Development of a novel chlamydia growth-monitoring method and its application for screening anti- and pro-chlamydial compounds

Sexually transmitted infections (STIs) are the most prevalent infectious diseases in the world. Among the STIs, Chlamydia trachomatis (C. trachomatis)-related infections are the most common. C. trachomatis serovars D-K cause pelvic inflammatory diseases and infertility, while the LGV serovars are the pathogens that cause lymphogranuloma venereum, an STI with systemic manifestations. C. trachomatis infections have also been linked to arthritis and spondyloarthritis. C. pneumoniae is a frequent cause of communityacquired pneumonia and is suspected of participating in the pathogenesis of chronic diseases, such as asthma and atherosclerosis. Human herpes simplex virus-1 (HHSV-1) and preferentially HHSV-2 genital infections are common viral STIs. The number of HHSV-2 seropositive people (15–49 years) was estimated as 417 million in 2012, with an 11.3% prevalence in the population. Besides the vesicular lesions of the urogenital and anal regions, HSV infections may lead to severe complications including encephalitis, meningitis and neonatal herpes infections. C. trachomatis and HHSV-2 pathogens preferentially cause persistent or latent infections either locally (C. trachomatis) and/or from the site of the primary infection, such as in the sacral ganglia for HSV-2 and joints for C. trachomatis. Because of the long-term presence of the pathogens, there is a possibility that their infectious cycles, including the active and persistent growths, are influenced by locally or systematically applied compounds, including nanomaterials. The standard method for counting chlamydiae is immunofluorescence staining and manual counting of chlamydial inclusions. High- or medium-throughput estimation of the reduction in chlamydial inclusions should be the basis of testing antichlamydial compounds and other drugs that positively or negatively influence chlamydial growth, yet low-throughput manual counting is the common approach. Titanium dioxide (TiO2) is the naturally occurring oxide of titanium. TiO2 exists in three most common forms: rutile, anatase and brookite. Titanium dioxide, in the anatase form, is a photocatalyst under ultraviolet (UV) light. The positive holes oxidize water to create hydroxyl radicals, by the strong oxidative potential. TiO2 nanoparticles have a strong bactericidal effect. The TiO2 reacts by photocatalysis with water to release the hydroxyl radical with subsequent formation of superoxide. The reactive oxygen species can then synergistically act by attacking polyunsaturated phospholipids in bacteria and catalyzed site-specific DNA damage via generation of H2O2. TiO2 NPs are applied as a food additive or a drug delivery vehicle and we wanted to test their interactions with C.trachomatis and HHSV-2 to test their efficacy in non-activated form

Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis

Chlamydia trachomatis (C. trachomatis) is an obligate intracellular bacterium linked to ocular and urogenital infections with potentially serious sequelae, including blindness and infertility. First-line antibiotics, such as azithromycin (AZT) and doxycycline, are effective, but treatment failures have also been reported. Encapsulation of antibiotics in liposomes is considered an effective approach for improving their local effects, bioavailability, biocompatibility and antimicrobial activity. To test whether liposomes could enhance the antichlamydial action of AZT, we encapsulated AZT in different surface-charged elastic liposomes (neutral, cationic and anionic elastic liposomes) and assessed their antibacterial potential against the C. trachomatis serovar D laboratory strain as well as the clinical isolate C. trachomatis serovar F. A direct quantitative polymerase chain reaction (qPCR) method was used to measure chlamydial genome content 48 h post infection and to determine the recoverable chlamydial growth. All the liposomes efficiently delivered AZT to HeLa 229 cells infected with the laboratory Chlamydia strain, exhibiting the minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of AZT even 4-8-fold lower than those achieved with the free AZT. The tested AZT-liposomes were also effective against the clinical Chlamydia strain by decreasing MIC values by 2-fold relative to the free AZT. Interestingly, the neutral AZT-liposomes had no effect on the MBC against the clinical strain, while cationic and anionic AZT-liposomes decreased the MBC 2-fold, hence proving the potential of the surface-charged elastic liposomes to improve the effectiveness of AZT against C. trachomatis

