Correlation between rises in Chlamydia pneumoniae-specific antibodies, platelet activation and lipid peroxidation after percutaneous coronary intervention.

We recently showed that Chlamydia pneumoniae activates platelets in vitro, with an associated oxidation of low-density lipoproteins. The aim of this study was to investigate whether C. pneumoniae is released during percutaneous coronary intervention (PCI) and, thereby, causes platelet activation and lipid peroxidation. Seventy-three patients undergoing coronary angiography and following PCI or coronary artery bypass graft (CABG) and 57 controls were included in the study. C. pneumoniae antibodies, serotonin and lipid peroxidation were measured before and 24 h, 1 month and 6 months after angiography. The results show that serum C. pneumoniae IgA concentrations were significantly higher in patients than in the controls. Furthermore, in 38% of the C. pneumoniae IgG positive patients, the C. pneumoniae IgG concentration increased 1 month after PCI. The levels of C. pneumoniae IgG antibodies 1 month after PCI correlated with plasma-lipid peroxidation (r = 0.91, P < 0.0001) and platelet-derived serotonin (r = 0.62, P = 0.02). There was no elevation in the total serum IgG 1 month after PCI. In conclusion, the present results suggest that PCI treatment of coronary stenosis releases C. pneumoniae from the atherosclerotic lesions, which leads to platelet activation and lipid peroxidation.

Expression of chemokines and adhesion molecules in human coronary artery endothelial cells infected with Chlamydia (Chlamydophila) pneumoniae.

Chlamydia pneumoniae has during recent years been associated with cardiovascular disease and atherosclerosis. Chemokines, leukocyte adhesion proteins and metalloproteinases are significant for chemotaxis and attachment of leukocytes to vessel walls, and for stability of atherosclerotic plaques. To determine the ability of C. pneumoniae to elicit inflammation in a relevant target host cell, we infected human coronary artery endothelial cells (HCAEC) with a clinical isolate of C. pneumoniae. Extracellular release of five chemokines, two adhesion proteins and a metalloproteinase was measured at different time points after infection using a cytometric bead assay and ELISA. Secretion of IL-8, MCP-1, MIG, IP-10 and ICAM-1 was significantly increased 48 h after C. pneumoniae infection of HCAEC in comparison with uninfected controls. Release of RANTES occurred already 6 h after infection. C. pneumoniae did not elicit release of E-selectin or MMP-1. We conclude that C. pneumoniae induces expression of proinflammatory components in HCAEC, which would promote migration of leukocytes towards endothelial cells. This suggests that C. pneumoniae initiates and propagates vascular inflammation in ways that contribute to coronary artery disease.

Leucocyte esterase testing of first-voided urine and urethral and cervical smears to identify Mycoplasma genitalium-infected men and women.

Leucocyte esterase (LE) in first-voided urine (FVU) and presence of leucocytes in urethral and cervical smears were evaluated to identify Mycoplasma genitalium infection in 416 men and 417 women attending Department of Genitourinary Medicine. M. genitalium was diagnosed in FVU specimens by realtime polymerase chain reaction. The prevalence of M. genitalium was 6.5% in women and 6.7% in men. In total, 88.5% (23/26) of M. genitalium-infected men were identified by a combination of urethral smear and the LE test. In women, the combination of urethral and/or cervical smears and/or a positive LE test identified 91.3% (21/23) of M. genitalium-infected patients. Organism load in FVU correlated significantly with presence of urethritis (> or =4 leucocytes per high-power field) in men. A combination of LE testing of urine and urethral and/or cervical smears can be used as screening tests to select patients for specific M. genitalium testing. By this strategy, about 10% of infected individuals will remain undetected.

Urine-based testing for Chlamydia trachomatis using polymerase chain reaction, leucocyte esterase and urethral and cervical smears.

