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.

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).

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.

Expression of Chlamydia muridarum plasmid genes and immunogenicity of pGP3 and pGP4 in different mouse strains.

Chlamydia muridarum carries a cryptic plasmid (pMoPn) of 7.5kb, which encodes seven genes. Our aims were to describe the transcriptional pattern of the pMoPn genes in C. muridarum-infected mice and to evaluate the host immune responses against pGP3 and pGP4 proteins. BALB/c and C57BL/6N female mice were inoculated intranasally with C. muridarum and sacrificed at different time points, and the total RNA was extracted from the lung suspensions to determine the levels of expression of the different plasmid genes by RT qPCR. The supernatants of the lungs were subjected to the quantitation of recoverable C. muridarum. TCA04 and TCA05, which encode pGP3 and pGP4, respectively, were amplified by PCR and cloned into the pET vector. The proteins were overexpressed in E. coli HB101 and purified. Selected groups of BALB/c and C57BL/6N mice were infected with C. muridarum 1-3 times. The humoral immune responses in the sera of the mice to the proteins encoded by TCA04 and TCA05 were tested by Western blotting, and the cellular immune responses were assessed in lymphocyte proliferation assays. The proteins recognized by the mouse sera were further analysed by a LC/MSMS technique. The kinetics of C. muridarum growth were similar in the mouse strains used, but the pathogen burden was higher in the BALB/c mice in the late phase of infection. All the plasmid genes in the BALB/c mice showed an increased level of expression on day 7, whereas the expression of the same genes did not change on day 7 in the C57BL/6N mice. The levels of expression of the plasmid genes were higher in the C57BL/6N mice at later time points. In Western blot assays, the sera of the singly infected C57BL/6N mice reacted with the monomeric form of pGP3, whereas the sera of the singly infected BALB/c mice reacted with the trimeric form of pGP3. The sera of the multiply infected C57BL/6N mice also recognized pGP4. Similarly to the humoral immune response, cellular immune responses to pGP3 and pGP4 were detected in the C. muridarum-infected C57BL/6N mice, but the spleen cells of BALB/c mice responded with proliferation only to the pGP3 protein. These results suggest that the proteins encoded by pMoPn genes may modulate the host immune response during C. muridarum infection, and that the evolved immune response against plasmid proteins, similarly to that against other chlamydial proteins, depends on the genetic background of the host.

Generation of targeted Chlamydia trachomatis null mutants.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reverse-genetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ-induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.

Chlamydophila pneumoniae re-infection triggers the production of IL-17A and IL-17E, important regulators of airway inflammation.

OBJECTIVE: Investigation of the effects of interleukin (IL)-17 cytokines in Chlamydophila pneumoniae-infected mice. METHODS: Mice were infected with C. pneumoniae once or three times and the expression of IL-17 cytokines was followed by RT qPCR from day 1 to day 28 after infection and re-infection. After the treatment of mice with anti-IL-17A, ELISA was used to detect the differences in cytokine and chemokine production. The number and phenotype of the IL-17A-producing cells were determined by ELISPOT. RESULTS: Chlamydophila pneumoniae induced IL-17A and IL-17F from day 2 after infection, and their levels remained elevated on day 28. The expression of IL-17C, IL-17D and IL-17E mRNA did not change significantly in response to a single infection. The in vivo neutralization of IL-17A resulted in a higher C. pneumoniae burden in the mouse lungs, a decreased cell influx, and diminished chemokine levels. The phenotype of IL-17A-producing cells was CD4(+). The re-infection of mice led to an increased expression of IL-17E mRNA. CONCLUSION: These results facilitate an understanding of the early inflammatory response after C. pneumoniae infection and suggest that C. pneumoniae re-infection induces the production of a high amount of IL-17E, which has an important role in the pathogenesis of allergic pulmonary diseases.

Immunization with a combination of 2 peptides derived from the C5a receptor significantly reduces early atherosclerotic lesion in Ldlr(tm1Her) Apob(tm2Sgy) J mice.