Vaginal Gel Component Hydroxyethyl Cellulose Significantly Enhances the Infectivity of Chlamydia trachomatis Serovars D and E

The transmission of the urogenital serovars of Chlamydia trachomatis can be significantly influenced by vaginal gels. Hydroxyethyl cellulose is a commonly used gelling agent that can be found in vaginal gels. Hydroxyethyl cellulose showed a concentration-dependent growth-enhancing effect on C. trachomatis serovars D and E, with a 26.1-fold maximal increase in vitro and a 2.57-fold increase in vivo

High-resolution Imaging of Myeloperoxidase Activity Sensors in Human Cerebrovascular Disease

Progress in clinical development of magnetic resonance imaging (MRI) substrate-sensors of enzymatic activity has been slow partly due to the lack of human efficacy data. We report here a strategy that may serve as a shortcut from bench to bedside. We tested ultra high-resolution 7T MRI (microMRI) of human surgical histology sections in a 3-year IRB approved, HIPAA compliant study of surgically clipped brain aneurysms. microMRI was used for assessing the efficacy of MRI substrate-sensors that detect myeloperoxidase activity in inflammation. The efficacy of Gd-5HT-DOTAGA, a novel myeloperoxidase (MPO) imaging agent synthesized by using a highly stable gadolinium (III) chelate was tested both in tissue-like phantoms and in human samples. After treating histology sections with paramagnetic MPO substrate-sensors we observed relaxation time shortening and MPO activity-dependent MR signal enhancement. An increase of normalized MR signal generated by ultra-short echo time MR sequences was corroborated by MPO activity visualization by using a fluorescent MPO substrate. The results of microMRI of MPO activity associated with aneurysmal pathology and immunohistochemistry demonstrated active involvement of neutrophils and neutrophil NETs as a result of pro-inflammatory signalling in the vascular wall and in the perivascular space of brain aneurysms

Indoleamine 2,3-Dioxygenase Activity in Chlamydia muridarum and Chlamydia pneumoniae Infected Mouse Lung Tissues

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Azithromycin-liposomes as a novel approach for localized therapy of cervicovaginal bacterial infections

Background: Efficient localized cervicovaginal antibacterial therapy, enabling the delivery of antibiotic to the site of action at lower doses while escaping systemic drug effects and reducing the risk of developing microbial resistance, is attracting considerable attention. Liposomes have been shown to allow sustained drug release into vaginal mucosa and improve delivery of antibiotics to bacterial cells and biofilms Azithromycin (AZI), a potent broad-spectrum macrolide antibiotic, has not yet been investigated for localized therapy of cervicovaginal infections, although it is administered orally for the treatment of sexually transmitted diseases. Encapsulation of AZI in liposomes could improve its solubility, antibacterial activity, and allow the prolonged drug release in the cervicovaginal tissue, while avoiding systemic side effects. Purpose: The objective of this study was to develop AZI-liposomes and explore their potentials for treating cervicovaginal infections. Methods: AZI-liposomes that differed in bilayer elasticity/rigidity and surface charge were prepared and evaluated under simulated cervicovaginal conditions to yield optimized liposomes, which were assessed for antibacterial activity against several planktonic and biofilm-forming Escherichia coli strains and intracellular Chlamydia trachomatis, ex vivo AZI vaginal deposition/penetration, and in vitro cytotoxicity toward cervical cells. Results: Negatively charged liposomes with rigid bilayers (CL-3), propylene glycol liposomes (PGL-2) and deformable propylene glycol liposomes (DPGL-2) were efficient against planktonic E. coli ATCC 700928 and K-12. CL-3 was superior for preventing the formation of E. coli ATCC 700928 and K-12 biofilms, with IC50 values (concentrations that inhibit biofilm viability by 50%) up to 8-fold lower than those of the control (free AZI). DPGL-2 was the most promising for eradication of already formed E. coli biofilms and for treating C. trachomatis infections. All AZI-liposomes were biocompatible with cervical cells and improved localization of the drug inside vaginal tissue compared with the control. Conclusion: The performed studies confirm the potentials of AZI-liposomes for localized cervicovaginal therapy.Peer reviewe