Carnosic Acid Inhibits Herpes Simplex Virus Replication by Suppressing Cellular ATP Synthesis.

Acquiring resistance against antiviral drugs is a significant problem in antimicrobial therapy. In order to identify novel antiviral compounds, the antiviral activity of eight plants indigenous to the southern region of Hungary against herpes simplex virus-2 (HSV-2) was investigated. The plant extracts and the plant compound carnosic acid were tested for their effectiveness on both the extracellular and intracellular forms of HSV-2 on Vero and HeLa cells. HSV-2 replication was measured by a direct quantitative PCR (qPCR). Among the tested plant extracts, Salvia rosmarinus (S. rosmarinus) exhibited a 90.46% reduction in HSV-2 replication at the 0.47 µg/mL concentration. Carnosic acid, a major antimicrobial compound found in rosemary, also demonstrated a significant dose-dependent inhibition of both extracellular and intracellular forms of HSV-2. The 90% inhibitory concentration (IC90) of carnosic acid was between 25 and 6.25 µg/mL. Proteomics and high-resolution respirometry showed that carnosic acid suppressed key ATP synthesis pathways such as glycolysis, citrate cycle, and oxidative phosphorylation. Inhibition of oxidative phosphorylation also suppressed HSV-2 replication up to 39.94-fold. These results indicate that the antiviral action of carnosic acid includes the inhibition of ATP generation by suppressing key energy production pathways. Carnosic acid holds promise as a potential novel antiviral agent against HSV-2.

Indoleamine 2,3-Dioxygenase Cannot Inhibit Chlamydia trachomatis Growth in HL-60 Human Neutrophil Granulocytes.

Aims: Neutrophil granulocytes are the major cells involved in Chlamydia trachomatis (C. trachomatis)-mediated inflammation and histopathology. A key protein in human intracellular antichlamydial defense is the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) which limits the growth of the tryptophan auxotroph Chlamydia. Despite its importance, the role of IDO in the intracellular defense against Chlamydia in neutrophils is not well characterized. Methods: Global gene expression screen was used to evaluate the effect of C. trachomatis serovar D infection on the transcriptome of human neutrophil granulocytes. Tryptophan metabolite concentrations in the Chlamydia-infected and/or interferon-gamma (IFNG)-treated neutrophils were measured by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results: Our results indicate that the C. trachomatis infection had a major impact on neutrophil gene expression, inducing 1,295 genes and repressing 1,510 genes. A bioinformatics analysis revealed that important factors involved in the induction of neutrophil gene expression were the interferon-related transcription factors such as IRF1-5, IRF7-9, STAT2, ICSB, and ISGF3. One of the upregulated genes was ido1, a known infection- and interferon-induced host gene. The tryptophan-degrading activity of IDO1 was not induced significantly by Chlamydia infection alone, but the addition of IFNG greatly increased its activity. Despite the significant IDO activity in IFNG-treated cells, C. trachomatis growth was not affected by IFNG. This result was in contrast to what we observed in HeLa human cervical epithelial cells, where the IFNG-mediated inhibition of C. trachomatis growth was significant and the IFNG-induced IDO activity correlated with growth inhibition. Conclusions: IDO activity was not able to inhibit chlamydial growth in human neutrophils. Whether the IDO activity was not high enough for inhibition or other chlamydial growth-promoting host mechanisms were induced in the infected and interferon-treated neutrophils needs to be further investigated.

Stochastic Modeling of In Vitro Bactericidal Potency.

We provide a Galton-Watson model for the growth of a bacterial population in the presence of antibiotics. We assume that bacterial cells either die or duplicate, and the corresponding probabilities depend on the concentration of the antibiotic. Assuming that the mean offspring number is given by [Formula: see text] for some [Formula: see text], where c stands for the antibiotic concentration we obtain weakly consistent, asymptotically normal estimator both for [Formula: see text] and for the minimal inhibitory concentration, a relevant parameter in pharmacology. We apply our method to real data, where Chlamydia trachomatis bacterium was treated by azithromycin and ciprofloxacin. For the measurements of Chlamydia growth quantitative polymerase chain reaction technique was used. The 2-parameter model fits remarkably well to the biological data.

