Ambroxol Treatment Suppresses the Proliferation of Chlamydia pneumoniae in Murine Lungs.

Ambroxol (Ax) is used as a mucolytics in the treatment of respiratory tract infections. Ax, at a general dose for humans, does not alter Chlamydia pneumoniae growth in mice. Therefore, we aimed to investigate the potential anti-chlamydial effect of Ax at a concentration four timed higher than that used in human medicine. Mice were infected with C. pneumoniae and 5-mg/kg Ax was administered orally. The number of recoverable C. pneumoniae inclusion-forming units (IFUs) in Ax-treated mice was significantly lower than that in untreated mice. mRNA expression levels of several cytokines, including interleukin 12 (IL-12), IL-23, IL-17F, interferon gamma (IFN-γ), and surfactant protein (SP)-A, increased in infected mice treated with Ax. The IFN-γ protein expression levels were also significantly higher in infected and Ax-treated mice. Furthermore, the in vitro results suggested that the ERK 1/2 activity was decreased, which is essential for the C. pneumoniae replication. SP-A and SP-D treatments significantly decreased the number of viable C. pneumoniae IFUs and significantly increased the attachment of C. pneumoniae to macrophage cells. Based on our results, a dose of 5 mg/kg of Ax exhibited an anti-chlamydial effect in mice, probably an immunomodulating effect, and may be used as supporting drug in respiratory infections caused by C. pneumoniae.

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.

Chlamydia pneumoniae Infection Exacerbates Atherosclerosis in ApoB100only/LDLR-/- Mouse Strain.

AIMS: Hyperlipidaemia model animals have been used to elucidate the role of Chlamydia pneumoniae (Cpn) infection in atherosclerosis. The aims of this study were to investigate the proatherogenic effect of multiple Cpn infections in ApoB100only/LDLR-/- mice which based on lipid profile can be regarded as the most suitable mouse model of human hypercholesterolemia and to compare the lesion development to that in a major atherosclerosis model ApoE-/- mice. METHODS AND RESULTS: Aorta samples of ApoB100only/LDLR-/- mice infected three times with Cpn were subjected to morphometric analyses. Morphometric evaluation disclosed that Cpn infections exacerbated atherosclerosis development in the aortic root and descending aorta of the mice fed with normal diet. Viable Cpn was detected in the ascending aorta by RT-PCR. Chlamydial 16SrRNA expression showed the presence of viable Cpn in the aorta of infected animals. A similar rate of acceleration of atherosclerosis was observed when the infection protocol was applied in ApoB100only/LDLR-/- and in ApoE-/- mice. CONCLUSION: Similar to ApoE-/- mice, ApoB100only/LDLR-/- mice with more human-relevant serum lipoprotein composition develop increased atherosclerosis after Cpn infections; thus this mouse strain can be used as a model of infection-related atherosclerosis enhancement and can provide further evidence for the proatherogenic influence of Cpn in mice.

Chryseobacterium gleum - a novel bacterium species detected in neonatal respiratory tract infections.

We report three patients with early neonatal infections. All patients had respiratory tract involvement with increased inflammation markers. Chryseobacterium gleum was cultured from the stomach content aspirated on arrival at the Neonatal intensive Care Unit and it was identified with the help of a Microflex™ MALDI Biotyper mass spectrometer (Bruker-Daltonik, Fremont, CA). Recovery could be achieved with ciprofloxacin treatment. We consider our cases a possible new clinical presentation of a rare human pathogen.

Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae.

BACKGROUND: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. RESULTS: Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway(®) 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway(®) entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. CONCLUSION: This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.

Expression of heteromeric glycine receptor-channels in rat spinal cultures and inhibition by neuroactive steroids.

Ionotropic glycine receptors were studied in cultured spinal cord neurons prepared from 17-day-old rat embryos, using whole-cell patch clamp electrophysiology. Glycine receptors of 3-17 days in vitro were characterized via subtype-specific channel blockade by micromolar picrotoxin and cyanotriphenylborate, as well as nanomolar strychnine. Potentiation by nanomolar tropisetron indicated coexpression of beta with alpha subunits. The neuroactive steroids pregnenolone sulfate and dehydroepiandrosterone sulfate, as well as alphaxalone and its 3beta epimer betaxalone inhibited the chloride current with IC(50) values of 19, 46, 16 and 208 microM, respectively, with no potentiation. Reverse transcription polymerase chain reaction and immunocytochemistry demonstrated mRNAs and proteins of alpha1, alpha2, alpha3 and beta subunits in rat spinal cord cultures. In conclusion, neuroactive steroids, both positive and negative modulators of gamma-aminobutyric-acid(A) receptors, inhibited heteromeric glycine receptors at micromolar concentrations.

Flow cytometry-based method to analyze the change in Tau phosphorylation in a hGSK-3beta and hTau over-expressing EcR-293 cell line.

Neurofibrillary tangles are composed of insoluble aggregates of microtubule-associated protein Tau. In the pathology of Alzheimer's disease (AD), accumulation of hyperphosphorylated Tau results in formation of paired helical filaments. One of the main candidate to hyperphosphorylate Tau in AD is glycogen synthase kinase 3beta (GSK-3beta). Here we introduce a non-neuronal cell line, stably co-expressing human Tau and GSK-3beta proteins, where the effect of potential kinase inhibitors on Tau phosphorylation can be monitored. The aim of our study was to establish a new flow-cytometry-based method to quantitatively analyze the changing of Tau phosphorylation, which is a suitable alternative to the well-accepted but non-quantitative Western blot technique. Our results demonstrate that the flow cytometry-based method is a convenient tool to analyze the effect of GSK-3beta inhibitors on Tau phosphorylation. This new approach provides appropriate throughput for screening purposes in preclinical research for characterization of GSK-3beta inhibitors, as potential drug candidate to cure Alzheimer's disease.