The performance of Roche polymerase chain reaction (PCR) Amplicor to detect Chlamydia trachomatis in first-voided urine specimens from 422 males and 456 females attending two clinics for sexually transmitted infections was evaluated in comparison with cultures of urethral and cervical specimens. At the same time, the ability of leucocyte esterase (LE) in first-voided urine and the presence of leucocytes in urethral and cervical smears to identify C. trachomatis-infected individuals based on PCR and culture was determined. The prevalence of C. trachomatis infection was 10.9% in men and 7.7% in women. Sensitivity, specificity, positive predictive value and negative predictive value of Amplicor was 93.5%, 99.7%, 97.7% and 99.2% in males and 91.4%, 99.5%, 94.1% and 99.3% in females. All Chlamydia-infected men were identified by means of a combination of urethritis (4 leucocytes in the urethral smear) and/or a positive LE test in urine, although the specificity was only 42.2%. In women, the combination of urethritis and/or cervicitis and/or a positive LE test identified 85.7% of Chlamydia-infected patients with a specificity of 38.2%. It is concluded that a combination of urethral and/or cervical smears and LE testing of urine can be used as a screening test to select patients, especially males, for specific C. trachomatis testing.

Infection of human endothelial cells with Staphylococcus aureus induces transcription of genes encoding an innate immunity response.

Staphylococcus aureus is a gram-positive bacterium frequently isolated from patients with bloodstream infections. Endothelial cells (EC) play an important role in host defence against bacteria, and recent reports have shown that infection of EC with S. aureus induces expression of cytokines and cell surface receptors involved in activating the innate immune response. The ability of S. aureus to invade nonphagocytic cells, including EC, has been documented. However, the knowledge of the role of EC in pathogenesis of S. aureus infection is still limited. In this study, we investigate the gene-expression program in human EC initiated by internalized S. aureus, using microarray analysis. We found 156 genes that were differentially regulated at least threefold, using arrays representing 14,239 genes. Many of the upregulated genes code for proteins involved in innate immunity, such as cytokines, chemokines and cell adhesion proteins. Other upregulated genes encode proteins involved in antigen presentation, cell signalling and metabolism. Furthermore, intracellular bacteria survived for days without inducing EC death.

Clinical isolates of Staphylococcus aureus vary in ability to stimulate cytokine expression in human endothelial cells.

Human umbilical vein endothelial cells (HUVEC) were infected for 24 h with 18 well-characterized Staphylococcus aureus isolates, and the supernatants from infected HUVEC were analysed for interleukin (IL)-1beta, tumour necrosis factor-alpha, IL-6, IL-8, IL-10, IL-12p70, growth-related oncogene (GRO)-alpha, granulocyte macrophage colony-stimulating factor (GM-CSF) and regulated upon activation, normal T cell expressed and secreted (RANTES) by immunoassay. All staphylococcal isolates induced the expression of IL-6, IL-8, GRO-alpha, GM-CSF and RANTES. The magnitude of cytokine expression varied between isolates. Staphylococcus aureus inducing high expression of one of these cytokines also showed simultaneous high expression of the other four, indicating a common mechanism for the ability of individual S. aureus to induce expression of these cytokines. No direct correlation between cytokine expression and adhesion of S. aureus to HUVEC was observed, indicating that bacterial properties besides adhesion contribute to the activation of HUVEC.

Microorganisms and volatile organic compounds in airborne dust from damp residences.

Airborne dust samples from damp (n = 9) and control (n = 9) residences were analyzed for microorganisms (molds and bacteria), bacterial markers (3-hydroxy fatty acids and muramic acid), and adsorbed volatile organic compounds (VOCs). The number of mold species was greater in the damp residences than in the controls (23 vs.18) and nine mold species were found only in damp residences. The levels of 3-hydroxy fatty acids and muramic acid correlated better in damp residences than in controls, indicating that damp conditions affect the bacterial flora of airborne dust. Identifications made by culture and microscopy of the major molds found, i.e. Aspergillus, Cladosporium, and Penicillum, coincided with the identification of VOCs known to be produced by these species. A number of additional VOCs irritating to the skin, eyes, or respiratory tract were also found. The results from this pilot study illustrate the diversity of microorganisms and VOCs present in the indoor environment and suggest that analysis of airborne dust may help to assess human exposure to microorganisms and chemical compounds.

