In Vitro and In Vivo Screening of Wild Bitter Melon Leaf for Anti-Inflammatory Activity against Cutibacterium acnes.

Cutibacterium acnes (formerly Propionibacterium acnes) is a key pathogen involved in the development and progression of acne inflammation. The numerous bioactive properties of wild bitter melon (WBM) leaf extract and their medicinal applications have been recognized for many years. In this study, we examined the suppressive effect of a methanolic extract (ME) of WBM leaf and fractionated components thereof on live C. acnes-induced in vitro and in vivo inflammation. Following methanol extraction of WBM leaves, we confirmed anti-inflammatory properties of ME in C. acnes-treated human THP-1 monocyte and mouse ear edema models. Using a bioassay-monitored isolation approach and a combination of liquid-liquid extraction and column chromatography, the ME was then separated into n-hexane, ethyl acetate, n-butanol and water-soluble fractions. The hexane fraction exerted the most potent anti-inflammatory effect, suppressing C. acnes-induced interleukin-8 (IL-8) production by 36%. The ethanol-soluble fraction (ESF), which was separated from the n-hexane fraction, significantly inhibited C. acnes-induced activation of mitogen-activated protein kinase (MAPK)-mediated cellular IL-8 production. Similarly, the ESF protected against C. acnes-stimulated mouse ear swelling, as measured by ear thickness (20%) and biopsy weight (23%). Twenty-four compounds in the ESF were identified using gas chromatograph-mass spectrum (GC/MS) analysis. Using co-cultures of C. acnes and THP-1 cells, ß-ionone, a compound of the ESF, reduced the production of IL-1ß and IL-8 up to 40% and 18%, respectively. ß-ionone also reduced epidermal microabscess, neutrophilic infiltration and IL-1ß expression in mouse ear. We also found evidence of the presence of anti-inflammatory substances in an unfractionated phenolic extract of WBM leaf, and demonstrated that the ESF is a potential anti-inflammatory agent for modulating in vitro and in vivo C. acnes-induced inflammatory responses.

Anti-Inflammatory and Antibacterial Activity Constituents from the Stem of Cinnamomum validinerve.

One new dibenzocycloheptene, validinol (1), and one butanolide firstly isolated from the natural source, validinolide (2), together with 17 known compounds were isolated from the stem of Cinnamomum validinerve. Among the isolates, lincomolide A (3), secosubamolide (7), and cinnamtannin B1 (19) exhibited potent inhibition on both superoxide anion generation (IC50 values of 2.98 ± 0.3 µM, 4.37 ± 0.38 µM, and 2.20 ± 0.3 µM, respectively) and elastase release (IC50 values of 3.96 ± 0.31 µM, 3.04 ± 0.23 µM, and 4.64 ± 0.71 µM, respectively) by human neutrophils. In addition, isophilippinolide A (6), secosubamolide (7), and cinnamtannin B1 (19) showed bacteriostatic effects against Propionibacterium acnes in in vitro study, with minimal inhibitory concentration (MIC) values at 16 µg/mL, 16 µg/mL, and 500 µg/mL, respectively. Further investigations using the in vivo ear P. acnes infection model showed that the intraperitoneal administration of the major component cinnamtannin B1 (19) reduced immune cell infiltration and pro-inflammatory cytokines TNF-α and IL-6 at the infection sites. The results demonstrated the potential of cinnamtannin B1 (19) for acne therapy. In summary, these results demonstrated the anti-inflammatory potentials of Formosan C. validinerve during bacterial infections.

Disulfiram, an alcohol dependence therapy, can inhibit the in vitro growth of Francisella tularensis.

Disulfiram (DSF) can help treat alcohol dependency by inhibiting aldehyde dehydrogenase (ALDH). Genomic analysis revealed that Francisella tularensis, the causative agent of tularemia, has lost all but one ALDH-like domain and that this domain retains the target of DSF. In this study, minimum inhibitory concentration (MIC) assays demonstrated that both DSF and its primary metabolite diethyldithiocarbamate (DDC) have strong antimicrobial activity against F. tularensis strain SCHU S4, with the MIC of DSF determined as 2 µg/mL in comparison with 8 µg/mL for DDC. The activity of DSF was further confirmed using an in vitro human macrophage infection assay. Francisella tularensis bacteria in DSF-treated cells were reduced in comparison with untreated and DDC-treated cells, comparable with that observed in doxycycline-treated cells. This suggests that DSF may be suitable for further investigation as an in vivo therapy for tularemia.

