CARDIOVASCULAR DISBASES
C. PNEUMONIAE SEROLOGY IN PATIENTS WITH CORONARY ARTERY DISEASE
Jan Kaehler1, Ralf Koester1, Marion Carstensen1, Sabine Dettlaff 2* and Thomas Meinertz1
department of Cardiology, Department of Microbiology, University Hospital Hamburg, diagnostic Division, medac GmbH, Hamburg, Germany
Elevated antibody titers against C.
pneumoniae have been associated with coronary artery disease, but few data are available regarding titers in patients with stable or unstable angina and under treatment with roxithromycin.
196 consecutive patients undergoing coronary angioplasty were evaluated regardless of antibody titers at inclusion in the context of a study investigating the effect of roxithromycin on restenosis. Indication for angioplasty was stable angina in 49% of patients and acute coronary syndrome in 44%
of patients. Antibodies against C. pneumoniae, chlamydial LPS and chlamydia heat shock protein 60 (cHSP60) were detected by
C. pneumoniae-sELlSA, Chlamydia-rELISA and cHSP-ELISA (medac, Hamburg, Germany).
The prevalences for both C.
pneumoniae-IgG antibodies and anti-LPS-IgG were very high: 82-93% and 53-73%
respectively, while IgA antibodies were detected in up to 79% of patients. In all patients groups, cHSP60-IgG antibody titers were positive in only 18-20%. Age, body mass index and coronary risk factors were only marginal associated with the prevalence of antibodies investigated.
Treatment with roxithromycin had no effect on any of the titers.
All antibody titers determined increased during the first 6 weeks of the follow-up. With comparable titers at inclusion, patients with a history of myocardial infarction (MI) and acute coronary syndrome had the most pronounced increase in titers, while patients without
previous MI and stable angina had the least pronounced increase in titers.
Patients undergoing coronary angioplasty have high antibody titers against C.
pneumoniae. Coronary angioplasty itself induces a small but long-lasting increase of antibody titers against C. pneumoniae, while treatment with roxithro-mycin has no effect on any of the titers determined.
ASSOCIATION BETWEEN THE DEVELOPMENT OF RESTENOSIS AFTER PTCA AND THE CHANGES AFTER PTCA IN THE ANTIBODY TITERS TO MICROBES INCRIMINATED IN THE PATHOMECHANISM OF ATHEROSCLEROSIS
Krisztina Heltai1, Fruzsina Petrovay2' Zoltán Kis2, Valéria Endresz3, Endre Ludwig4, Róbert Kiss5, Eva Gonczol2, István Préda5, István Valyi-Nagy5
'Department of Cardiology, Railway Hospital, Budapest, 2Division of Virology and Bacteriology, Bela Johan National Center for Epidemiology, Budapest, department of Microbiology and Immunobiology, University of Szeged, Szeged, 4St. Laszlo Hospital, Budapest, 5National Center of Medicine, Budapest, Hungary
Aim of the study: To establish whether PTCA results in the reactivation of certain pathogens in coronary plaques and, if so, whether the reactivation has a pathogenic role in the development of restenosis.
Blood samples were withdrawn from 31 patients immediately before and 4 and 14 days after PTCA. IgG and IgA antibody titers
against Chlamydia pneumoniae, cytomegalovirus (CMV), Epstein-Barr virus
and herpes simplex virus type 1 were determined by ELISA and microimmunofluorescence methods in dilutions of 1/16 to 1/2048. C. pneumoniae and CMV DNA detections were performed by PCR. The clinical courses of the patients were followed for 8 months. Symptoms of respiratory infections were not detected in the patients between the first and last samplings.
PTCA may reactivate chronic C.
pneumoniae and CMV infections, resulting in an increased risk of restenosis.
1. Increases in the C. pneumoniae IgG and IgA titers were observed in 3 patients, and similar increases in the CMV antibodies in 3 other patients. There were no changes in the Epstein-Barr virus and herpes simplex virus type 1 titers. 2. As concerns the 6 patients with restenosis, the antibody titers were increased in 3, while the antibody titers were also increased in 3 of the 25 patients without restenosis. The difference is statistically significant (p=0.01), though the number of patients is small. 3. The DNA detection tests are in progress.