PLUNC (palate, lung and nasal epithelial clone) proteins in human nasal lavage fluid.

PLUNC (palate, lung and nasal epithelial clone) is a newly discovered gene that is expressed in the upper respiratory tract and is suggested to be of importance in host defence against bacteria. We have identified two forms of the PLUNC protein in human nasal lavage fluid (NLF) using two-dimensional gel electrophoresis (2-DE) and MS. The apparent molecular masses and isoelectric points of these forms are 24.8 kDa/pI 5.4 and 25.1 kDa/pI 5.5. Notably, the 24.8 kDa/pI 5.4 form of PLUNC is an abundant protein in the 2-DE protein patterns of NLF from healthy subjects. Decreased levels of PLUNC were found in NLF from smokers and workers exposed to reactive epoxy chemicals, indicating that long-term exposure to airway irritants impairs the production of PLUNC in the upper respiratory tract. We have also investigated the presence of lipopolysaccharide (LPS)-binding proteins in NLF. Five proteins were found to adsorb to a LPS-coated surface; two of these proteins correspond to the two PLUNC forms, as judged by 2-DE pattern matching. For comparison, human saliva was found to contain a set of LPS-binding proteins with similar 2-DE spot positions (the same pIs but somewhat lower apparent molecular masses of approximately 20 kDa). These results indicate that PLUNC may be a new marker of airway inflammation and may play a part in the innate immune response, and that human saliva contains yet other members of the family of LPS-binding proteins.

Chlamydia trachomatis-induced apoptosis occurs in uninfected McCoy cells late in the developmental cycle and is regulated by the intracellular redox state.

Infections with the obligate intracellular bacterium Chlamydia trachomatis are characterized by avoidance of fusion between chlamydia-containing endosomes and lysosomes, bacterial persistence and development of post-infectious sequelae. In this report we show that C. trachomatis induces apoptosis in McCoy and HeLa cells. Apoptosis was monitored by three different techniques; enzyme-linked immunoassay (EIA) of fragmented nucleosomes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and flow cytometry of propidium iodide-stained cells. Apoptosis occurred in uninfected cells, was induced late in the chlamydial developmental cycle, beyond 24 h post-infection and was dependent on bacterial protein synthesis. Apoptosis was not significantly increased in infected, inclusion-containing cells. Treatment of cells with the antioxidants ascorbic acid (10 microM) and alpha-tocopherol (10 microM) reduced the degree of apoptosis. These results suggest that host cells infected with C. trachomatis generate proapoptotic stimuli that induce apoptosis in uninfected, neighbouring cells and that the redox state of the cell is a regulator in chlamydia-induced apoptosis.

Anti-lactoferrin antibodies and other types of anti-neutrophil cytoplasmic antibodies (ANCA) in reactive arthritis and ankylosing spondylitis.

Fifty-five serum samples from patients with reactive arthritis (ReA), 40 from patients with ankylosing spondylitis (AS) and three from patients with chronic sacroiliac joint arthritis were analysed for the presence of ANCA of IgG class by means of enzyme immunosorbent assay using lactoferrin (Lf), myeloperoxidase (MPO) and antigen extracted from azurophil granules ('alpha-antigen') containing proteinase 3 (PR3) as substrate. IgG-ANCA were found in 31 (56%) patients with ReA. Twenty-three (42%) had anti-Lf antibodies, nine (16%) had anti-MPO and eight (15%) had anti-alpha-antigen antibodies, none of which reacted with PR3. Only six (14%) AS or sacroiliac joint arthritis patients had ANCA (P < 0.001). Three (7%) had anti-Lf, two (5%) anti-MPO and two (5%) anti-alpha-antigen antibodies. Yersinia and Salmonella bacteria were separated by SDS-PAGE and blots were incubated with serum from rabbits immunized with human Lf. The hyperimmune serum recognized a band of 78 kD from both bacteria which was not seen when preimmune serum was used. The reaction to the 78-kD antigen could be completely inhibited when anti-Lf antibodies were absorbed on Lf coupled to cyanogen bromide-activated Sepharose, possibly indicating cross-reacting epitopes in Lf and enterobacterial antigen.

