Project leader: Professor PEKKA SAIKKU
Department of Medical Microbiology, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland, +358-50-3036572, pekka.saikku@oulu.fi

Doctoral students of the project:
Kari Poikonen, Dept. Medical Microbiology, University of Oulu, kari.poikonen@oulu.fi
Ying Yan, Dept. Medical Microbiology, University of Oulu, ying.yan@oulu.fi
Taina Korhonen, Dept. Medical Microbiology, University of Oulu, takorhon@mail.student.oulu.fi
Pirkka Vikatmaa, Helsinki University Hospital, pirkka.vikatmaa@hus.fi

Other researchers of the project:
Maija Leinonen, National Public Health Institute, maija.leinonen@ktl.fi
Sylvi Silvennoinen-Kassinen, Dept. Medical Microbiology, University of Helsinki, sylvi.silvennoinen-kassinen@oulu.fi
Mauri Lepäntalo, Helsinki University Hospital, mauri.lepantalo@hus.fi
Tuija Ikonen, Tampere University Hospital, tuija.ikonen@helsinki.fi

Key words: atherosclerosis, inflammation, Chlamydia pneumoniae, pathogen burden, genetic susceptibility

Results

In our research we concentrated mainly on C. pneumoniae, studied its presence in patient groups with occlusive and aneurysmal atherosclerosis, markers of innate immunity and the effect of their genetic polymorphism on chlamydial growth and infection. Due to numerous parameters measured and ongoing statistical survey, only some results of various combinations can be mentioned.
The study population consisted of patients with carotid artery disease (CAR), with abdominal aortic aneurysm (AAA), with aortic occlusive disease (ASO) and 393 healthy Finnish blood donors as controls

1. The presence of C. pneumoniae in atherosclerotic plaques.
Quite recently there have been attacks against the earlier reports on the presence of C. pneumoniae in atherosclerotic plaques, pointing to the possibility that especially PCR findings are vulnerable for contaminations. There were 99 patients, who had all the C. pneumoniae measurements for chronic infection done. Persistent C. pneumoniae infection was defined by anti- C. pneumoniae IgA titre, positive PCR result from the over 1000 C. pneumoniae PCR experiments, positive C. pneumoniae in situ hybridization result, and chlamydial LPS serum level of 2nd quartile or higher (_ 69,8 ng/ml).  When these markers of C. pneumoniae positivity were combined, 1.6 % of the patients were C. pneumoniae negative, 29.3 %  were positive for 1 marker, 42.4 %  were positive for 2 markers, 18.2 % were positive for 3 markers, and 1 % was positive for all of the 4 markers. Smaller pieces seem to be better starting material for DNA extraction than larger pieces. This may be due to lesser amount of human background DNA that possibly interferes with PCR. Using of several pieces cut from different parts of the atherosclerotic plaque may help, since C. pneumoniae is not evenly distributed in the plaques. It may also be advantageous to use freshly extracted DNA and RNase treatment and the PCR method need always to be nested. Electron microscopic studies verified our results. In all studied plaques forms resembling chronic chlamydial cells were found and they stained in immune electron microscopy with antichlamydial antibodies. Notably, other bacteria were absent in these EM preparations.

2. Individual differences in C. pneumoniae inclusion production
Adherent human mononuclear cells were isolated from buffy coats of 281 healthy blood donors and after growing for two weeks, infected with C. pneumoniae K7 strain, and further incubated for 76 h. The production of C. pneumoniae inclusions was highly variable, ranging individually from 0 to 3000 inclusions per culture. The presence of elevated anti- C. pneumoniae IgA (titre =40, p for trend = 0.051, X2-test) or IgG (titre = 128, p for trend = 0.048) or both associated positively with inclusion production, the p-value for this trend being 0.009 (X2-test). In the highest quartile, men produced more inclusions than women (mean ±S.E., 744±73 vs. 524±62 inclusions, respectively p=0.025). Some of the infected cell cultures were harvested after the 76 h incubation for DNA extraction. The amount of C. pneumoniae genomes and the number of human genomes in the culture was measured using a quantitative real-time PCR. The production of Chlamydia pneumoniae in cultivated human macrophages, measured as C. pneumoniae genomes/ human genome (Cpn/human), correlated with donors concentrations of chlamydial LPS (positively, p =0.035) and soluble CD14-recptor (negatively) in serum. The importance of CD14 tt genotype in C. pneumoniae is currently under debate. We noted highest production in cells with tt-genotype (p=0.018), setting the debate. The sensitivity of macrophages for production of C. pneumoniae in vitro was in accordance with antibodies and LPS concentrations in vivo.  Since macrophages were cultured in identical conditions, the noticed gender difference is suggested not to be due to different homeostasis in vivo, like hormonal status or levels of iron.

