Tanja Pessi

Atherosclerosis is an inflammatory disease with possible contributions from bacterial antigens. We aimed to investigate the role of oral bacteria as inducers of inflammatory cascades in smooth muscle cells from carotid endarterectomy patients (AthSMCs) and healthy controls (HSMCs). Inactivated Streptococcus mitis, S. sanguinis, S. gorgonii, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were used to stimulate inflammation in HSMCs and AthSMCs. Tumor necrosis factor-α (TNFα) was used as a positive control in all stimulations. Interleukin-6 (IL-6) levels were determined from cell culture supernatants and microRNA expression profiles from cells after 24 h of bacterial stimulation. Genome wide expression (GWE) analyses were performed after 5 h stimulation. All studied bacteria induced pro inflammatory IL-6 production in both SMCs. The most powerful inducer of IL-6 was A. actinomycetemcomitans (p < 0.001). Of the 84 studied miRNAs, expression of 9 miRNAs differed significantly (p ≤ 0.001) between HSMCs and AthSMCs stimulated with inactivated bacteria or TNFα. The data was divided into two groups: high IL-6 producers (A. actinomytectemcomititans and TNFα) and low IL-6 producers (streptococcal strains and P. gingivalis). The expression of 4 miRNAs (miR-181-5p, −186-5p, −28-5p and −155-5p) differed statistically significantly (p < 0.001) between healthy HSMCs and AthSMCs in the low IL-6 producer group. According to multidimensional scaling, two gene expression clusters were seen: one in HSMCs and one AthSMCs. Our results suggest that inactivated oral bacteria induce inflammation that is differently regulated in healthy and atherosclerotic SMCs.

The liver is the first line of defence against continuously occurring influx of microbial-derived products and bacteria from the gut. Intestinal bacteria have been implicated in the pathogenesis of alcoholic liver cirrhosis. Escape of intestinal bacteria into the ascites is involved in the pathogenesis of spontaneous bacterial peritonitis, which is a common complication of liver cirrhosis. The association between faecal bacterial populations and alcoholic liver cirrhosis has not been resolved. Relative ratios of major commensal bacterial communities (Bacteroides spp., Bifidobacterium spp., Clostridium leptum group, Enterobactericaea and Lactobacillus spp.) were determined in faecal samples from post mortem examinations performed on 42 males, including cirrhotic alcoholics (n = 13), non-cirrhotic alcoholics (n = 15), non-alcoholic controls (n = 14) and in 7 healthy male volunteers using real-time quantitative PCR (RT-qPCR). Translocation of bacteria into liver in the autopsy cases and into the ascites of 12 volunteers with liver cirrhosis was also studied with RT-qPCR. CD14 immunostaining was performed for the autopsy liver samples. Relative ratios of faecal bacteria in autopsy controls were comparable to those of healthy volunteers. Cirrhotics had in median 27 times more bacterial DNA of Enterobactericaea in faeces compared to the healthy volunteers (p = 0.011). Enterobactericaea were also the most common bacteria translocated into cirrhotic liver, although there were no statistically significant differences between the study groups. Of the ascites samples from the volunteers with liver cirrhosis, 50% contained bacterial DNA from Enterobactericaea, Clostridium leptum group or Lactobacillus spp.. The total bacterial DNA in autopsy liver was associated with the percentage of CD14 expression (p = 0.045). CD14 expression percentage in cirrhotics was significantly higher than in the autopsy controls (p = 0.004). Our results suggest that translocation of intestinal bacteria into liver may be involved as a one factor in the pathogenesis of alcoholic liver cirrhosis.