CHLAMYDOPHILA PNEUMONIAE- SPECIFIC MRNA IS PRESENT IN AORTIC WALL BIOPSIES OF PATIENTS SUFFERING FROM STABLE OR UNSTABLE ANGINA PECTORIS
Marie Edvinsson, Eva Hjelm, Stefan Thelin, Goran Friman and Christina Nystrôm-Rosander
Infectious Diseases and Clinical Bacteriology, University Hospital, Uppsala, Sweden
Chlamydophila pneumoniae (Cp) might somehow be involved in the pathogenesis of atherosclerosis. Several studies have demonstrated a serological association between Cp and coronary artery disease and DNA from the bacteria has been found in various atheromatous vessels. However, only a few investigators have managed to culture Cp from atheromatous plaques. Instead of culturing, viable bacteria could be demonstrated by reverse transcriptase PCR (RT-PCR) against bacterial mRNA. We investigated the presence of Cp DNA and mRNA in aortic wall biopsies, obtained at surgery, from 24 patients with stable angina pectoris (SAP) and 20 patients with unstable angina pectoris (UAP).
DNA and RNA were extracted from the same biopsy using the RNA/DNA mini kit (Qiagen). cDNA was made using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Real time PCR directed against the MOMP gene was used to detect the presence of Cp DNA and mRNA in the biopsies. Patient sera were tested for Cp-specific IgM, IgG and IgA antibodies by the microimmunofluorescence technique.
13 (28%) of the biopsies (6 SAP; 7 UAP) were positive for Cp DNA, 8 (17%) were positive for Cp mRNA (5 SAP; 3 UAP) and 6 (12%) were positive for both DNA and mRNA (4JSAP; 2 UAP). All biopsies but one were positive for human cDNA encoding the GAPDH gene.
Department of Medical Sciences, Uppsala
That biopsy was negative also for Cp DNA and Cp mRNA. Results from the serology will be presented on the poster.
We have demonstrated the presence of Cp in the aortic wall of patients suffering from stable (SAP) or unstable (UAP) angina pectoris. Also, we have demonstrated Cp mRNA in 17% of these patients indicating that the bacteria were metabolically active. There were no significant differences in the frequencies of positivity for Cp DNA or Cp mRNA between the SAP and UAP groups.
The results support the hypothesis of an active role for jCp in. the pathogenesis of atherosclerosis.
COMPARISON OF CHLAMYDIA PNEUMONIA/:-SPECIFIC DNA DETECTION METHODS BY PCR AND TWO DIFFERENT RT-PCR METHODS
Judith Deák1, Judith Tóth1, F. Somogyvári1, Beatrix Kele1, Elisabeth Nagy1, R. Sipka2, B.A. Szabó2
'Department of Clinical Microbiology, department of Surgery, University of Szeged, Szeged, Hungary
The first evidence of C. pneumoniae in atherosclerotic plaques was reported in 1992 by Shor et al. The pathogenesis of artherosclerosis was described by Ross. In the various studies, C. pneumoniae has been detected in atherosclerotic lesions of different major arteries in very different distributions.
Our aim in this study was to detect C. pneumoniae-specific nucleic acid in atherosclerosis patients.
Internal carotid samples were collected from patients with significant internal carotid stenosis after eversion endarterectomy. The aortic aneurysm samples originated from the infrarenal region (>5 cm in diameter) after resection. Aortic samples were collected from the infrarenal region because of aortoiliacal occlusion or multiple stenosis between 1998 and 2003. Carotid and aortic samples were graded macroscopically on a scale of 1-4.
1. DNA was extracted with phenol-chloroform-isoamyl alcohol (Sigma) and then precipitated with ethanol after proteinase K (Sigma) incubation. Nested PCR was carried out as described by Cunningham et al. 2. The same nucleic acid isolation method was used for RT-PCR (SybrGreen) method as described by Apfalter et al. 3. The QIAmp nucleic acid
isolation method as described by Apfalter et al.
was used for the TaqMan PCR assay.