Epidemiological typing of Streptococcus pneumoniae from various sources in Sweden and India using Box A PCR fingerprinting.

Epidemiological typing of Streptococcus pneumoniae is essential to determine strain relatedness and also to trace resistant clones. The novel Box A PCR assay was used for characterization of S. pneumoniae isolates from two Scandinavian countries and to compare those from India on the Asian continent. In addition, the assay was employed to determine the clonality of 25 pneumococcal strains from an outbreak in a day-care centre in Linköping, Sweden. All 25 showed a unique pattern with 100% homology for 24 of them, thereby establishing the clonal nature of the outbreak. The pneumococcal strains involved in the outbreak belonged to serotype 9 and were resistant to penicillin, with an MIC value of 2 mg/l. Thirty-eight genotypes were obtained when the Box A results were analysed by computer (Molecular Analyst Software with GelCompar). The discriminatory index of the method was D=0.98, which indicates excellent performance. No major segregation of strains from the different geographical locations was observed when a lower level of similarity was used for typing (80%, 13 types). However, at the level chosen for genotyping, 95% (38 genotypes) there was a clear geographical segregation. No correlation between genotype and serotype was seen as strains from a common place of origin were most often of different serotypes. Computer-assisted analysis of the results of Box A PCR typing facilitated the evaluation.

Localization of intracellular Ca2+ stores in HeLa cells during infection with Chlamydia trachomatis.

Chlamydia trachomatis elementary bodies (EBs) enter epithelial cells within membrane-bound endosomes that aggregate with each other in a calcium-regulated process, but avoid fusion with lysosomes. Annexin III but not I translocates to chlamydial aggregates and inclusions. In this study, we localize the intracellular Ca2+ stores during the course of infection by analyzing the distribution of three intracellular Ca2+ store proteins: calreticulin, type-1 inositol-1,4, 5-trisphosphate receptor (IP3-R), and Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase type 2 (SERCA2) in HeLa cells infected with C. trachomatis serovar L2. In uninfected cells, immunofluorescence staining of the proteins showed a fine granular distributed pattern for all three proteins. After infection with C. trachomatis, calreticulin was found at the periphery of chlamydial aggregates and inclusions from 3 to 48 hours post-infection. In infected cells, SERCA2 was intimately associated with chlamydial inclusions after 3 and 24 hours, but not after 48 hours. Moreover, IP3-R was translocated to and colocalized with EB aggregates and chlamydial inclusions and had a distribution very similar to that of SERCA 2. After 24 hours incubation with chlamydiae, there was a local accumulation of [Ca2+]i (105+/-17 nM) in the proximity of chlamydial inclusions, compared to 50+/-13 nM in other parts of the cell cytoplasm. In the absence of extracellular Ca2+, this local accumulation of Ca2+ increased to 295+/-50 nM after adding 50 microM ATP, and to a similar extent after adding 100 nM thapsigargin (Tg). These data indicate that during infection of HeLa cells with chlamydiae, intracellular Ca2+ stores are redistributed, causing local accumulation of Ca2+ in the vicinity of chlamydial inclusions. These changes may trigger the association of certain proteins such as annexins with chlamydia-containing vesicles, and thereby regulation of membrane-membrane interaction during endosome aggregation and inclusion formation.

Impaired phagocytosis and opsonisation towards group B streptococci in preterm neonates.