OBJECTIVE: The goal of this study was to assess whether immunization of Ldlr(tm1Her) Apob(tm2Sgy) J mice with 2 peptides located at the N-terminus of the C5a receptor (C5aR), either alone or in combination, is effective in reducing atherosclerotic lesions. METHODS AND RESULTS: Five- to 6-week-old female Ldlr(tm1Her)Apob(tm2Sgy) J mice were immunized using a repetitive immunization multiple sites strategy with keyhole limpet hemocyanin-conjugated peptides derived from the C5aR, either alone (designated as C5aR-P1 [aa 1-21] and C5aR-P2 [aa 19-31]) or in combination (designated as C5aR-P1+C5aR-P2). Mice were fed a high-fat diet for 10 weeks. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants. Immunization of Ldlr(tm1Her)Apob(tm2Sgy) J mice with these peptides elicited high concentrations of antibodies against each peptide. Immunization with the single peptide inhibited plaque development. Combined inoculation with C5aR-P1+C5aR-P2 had an additive effect on reducing the lesion in the aorta sinus and descending aortas when compared with controls. This effect correlated with cellular infiltration and cytokine/chemokine secretion in the serum or in stimulated spleen cells as well as specific cellular immune responses when compared with controls. CONCLUSIONS: Immunization of mice with C5aR-P1 and C5aR-P2, either alone or in combination, was effective in reducing early atherosclerotic lesion development. The combined peptide is more potential than either epitope alone to reduce atherosclerotic lesion formation through the induction of a specific Treg cell response as well as blockage of monocyte differentiation into macrophages.

Isocitrate lyase encoding plasmids in BCG cause increased survival in ApoB100-only LDLR-/- mice.

We studied the role of isocitrate lyase in the interaction between Mycobacterium bovis BCG and mice. ApoB100-only LDLR-/- (B6;129S-ApoBtm2SgyLdlrtm1Her/J) mice were inoculated with M. bovis BCG harbouring plasmids carrying the gene for isocitrate lyase. The presence of ~29 times more copies of this gene resulted in a higher bacterial yield in the spleens and lungs of the infected mice. The spleen was 3-4 times heavier, and in the spleen the bacteria survived over 10 days longer than did the bacteria with the control plasmid. Propionate was less toxic for bacteria carrying icl plasmids in vitro. This recombinant BCG can be a possible vaccine candidate.

Chlamydophila pneumoniae induces production of the defensin-like MIG/CXCL9, which has in vitro antichlamydial activity.

CXC chemokines that lack the ELR motif, including the monokine induced by IFN-γ (MIG/CXCL9), the IFN-induced protein of 10 kDa (IP-10/CXCL10), and the IFN-inducible T-cell α-chemoattractant (I-TAC/CXCL11), have been shown to mediate the generation of type 1 immune responses and to possess defensin-like bactericidal effects. This study revealed that the infection of mice with Chlamydophila pneumoniae via the intranasal route resulted in the local expression of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. The expression of IP-10/CXCL10 and I-TAC/CXCL11 mRNA peaked on day 4. On day 7, the expression of MIG/CXCL9 mRNA in the infected lungs was increased 156-fold relative to that in the uninfected mouse lungs. MIG/CXCL9 was also detected at a protein level from day 1, with the highest concentration in the supernatants of the infected lungs on day 7. The expression of IFN-γ displayed similar kinetics. C. pneumoniae and its inactivated form also induced the production of MIG/CXCL9 in mouse fibroblasts and in the murine macrophage cell line J774A in vitro. Cotreatment of the tissue cultures with C. pneumoniae and different quantities of IFN-γ resulted in strong increases in MIG/CXCL9 production. Recombinant MIG/CXCL9 exerted dose-dependent antibacterial activity against C. pneumoniae. Significant antichlamydial activity of MIG/CXCL9 was observed after a 15-min incubation period. Chlamydial proteins at a molecular weight of 60 kDa were identified by Far-Western blot assay and liquid chromatography-tandem mass spectrometry as binding molecules of MIG/CXCL9. The results of these experiments suggest that MIG/CXCL9 might play an important role in the innate and acquired defense mechanisms against C. pneumoniae.

Transcriptome analysis indicates an enhanced activation of adaptive and innate immunity by chlamydia-infected murine epithelial cells treated with interferon γ.