Comparison of Solution Chemical Properties and Biological Activity of Ruthenium Complexes of Selected ß-Diketone, 8-Hydroxyquinoline and Pyrithione Ligands.

In this work, the various biological activities of eight organoruthenium(II) complexes were evaluated to reveal correlations with their stability and reactivity in aqueous media. Complexes with general formula [Ru(η6-p-cymene)(X,Y)(Z)] were prepared, where (X,Y) represents either an O,O-ligand (ß-diketone), N,O-ligand (8-hydroxyquinoline) or O,S-pyrithione-type ligands (pyrithione = 1-hydroxypyridine-2(1H)-thione) with Cl- or 1,3,5-triaza-7-phosphaadamantane (PTA) as a co-ligand (Z). The tested complexes inhibit the chlamydial growth on HeLa cells, and one of the complexes inhibits the growth of the human herpes simplex virus-2. The chlorido complexes with N,O- and O,S-ligands displayed strong antibacterial activity on Gram-positive strains including the resistant S. aureus (MRSA) and were cytotoxic in adenocarcinoma cell lines. Effect of the structural variation on the biological properties and solution stability was clearly revealed. The decreased bioactivity of the ß-diketone complexes can be related to their lower stability in solution. In contrast, the O,S-pyrithione-type complexes are highly stable in solution and the complexation prevents the oxidation of the O,S-ligands. Comparing the binding of PTA and the chlorido co-ligands, it can be concluded that PTA is generally more strongly coordinated to ruthenium, which at the same time decreased the reactivity of complexes with human serum albumin or 1-methylimidazole as well as diminished their bioactivity.

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.

Correlation between detergent activity and anti-herpes simplex virus-2 activity of commercially available vaginal gels.

OBJECTIVE: Herpes simplex virus-2 (HSV-2) infections are almost exclusively sexually transmitted. The presence of vaginal gels during sexual activity may have a significant positive or negative impact on viral transmission. Therefore we investigated three off-the-shelf vaginal lubricants and one pH restoring gel to evaluate their impact on HSV-2 replication. RESULTS: HeLa cells were infected with untreated virions and virions incubated with the particular gels. The accumulation of viral genomes was monitored by quantitative PCR (qPCR) method at 24 h post infection. Two of the tested gels had no significant effect on HSV-2 replication at the maximum applied concentration, while two had a strong inhibitory effect (~ 98% reduction of replication). The replication inhibitory effect was observed at various multiplicity of infection (MOI 0.4-6.4) and the two inhibitory gels were also capable of inhibiting the HSV-2 induced cytopathic effect on HeLa cells. The surface tension decreasing activity-an indication of detergent activity-was strongly correlated with the anti-HSV-2 activity of the gels (R2: 0.88). Our results indicate that off-the-shelf vaginal gels have a markedly different anti-HSV-2 activity that may influence HSV-2 transmission.

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.

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

Chlamydia trachomatis infections are the most prevalent sexually transmitted infections with potentially debilitating sequelae, such as infertility. Mouse models are generally used for vaccine development, to study the immune response and histopathology associated with Chlamydia infection. An important question regarding murine models is the in vivo identification of murine host genes responsible for the elimination of the murine and human Chlamydia strains. RNA sequencing of the Chlamydia muridarum infected BALB/c lung transcriptome revealed that several genes with direct antichlamydial functions were induced at the tissue level, including the already described and novel members of the murine interferon-inducible GTPase family, the CXCL chemokines CXCL9, CXCL11, immunoresponsive gene 1, nitric oxide synthase-2 (iNOS), and lipocalin-2. Indoleamine 2,3-dioxygenase 1-2 (IDO1-2) previously described potent antichlamydial host enzymes were also highly expressed in the infected murine lungs. This finding was novel, since IDO was considered as a unique human antichlamydial defense gene. Besides a lower level of epithelial cell positivity, immunohistochemistry showed that IDO1-2 proteins were expressed prominently in macrophages. Detection of the tryptophan degradation product kynurenine and the impact of IDO inhibition on Chlamydia muridarum growth proved that the IDO1-2 proteins were functionally active. IDO1-2 activity also increased in Chlamydia muridarum infected C57BL/6 lung tissues, indicating that this phenomenon is not mouse strain specific. Our study shows that the murine antichlamydial response includes a variety of highly up-regulated defense genes in vivo. Among these genes the antichlamydial effectors IDO1-2 were identified. The potential impact of murine IDO1-2 expression on Chlamydia propagation needs further investigation.