Ethanolic Extract of Origanum vulgare Suppresses Propionibacterium acnes-Induced Inflammatory Responses in Human Monocyte and Mouse Ear Edema Models.

Acne vulgaris (acne) is a common inflammatory skin disorder, and Propionibacterium acnes plays a major role in the development and progression of acne inflammation. Herbs possessing antimicrobial and anti-inflammatory activity have been applied as a medical option for centuries. In this study, we examined the suppressive effect of ethanolic oregano (Origanum vulgare) extract on live P. acnes-induced in vivo and in vitro inflammation. Following ethanol extraction of oregano leaves, four compounds with strong antioxidant activity, including rosmarinic acid, quercetin, apigenin, and carvacrol, were identified by high-performance liquid chromatography. Using the mouse ear edema model, we demonstrated that ethanol oregano extracts (EOE) significantly suppressed P. acnes-induced skin inflammation, as measured by ear thickness (32%) and biopsy weight (37%). In a separate study, using the co-culture of P. acnes and human THP-1 monocytes, EOE reduced the production of interleukin (IL)-8, IL-1ß and tumor necrosis factor (TNF)-α up to 40%, 37%, and 18%, respectively, as well as the expression of these three pro-inflammatory mediators at the transcriptional level. Furthermore, EOE inhibited the translocation of nuclear factor-kappa B (NF-κB) into the nucleus possibly by inactivating toll-like receptor-2 (TLR2). The suppressive effect of EOE on live P. acnes-induced inflammatory responses could be due, in part, to the anti-inflammatory and antioxidant properties, but not the anti-microbial effect of EOE.

Cure of tuberculosis using nanotechnology: An overview.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a major health issue of the present era. The bacterium inhabits the host macrophage and other immune cells where it modulates the lysosome trafficking protein, hinders the formation of phagolysosome, and blocks the TNF receptor-dependent apoptosis of host macrophage/monocytes. Other limitations such as resistance to and low bioavailability and bio-distribution of conventional drugs aid to their high virulence and human mortality. This review highlights the use of nanotechnology-based approaches for drug formulation and delivery which could open new avenues to limit the pathogenicity of tuberculosis. Moreover phytochemicals, such as alkaloids, phenols, saponins, steroids, tannins, and terpenoids, extracted from terrestrial plants and mangroves seem promising against M. tuberculosis through different molecular mechanisms. Further understanding of the genomics and proteomics of this pathogenic microbe could also help overcome various research gaps in the path of developing a suitable therapy against tuberculosis.

Treatment of human challenge and MDR strains of Neisseria gonorrhoeae with LpxC inhibitors.

Objectives: Inhibitors of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which catalyses the second step in the biosynthesis of lipid A, have been developed as potential antibiotics for Gram-negative infections. Our objectives were to determine the effect of LpxC inhibition on the in vitro survival and inflammatory potential of Neisseria gonorrhoeae. Methods: Survival of four human challenge strains was determined after treatment with two LpxC inhibitors for 2 and 4 h. To confirm results from treatment and assess their anti-inflammatory effect, the expression of TNF-α by human THP-1 monocytic cells infected with bacteria in the presence of the LpxC inhibitors was quantified. Cytotoxicity of inhibitors for THP-1 cells was evaluated by release of lactate dehydrogenase. Survival of five MDR strains was determined after 2 h of treatment with an LpxC inhibitor and the effect of co-treatment on MICs of ceftriaxone and azithromycin was examined. Results: The inhibitors had bactericidal activity against the four human challenge and five MDR strains with one compound exhibiting complete killing at ≥5 mg/L after either 2 or 4 h of treatment. Treatment of gonococci infecting THP-1 monocytic cells reduced the levels of TNF-α probably owing to reduced numbers of bacteria and a lower level of expression of lipooligosaccharide. Neither inhibitor exhibited cytotoxicity for THP-1 cells. The MIC of azithromycin was slightly lowered by sublethal treatment of two MDR strains with an LpxC inhibitor. Conclusions: Our in vitro results demonstrated promising efficacy of LpxC inhibition of N. gonorrhoeae that warrants further investigation particularly owing to the rise in MDR gonorrhoea.