We recently reported that large amounts of oral bacterial DNA can be found in thrombus aspirates of myocardial infarction patients. Some case reports describe bacterial findings in pericardial fluid, mostly done with conventional culturing and a few with PCR; in purulent pericarditis, nevertheless, bacterial PCR has not been used as a diagnostic method before. To find out whether bacterial DNA can be measured in the pericardial fluid and if it correlates with pathologic-anatomic findings linked to cardiovascular diseases. Twenty-two pericardial aspirates were collected aseptically prior to forensic autopsy at Tampere University Hospital during 2009-2010. Of the autopsies, 10 (45.5%) were free of coronary artery disease (CAD), 7 (31.8%) had mild and 5 (22.7%) had severe CAD. Bacterial DNA amounts were determined using real-time quantitative PCR with specific primers and probes for all bacterial strains associated with endodontic disease (Streptococcus mitis group, Streptococcus anginosus group, Staphylococcus aureus/Staphylococcus epidermidis, Prevotella intermedia, Parvimonas micra) and periodontal disease (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatus, and Dialister pneumosintes). Of 22 cases, 14 (63.6%) were positive for endodontic and 8 (36.4%) for periodontal-disease-associated bacteria. Only one case was positive for bacterial culturing. There was a statistically significant association between the relative amount of bacterial DNA in the pericardial fluid and the severity of CAD (p=0.035). Oral bacterial DNA was detectable in pericardial fluid and an association between the severity of CAD and the total amount of bacterial DNA in pericardial fluid was found, suggesting that this kind of measurement might be useful for clinical purposes.

Background—: Infectious agents, especially bacteria and their components originating from the oral cavity or respiratory tract, have been suggested to contribute to inflammation in the coronary plaque, leading to rupture and the subsequent development of coronary thrombus. We aimed to measure bacterial DNA in thrombus aspirates of patients with ST-segment–elevation myocardial infarction and to check for a possible association between bacteria findings and oral pathology in the same cohort. Methods and Results—: Thrombus aspirates and arterial blood from patients with ST-segment–elevation myocardial infarction undergoing primary percutaneous coronary intervention (n=101; 76% male; mean age, 63.3 years) were analyzed with real-time quantitative polymerase chain reaction with specific primers and probes to detect bacterial DNA from several oral species and Chlamydia pneumoniae. The median value for the total amount of bacterial DNA in thrombi was 16 times higher than that found in their blood samples. Bacterial DNA typical for endodontic infection, mainly oral viridans streptococci, was measured in 78.2% of thrombi, and periodontal pathogens were measured in 34.7%. Bacteria-like structures were detected by transmission electron microscopy in all 9 thrombus samples analyzed; whole bacteria were detected in 3 of 9 cases. Monocyte/macrophage markers for bacteria recognition (CD14) and inflammation (CD68) were detected in thrombi (8 of 8) by immunohistochemistry. Among the subgroup of 30 patients with myocardial infarction examined by panoramic tomography, a significant association between the presence of periapical abscesses and oral viridans streptococci DNA–positive thrombi was found (odds ratio, 13.2; 95% confidence interval, 2.11–82.5; P =0.004). Conclusions—: Dental infection and oral bacteria, especially viridans streptococci, may be associated with the development of acute coronary thrombosis.

Post mortem or even normal changes during life occurring in major gut bacterial populations are not known. We investigated Bacteroides sp., Bifidobacterium sp., Clostridium leptum, Clostridium coccoides, Streptococcus sp., Lactobacillus sp. and Enterobacteriacaea ratios in 7 fecal samples from healthy volunteers and in 61 autopsies rectum and cecum samples and studied the effect of post mortem time using quantitative real-time PCR. Bacterial ratios in stool samples from volunteers and rectum samples from autopsy cases were similar and did not change significantly up to 5 days post mortem. In cecum, significant post mortem time-dependent differences were observed in ratios of Bacteroides sp. (p = 0.014) and Lactobacillus sp. (p = 0.024). Our results showed that ratios of Bacteroides sp., Bifidobacterium sp., Clostridium leptum, Clostridium coccoides, Streptococcus sp., Lactobacillus sp. and Enterobacteriacaea can be investigated in autopsy rectum samples up to 5 days after death.

