Isolation of Chlamydia Pneumoniae from Serum Samples of the Patients with Acute Coronary Syndrome

BACKGROUND: Limited body of evidence suggests that lipopolysaccharide of C. pneumoniae as well as C. pneumoniae-specific immune complexes can be detected and isolated from human serum. The aim of this study was to investigate the presence of viable elementary bodies of C.pneumoniae in serum samples of patients with acute coronary syndrome and healthy volunteers.MATERIAL AND METHODS: Serum specimens from 26 healthy volunteers and 56 patients with acute coronary syndrome were examined subsequently by serological (C.pneumoniae-specific IgA and IgG), PCR-based and bacteriological methods. Conventional, nested and TaqMan PCR were used to detect C.pneumoniae genetic markers (ompA and 16S rRNA) in DNA from serum specimens extracted with different methods. An alternative protocol which included culturing high-speed serum sediments in HL cells and further C.pneumoniae growth evaluation with immunofluorescence analysis and TaqMan PCR was established. Pellet fraction of PCR-positive serum specimens was also examined by immunoelectron microscopy.RESULTS: Best efficiency of final PCR product recovery from serum specimens has been shown with specific C. pneumoniae primers using phenol-chloroform DNA extraction protocol. TaqMan PCR analysis revealed that human serum of patients with acute coronary syndrome may contain genetic markers of C. pneumoniae with bacterial load range from 200 to 2000 copies/ml serum. However, reliability and reproducibility of TaqMan PCR were poor for serum specimens with low bacterial copy number (<200 /ml). Combination of bacteriological, immunofluorescence and PCR- based protocols applied for the evaluating HL cells infected with serum sediments revealed that 21.0 % of the patients with acute coronary syndrome have viable forms C.pneumoniae in serum. The detection rate of C.pneumoniae in healthy volunteers was much lower (7.7%). Immunological profile of the patients did not match accurately C.pneumoniae detection rate in serum specimens. Elementary bodies of C.pneumoniae with typical ultrastructural characteristics were also identified in serum sediments using immunoelectron microscopy.Conclusions: Viable forms C. pneumoniae with typical electron microscopic structure can be identified and isolated from serum specimens of the patients with acute coronary syndrome and some healthy volunteers. Increased detection rate of C. pneumoniae in serum among the patients with an acute coronary syndrome may contribute towards enhanced pro-inflammatory status in cardiovascular patients and development of secondary complications of atherosclerosis.     

<Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>growth inhibition and restoration of LDL-receptor level in HepG2 cells treated with mevastatin

Background  

Perihepatitis is rare but consistently occurring extragenital manifestation of untreated Chlamydia trachomatis infection. Despite of possible liver involvement in generalized C. trachomatis infection, the ability of the pathogen to propagate in the hepatic cells and its impact on liver functions is not thoroughly investigated. The effect of mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, on C. trachomatis growth in human hepatoma cell line HepG2 has been studied. Bacterial growth was assessed by immunostaining with FITC-labeled monoclonal antibody against chlamydial lipopolysaccharide and by RT-PCR for two chlamydial genetic markers (16S rRNA and euo).     

Synthesis and Anti-Chlamydial Activity of Substituted N-Benzylanilines

Pharmaceutical Chemistry Journal - Eleven substituted N-benzylanilines were synthesized. Their anti-chlamydial activity was studied. A water-soluble hydrochloride was prepared from one of the lead...     

ApoB-containing lipoproteins promote infectivity of chlamydial species in human hepatoma cell line

