light microscopy and TEM. [3H]L-aspartic acid was incorporated into high molecular weight compounds of demineralized particles. PCR amplification of 16S rDNA sequences from the particles was unsuccessful by traditional protocols. Identification of nanobacteria-like particles at the lesion supports, but does not by itself prove the hypothesis that these agents contribute to the pathogenesis of atherosclerosis, especially vascular calcifications.

Treponema denticola and Porphyromonas gingivalis have been identified in atheromatous plaques of two patients suffering from atherosclerosis by PCR and fluorescence in situ hybridization (FISH). The use of the FISH technique suggested that these periodontopathic micro-organisms might be metabolically active within the wall of arteries, under the atherosclerotic lesion.

Background: Herpes virus infections are suspected to be involved in the pathogenesis of atherosclerosis., Objective and Method: Viral DNA of herpes simplex virus (HSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) was analyzed by real-time PCR on 48 biopsies from atherosclerotic plaques extracted by end-arterectomy (46 coronary arteries, 2 carotid arteries), and in tissue from non-atherosclerosis vessels from the same patient as controls (23 internal mammary arteries, 43 saphenous veins)., Results: HSV-1 DNA was detected significantly more frequently in plaques (35%) than in control veins (9%, P = 0.006). However, the frequency of HSV-1 DNA detection in the internal mammary artery grafts was as high as in plaques (22%, P = 0.28). CMV and EBV DNA were exclusively found in plaques but not in controls, with 10% for CMV (P = 0.06 versus veins, P = 0.17 versus graft arteries) and 2% for EBV (P = 1.0), respectively. HSV-2 was neither detected in plaques nor in controls. Herpes viral DNA was significantly associated only with arterial hypertension but not with other classical risk factors (P = 0.02), in accordance with the hypothesis that herpes viral infection may alter the vessel wall., Conclusion: We conclude that herpes viral infections may have a role in atherosclerosis and that the presence of herpes viral DNA in the grafts used for bypass surgery might constitute a potential risk for atherosclerosis or restenosis.

To investigate the presence of infectious agents in human atherosclerotic arterial tissues. Atherosclerotic plaques were removed from 128 patients undergoing carotid endarterectomy or other bypass procedures for occlusive disease, and from twenty normal arterial wall samples, obtained from transplant donors with no history of diabetes, hypertension, smoking, or hyperlipidemia. Using the polymerase chain reaction (PCR) or reverse transcription-PCR, these samples were analyzed for the presence of Chlamydia pneumoniae, cytomegalovirus, enterovirus, adenovirus, herpes simplex viruses types 1 and 2, and Epstein-Barr virus. The amplicons were then sequenced, and phylogenetic analyses were performed. Enteroviral RNA was found in 22 of 128 atherosclerotic vascular lesions (17.2%), and C. pneumoniae and cytomegalovirus were each found in 2 samples (1.6%). In contrast, adenovirus, herpes simplex viruses, and Epstein-Barr virus were not identified in any of the atherosclerotic samples. Enterovirus was detected in 6/24 (25.0%) aortas, 7/33 (21.2%) carotid arteries, 6/40 (15.0%) femoral arteries, and 3/31 (9.7%) radial arteries of patients with chronic renal failure. There were no infectious agents detected in any of the control specimens. Using phylogenetic analysis, the enterovirus isolates were clustered into 3 groups, arranged as echovirus 9 and coxsackieviruses B1 and B3. Enteroviral RNA was detected in 17.2% of atherosclerotic plaques, but was not observed in any of the control specimens. This suggests a connection between enteroviral infection and atherosclerosis. These findings differ from those of other studies, which found more frequent incidence of C. pneumoniae and cytomegalovirus infection in atherosclerotic plaques.

