Genotypic and phenotypic characterization of carriage and invasive disease isolates of Neisseria meningitidis in Finland.

The relationship between carriage and the development of invasive meningococcal disease is not fully understood. We investigated the changes in meningococcal carriage in 892 military recruits in Finland during a nonepidemic period (July 2004 to January 2006) and characterized all of the oropharyngeal meningococcal isolates obtained (n = 215) by using phenotypic (serogrouping and serotyping) and genotypic (porA typing and multilocus sequence typing) methods. For comparison, 84 invasive meningococcal disease strains isolated in Finland between January 2004 and February 2006 were also analyzed. The rate of meningococcal carriage was significantly higher at the end of military service than on arrival (18% versus 2.2%; P < 0.001). Seventy-four percent of serogroupable carriage isolates belonged to serogroup B, and 24% belonged to serogroup Y. Most carriage isolates belonged to the carriage-associated ST-60 clonal complex. However, 21.5% belonged to the hyperinvasive ST-41/44 clonal complex. Isolates belonging to the ST-23 clonal complex were cultured more often from oropharyngeal samples taken during the acute phase of respiratory infection than from samples taken at health examinations at the beginning and end of military service (odds ratio [OR], 6.7; 95% confidence interval [95% CI], 2.7 to 16.4). The ST-32 clonal complex was associated with meningococcal disease (OR, 17.8; 95% CI, 3.8 to 81.2), while the ST-60 clonal complex was associated with carriage (OR, 10.7; 95% CI, 3.3 to 35.2). These findings point to the importance of meningococcal vaccination for military recruits and also to the need for an efficacious vaccine against serogroup B isolates.

Association of IL-6 and IL-6R gene polymorphisms with susceptibility to respiratory tract infections in young Finnish men.

Interleukin-1 (IL-6) is an important mediator of inflammatory response in the respiratory tract during an infection, and the action of IL-6 is mediated by an IL-6 receptor. Several polymorphisms in the IL-6 and IL-6R genes have been associated with different inflammatory disease states. We studied the association between 2 IL-6 (IL6A and IL6B) and 5 IL-6R gene polymorphisms (IL6R1 to IL6R5) and respiratory infections in 511 Finnish military recruits whose respiratory infectious episodes were followed during 6 months of service. A promoter polymorphism of the IL-6R gene, IL6R1 (-183G/A), and two intron 1 polymorphisms, IL6R2 (A/G) and IL6R3 (T/A), were associated with infections. The strongest associations were found for the IL6R1 and IL6R2 polymorphisms, which were in the same linkage disequilibrium block. Conscripts with the A/A (IL6R1), G/G (IL6R2), and A/A (IL6R3) genotypes had an increased risk for respiratory infections during service as follows: odds ratio (OR) 1.72, 95% confidence interval (CI) 1.35-2.19; OR 1.66, 95% CI 1.23-2.26; and OR 1.23, 95% CI 0.98-1.55, respectively. IL-6 gene polymorphism IL6A (-174C/G) was associated with infections only in combination with an IL-6R polymorphism. Our data suggest that polymorphisms in the 5' area of the IL-6R gene may be associated with increased susceptibility to respiratory infections.

Low mannose-binding lectin levels and MBL2 gene polymorphisms associate with Chlamydia pneumoniae antibodies.

OBJECTIVE: Mannose-binding lectin (MBL) has been shown to inhibit infection of host cells by Chlamydia pneumoniae in vitro. We studied if MBL levels and MBL2 polymorphisms associate with the presence of C. pneumoniae antibodies in vivo. MATERIALS AND METHODS: Single nucleotide polymorphisms (SNPs) of the MBL2 gene (promoter alleles H/L, X/Y and P/Q; and exon 1 variant alleles B, C and D and wild-type allele A) were genotyped and serum MBL concentrations and C. pneumoniae IgG, IgA and IgM antibodies were analysed in 889 Finnish military recruits. RESULTS: An MBL level below the median concentration and the MBL2 P/P genotype were significant risk factors of IgG or IgA seroconversions or the presence of IgM antibodies during military service (adjusted odds ratio (OR) 1.5; 95% confidence interval (CI) 1.1-2.1 and OR 1.5; 95% CI 1.0-2.2, respectively). In addition, the promoter Y/Y (OR 1.6; 95% CI 1.1-2.3) and exon 1 variant allele genotypes (OR 1.4; 95% CI 1.0-2.0) were possibly associated with elevated antibodies. CONCLUSIONS: These results suggest, for the first time, that low serum MBL levels and MBL2 polymorphisms may associate with elevated C. pneumoniae antibodies and seroconversions and thus support the previous findings in vitro.