AIM: To evaluate the direct binding of two main chlamydial biovars (C. trachomatis and C. pneumoniae) to plasma lipoproteins and its effect on chlamydial infection rate in human hepatoma cell line (HepG2 cells).METHODS: Murine plasma lipoproteins were fractionated and isolated using fast-performance liquid chromatography (FPLC), spotted on nitrocellulose membrane and incubated with chlamydial suspensions. Direct binding of chlamydial particles to lipoprotein fractions has been studied using lipopolysaccharide-specific antibodies in immuno-dot blot binding assay and immunoprecipitation analysis. Immunostaining protocol as well as flow cytometry analysis have been employed to study the infectivity rate of chlamydial species in HepG2 cells.RESULTS: Elementary bodies of both C. trachomatis and C. pneumoniae bind ApoB-containing fractions of plasma lipoproteins. That binding becomes stronger when heat-denatured FPLC fractions are used, suggesting a primary role of apolipoproteins in interaction between chlamydial particle and lipoprotein. Both chlamydial biovars efficiently propagate in human hepatoma cell line - HepG2 cells even in serum free conditions forming late-stage inclusion bodies and releasing extracellular elementary bodies. Preincubation of C. trachomatis and C. pneumoniae with native ApoB-containing lipoproteins enhances the rate of chlamydial infection in HepG2 cells.CONCLUSION: A productive infection caused by C. trachomatis and C. pneumoniae may take place in human-derived hepatocytes revealing hepatic cells as possible target in chlamydial infection. Obtained results may suggest the participation of lipoprotein receptors in the mechanism of attachment and/or entry of chlamydial particles into target cells.     

The role of the type-III secretion system of Gram-negative bacteria in the regulation of chronic infections

This review article focuses on the discussion about the role of the type III secretion system (T3SS), which was found in several Gram-negative bacteria, in the development of chronic infectious processes. The latest investigations on this issue have revealed that most severe chronic somatic diseases derive from prolonged chronic inflammation induced by various infectious agents. The T3SS may play a crucial role in the transition of an infection from an acute form to persistent one. Numerous clinical and bacteriological studies have shown that pathogenic microorganisms are persistent in a form resistant to various antibiotics. Therefore, one of the most promising goals for the development of novel antibiotics is the T3SS, which transports bacterial pathogenic factors directly into the eukaryotic cell. The functioning of the T3SS is essential for occurrence and development of both acute and chronic infectious processes.     

Chlamydia trachomatis proteasome protein as a significant pathogenicity factor

Sexually transmitted diseases are a global problem. These infections may lead to acute or chronic diseases. Chlamydia trachomatis is a dangerous and widespread pathogenicity factor that is not sensitive to conventional drugs and has no obvious symptoms. CPAF protein is a leading factor of pathogenesis. This protein inhibits the signaling pathways of the host cell and supports long survival of the pathogen in the host cell. The goal of this work was to review the general properties of the proteosome chlamydia CPAF protein, its functions, and role in pathology. The role of CPAF protein in the antichlamydia immune reaction is discussed. The prospects of the development of a promising antichlamydia vaccine, as well as new effective antichlamydia, drugs are also discussed.     

Development of pulmonary chlamydia infection in inbred mice strains differentiated by genetically determined sensitivity to tuberculosis infection

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. Susceptibility does not depend upon the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by a few interacting QTL mapped to chromosomes 3, 9 and 17. To find out whether tuberculosissusceptible I/St mice are susceptible to other intracellular bacteria, we investigated taxonomically distant pathogen, Chlamydia pneumoniae. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former are more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St 9.2 ± 1.2 days, A/Sn − 22.0 ± 2.0 days (p < 0.001). To estimate the degree of chlamydial multiplication in the lungs, we established a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite’s genome equivalents in infected tissue from 1 to 16 days after challenge. Interstrain difference of chlamydia burden in lungs we obtained after 24 hours after infection only. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection, and the numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia, despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p < 0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-α and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p < 0.05). Because the prominent differences in survival time did not correlate with permanent differences in bacterial multiplication, we suggest that both infections trigger fatal pathological processes whose dynamics depend strongly upon the host genetics.     

Effect of therapy with antibiotics on lipid metabolism and antioxidant reserve of patients with ischemic heart disease during Chlamydia pneumoniae infection

Effect of therapy with azithromycin and doxycycline on lipid metabolism, processes of lipid peroxidation and state of antioxidant defense was studied in patients with ischemic heart disease with elevated titer of antibodies to Chlamydia Pneumonia. Therapy with azithromycin (500 mg/day for 2 months) was associated with lowering of antibody titer, moderate improvement of lipid spectrum, marked decrease of activity of lipid peroxidation and augmentation of blood plasma antioxidant reserve. There were no such changes in a group of doxycycline treated patients. This difference can be attributed to more pronounced antimicrobial activity of azithromycin against Chlamydophila Pneumonia and its intrinsic antioxidant properties.