Background: The purpose of the study was to: 1) investigate carotid atherosclerotic plaque morphology and immunohistochemistry (IHC) with respect to the localization sites of Chlamydia pneumoniae, 2) find correlations between plaque morphology and clinical sonographical characteristics, and 3) determine the correlation between abundance of C. pneumoniae and complexity of the plaque., Material/methods: 200 patients with asymptomatic (A=59) and symptomatic (S=141) courses of carotid stenosis admitted to the Neurology and Neurosurgery Clinic for TIA or acute ischemic stroke were studied. For IHC the atherosclerotic plaques were incubated with primarily monoclonal anti-Chlamydia pneumoniae antibody clone RP402, code no. M6600, diluted 1:50 (DAKO). Statistical evaluation was performed., Results: 34 (17%) homogeneous (HO), or stable, and 166 (83%) heterogeneous (HE), or unstable, plaques were divided into 3 HO and 5 HE subtypes according to the number of histopathological signs and prevalence of inflammatory cell types, having different sonographical characteristics and plaque thickness. Complex plaques of both A and S patients have activation signs for chronic inflammation, phagocytosis, and atheromatosis: all three of these in HE plaque subtypes, and only for atheromatosis in HO plaques., Conclusions: 1) C. pneumoniae IHC signals were found in every kind of phagocyte (histiocytes, macrophages, foreign body macrophages, foam cells, PMNs) located in the fibrous cap, atheroma (necrotic core), and especially at the boundary of fibrous cap/atheroma; 2) complexities were found, so plaque instability signs strongly correlate with abundance of C. pneumoniae (p<0.001); 3) sufficient correlation between plaque thickness and disease symptomaticity (p<0.01) was found.

Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates. In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C. pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium. A sensitive nested PCR assay detected C. pneumoniae DNA in all patients at one or more locations within the plaque. On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C. pneumoniae DNA present. A patchy distribution of C. pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C. pneumoniae DNA present. If a single random 30- microm-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C. pneumoniae DNA in a patient with carotid artery disease. A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives. The low concentration and patchy distribution of C. pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.

In this study, using scanning and transmission electron microscope, we attempt to evaluate ultrastructural alterations of endothelial cells, macrophages and smooth muscles cells. The inflammatory process has an essential impact on the development of Chlamydia infection. Specimens from human carotid were obtained from patients who underwent endarterectomy. For examination under scanning and transmission electron microscope vessel sections were fixed in paraformaldehyde and glutaraldehyde. We analysed alterations of endothelial cells covering advanced atherosclerotic plaque in carotid using scanning electron microscope. Smooth muscle cells had undergone the heaviest proliferation among the cells on artery wall. In the tested material we detect diversified morphological forms of Chlamydia sp. We found that one of the pathogens that may lead to atherosclerotic lesions is Chlamydia pneumoniae.

Background and Purpose: There is growing clinical and experimental evidence that infections with Chlamydia pneumoniae might contribute to the development and progression of atherosclerosis. However, studies detecting the pathogen in atherosclerotic lesions examined either only atherosclerotic vessels or control vessels without atherosclerosis obtained from a different group of individuals. We analyzed atherosclerotic plaques of the carotid artery, samples of apparently healthy greater saphenous veins, and circulating leukocytes from the same individual patients for the presence of C pneumoniae., Methods: From each of 46 patients undergoing carotid endarterectomy for symptomatic carotid artery stenosis, these samples were analyzed by nested polymerase chain reaction for C pneumoniae-specific DNA. Furthermore, we determined IgA and IgG titers specific for the pathogen and plasma levels of C-reactive protein in these patients., Results: C pneumoniae DNA was detected in 86.9% of the leukocytes and in 82.6% of the atherosclerotic plaques but in only 6.5% of the saphenous veins. In 85% of patients who also had leukocytes positive for C pneumoniae, the atherosclerotic plaques were positive and the saphenous veins were negative. The presence of C pneumoniae-specific DNA in leukocytes significantly coincided with the presence of the respective DNA in the plaques of the carotid arteries (P=0.0002). No association between the presence of C pneumoniae and specific IgA or IgG levels was seen. C-reactive protein levels were significantly higher in patients with chlamydia-positive atherosclerotic plaques and with positive leukocytes than in patients with negative plaques of the carotid arteries or negative leukocytes, respectively (P<0.01, P<0.05)., Conclusions: Our observation of >80% incidence of C pneumoniae in atherosclerotic plaques of the carotid artery does not prove causality between an infection with the pathogen and the development of atherosclerosis. It must be emphasized, however, that >90% of apparently healthy saphenous veins were negative for C pneumoniae. Given the structural and functional differences between veins and arteries, careful interpretation of our results regarding a possible causative role of C pneumoniae seems warranted.