The sensitivity of RT-PCR (SybrGreen) proved to be higher than that of conventional nested PCR. The QIAmp nucleic acid isolation method combined with the TaqMan PCR assay furnished not only higher sensitivity, but also quantitative results.
References
Apfalter, P., Barousch, W., Nehr, M., Makristathis, A., Willinger, B., Rotter, M., Hirschl, A., M.: Comparison of a new quantitative ompA-based real-time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens
with four conventional PCR assays, Journal of Clinical Microbiology, 2003. 41, 592-600.
Cunningham A., Johnston S., Julious S., Sillis M., Ward M.E.: The role of Chlamydia pneumoniae and other pathogens in acute
episodes of asthma in children. In: Chlamydial infections. Ed: Orfila J., Byrne G.I., Chernesky M.A., Grayston J.T., Jones R.B., Ridgway G.L., Saikku P., Schachter J., Stamm W.E., Stephens R.S., Bologna, 1994, ISBN: 88 85040 92 63.
Results of different PCR methods in different patient groups
N Nested Nested RT-PCR RT-PCR RT-PCR RT-PCR PCR- PCR- SybrGreen- SybrGreen- TaqMan- TaqMan-positive negative positive negative positive negative
Carotid 58 4 54 6 52 10 48
stenosis
Aortic 10 0 10 0 10 0 10
aneurysm
Aortic 10 0 10 0 10 0 10
occlusion or stenosis
Total 78 4 74 6 72 10 68
CHLAMYDIA PNEUMONIAE AND CORONARY ATHEROSCLEROSIS: AN ASSOCIATION STUDY USING PCR ASSAY, ANTIGEN DETECTION AND SEROLOGY
Gita Satpathy, Anil Bhan, Anjana Sharma, U Kar and SK Panda All India Institute of Medical Sciences, New Delhi, INDIA
To determine the association of C.
pneumoniae with coronary atherosclerosis using agent detection by PCR and antigen detection assays and antibody detection by microimmunofluorescence assay.
Specimens of seventy two atherosclerotic plaques from coronary arteries from patients of coronary heart disease, collected during coronary artery bypass grafting were tested for presence of C.
pneumoniae genome using a nested PCR assay and antigen detection by monoclonal based immunofluorescence assay. Presence of species specific C. pneumoniae antibodies were also tested in these patients using microimmunofluorescence assay using a kit from Prof. Darougar, UK. Seventy two age and sex matched controls were also tested for C.
pneumoniae species specific antibodies.
In none of these 72 atherosclerosis plaque specimens C. pneumoniae could be detected by PCR assay or by immunofluorescence assay. C. pneumoniae antibodies at a titre of >1:16 were detected in 65% of these 72 CHD patients. However 63%
of the controls also had C. pneumoniae antibodies.
We failed to get any positive association of C. pneumoniae with coronary artery disease in these groups of patients.
CHLAMYDIA PNEUMONIAE INFECTION DOES NOT INCREASE ATHEROSCLEROTIC LESIONS IN THE ASCENDING AORTA OF APOE-DEFICIENT MICE
Christian Wahl*, Philipp Duerr, Sonja Maier, Ulrike Simnacher, Reinhard Marre, and Andreas Essig
Department of Medical Microbiology and Hygiene, University of Ulm, Germany Chlamydia pneumoniae (C.
pneumoniae) has been associated with arteriosclerosis. In this study we investigated the influence of repeated C. pneumoniae-infection of the respiratory tract for the progression of arteriosclerotic lesion in the ascending aorta of apolipoprotein E (apoE) deficient mice.
For experiments eight-weeks old male apoE-deficient mice on C57BL/6J background were used. The mice were infected intranasally with lxlO6 inclusion-forming units (IFU) 3 times at 2-weeks intervals and killed 6 weeks later. After Elastica van Gieson's staining of sections from the ascending aorta, the lesion areas in the histological cross-sections were measured morphometrically. To detect chlamydial DNA, MOMP-PCR and LightCycler-PCR were performed.
Immunohistochemistry stainings were also performed to detect macrophages and chlamydial antigen in atherosclerotic lesions.