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.

Phenanthrenes from Juncus Compressus Jacq. with Promising Antiproliferative and Anti-HSV-2 Activities.

Juncaceae species are rich sources of phenanthrenes. The present study has focused on the isolation and structure determination of biologically active components from Juncus compressus. Eleven compounds (nine phenanthrenes and two flavonoids) have been isolated from the plant by the combination of different chromatographic methods. Two compounds (compressins A (Compound 1) and B (Compound 2)) are novel natural products, while seven phenanthrenes (effusol (Compound 3), effususol (Compound 4), juncusol (Compound 5), 2-hydroxy-1-methyl-4-oxymethylene-5-vinyl-9,10-dihydrophenanthrene (Compound 6), 7-hydroxy-1-methyl-2-methoxy-5-vinyl-9,10-dihydrophenanthrene (Compound 7), effususin A (Compound 8), and dehydroeffusol (Compound 9)), and two flavonoids (apigenin (Compound 10) and luteolin (Compound 11) were isolated for the first time from the plant. Compressin B (Compound 2) is a dimeric phenanthrene, in which two juncusol monomers (Compound 5) are connecting through their C-3 atoms. The structure elucidation of the isolated compounds was carried out using 1D, 2D NMR spectroscopic methods and HR-MS measurements. In vitro investigation of the antiproliferative effect of the phenanthrenes on two cervical (HeLa and SiHa) and an ovarian human tumor cell line (A2780) revealed that compounds have remarkable antiproliferative activity, mainly on the HeLa cell line. Moreover, juncusol (Compound 5) proved to possess significant antiviral activity against the herpes simplex 2 virus (HSV-2).

Characterization of a proteolytically stable D-peptide that suppresses herpes simplex virus 1 infection: implications for the development of entry-based antiviral therapy.

Uncontrolled herpes simplex virus 1 (HSV-1) infection can advance to serious conditions, including corneal blindness or fatal encephalitis. Here, we describe a highly potent anti-HSV-1 peptide (DG2) that inhibits HSV-1 entry into host cells and blocks all aspects of infection. Importantly, DG2 is highly resistant to proteases and shows minimal toxicity, paving the way for prophylactic or therapeutic application of the peptide in vivo.

Application of DNA chip scanning technology for automatic detection of Chlamydia trachomatis and Chlamydia pneumoniae inclusions.

Chlamydiae are obligate intracellular bacteria that propagate in the inclusion, a specific niche inside the host cell. The standard method for counting chlamydiae is immunofluorescent 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. To overcome the time-consuming and subjective manual counting, we developed an automatic inclusion-counting system based on a commercially available DNA chip scanner. Fluorescently labeled inclusions are detected by the scanner, and the image is processed by ChlamyCount, a custom plug-in of the ImageJ software environment. ChlamyCount was able to measure the inclusion counts over a 1-log-unit dynamic range with a high correlation to the theoretical counts. ChlamyCount was capable of accurately determining the MICs of the novel antimicrobial compound PCC00213 and the already known antichlamydial antibiotics moxifloxacin and tetracycline. ChlamyCount was also able to measure the chlamydial growth-altering effect of drugs that influence host-bacterium interaction, such as gamma interferon, DEAE-dextran, and cycloheximide. ChlamyCount is an easily adaptable system for testing antichlamydial antimicrobials and other compounds that influence Chlamydia-host interactions.