The role of immune inflammation at atherogenesis is studied in the paper. Two etiological factors of atherosclerosis pathogeny are under examination: the role of modified low density lipoproteins (mLDL) and essential parasites (Chlamydia pneumoniae and Cytomegaloviruses). Generality of immune response during persistent infection into a blood vessel wall and deposit or formation of mLDL are discussed. The point of view is substantiated that the development of atherosclerotic damages of blood vessels is speeded up by a combination of the two mentioned etiological factors.

Background and Purpose: An elevated serum level of C-reactive protein, an inflammatory marker, is an independent predictor of stroke and coronary artery disease. To determine whether chronic infection with Chlamydia pneumoniae, which has been identified in atherosclerotic plaques, is responsible for systemic inflammation, we studied the association between serum C-reactive protein levels and infection of carotid artery atherosclerotic plaque with viable C pneumoniae., Methods: Serum C-reactive protein levels were obtained before endarterectomy for carotid artery stenosis. Plaques were tested for C pneumoniae mRNA, an indicator of viability, and DNA by polymerase chain reaction of DNA and cDNA, respectively., Results: Forty-eight samples were studied, of which 18 (38%; 95% CI, 23 to 50) were infected with viable C pneumoniae as evidenced by isolated chlamydial mRNA. All 18 of these samples, plus 1 additional sample, were positive for chlamydial DNA. Serum C-reactive protein levels were higher in those with viable C pneumoniae compared with those without infection (median, 8 mg/L versus undetectable; P=0.045 by Wilcoxon rank-sum test). In multivariable models, the only independent predictor of the presence of viable C pneumoniae was a detectable C-reactive protein level (odds ratio, 4.2; 95% CI, 1.1 to 17; P=0.04)., Conclusions: Viable C pneumoniae are present in a substantial portion of carotid artery atherosclerotic plaques and are associated with increased serum C-reactive protein levels. These findings may explain the link between elevated C-reactive protein levels and the risk of cardiovascular disease and stroke but should be reproduced in a larger cohort.

Purpose: Several recent articles indicate the renewed interest that Chlamydia pneumoniae is associated with and contributes to arterial diseases. Associations have been established for coronary arteries, carotid arteries and abdominal aortic aneurysms by serological studies, immunofluorescence and DNA-specific testing. The purpose of this study was to determine the presence of C. pneumoniae in coronary artery plaques, carotid artery plaques and old vein grafts that were harvested at the time of surgery., Methods and Materials: Interventions were carried out on patients with carotid artery disease and coronary disease based upon their anatomic findings and symptomatology. Plaques were removed from the carotid arteries with standard endarterectomy techniques. The coronary plaques were removed utilizing blunt dissection to peel the plaque from the coronary artery. Old vein grafts were harvested during repeat surgery by excising the vein grafts. The tissue specimens were analyzed by polymerase chain reaction (PCR)., Results: Ninety-nine specimens from 68 consecutive patients with advanced coronary atherosclerosis with or without cerebrovascular disease were analyzed. We failed to find C. pneumoniae in any of the vascular tissue., Conclusions: A large cooperative study involving surgical specimen analysis is needed to assess the role of C. pneumoniae in the etiology of atherosclerosis.

This study was designed to determine the prevalence of Chlamydia pneumoniae in carotid artery plaques. Although there have been numerous studies evaluating coronary plaques for this bacterium fewer studies have assessed noncoronary vasculature. In addition we wished to evaluate whether correlation exists between the presence of C. pneumoniae in carotid plaques and established risk factors for atherosclerosis. Sixty intact carotid artery plaques removed during surgery (carotid endarterectomy) were formalin-fixed and paraffin-embedded according to conventional techniques. These samples were evaluated by polymerase chain reaction analysis to detect presence of C. pneumoniae DNA. Results were tabulated and compared against established risk factors for atherosclerosis: diabetes, hypertension, hyperlipidemia, age, and smoking. Forty-two (70.0%) of the 60 plaques that were evaluated tested positive for the presence of C. pneumoniae DNA by polymerase chain reaction analysis. In the sample defined as being from heavy smokers (greater than 15-pack-year history) 33 (94.3%) of 35 plaques tested positive whereas two (5.7%) tested negative. This correlation demonstrated statistical significance (P = 1.36 x 10(-6), two-tailed Fisher exact test). Presence of C. pneumoniae in carotid plaques demonstrated no statistically significant correlation with diabetes, hypertension, or hyperlipidemia. Age as a risk factor was examined but not statistically evaluated because of the narrow range within our patient sample. Analysis of the data reveals that C. pneumoniae is present in large numbers of atheromatous plaques as is consistent with emerging data. What is interesting though is that 33 (94.3%) of the 35 smokers had plaques that tested positive for the bacterium as opposed to only nine (36.0%) of the 25 nonsmokers. Identification of specific populations exhibiting a high prevalence of C. pneumoniae may serve to focus future studies. Ongoing investigation will seek to determine whether C. pneumoniae plays an active role in the pathogenesis of atherosclerosis.