Induction of Interleukin 10 by Borrelia burgdorferi Is Regulated by the Action of CD14-Dependent p38 Mitogen-Activated Protein Kinase and cAMP-Mediated Chromatin Remodeling.

Host genotype influences the severity of murine Lyme borreliosis, caused by the spirochetal bacterium Borrelia burgdorferi C57BL/6 (B6) mice develop mild Lyme arthritis, whereas C3H/HeN (C3H) mice develop severe Lyme arthritis. Differential expression of interleukin 10 (IL-10) has long been associated with mouse strain differences in Lyme pathogenesis; however, the underlying mechanism(s) of this genotype-specific IL-10 regulation remained elusive. Herein we reveal a cAMP-mediated mechanism of IL-10 regulation in B6 macrophages that is substantially diminished in C3H macrophages. Under cAMP and CD14-p38 mitogen-activated protein kinase (MAPK) signaling, B6 macrophages stimulated with B. burgdorferi produce increased amounts of IL-10 and decreased levels of arthritogenic cytokines, including tumor necrosis factor (TNF). cAMP relaxes chromatin, while p38 increases binding of the transcription factors signal transducer and activator of transcription 3 (STAT3) and specific protein 1 (SP1) to the IL-10 promoter, leading to increased IL-10 production in B6 bone marrow-derived monocytes (BMDMs). Conversely, macrophages derived from arthritis-susceptible C3H mice possess significantly less endogenous cAMP, produce less IL-10, and thus are ill equipped to mitigate the damaging consequences of B. burgdorferi-induced TNF. Intriguingly, an altered balance between anti-inflammatory and proinflammatory cytokines and CD14-dependent regulatory mechanisms also is operative in primary human peripheral blood-derived monocytes, providing potential insight into the clinical spectrum of human Lyme disease. In line with this notion, we have demonstrated that cAMP-enhancing drugs increase IL-10 production in myeloid cells, thus curtailing inflammation associated with murine Lyme borreliosis. Discovery of novel treatments or repurposing of FDA-approved cAMP-modulating medications may be a promising avenue for treatment of patients with adverse clinical outcomes, including certain post-Lyme complications, in whom dysregulated immune responses may play a role.

HLA-B27 Correlates with the Intracellular Elimination, Replication, and Trafficking of Salmonella Enteritidis Collected from Reactive Arthritis Patients.

BACKGROUND The aim of this study was to explore the correlation between HLA-B27 and the intracellular elimination, replication, and trafficking of Salmonella enteritidis (S. enteritidis) collected from patients with reactive arthritis. MATERIAL AND METHODS Confocal microscopy, flow cytometry, and sandwich enzyme-linked immunosorbent assay (ELISA) were employed in this study to evaluate the localization of proteins of interest, to assess the intracellular trafficking of S. enteritidis, and to measure the production of cytokines of interest. RESULTS HLA-B27 was negatively associated with intracellular S. enteritidis elimination in healthy human monocytes/macrophages. In S. enteritidis infected monocytes/macrophages, HLA-27B was also negatively correlated with bacteria elimination but positively related to bacteria replication. S. enteritidis did not co-localize with NRAMP1 and LAMP1/2 in HLA-B27 cells. S. enteritidis did not co-exist with transferrin or dextran within HLA-B27 and A2 cells. CONCLUSIONS HLA-B27 is closely associated with the intracellular elimination and replication of S. enteritidis. Replicated bacteria in HLA-B27 monocytic cells were located within unique vacuoles rather than disturbing host endocytosis.

Linezolid as treatment for pulmonary Mycobacterium avium disease.