Pneumococcal carriage is more common in asthmatic than in non-asthmatic young men.

INTRODUCTION: The aim was to investigate the prevalence of oropharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Neisseria meningitidis and beta-haemolytic streptococci among asthmatic and non-asthmatic young Finnish men and to identify putative risk factors. OBJECTIVES: A total of 224 asthmatics and 668 non-asthmatic men (mean age 19.6 years) from two intakes of conscripts to the Kainuu Brigade, Finland in July 2004 and January 2005 were enrolled upon entering military service. METHODS: Oropharyngeal specimens were examined for bacteria by routine culture methods. All the participants filled in questionnaires concerning risk factors for asthma and respiratory infections. RESULTS: S. pneumoniae (48 cases, 5.4%), Group A streptococci (16, 1.8%), H. influenzae (45, 5.0%), M. catarrhalis (24, 2.7%) and N. meningitidis (20, 2.2%) were isolated from the 892 participants. Ten putative risk factors for oropharyngeal colonization (asthma, atopy, allergic rhinitis, smoking, current use of asthma medication, history of adeno/tonsillectomy, level of highly sensitive C-reactive protein, peak expiratory flow, results of a 12-min running test and body mass index) were evaluated. The only significant risk factor for S. pneumoniae carriage was asthma (OR, 2.04; 95% CI 1.12 to 3.72). CONCLUSIONS: Pneumococcal carriage is more common in asthmatic than in non-asthmatic young men.

Rapamycin can inhibit the development of Chlamydia pneumoniae, which might partly contribute to the prevention of in-stent restenosis.

BACKGROUND: Rapamycin, an immunosuppressive and antiproliferative drug, is used to prevent neointima formation to reduce the risk of in-stent restenosis with rapamycin eluting-stents. Chronic infection of Chlamydia pneumoniae has been associated with cardiovascular diseases, and could play an important role in the mechanism of restenosis. We examined the effect of rapamycin on the growth of C. pneumoniae in cell cultures. METHODS: HL cell monolayers were inoculated with C. pneumoniae CWL029 or C. trachomatis L2. Different concentrations of rapamycin were present in the culture medium continuously or for 8-hour periods. After incubation the infected cells were repassaged to fresh HL cell monolayers and incubated in the medium without rapamycin. The newborn inclusions from both passages were checked by fluorescent microscope or electron microscope. RESULTS: The presence of 23 microg/ml rapamycin restricted over 90% of the growth of C. pneumoniae. Continuous presence of 11 microg/ml rapamycin inhibited the growth of C. pneumoniae up to 80% and caused smaller inclusions, fewer chlamydial particles and fewer matured EBs. 11 microg/ml rapamycin presented in first passage caused the reduction of C. pneumoniae to 57% at first passage and 24% at second passage. CONCLUSIONS: Sufficient rapamycin can inhibit the growth of C. pneumoniae effectively, but it should be applied at the early stage of the chlamydial infections. Rapamycin eluting-stents can induce a high enough local concentration of rapamycin. This provides a possibility for us to suppose that the beneficial effect of rapamycin in preventing in-stent restenosis might partly be explained by its inhibitory effects on the growth of C. pneumoniae.

Quantification of Chlamydia pneumoniae in cultured human macrophages and HL cells: comparison of real-time PCR, immunofluorescence and ELISA methods.