Background: Chlamydia pneumoniae has been identified in atherosclerotic plaques of patients with cerebrovascular and cardiovascular disease. However, the direct causative effect of C pneumoniae infection in the activation of atherosclerotic plaque to a prothrombotic state remains to be established. The aim of the present study is to examine the correlation between intraplaque presence of chlamydiae and symptomatic carotid disease in humans., Methods: Plaques from 37 symptomatic and 57 asymptomatic consenting patients undergoing carotid endarterectomy were snap-frozen, and the tissue was prepared for polymerase chain reaction analysis for Chlamydia pneumoniae per Institutional Review Board-approved protocol. Blood was drawn from each patient at the time of surgery for serological analysis., Results: The overall rate of plaques positive for C pneumoniae was 14.82%, with 5 of 37 (13.5%) plaques from symptomatic patients and 9 of 57 (15.8%) from asymptomatic patients, which revealed a definitive presence of the organism. No association existed between C pneumoniae presence and symptomatic disease (P:=1.0). Also, no association existed between presence of C pneumoniae and severity of stenosis. Finally, seropositivity for anti-chlamydial IgG, IgA, and IgM anti-chlamydial antibodies did not correlate with identification of C pneumoniae in the plaques. However, high-serum anti-chlamydial IgA levels (>/=1:128) were associated with occurrence of symptomatic disease (P=0.03; odds ratio, 2.86; 95% CI, 1.12 to 7.28)., Conclusions: Presence of C pneumoniae as a single factor does not appear to be sufficient to explain the occurrence of cerebrovascular symptoms. Low sensitivity of seropositivity for IgG, IgA, or IgM associated with PCR-identified C pneumoniae presence in the plaque makes it unlikely to be valuable as the single determining factor for actively infected plaque. Association of high-level anti-chlamydial IgA with symptomatic disease suggests that chronic or acute chlamydial infection anywhere in the body could play a role in atherosclerotic plaque activation and be used as a marker to target populations in future stroke prevention trials.

Samples of atherosclerotic tissue from 58 patients undergoing carotid surgery were analysed by tissue culture and PCR for Chlamydia pneumoniae; PCR was performed to detect Omp1, 16S rRNA and HSP-70 genes. To understand the active pathogenic role of C. pneumoniae, a reverse transcriptase-PCR (RT-PCR) assay was applied to detect the specific RNAs expressed either in the replicative form, or in the cryptic form found in chronic infection. The C. pneumoniae omp1 gene, encoding the major outer-membrane protein (MOMP), was detected in 13 of 58 samples. Among these, the result was confirmed in 11 samples after amplification of a further target, the 16S rRNA, and the presence of the HSP-70 gene, encoding heat-shock protein 70, was revealed in only five cases. All the samples were negative for evidence of specific RNAs by RT-PCR. The presence of genomic DNA and absence of specific RNAs in atherosclerotic tissue samples suggests a lack of an active metabolic or persistent infective role for C. pneumoniae. Thus, traces of C. pneumoniae DNA in these samples could be due to a degradative pathway of the host defensive cellular and biochemical mechanisms.