AIMS: To study the chemiluminescence response in polymorphonuclear leucocytes (PMNL) at different stages of maturity and the opsonic capacity of sera with defined titres of anti-capsular type III antibodies, after exposure to serotype III group B streptococci (GBS). The influence of GBS type III capsule expression on PMNL chemiluminescence response was also investigated. METHODS: Two clinical isolates of serotype III GBS and two serotype III reference strains which form isogenic variants with high and low amounts of capsule substance, respectively, were used. PMNL and sera were obtained from adult healthy blood donors, full term neonates, and preterm neonates. RESULTS: PMNL from premature infants showed a significantly lower chemiluminescence response (p < 0.0001) than the PMNL from adults and neonates, while the chemiluminescence response with adult, neonatal, and preterm sera gradually diminished. In the presence of a serum pool with a standardised complement value, raised (> 10 mg/l), rather than low (< 1.0 mg/l) anti-III antibody titres induced a higher chemiluminescence response to the capsule expressing variant. When GBS were cultured at pH 5.0, the bacteria had a higher buoyant density, reflecting decreased expression of capsule substance compared with bacteria grown at pH 7.4. Concomitantly, there was a substantial increase in chemiluminescence response for all isolates cultured at the lower pH, except for the capsule deficient mutant. CONCLUSIONS: PMNL function and opsonic capacity are significantly impaired in neonates and correlate with maturation of the newborn child. The combined defect in cellular and humoral defences in preterm neonates may contribute to their increased susceptibility to GBS infection. Growth conditions for GBS, simulating different in vivo environments, greatly affect capsule expression and resistance to phagocytosis.

Secretion of IL-6, IL-8 and G-CSF by human endothelial cells in vitro in response to Staphylococcus aureus and staphylococcal exotoxins.

The capacity of endothelial cells to produce and release cytokines (IL-6, IL-8 and G-CSF) in response to exposure to Staphylococcus aureus strains or staphylococcal exotoxins (alpha-toxin, enterotoxin A and TSST-1) was investigated. An endothelial cell culture model of human umbilical vein endothelial cells (HUVEC) was used. Five out of ten clinical isolates of S. aureus were found to induce cytokine production and release from endothelial cells. Four of the five isolates that induce cytokine release produced enterotoxin A, B, C, D and/or TSST-1, compared with two of those that did not induce release. Purified staphylococcal exotoxins (1 pg/ml-1 microg/ml) did not act as primary stimuli and induced no detectable cytokine secretion. When endothelial cells were prestimulated with IL-1beta or TNF alpha at a concentration of 1 ng/ml for 2 h, IL-1beta served as a potent primary stimulus for IL-6, IL-8 and G-CSF production, whereas TNF alpha did not induce any significant cytokine release during the subsequent 24 h. A further increase in IL-6 and G-CSF release, but not of IL-8, was observed when IL-1beta prestimulated cells were exposed to alpha-toxin or TSST-1. However, to potentiate cytokine production (IL-6 and IL-8) by SEA, both IL-1beta and the toxin had to be present simultaneously. Our data show that S. aureus, but not staphylococcal exotoxins, have the capacity to act as primary stimuli of endothelial cells and induce production and release of cytokines. IL-1beta may prime HUVEC to release IL-6, IL-8 and G-CSF prior to subsequent stimulation with staphylococcal exotoxins.

Penetration of group B streptococci through polarized Madin-Darby canine kidney cells.

Group B streptococci (GBS) are one of the major causes of invasive neonatal infection. The pathogenesis of early onset disease is a multistep process. Adhesion of GBS to eucaryotic cells is considered to be an important step for the establishment of infection. Subsequent to adhesion, GBS invade cells and give rise to septicemia and meningitis. To investigate passage of GBS across epithelial cell linings we examined the interaction between bacteria and Madin-Darby canine kidney (MDCK) cells. When grown on permeable support, these cells form a polarized epithelial monolayer with an apical-to-basolateral orientation, which more reflects the in vivo situation compared with conventionally cultured cells. Our results show that GBS are translocated in vacuoles from the apical to the basolateral surface of MDCK cells in a temperature-dependent process. The passage of GBS through the cells is selective with only small numbers of bacteria penetrating in the basolateral-to-apical direction. Transcytosis of GBS starts before decrease in transepithelial resistance of the monolayer. These data suggest a mechanism for traversal of GBS over intact chorioamniotic membranes and from alveoli into the circulation of the fetus.