OBJECTIVES: To identify the pharmacokinetic/pharmacodynamic parameters and exposures of linezolid in the treatment of pulmonary Mycobacterium avium complex (MAC) disease. METHODS: Human-derived monocytes infected with MAC were inoculated into hollow-fibre systems for dose-effect and dose-scheduling studies. We mimicked linezolid concentration-time profiles achieved in adult human lungs treated for 28 days. Sampling to confirm that the intended linezolid pharmacokinetics had been achieved, and for enumeration of MAC colony-forming units, was performed based on repetitive sampling from each system over the 28 days. We then performed 10 000 patient Monte Carlo simulations to identify doses associated with optimal effect in the clinic. RESULTS: Linezolid achieved a hitherto unprecedented feat of at least 1.0 log10 cfu/mL reduction. Efficacy was most closely linked to the AUC0-24/MIC ratio. The AUC0-24/MIC ratio associated with no change in bacterial burden or bacteriostasis was 7.82, while that associated with 1.0 log10 cfu/mL kill was 42.06. The clinical dose of 600 mg/day achieved or exceeded the bacteriostasis exposure in 98.73% of patients. The proportion of 10 000 patients treated with the standard 1200 mg/day who achieved the exposure for 1.0 log10 cfu/mL kill was 70.64%, but was 90% for 1800 mg/day. The proposed MIC breakpoint for linezolid is 16 mg/L, with which 49%-80% of clinical isolates would be considered resistant. CONCLUSIONS: Linezolid is associated with a bactericidal effect in pulmonary MAC that is greater than that seen with other recommended drugs. However, because of the MIC distribution, doses that would optimize the bactericidal effect would be associated with a high adverse event rate.

Effect of 1,25-dihydroxyvitamin D3 on the expression of mannose receptor, DC-SIGN and autophagy genes in pulmonary tuberculosis.

1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a powerful immuno-modulator, which enhances expression of antimicrobial peptides and induces autophagy in monocytes/macrophages. Since 1,25(OH)2D3 increases the phagocytic potential of monocytes/macrophages, we have explored the effect of 1,25(OH)2D3 on the expression of receptors such as mannose receptor (CD206) and DC-SIGN (CD209) as well as autophagy genes such as ATG5 and Beclin-1 (BECN1) in monocytes/macrophages of healthy controls (HCs) and pulmonary tuberculosis (PTB) patients with and without cavitary disease. Peripheral blood mononuclear cells (PBMCs) were isolated from 40 HCs and 40 PTB patients and were cultured for 72 h with Mtb in the presence or absence of 1,25(OH)2D3 at 10(-7) M concentration. 1,25(OH)2D3 significantly upregulated the expression of mannose receptor, ATG5 and BECN1; whereas DC-SIGN expression was suppressed in Mtb infected cells of both study groups (p < 0.05). The 1,25(OH)2D3-induced expression of CD206, ATG5 and BECN1 genes was lower in PTB patients compared to HCs, whereas expression of these genes was impaired in PTB patients with cavitary disease. Moreover, the relative expression of ATG5 and BECN1 was positively correlated with monocyte/macrophage phagocytosis and cathelicidin antimicrobial peptide gene expression in HCs and PTB patients (p < 0.05). Our study results suggest that vitamin D supplementation in PTB patients without cavitary disease could enhance innate immune functions and may help to control intracellular growth of mycobacteria in macrophages.

RX-P873, a Novel Protein Synthesis Inhibitor, Accumulates in Human THP-1 Monocytes and Is Active against Intracellular Infections by Gram-Positive (Staphylococcus aureus) and Gram-Negative (Pseudomonas aeruginosa) Bacteria.