Chlamydia pneumoniae is an intracellular gram-negative bacterium, which replicates only in eukaryotic cells. Quantification of C. pneumoniae in cell culture is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally, this has been performed by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary, and multiple inclusions are also seen. Therefore, methods are needed to quantify exact amounts of C. pneumoniae in cells. Here, we describe a new method based on the real-time PCR quantification of chlamydial genomes adjusted to the number of human genomes in cultures. In human epithelial (HL) cell cultures, the C. pneumoniae inclusion numbers and the ratio of C. pneumonia genomes/human genome (Cpn/Hum) correlated significantly (r = 0.978, p < 0.001); thus with HL cells, both methods are usable. However, in macrophage cultures, the correlation was weaker (r = 0.133, p = 0.036) and we recommend PCR quantification for exact measurements.

Smoking status interacts with the association between mannose-binding lectin serum levels and gene polymorphism and the carriage of oropharyngeal bacteria.

Mannose-binding lectin (MBL) role in the carriage of oropharyngeal bacteria is not known. We investigated the association of smoking, MBL2 polymorphisms, and MBL concentrations with oropharyngeal carriage of respiratory bacteria in young men. Oropharyngeal specimens, MBL concentrations, and MBL2 gene polymorphisms were measured in 124 asthmatic and 394 nonasthmatic Finnish military recruits. The carriage rates of S. pneumoniae (p = 0.002), N. meningitidis (p = 0.005), and beta-hemolytic streptococci (p < 0.001) throughout the military service were significantly higher among smokers than in nonsmokers. An MBL level below the median proved to be a significant risk factor for the carriage of N. meningitidis (odds ratio [OR] = 1.9; 95% confidence interval [CI] 1.0-3.6) and beta-hemolytic streptococci (OR = 2.0; 95% CI 1.2-3.2) in the nonsmokers and a borderline significant risk factor for the carriage of S. pneumoniae (OR = 1.5; 95% CI 0.9-2.6), whereas low MBL levels producing MBL2 haplotypes (LXA/LXA, LXA/O, HYA/O, LYA/O, O/O) seemed to be associated with the carriage of N. meningitidis (OR = 1.8; 95% CI 1.0-3.4) and S. pneumoniae (OR = 1.6; 95% CI 0.9-2.7). Thus, MBL deficiency may predispose nonsmokers to oropharyngeal carriage of these bacteria. We hypothesize that the major factor contributing to elevated bacterial carriage in smokers might be increased bacterial adherence to epithelial cells, which obscures the effect of MBL.

Cold temperature and low humidity are associated with increased occurrence of respiratory tract infections.

OBJECTIVE: The association between cold exposure and acute respiratory tract infections (RTIs) has remained unclear. The study examined whether the development of RTIs is potentiated by cold exposure and lowered humidity in a northern population. METHODS: A population study where diagnosed RTI episodes, outdoor temperature and humidity among conscripts (n=892) were analysed. RESULTS: Altogether 643 RTI episodes were diagnosed during the follow-up period. Five hundred and ninety-five episodes were upper (URTI) and 87 lower (LRTI) RTIs. The mean average daily temperature preceding any RTIs was -3.7+/-10.6; for URTI and LRTI they were -4.1+/-10.6 degrees C and -1.1+/-10.0 degrees C, respectively. Temperature was associated with common cold (p=0.017), pharyngitis (p=0.011) and LRTI (p=0.048). Absolute humidity was associated with URTI (p<0.001). A 1 degrees C decrease in temperature increased the estimated risk for URTI by 4.3% (p<0.0001), for common cold by 2.1% (p=0.004), for pharyngitis by 2.8% (p=0.019) and for LRTI by 2.1% (p=0.039). A decrease of 1g/m(-3) in absolute humidity increased the estimated risk for URTI by 10.0% (p<0.001) and for pharyngitis by 10.8% (p=0.023). The average outdoor temperature decreased during the preceding three days of the onset of any RTIs, URTI, LRTI or common cold. The temperature for the preceding 14 days also showed a linear decrease for any RTI, URTI or common cold. Absolute humidity decreased linearly during the preceding three days before the onset of common cold, and during the preceding 14 days for all RTIs, common cold and LRTI. CONCLUSIONS: Cold temperature and low humidity were associated with increased occurrence of RTIs, and a decrease in temperature and humidity preceded the onset of the infections.