Increase of staphylococci in neonatal septicaemia: a fourteen-year study.

All cases of neonatal septicaemia during 1981-94 were studied at Orebro Medical Centre Hospital, Sweden. One hundred and thirty-two children fulfilled laboratory and clinical criteria for neonatal septicaemia and were included. Staphylococcus aureus (n = 41), Group B streptococcus (GBS) (n = 32) and coagulase-negative staphylococci (CoNS) (n = 27) were the dominating aetiologies. The annual incidence of septicaemia increased significantly, from 2.3 cases during the first 7-year period to 3.3 per 1000 live births during 1988-94. This increase was caused by S. aureus and CoNS, which mainly affected premature children and had an onset more than 48 h after delivery. GBS, on the other hand, slightly decreased and affected full-term children within 48 h. The overall mortality was 11%. CoNS isolated during the latter 7-year period were more resistant to antibiotics than those isolated during 1981-87; resistance to methicillin increased from 14 to 45% and to gentamicin from 0 to 20%. These changes in aetiology and antibiotic susceptibility should be considered when selecting antibiotic treatment in neonatal septicaemia.

Detection and identification of bacteria using in-house broad range 16S rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA genes.

Broad range PCR amplification and genus-specific 16S ribosomal DNA hybridization was used to demonstrate that Chlamydia, Helicobacter and Mobiluncus hybridization probes, located within variable regions V3, V4, and V9 of the 16S rDNA, specifically bound to the corresponding PCR product obtained from pure cultures of the three genera. The sensitivity of the assay was determined by analysis of C. trachomatis serially diluted in urine. The detection limit was 1-10 elementary bodies using a hybridization probe derived from the variable region V3 of the 16S rRNA gene. A PCR product was furthermore formed in urine specimens not containing C. trachomatis, showing amplification of Chlamydia also in the presence of DNA from the resident urethral flora that competes for annealing sites. Analysis of a restricted number of male urine specimens using the C. trachomatis-specific probe showed complete agreement with culture and a commercially available PCR kit. Our method not only has the capacity to detect C. trachomatis in microbiologically mixed urine samples but also the potential advantage of identifying other bacterial pathogens from the same PCR product by varying the hybridization probes.

Adhesion to and invasion of HeLa cells by Helicobacter pylori.

Eight clinical isolates and two reference strains of Helicobacter pylori were studied with regard to their interactions with HeLa cells. All the isolates adhered poorly to HeLa cells and the number of invasive bacteria was very low. No correlation was found between the adherence and invasiveness of the isolates on one hand, and the corresponding patients having ulcer or non-ulcer disease, or the ability of the strains to produce cytotoxin and to induce an oxidative burst of human polymorphonuclear leukocytes without opsonins, on the other. These results indicate that invasion of epithelial cells would play no important role in the pathogenesis of infections caused by H. pylori.

Occurrence of pneumococci with resistance or decreased susceptibility to penicillin in southeast Sweden.

The minimal inhibitory concentrations (MICs) of 14 beta-lactam and non-beta-lactam antibiotics were determined for all pneumococci with intermediate susceptibility (I), (n = 26) or resistance (R), (n = 15) to penicillin G isolated at the Clinical Microbiology Laboratory, University Hospital, Linköping, Sweden during 1994. These isolates accounted for 3% of all pneumococcal isolates. The results were compared with those of 26 penicillin-susceptible isolates. The MICs of all tested beta-lactam antibiotics increased with MICs of penicillin G. The least increase and the lowest MICs of these agents were recorded for cefotaxime and imipenem. 27% of I- and R-strains were multiple-resistant, most often to tetracycline, trimethoprim-sulfametoxazole, erythromycin, chloramphenicol and clindamycin. All strains were susceptible to vancomycin and rifampicin. I-strains belonged to at least 5 different serotypes. However, 12 of the 15 R-strains were serotype 9 and 6 of these were recovered during contact tracing, indicating spread of a single clone within day-care centres.