The pyrrolocytosine RX-P873, a new broad-spectrum antibiotic in preclinical development, inhibits protein synthesis at the translation step. The aims of this work were to study RX-P873's ability to accumulate in eukaryotic cells, together with its activity against extracellular and intracellular forms of infection by Staphylococcus aureus and Pseudomonas aeruginosa, using a pharmacodynamic approach allowing the determination of maximal relative efficacies (Emax values) and bacteriostatic concentrations (Cs values) on the basis of Hill equations of the concentration-response curves. RX-P873's apparent concentration in human THP-1 monocytes was about 6-fold higher than the extracellular one. In broth, MICs ranged from 0.125 to 0.5 mg/liter (S. aureus) and 2 to 8 mg/liter (P. aeruginosa), with no significant shift in these values against strains resistant to currently used antibiotics being noted. In concentration-dependent experiments, the pharmacodynamic profile of RX-P873 was not influenced by the resistance phenotype of the strains. Emax values (expressed as the decrease in the number of CFU from that in the initial inoculum) against S. aureus and P. aeruginosa reached more than 4 log units and 5 log units in broth, respectively, and 0.7 log unit and 2.7 log units in infected THP-1 cells, respectively, after 24 h. Cs values remained close to the MIC in all cases, making RX-P873 more potent than antibiotics to which the strains were resistant (moxifloxacin, vancomycin, and daptomycin for S. aureus; ciprofloxacin and ceftazidime for P. aeruginosa). Kill curves in broth showed that RX-P873 was more rapidly bactericidal against P. aeruginosa than against S. aureus. Taken together, these data suggest that RX-P873 may constitute a useful alternative for infections involving intracellular bacteria, especially Gram-negative species.

Inflammasome priming is similar for francisella species that differentially induce inflammasome activation.

Inflammasome activation is a two-step process where step one, priming, prepares the inflammasome for its subsequent activation, by step two. Classically step one can be induced by LPS priming followed by step two, high dose ATP. Furthermore, when IL-18 processing is used as the inflammasome readout, priming occurs before new protein synthesis. In this context, how intracellular pathogens such as Francisella activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared Francisella strains that differ in virulence and ability to induce inflammasome activation for their relative effects on step one vs. step two. When using the rapid priming model, i.e., 30 min priming by live or heat killed Francisella strains (step 1), followed by ATP (step 2), we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between Francisella strains (F. novicida, F. holarctica -LVS and F. tularensis Schu S4). This priming is fast, independent of bacteria viability, internalization and phagosome escape, but requires TLR2-mediated ERK phosphorylation. In contrast to their efficient priming capacity, Francisella strains LVS and Schu S4 were impaired in inflammasome triggering compared to F. novicida. Thus, observed differences in inflammasome activation by F. novicida, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.

The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes.

Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired). Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients' peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients' phagocytes' ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy.

Disruption of mycobactin biosynthesis leads to attenuation of Mycobacterium tuberculosis for growth and virulence.

BACKGROUND: Low iron availability in the host upregulates the mbt gene cluster of Mycobacterium tuberculosis, which is responsible for mycobactin biosynthesis. However, the biological significance of mycobactins in the growth of this pathogen and in disease progression has not been elucidated. METHODS: We have disrupted the mbtE gene (Rv2380c) in the mbt cluster to evaluate the importance of mycobactin biosynthesis in the growth and virulence of M. tuberculosis. RESULTS: The mbtE mutant (MtbΔmbtE) was unable to synthesize mycobactins, displayed an altered colony morphology, and was attenuated for growth in broth culture and in macrophages. Transmission electron microscopy revealed that MtbΔmbtE displayed an altered cell wall permeability. The growth characteristics and colony morphology of MtbΔmbtE were similar to wild type when the medium was supplemented with mycobactins or when MtbΔmbtE was genetically complemented with the mbtE gene. Moreover, guinea pigs infected with MtbΔmbtE exhibited a significantly reduced bacillary load and histopathological damage in the organs, in comparison to M. tuberculosis-infected animals. CONCLUSIONS: This study highlights the importance of mycobactins in the growth and virulence of M. tuberculosis and establishes the enzymes of mycobactin biosynthesis as novel targets for the development of therapeutic interventions against tuberculosis.

Intracellular forms of menadione-dependent small-colony variants of methicillin-resistant Staphylococcus aureus are hypersusceptible to ß-lactams in a THP-1 cell model due to cooperation between vacuolar acidic pH and oxidant species.