The serum levels of C. pneumoniae-specific antibody titers were determined by microimmunofluorescence test using C.
pneumoniae elementary bodies as antigen.
C. pneumoniae has no influence in the progression of arteriosclerotic lesions in the ascending aorta of apoE-deficient mice. As demonstrated by determination of the P-value (P=0,892), there is no significance in the lesion size between C. pneumoniae and mock-infected mice. Each group contains 23 mice.
To detect chlamydial antigen in aortic atherosclerotic lesions, a mouse anti-chlamydial lipopolysaccharide (LPS) antibody was used on paraffin sections. Neither in C.
pneumoniae-infected nor in mock-infected mice was chlamydial LPS antigen detected within atherosclerotic lesions.
To examine the degree of vascular inflammation and changes in the composition of subpopulations of infiltrated macrophages, paraffin sections were stained for CD59 and MoMa. CD59 is a foam cell marker, whereas MoMa is a monocyte and macrophage marker.
There were no significant differences of CD59+ (P=0,412) and MoMa+ (P=0,886) cell infiltration in lesions of C. pneumoniae-infected versus mock-pneumoniae-infected apoE-deficient mice.
To examine, if C. pneumoniae can generate a systemic dissemination in apoE-deficient mice two different PCR methods were used. In only one mice could we detect chlamydial DNA by PCR. This LightCycler-PCR positive result could not be verified by nested MOMP-PCR, indicating that this probe must be handled as unclear.
Only infected mice developed an IgG-titer between 1:256 and 1:4096 as quantified 10 weeks after first infection, whereas all mock infected mice have a titer of <1:32 .
In our study we could demonstrate, that C. pneumoniae has no influence in the progression of arteriosclerotic lesions in the ascending aorta of apoE-deficient mice.
RELATIONSHIP BETWEEN CHLAMYDIA PNEUMONIAE SEROPOSITIVITY AND AGE-RELATED MACULAR DEGENERATION, IN PATIENTS WITH CORONARY ARTERY DISEASE
Annamária Nagy*, Andrea Facskó*, Judith Deák**, István Édes***, András Berta*
Department of Ophthalmology*, University of Debrecen, Department of Clinical Microbiology**, University of Szeged, Department of Cardiology***, University of Debrecen, Hungary
Age-related macular degeneration (AMD) is a leading cause of blindness in the well-developed countries. Many risk factors have been established, but the etiopathological background is still not completely clear.
Among the potential risk factors, atherosclerosis is one of the proven causes of AMD. There is increasing evidence in support of the role of chronic Chlamydia pneumoniae (C. pneumoniae) infection in the pathogenesis of general atherosclerosis and coronary artery disease (CAD). The aim of the present study was to obtain evidence on the possible role of chronic C. pneumoniae infection in AMD.
C. pneumoniae titers were determined with a microimmunofluorescence (MIF) method (Focus Technologies, Cypress, USA) in the serum of 30 patients with coronarographically-proven CAD, and also in 24 cataract patients with no clinical signs of CAD (controls). The patients in both groups were examined clinically for the presence of AMD. Statistical analysis was carried out by means of Chi-square analysis: a level of p<0.05 was regarded as statistically significant.
AMD was detected in 12 patients in the CAD group, and in 6 patients in the control group. There was no statistically significant difference in C. pneumoniae seropositivity between the CAD and control groups.
The results of the study do not support the assumption that chronic C. pneumoniae infection plays a role in the development of AMD. However, these limited observations and the results of others justify further studies on the role of C. pneumoniae infection in
AMD, and the authors plan to extend this examination to a larger number of subjects, with the use of additional laboratory techniques.