Mannose-binding lectin concentrations, MBL2 polymorphisms, and susceptibility to respiratory tract infections in young men.

BACKGROUND: Mannose-binding lectin (MBL) is an important component of innate immunity, and its deficiency is associated with susceptibility to recurrent infections. METHODS: This exploratory study investigated the association of serum MBL concentrations and MBL2 gene polymorphisms with respiratory tract infections in young men. We genotyped 6 single-nucleotide polymorphisms (SNPs) in the promoter region (alleles H/L, X/Y, and P/Q) and exon 1 (variant alleles B, C, and D and wild-type allele A) of the MBL2 gene by real-time polymerase chain reaction and measured serum MBL concentrations in 111 Finnish military recruits with asthma and 362 without. RESULTS: An MBL level below the median concentration was a significant risk factor for infections (asthma status-adjusted odds ratio [OR], 2.5 [95% confidence interval {CI}, 1.4-4.5]). Among the 6 SNPs, there was a significant association between the promoter Y/Y genotype and infections (OR, 2.3 [95% CI, 1.2-4.4]) and a borderline significant association between exon 1 variant alleles and infections (OR, 1.7 [95% CI, 0.9-3.1]), after adjustment for asthma status. CONCLUSION: These preliminary results suggest, for the first time, an association between MBL level and respiratory tract infections in young men and a possible association between infections and MBL2 polymorphisms.

Selective cyclooxygenase inhibitors prevent the growth of Chlamydia pneumoniae in HL cells.

The effects of the selective cyclooxygenase (COX) inhibitors SC-560 and PTPBS were studied in Chlamydia pneumoniae-infected HL cell cultures. Chlamydia pneumoniae growth and viability were assessed by quantifying inclusions and re-passages. COX-1 and COX-2 mRNA expression in HL cells during chlamydial infection was quantified with real-time polymerase chain reaction. SC-560 (10 microg/mL) and PTPBS (18 microg/mL) completely inhibited the growth of C. pneumoniae and the effect was dose-dependent between 4-9 microg/mL and 2-16 microg/mL, respectively. Inclusion size was reduced from 11.5+/-1.3 microm to 1.9+/-0.7 microm in the presence of the drugs. Removing the drugs returned the size to normal and increased the number of inclusions. Selective COX inhibitors appear to have a chlamydiostatic but not chlamydiacidic effect; they inhibit the growth of C. pneumoniae in vitro but do not prevent infection or eradicate C. pneumoniae from host cells.

Risk factors for acute respiratory tract illness in military conscripts.

BACKGROUND AND OBJECTIVE: Acute respiratory tract infections are the leading cause of missed service days among military conscripts. The aim of this study was to identify factors that possibly predicted and contributed to frequent respiratory tract infections among military conscripts. METHODS: Data on episodes of respiratory illness were collected during the 180-day period of military service in Kajaani, Finland, between July 2004 and July 2005. RESULTS: There were 518 military conscripts recruited, 124 of whom had a diagnosis of asthma. Conscripts with frequent (three or more) infections were more often atopic or suffered from allergic rhinitis or asthma. Overweight (BMI >/= 25 kg/m(2)) and previous respiratory tract infections were the two independent risk factors for frequent respiratory infections. Overall, 4.8% of those who had no risk factors, 10. 3% of those with one risk factor and 35.7% of those with two risk factors suffered from frequent respiratory infections (P for trend < 0.001). CONCLUSIONS: Overweight (BMI >/= 25 kg/m(2)) and previous respiratory tract infections are risk factors for frequent respiratory tract infections in young men during military service.

Training improves physical fitness and decreases CRP also in asthmatic conscripts.