Use of combined oral contraceptives (COCs) is known to increase concentrations of C-reactive protein (CRP), an important predictor of cardiovascular disease. The inflammatory nature of the disease is well acknowledged. The aim of this study was to find out whether the metabolic, lifestyle and genetic determinants of CRP differ between women who use COCs and those who do not use any hormonal contraceptives (non-users). A total of 1,257 women (24-39 years) participated in the ongoing Cardiovascular Risk in Young Finns Study, a population based cross-sectional follow-up study. Use of hormonal contraceptives was determined by questionnaire. Plasma CRP and other cardiovascular risk factors were measured; five CRP gene polymorphisms were genotyped (-717A>G, -286C>T>A, +1059G>C, +1444C>T and +1846G>A) and CRP haplotypes were constructed. Multivariate regression analysis revealed that BMI and leptin were the main determinants of CRP in non-users, whereas in COC users the main determinants were BMI, leptin and triglycerides. The median CRP and triglyceride values were significantly higher in COC users than in non-users. The correlations between triglyceride and CRP were tested separately in different COC users in accordance with progestagen content and dosage, the analysis revealing significant association only in women using a high dosage of progestagen or cyproterone. The haplotypes of CRP gene had no significant association with CRP concentration in COC users, while independent effects on CRP were found in non-users. Our study suggests that use of COCs alters the metabolic determinants and genetic regulation of CRP.

Background: Both genetic and environmental factors, e.g. early childhood infections, have a role in the pathogenesis of atopic diseases. Objective: To examine simultaneously the strength and possible interactions of two known such factors, IL4 genetics and Helicobacter pylori infection, on the risk of atopy and asthma. Methods: Gene polymorphism analyses and skin prick tests (SPT) were determinedin 245 adult asthmatics and 405 nonasthmatic controls of population-based case-control study. SPTs were used as an indicator of atopy. H. pylori infection was verified by detecting anti-H. pylori IgG antibodies in sera. Results: A significant negative association was seen between the presence H. pylori antibodies and SPT positivity (≧1 positive reactions) in both asthmatics and controls (p = 0.002 and p = 0.025, respectively) but the effect of IL-4 polymorphism (SNP –590C/T) was nonsignificant in both groups (p = 0.071 and p = 0.072, respectively). However, IL4 genetics had an effect on susceptibility to H. pylori: asthmatics carrying the IL4 –590 allele T had a diminished risk to be H. pylori infected (OR 0.485 95%CI 0.287–0.819). This effect was not seen in controls. Logistic regression analysis indicated that H. pylori and IL4 effects on atopy risk are not interdependent. Conclusions: This study showed that the effect of H. pylori infection on atopy risk is stronger than that of IL4 genetics. There is no interaction between these factors on the pathogenesis of atopy suggesting that these factors have distinct immunopathogenetic mechanisms. However, the genetic effect may modify the role of infective agents by effecting on susceptibility to disease.

C-reactive protein (CRP) is a sensitive marker of inflammation induced by both IL-6 and IL-1. Thus, genetic variation in these genes could be associated with the variety in C-reactive protein levels, and therefore with the severity of the entire inflammatory response. Even a subtle elevation in baseline CRP levels in healthy individuals has been found to significantly increase the risk for cardiovascular diseases. Therefore, to find out the possible role of pro-inflammatory cytokines in CRP baseline regulation we conducted a study of 338 healthy blood donors whose CRP levels were determined and whose single nucleotide polymorphisms of IL1A(C/T)-889, IL1B(C/T)-511, IL1B(C/T) + 3954, IL6(G/C)-174 and ILRN (a VNTR) both genotyped and haplotyped. The data revealed an association between CRP levels and the IL1B + 3954 genotype. Also, the bilocus haplotype IL1B-511*1/IL1B + 3954*2 was more frequent in subjects with below median CRP levels (< 0.72 mg/l), and composite genotype analysis of IL1B-511/IL1B + 3954 supported this finding. Our findings suggest that in healthy people, basal CRP levels are regulated by IL1B but not by IL6 genetics.