3. Marker molecules.
Serum concentrations of interleukin 6 (IL-6), C-reactive protein (CRP), soluble CD14 (sCD14) LPS-binding protein (LBP) and levels of anti- human heat-shock protein 60 (hHsp60) IgA and IgG antibodies were measured. All marker molecules were elevated in patient groups, but there were differences between gender and various disease groups. Nearer statistical calculations are under way.

4. Gene polymorphism.
Gene polymorphisms of interleukin-6 (IL-6), lipopolysaccharide binding protein (LBP), CD14, toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), and apolipoprotein E (ApoE) genes were studied.  There were relatively more anti- C. pneumoniae IgG positive individuals among the carriers of the LPB genotype Phe/Phe than among the carriers of the genotype Leu/Phe (p=0.011). Median LBP concentrations in controls for the genotypes were: 10.2 µg/ml for the Phe/Phe, 14.5 µg/ml for the Leu/Phe, and 7.1 µg/ml for the Leu/Leu, p < 0,001. In the AAA group, there were relatively more carriers of the CD14 genotypes TT and CT than in the control group, p=0.039. In the CAR group, there were relatively more C. pneumoniae DNA positives among the carriers of E4 than among the non-carriers (p=0.037). In the CAR group, there were relatively more C. pneumoniae in situ positives among the carriers of E4 than among the non-carriers (p=0.019).  Other statistically significant differences were not found.

5. Conclusions
The results shown in here are results of rather preliminary statistical analysis. Multifactorial models will be applied in order to find out which associations remain statistically significant when other factors are taken into account. Association with different histological pictures has also so far not been done.  Results of this study suggest that C. pneumoniae  infections are associated with atherosclerotic diseases. Negative results in some laboratories seem to be due to the difficulties in diagnosis of the agent. This study also implies that presence of this infectious agent promotes inflammatory and autoimmune reactions and may thus promote pathogenesis of atherosclerosis. Many of the polymorphisms studied here were associated with markers of infection, and with serum levels of the other marker molecules studied here. The collected material has already partly been studied for dental pathogens, with positive results. For further studies a Finnish C. pneumoniae strain has been sequenced, chip technology and proteomics have been started.

Selected publications:

Sävykoski T, Harju T, Paldanius M, Kuitunen H, Bloigu A, Wahlström E, Rytilä P, Kinnula V, Saikku P, Leinonen M. Chlamydia pneumoniae infection and inflammation in adults with asthma. Respiration, 71:120-5, 2004

Erkkilä L, Laitinen K, Haasio K, Tiirola T, Jauhiainen M, Lehr HA, Aalto-Setälä K, Saikku P, Leinonen M. Heat shock protein 60 autoimmunity and early lipid lesions in chholesterol-fed C57BL/6Jbom mice during Chlamydia pneumoniae infection. Atherosclerosis, 177:321-328, 2004

Yan Y, Silvennoinen-Kassinen S, Leinonen M, Saikku P. Methodological aspects affecting the infectivity of Chlamydia pneumoniae in cell cultures in vitro. J Microbiol Methods. Apr;61(1):127-30, 2005

Törmäkangas L, Erkkilä L, Korhonen T, Tiirola T, Bloigu A, Saikku P, Leinonen M. Effects of repeated Chlamydia pneumoniae inoculations on aortic lipid accumulation and inflammatory response in C57BL/6J mice. Infec Immun 73:6458-6466, 2005

Yan Y, Silvennoinen-Kassinen S, Leinonen M, Saikku P. Inhibitory effect of heparan sulfate-like glycosaminoglycans on the infectivity of Chlamydia pneumoniae in HL cells varies between strains. Microbes Infect. 2006 Jan 17;

Harju T, Leinonen M, Nokso-Koivisto J, Korhonen T, Räty R, He Q, Hovi T, Mertsola J, Bloigu A, Rytilä P, Saikku P. Infection load in stable asthma: Non-random distribution of pathogenic bacteria and viruses in induced sputum or pharyngeal secretions of adults with stable asthma. Thorax 2006, (in press)

An abstract of the research plan (January 2003)