Background and Purpose: Several lines of evidence point toward a relationship between infection and atherosclerotic vascular disease. Thus, infection and inflammation often precede ischemic neurological events. Transient alterations in coagulation and direct arterial invasion by certain microorganisms have been reported. Helicobacter pylori infection is the major cause of peptic ulcer disease and appears to be a risk factor for ischemic cerebrovascular disease. However, in contrast to other chronic infectious agents, H pylori has not been consistently isolated from atherosclerotic lesions., Methods: We investigated the presence of H pylori in 38 atherosclerotic plaques obtained at carotid endarterectomy by using morphological and immunohistochemical techniques and a highly sensitive polymerase chain reaction method. We performed immunohistochemical detection of intercellular adhesion molecule-1, a marker related to inflammatory cell response. We also examined 7 carotid arteries obtained at autopsy from subjects without carotid atherosclerosis., Results: H pylori DNA was found in 20 of 38 atherosclerotic plaques. Ten of the H pylori DNA-positive plaques also showed morphological and immunohistochemical evidence of H pylori infection. None of 7 normal carotid arteries was positive for H pylori. Intercellular adhesion molecule-1 was expressed in 75% of H pylori-positive plaques and in 22% of H pylori-negative plaques. The presence of the microorganism was associated with male sex but was independent of age, vascular risk factor profile, and prior neurological symptoms., Conclusions: H pylori is present in a substantial number of carotid atherosclerotic lesions and is associated with features of inflammatory cell response. This study provides additional evidence of the relationship between H pylori infection and atherosclerotic disease.

Background and Purpose: Chronic infection with Chlamydia pneumoniae, Helicobacter pylori, cytomegalovirus (CMV), and herpes simplex virus (HSV) has been implicated in the pathogenesis of atherosclerosis. The carotid intima-media thickness (IMT) can be taken to indicate early atherosclerosis, the presence of a carotid stenosis is a marker of a manifest carotid atherosclerosis, and an increase in arterial stiffness is used as marker of structural and functional changes in an atherosclerotic vessel wall., Methods: In 504 patients (75% men; mean age 62.9 [SD 10] years), we measured the IMT and the elastic pressure modulus (EP; n=445) of the common carotid artery and the prevalence of a internal or external carotid artery stenosis. Blood samples were taken, and antibodies against C pneumoniae, H pylori, CMV, and HSV types 1 and 2 were evaluated. Statistical evaluation was performed with regression procedures and multivariate logistic regression analyses., Results: Seropositivity for C pneumoniae was an independent predictor for a combined end point of highest category of IMT and carotid artery stenosis (OR 1.8, 95% CI 1.1 to 3.1; adjusted) for IgG titers. Independently, CMV increased the risk for the combined end point (OR 1.7, 95% CI 1.1 to 2.8; adjusted) for IgG titers and for IgA titers (OR 2.3, 95% CI 1.1 to 4. 9; adjusted). We found a significant correlation between IgG antibodies against CMV and EP; HSV type 2 IgG titers were associated with IMT and carotid stenosis, but the latter results were no longer significant after adjustment. There was no association with H pylori or HSV type 1., Conclusions: We found a significant association of IgG antibodies against C pneumoniae and CMV with early and advanced carotid atherosclerosis. CMV was also correlated to functional changes of the carotid artery, but this could not be confirmed after adjustment.

Italian investigations have shown an association between Chlamydia pneumoniae infection and atherosclerosis. With the use of several diagnostic techniques, including serology, a microimmunofluorescence test, and nucleic acid amplification methods, a temporal association was found between acute C. pneumoniae reinfection and acute myocardial infarction, suggesting that an acute infection superimposed on a chronic or latent infection may trigger the onset of acute myocardial infarction. C. pneumoniae but not Helicobacter pylori or Mycoplasma pneumoniae was found in atherosclerotic plaques of abdominal aortic aneurysms and the carotid artery. A reverse transcriptase-polymerase chain reaction process confirmed the presence of viable C. pneumoniae in carotid atheromas. Nucleic amplification of peripheral blood mononuclear cells may enable the identification of subjects carrying C. pneumoniae in the vascular wall. Macrolide treatment reduced fibrinogen and C-reactive protein plasma levels and C. pneumoniae burden in patients with atherosclerotic diseases.

The positivity rate and localization of Chlamydia pneumoniae were investigated in atherosclerotic and nonatherosclerotic tissues by immunohistochemistry, polymerase chain reaction, and cell culture. In total, 67 atheromatous plaques from Japanese symptomatic patients and 110 nonatherosclerotic tissues and organs were evaluated. Of these, 62% of atherosclerotic plaques from symptomatic patients were infected with C. pneumoniae compared with just 2% of nonatherosclerotic tissues. Immunohistochemically stained C. pneumoniae were found most often in smooth muscle cells, less often in macrophages, and in a few endothelial cells.