BACKGROUND: Interferon γ (IFN­Î³) is the major cytokine involved in the elimination of Chlamydia infection. Despite its importance, the combined effect of Chlamydia infection and IFN­Î³ on the gene expression of murine epithelial cells has only partially been described. METHODS: The DNA chip method was used to evaluate the impact of IFN­Î³ and both the human strain Chlamydia trachomatis L2 infection and the murine strain Chlamydia muridarum infection on the transcriptome of murine epithelial cells. RESULTS: The gene expression analysis revealed that IFN­Î³ had an enhancing effect on both the up­regulation and down­regulation of the epithelial gene expression. The influenced gene functional classes included cytokine and chemokine expression, antigen presentation, apoptosis, and genes involved in basic metabolic processes such as fatty acid oxidation. We also detected the up­regulation of various genes that could be directly antichlamydial, such as members of the p47 GTPase family, inducible nitric oxide synthase, and monokine induced by IFN­Î³ (MIG). As a functional validation of DNA chip data, we measured the antichlamydial effect of MIG on the extracellular form of Chlamydia. CONCLUSIONS: Our results show that IFN­Î³ is a key cytokine that primes epithelial cells to activate adaptive and innate immunity and to express antichlamydial effector genes both intracellularly and extracellularly.

Beneficial Immunomodulatory Effects of Fluticasone Propionate in Chlamydia pneumoniae-Infected Mice.

The associations between inhaled corticosteroid (ICS) use and pulmonary infections remains controversial. Chlamydia pneumoniae (C. pneumoniae) accounts for asthma exacerbations; however, there are no data regarding ICS effects on C. pneumoniae infections. Thus, we investigated whether fluticasone propionate (FP) or budesonide (BUD) could affect C. pneumoniae infection in vitro and in vivo, focusing on the possible mechanisms that lead to potential anti-chlamydial outcomes. We performed direct qPCR to detect C. pneumoniae growth in infected, FP-treated, and BUD-treated A549 cells. Furthermore, FP or BUD was administered by inhalation to C. pneumoniae-infected mice. The recoverable C. pneumoniae was determined by indirect immunofluorescence. Expression levels of interferon (IFN)-γ and IFN-γ inducible chemokines were assessed by qPCR. We measured the protein concentrations of IFN-γ and of other cytokines that potentially participate in the anti-chlamydial response by ELISA. We found that FP treatment suppressed Chlamydia growth in A549 cells and in mice. Higher levels of IFN-γ gene expression were observed in FP-treated mice compared to the untreated and BUD-treated mice (p < 0.0001). IFN-γ and anti-chlamydial protein MIG/CXCL9 values were significantly higher after FP inhalation. Collectively, FP, but not BUD, suppressed C. pneumoniae growth in vitro and in vivo, which was likely due to the enhanced IFN-γ related responses.

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.

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.

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.

Chlamydia pneumoniae Influence on Cytokine Production in Steroid-Resistant and Steroid-Sensitive Asthmatics.

Medications for asthma management consisting of inhaled corticosteroids act by controlling symptoms. However, some patients do not respond to steroid treatment due to immunological factors at the cytokine level. Chlamydia pneumoniae (C. pneumoniae) infection is strongly implicated in asthma pathogenesis, causing altered immune responses. We investigated the association of C. pneumoniae serostatus with the production of certain cytokines by peripheral blood mononuclear cells (PBMCs) of steroid-resistant and -sensitive asthmatic patients. Our most important findings are the following: In the case of C. pneumoniae seropositive patients we detected pronounced spontaneous interleukin (IL)-10 secretion and, in the case of steroid-resistant patients, IL-10 secretion was at a significantly higher level as compared with in-sensitive patients (p < 0.01). Furthermore, steroid-resistant seropositive patients produced a significantly higher level of IL-10 spontaneously and under antigen stimulation as compared with steroid-resistant seronegative individuals (p < 0.05). Concerning spontaneous TNF-α secretion by C. pneumoniae seropositive asthmatics, we observed that steroid-resistant patients produced significantly more of this cytokine than steroid-sensitive patients. In the steroid-resistant patients' sera, a remarkably high MMP-9 concentration was associated with C. pneumoniae seronegativity. Our study revealed that the differences in the cytokine production in steroid-sensitive and -resistant asthmatic patients can be influenced by their C. pneumoniae serostatus.

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.

Adjuvant modulation of the immune response of mice against the LcrE protein of Chlamydophila pneumoniae.