To study the respiratory and physical health of young men, 224 asthmatic and 668 non-asthmatic military conscripts were recruited from the intake groups of July 2004 and January 2005 in Kajaani, Finland. Factors affecting respiratory health were elicited by a questionnaire at the beginning of the service, and results of high sensitive C-reactive protein (hsCRP) determination, peak expiratory flow (PEF), and 12-minute running test were collected at the beginning and the end of the service. Respiratory infections were diagnosed by a study physician. Upon entering military service, asthmatics had frequent exercise- and cold-related asthma symptoms (69.6% and 76.3%), and 48% of them had no medication for asthma. At the beginning, 25.8% of asthmatics and 19.1% of non-asthmatics had a poor result of less than 2,200 m (p = 0.05) in the 12-minute running test, and after 180 to 362 days of service, the corresponding percentages were 11.7% and 9.7% (p = 0.434). The levels of hsCRP, a marker of low-grade systemic inflammation, decreased significantly among both asthmatics, 1.5 (p = 0.001), and non-asthmatics, 1.6 mg/L (p < 0.001). Asthmatic men had 0.2 and non-asthmatics 0.1 respiratory infections per month (p < 0.001). In summary, asthmatic conscripts can enhance their physical fitness by training similarly to non-asthmatic ones. Their levels of hsCRP also decrease.

Asthmatic persons are prone to the persistence of Chlamydia pneumoniae antibodies.

Acute Chlamydia pneumoniae infection may initiate asthma or worsen asthmatic symptoms. In crowded conditions, such as military service, young men are susceptible to respiratory infections, including C. pneumoniae. We recruited 127 asthmatic and 391 nonasthmatic military conscripts, followed up their respiratory tract infections and the kinetics of serum C. pneumoniae antibodies, and assessed the association between C. pneumoniae and asthma during 6 months of military service in 2 intake groups. During the 6-month period, in the July intake group, IgG antibody prevalence decreased from 60.3% to 43.8% in asthmatic and from 55.6% to 22.6% in nonasthmatic conscripts. In the January intake group, IgG antibody prevalence increased from 38.3% to 48.4% in asthmatic and from 37.2% to 43% in nonasthmatic recruits. IgG and IgA antibodies persisted more often in the asthmatic groups. In conclusion, the prevalence of IgG antibodies showed seasonal variation. Military recruits seem to be most vulnerable to C. pneumoniae infections during the period from January to June. The antibody titer changes were more rapid than previously thought.

Chlamydial lipopolysaccharide is present in serum during acute coronary syndrome and correlates with CRP levels.

Infections, Chlamydia pneumoniae as a major candidate, have been suggested to participate in inflammatory processes ultimately leading to atherosclerosis. In the present study we measured serum levels of chlamydial lipopolysaccharide (cLPS) and highly sensitive C-reactive protein (hsCRP) in the acute coronary syndrome (ACS) patients (n=145). During ACS, both cLPS and hsCRP were elevated and significant correlation (P=0.003, r=0.25) between them was observed. Both cLPS and hsCRP levels decreased after the event and correlation remained significant during the follow-up period. Our results suggest that cLPS is liberated from the damaged tissue persistently infected with C. pneumoniae during the ACS event. The significant correlation between cLPS and hsCRP levels further point to the possibility that both levels reflect the magnitude of tissue damage.

Rapid genotyping of lipopolysaccharide-binding protein (LBP) C(1341)-->T (Leu(436)-->Phe) polymorphism by LightCycler real-time PCR.

Lipopolysaccharide (LPS) is an exdotoxin found in the outer membrane of gram-negative bacteria. In circulation, LPS is bound by LPS-binding protein (LBP), which participates in cell activation by transferring LPS to CD14 and Toll-like receptor 4. A high LPS concentration may give rise to an exaggerated immune response, which may lead to septic shock during septicemia. However, LBP also neutralizes and removes LPS by transferring it to plasma lipoproteins. Recently, the presence of an amino acid-changing polymorphism in the LBP gene was reported, which, in men, was associated with sepsis and its severity and with myocardial infarction. Here, we describe a new LightCycler real-time PCR method for genotyping this LBP C(1341)-->T (Leu(436)-->Phe) polymorphism. In our study population of 393 Finnish blood donors, the genotype frequencies were: 86% TT, 13% CT and 1% CC.