CHLAMYDIAL LPS, CIRCULATING LPS BINDING PROTEINS AND C-REACTIVE PROTEIN IN HEALTHY BLOOD DONORS
Korhonen T1'2*, Jounio U2, Tiirola T2, Jauhiainen M2, Silvennoinen-Kassinen S1, Leinonen M2 and Saikku P1
1 Dept. of Medical Microbiology, University of Oulu, 2National Public Health Institute, Oulu and Helsinki, Finland
Chlamydia pneumoniae is capable to multiply and survive in e.g. macrophages, endothelial and smooth-muscle cells, that have important role in the generation and progression of atherosclerosis. Chlamydial LPS (cLPS) induces macrophage foam cell formation and production of cytokines and growth factors. In this work, we studied the association between circulating cLPS, circulating LPS binding proteins - soluble CD 14 (sCD14) and LPS binding protein (LBP) - and C-reactive protein (CRP), a marker of systemic inflammation. cLPS levels were also compared to C. pneumoniae IgA antibody titres. Because the genetic background of a host is likely to modify immune response, we also studied gene polymorphisms that might affect LPS recognition.
The polymorphisms tested were LBP 1341T—>C (Phe436—>Leu), Toll Jike receptor 4 (TLR4) 896A-4G (Asp299-)Gly), CD14 -260C->T, and apolipoprotein E (ApoE) alleles E 2, 3 and 4.
Sera and buffy coats of 387 healthy blood donors (265 men and 122 women) were obtained from Finnish Red Cross Blood Service. Serum levels of CRP, sCD14 and LBP were measured by commercial ELISA methods (Medix Biochemica, HyCult). Quantitation of cLPS (OD values) was performed by an LBP-based ELISA and C.
pneumoniae IgA antibody titres by microimmunofluorescence test. DNA was extracted from buffy coats by a commercial kit (Qiagen). Polymorphisms were genotyped either by Light Cycler Real Time or conventional PCR.
For statistical analysis X2 and Mann-Whitney U tests, Spearman's correlaton and binary logistic regression were used. The ApoE polymorphism was cathegorized as either having an ApoE4 allele or not. TLR 4 polymorphism genotype GG and LBP polymorphism genotype CC were so rare that they were omitted from the final analysis.
High C. pneumoniae IgA titres were associated with hig levels of cLPS (Md = 0.05 OD, interquartile range 0.05 for IgA > 20, and Md = 0.03 OD, interquartile range 0.07 IgA < 20) in
M-W test Z=-2.185 and p=0,029. In a binary logistic regression analysis for LBP, risk factors for having LBP levels higher than median (10.6 mg/1) were OR=8.8 (95 % CI 2.3-6.0) for CRP > median (0.6 mg/1), OR=2.1 (95 % CI 1.3-3.4) for sCD14 >
median (1.6 mg/1), OR=2.0 (95 % CI 1.3-3.3) for cLPS > median (OD =0.03), OR=2.6 (95 % CI 1.2-3.3) for LBP polymorphism genotype CT and OR=2.3 (95% CI 1.2-4.3) for TLR4 polymorphism genotype AA. In a corresponding analysis for sCD14, risk factors for having levels higher than median were OR=2.2 (95 % CI 1.4-3.5) for LBP, OR=2.3 (95 % CI 1.4-3.7) for ApoE polymorphic isoform E4, OR=1.9 (95 % CI 1.2-2.9) for CD14 polymorphism genotype TT/CT, OR=2.2 (95 % CI 1.1-4.5) for LBP polymorphism genotype CT, and OR=1.8 (95 % CI 1.1-3.0) for male sex. cLPS over median was associated with sCD14 levels under median with OR=0.5 (95 % CI 0.3-0.8).
Higher C. pneumoniae IgA antibody titres and higher cLPS levels were associated, suggesting that cLPS likely originated from C.
pneumoniae. LBP levels were in direct relation to CRP ja sCD14 levels suggesting that the production of these proteins is somehow synchronized on their expression/secretion levels.
All these molecules are acute phase proteins and, moreover, LPB and sCD14 are specific LPS binding proteins. Interestingly cLPS levels were in direct relation to LBP levels but inversely related to sCD14 levels. cLPS levels were low in these healthy subjects, so infrequency of positive (OD
>0,15) values may have affected analysis.
However, it is also possible that when cLPS level is high, more LPS is capable to interact with sCD14 directing sCD14-cLPS complexes to the cell surface, thus reducing free sCD14 levels in the serum. LBP and sCD14 levels also seem to be affected by polymorphisms of LBP, TLR4, CD 14 and ApoE genes.