LcrE protein is a TTSS component of Chlamydophila pneumoniae. The immunogenicity and protective effect of recombinant LcrE protein combined either with Freund's or Alum adjuvant were investigated in mice. The immunization with both protocols resulted in a significant reduction of the number of viable C. pneumoniae in the lungs after challenge. Lower IgG2a/IgG1 ratio in Alum-immunized mice suggested a shift towards Th2 type immune response, but the presence of LcrE-specific IFN-gamma-producing cells in LcrE+Alum-immunized mice also indicates Th1 type response. LcrE-specific IgA level was higher in both the sera and the lungs after using Freund's adjuvant. Phenotype of LcrE-specific IFN-gamma-producing cells was CD4+ in Alum- and Freund's-immunized mice, but CD8+ cells were also detected in Freund's-immunized mice. These results confirm that LcrE induces protective immunity in mice. The results also show that Alum is able to activate the CD4+ cell-based cellular immunity, thus it can be regarded as an alternative adjuvant during vaccine screening and a useful adjuvant in a potential protein vaccine against C. pneumoniae infection.

The Opposite Effects of Kynurenic Acid and Different Kynurenic Acid Analogs on Tumor Necrosis Factor-α (TNF-α) Production and Tumor Necrosis Factor-Stimulated Gene-6 (TSG-6) Expression.

Purpose: The investigation of anti-inflammatory and immunosuppressive functions of Kynurenic acid (KYNA) is now in focus. There is also substantial evidence that TSG-6 has an anti-inflammatory activity. Therefore, in the present study, we compared the effects of newly synthetized KYNA analogs on the TNF-α production in U-937 monocytic cells in correlation with the effects on the TSG-6 expression. Methods: TNF-α production was measured by ELISA, the TSG-6 expression was determined by RTqPCR method. As cytokine inducers Staphylococcus aureus and Chlamydia pneumoniae were used. Results: KYNA and KYNA analogs attenuated TNF-α production and increased TSG-6 mRNA expression in U-937 cells stimulated by heat inactivated Staphylococcus aureus. In contrast, KYNA and some of the KYNA analogs increased the TNF-α production of C. pneumoniae infected U-937 cells; however, the newly synthetized analogs (SZR104, SZR 105, and SZR 109) exerted significant inhibitory effects on the TNF-α synthesis. The inhibitory and stimulatory effects correlated inversely with the TSG-6 expression. Conclusions: TSG-6 expression following activation with bacterial components could participate in the suppression of inflammatory cytokines, such as TNF-α, We suppose that the elevation of the TSG-6 expression by KYNA and especially by new KYNA analogs might be one of the mechanisms that are responsible for their suppressive effect on TNF-α production as a feedback mechanism. KYNA and KYNA analogs have an important role in influencing TSG-6 expression, and there is a possible benefit of targeting TSG-6 expression by kynurenines in inflammatory conditions following infections.

Antifibrotic effect of mitomycin-C on human vocal cord fibroblasts.

OBJECTIVE: Acquired laryngotracheal stenosis is a potentially life-threatening situation and a very difficult and challenging problem in laryngology. Therefore, new trends and innovative approaches based on antifibrotic drugs and minimally invasive regimens are being developed to attenuate laryngotracheal fibrosis and scarring. The purpose of this study was to examine the efficacy of mitomycin-C (MMC) to reverse the transforming growth factor (TGF)-ß-induced differentiation of MRC-5 fibroblast and human primary vocal cord fibroblasts to reveal the possible applicability of MMC to laryngotracheal fibrotic conditions. METHODS: Human primary fibroblast cells were isolated from vocal cord specimens of patients undergoing total laryngectomy. The established primary vocal cord fibroblast cell cultures as well as the MRC-5 human fibroblast cells were treated with 5 ng/mL TGF-ß alone and then with 0.5 µg/mL MMC for 24 hours. Differentiation of fibroblasts was characterized by α-smooth muscle actin (α-SMA) immunhistochemistry, Western blot analysis, and real-time polymerase chain reaction. Cell motility was assessed by wound-healing assay. RESULTS: Elevated α-SMA mRNA and protein expression as well as increased cell motility were observed upon TGF-ß exposures. However, after MMC treatments the TGF-ß-induced fibroblasts exhibited a significant decrease in α-SMA expression and wound-healing activity. Therefore, TGF-ß-stimulated fibroblast-myofibroblast transformation was reversed at least in part by MMC treatment. Histopathological examinations of tissue specimens of a laryngotracheal stenosis patient supported these findings. CONCLUSION: Antifibrotic effects of MMC were demonstrated on the human MRC-5 cell line and on primary vocal cord fibroblast cultures. These results verify that MMC can be used with success to reverse upper airway stenosis by reverting the myofibroblast phenotype. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E255-E262, 2019.