The association between C-reactive protein levels and depression: Results from the northern Finland 1966 birth cohort study.

BACKGROUND: To investigate whether depressive episodes (previous, current single, and recurrent) are associated in both genders with highly sensitive C-reactive protein (hs-CRP) levels, earlier recommended for risk assessment of cardiovascular disease. The impact of the severity of current single and recurrent depressive episodes on this putative association was also investigated. METHODS: The genetically homogeneous Northern Finland 1966 Birth Cohort was followed until age 31, when, in a cross-sectional setting (n = 5269), the highly sensitive enzyme immunoassay (hs-EIA) method was used to measure CRP concentration. Depressive episodes were defined through mailed questionnaires, including Hopkins Symptom Checklist-25 (HSCL-25) and information on self-reported, doctor-diagnosed depression. RESULTS: After adjusting for confounders, logistic regression analyses showed that in male subjects, elevated hs-CRP levels (> or =1.0 mg/L) increased the probability for severe current and recurrent depressive episodes 1.7-fold and 3.1-fold, respectively. Correspondingly, an hs-CRP level of >3.0 mg/L increased the probability for recurrent depression up to 4.1-fold. In female subjects, no statistically significant associations were found. CONCLUSIONS: Our results support the hypothesis that an activation of systemic inflammatory processes may contribute to the pathophysiology of severe depression in men. Further investigations are needed regarding the impact of our findings on diagnostic/treatment strategies concerning severe and, especially recurrent, depression in men.

Inhibitory effect of heparan sulfate-like glycosaminoglycans on the infectivity of Chlamydia pneumoniae in HL cells varies between strains.

Glycosaminoglycans are known to participate in the attachment of several chlamydial strains. We studied the effect of heparin, enoxaparin, low-molecular-weight heparin, chondroitin sulfate A, and heparinase I on the infectivity of Chlamydia pneumoniae strain CWL029 and two Finnish isolates, Kajaani 7 and Parola, in an HL cell line which is epithelial in origin. Two Chlamydia trachomatis strains, L2 and E, were used for comparison. The infectivity of all C. pneumoniae strains and C. trachomatis serovar E was inhibited not only by heparin derivatives but also by chondroitin sulfate A and heparinase treatment. Treatment of host cells with heparin derivatives and heparinase was also inhibitory. Different chlamydial strains and species seem, however, to vary in their ability to use heparin in their attachment to host cells.

Novel enzyme immunoassay utilizing lipopolysaccharide-binding protein as a capture molecule for the measurement of chlamydial lipopolysaccharide in serum.

Chlamydia pneumoniae causes respiratory tract infections. It has a tendency to cause persistent infections, which have been associated with several chronic diseases (e.g., atherosclerosis). At present, there is no reliable method for the diagnosis of chronic C. pneumoniae infection. We developed a novel enzyme immunoassay (EIA) for the quantification of chlamydial lipopolysaccharide (cLPS) in human serum. Serum cLPS was solubilized with detergent and then captured by LPS-binding protein (LBP). LBP-LPS complexes were bound to the solid phase with anti-cLPS monoclonal antibody, and the bound complexes were detected with anti-LBP antibodies. The new method was used to quantify serum cLPS in acute coronary syndrome (ACS) patients (n = 102) and their healthy controls. cLPS was detected in 77.5% of ACS patients and in 52% of controls (P < .001) with geometric mean concentrations of 1.87 and 0.61 microg/mL (P < .001), respectively. The novel cLPS EIA method will provide a potential diagnostic tool for C. pneumoniae infection.

Methodological aspects affecting the infectivity of Chlamydia pneumoniae in cell cultures in vitro.

The effect of culture medium, washing, temperature, and sonication on the infectivity of Chlamydia pneumoniae for HL cells in experiments with heparin or heparinase treatment was studied. The presence of fetal calf serum (FCS) was necessary, and simple washing of elementary bodies increased their infectivity titer. Prolonged sonication was detrimental. Detailed description of the experimental conditions is necessary in studies of chlamydial attachment and infectivity.