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors.

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.

Role of altered structure and function of NMDA receptors in development of alcohol dependence.

Long-term alcohol exposure gives rise to development of physical dependence on alcohol in consequence of changes in certain neurotransmitter functions. Accumulating evidence suggests that the glutamatergic neurotransmitter system, especially the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol's action, since ethanol is a potent inhibitor of the NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory "upregulation" of NMDAR mediated functions supposedly contributing to the occurrence of ethanol tolerance, dependence as well as the acute and delayed signs of ethanol withdrawal.Recently, expression of different types of NMDAR subunits was found altered after long-term ethanol exposure. Especially, the expression of the NR2B and certain splice variant forms of the NR1 subunits were increased in primary neuronal cultures treated intermittently with ethanol. Since NMDA ion channels with such an altered subunit composition have increased permeability for calcium ions, increased agonist sensitivity, and relatively slow closing kinetics, the abovementioned alterations may underlie the enhanced NMDAR activation observed after long-term ethanol exposure. In accordance with these changes, the inhibitory potential of NR2B subunit-selective NMDAR antagonists is also increased, demonstrating excellent potency against alcohol withdrawal-induced in vitro cytotoxicity. Although in vivo data are few with these compounds, according to the effectiveness of the classic NMDAR antagonists in attenuation, not only the physical symptoms, but also some affective and motivational components of alcohol withdrawal, novel NR2B subunit selective NMDAR antagonists may offer a preferable alternative in the pharmacotherapy of alcohol dependence.

Inducible expression and pharmacology of recombinant NMDA receptors, composed of rat NR1a/NR2B subunits.

An ecdysone-inducible mammalian expression system was used to study expression of recombinant N-methyl-D-aspartate (NMDA) receptors. Human embryonic kidney (HEK) 293 cells expressing the regulatory vector pVgRXR (EcR 293 cells) were transfected with rat NR1a and NR2B cDNAs using the inducible vector pIND (Invitrogen). Inducible expression of the NR2B subunit in cell clone designated EcR/rNR1a2B was investigated using quantitative RT-PCR and flow cytometry based immunocytochemical methods. The mRNA level of the NR2B subunits in EcR/rNRa2B cells was dependent on the concentration of the ecdysone analogue inducing agent, muristerone A (MuA). Similarly, NR2B subunit protein expression was higher in cells pre-treated with the inducing agent. Functionally active NMDA receptors were also detected in EcR/rNR1a2B cells after MuA induction. In presence of the inducing factor, NMDA-evoked ion currents as well as increase in cytoplasmic calcium-concentrations were measured using whole-cell patch clamp and fluorometric calcium measuring techniques. The pharmacological profile of the expressed NMDA receptors was characterised by comparing the inhibitory activity of several NR2B subunit selective NMDA antagonists in EcR/rNR1a2B cells with that observed in primary cultures of rat cortical neurones. Whereas the efficacies of the NR2B subunit selective NMDA antagonists were similar in EcR/rNR1a2B cells and in neurones, their maximal inhibitory effects were significantly higher in cells expressing NR1a/NR2B recombinant receptors. This study demonstrates that recombinant NMDA receptors can be expressed in an inducible way in non-neuronal cell lines using the ecdysone-inducible mammalian expression system. Such cell lines can be suitable tools in high throughput functional screening for potential subtype selective modulators of the NMDA receptor.

Differential alterations in the expression of NMDA receptor subunits following chronic ethanol treatment in primary cultures of rat cortical and hippocampal neurones.

In our previous experiments, severe cellular damages and neuronal cell loss were observed following 24h of alcohol withdrawal in primary cultures of rat cortical neurones pre-treated with ethanol (50-200 mM) repeatedly for 3 days. Increased NMDA induced cytosolic calcium responses and excitotoxicity were also demonstrated in the ethanol pre-treated cultures. Thus, the enhancement in functions of NMDA receptors was supposed to be involved in the adaptive changes leading to the neurotoxic effect of alcohol-withdrawal. In this study, we investigated the effect of the 3-day repeated ethanol (100 mM) treatment on the function and subunit composition of the NMDA receptors. Here, we demonstrate that the maximal inhibitory effect of ethanol was significantly increased after ethanol pre-treatment. Similarly, the inhibitory activity of the NR2B subunit selective antagonists threo-ifenprodil, CP-101,606 and CI-1041 was also enhanced. On the contrary, the efficiency of the channel blocker agent MK-801 and the glycine-site selective antagonist 5,7-dichlorokynurenic acid was the same as in control cultures. According to these observations, a shift in subunit expression in favour for the NR2B subunit was suggested. Indeed, we provided evidence for increased expression of the NR2B and the C1 and C2' cassette containing splice variant forms of the NR1 subunit proteins in ethanol pre-treated cultures in further experiments using a flow cytometry based immunocytochemical method. These changes may constitute the basis of the increased NMDA receptor functions and subsequently the enhanced sensitivity of ethanol pre-treated cortical neurones to excitotoxic insults resulting in increased neuronal cell loss after ethanol withdrawal. Such alterations may play a role in the neuronal adaptation to ethanol as well as in the development of alcohol dependence, and might cause neuronal cell loss in certain areas of the brain during alcohol withdrawal.