OBJECTIVES: Phagocytosed methicillin-resistant Staphylococcus aureus (MRSA) are susceptible to ß-lactams because of an acid-induced conformational change of penicillin-binding protein (PBP) 2a within phagolysosomes. We have examined whether this mechanism applies to menD and hemB small-colony variants (SCVs) of the COL MRSA strain, using cloxacillin, meropenem, doripenem, and vancomycin as comparator. METHODS: Intracellularly, the change in cfu from post-phagocytosis inoculum was measured after 24 h of incubation with antibiotics combined or not with N-acetylcysteine (NAC; oxidant species scavenger); the relative potency (C(s)) was calculated from the Hill equation of concentration-response curves. Extracellularly, the effect of a pre-incubation with H(2)O(2) was determined on MICs and killing at pH 7.4 and 5.5. RESULTS: Intracellularly, the ß-lactam C(s) was similar for the COL strain and the hemB mutant and not modified or slightly decreased (2- to 16-fold) by NAC. In contrast, the C(s) was 100- to 900-fold lower for the menD mutant, but similar to that for the COL strain when NAC was present. Extracellularly, ß-lactam MICs were markedly reduced at pH 5.5 for the parental strain and the haemin-supplemented hemB mutant, with limited additional effect of pre-incubation with H(2)O(2). In contrast, MICs remained elevated at pH 5.5 for the menD mutant (supplemented with menadione sodium bisulphite or not), but were 7-10 dilutions lower after pre-incubation with H(2)O(2). Vancomycin MICs were unaltered in all conditions, with no marked effect of NAC on C(s). CONCLUSIONS: Cooperation between acidic pH and oxidant species confers high potency to ß-lactams against intracellular forms of menD SCVs of MRSA.

Pharmacodynamic evaluation of the activity of antibiotics against hemin- and menadione-dependent small-colony variants of Staphylococcus aureus in models of extracellular (broth) and intracellular (THP-1 monocytes) infections.

Staphylococcus aureus small-colony variants (SCVs) persist intracellularly, which may contribute to persistence/recurrence of infections and antibiotic failure. We have studied the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the COL methicillin-resistant S. aureus (MRSA) strain and the antibiotic pharmacodynamic profile against extracellular (broth) and intracellular (human THP-1 monocytes) bacteria. Compared to the parental strain, SCVs showed slower extracellular growth (restored upon medium supplementation with menadione or hemin), reduced phagocytosis, and, for the menD SCV, lower intracellular counts at 24 h postinfection. Against extracellular bacteria, daptomycin, gentamicin, rifampin, moxifloxacin, and oritavancin showed similar profiles of activity against all strains, with a static effect obtained at concentrations close to their MICs and complete eradication as maximal effect. In contrast, vancomycin was not bactericidal against SCVs. Against intracellular bacteria, concentration-effect curves fitted sigmoidal regressions for vancomycin, daptomycin, gentamicin, and rifampin (with maximal effects lower than a 2-log decrease in CFU) but biphasic regressions (with a maximal effect greater than a 3-log decrease in CFU) for moxifloxacin and oritavancin, suggesting a dual mode of action against intracellular bacteria. For all antibiotics, these curves were indistinguishable between the strains investigated, except for the menD mutant, which systematically showed a lower amplitude of the concentration-effect response, with markedly reduced minimal efficacy (due to slower growth) but no change in maximal efficacy. The data therefore show that the maximal efficacies of antibiotics are similar against normal-phenotype and menadione- and hemin-dependent strains despite their different intracellular fates, with oritavancin, and to some extent moxifloxacin, being the most effective.

Divergent modulation of Chlamydia pneumoniae infection cycle in human monocytic and endothelial cells by iron, tryptophan availability and interferon gamma.

Chlamydia pneumoniae is an obligatory intracellular bacterium causing chronic inflammatory diseases in humans. We studied the role of the nutritive factors, iron and tryptophan, towards the course of infection and immune response pathways in C. pneumoniae infected endothelial cells and monocytes. Human endothelial (EA.hy923) and monocytic cells (THP-1) were infected with C. pneumoniae, supplemented with iron or 1-methyltryptophan (1-MT), an inhibitor of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO), and subsequently stimulated with IFN-gamma or left untreated. The number of infected cells, the morphology and quantity of C. pneumoniae inclusion bodies, IDO activity and innate immune effector pathways were analysed. While neither iron challenge, IDO inhibition or IFN-gamma treatment had a significant effect on C. pneumoniae morphology or numbers within THP-1 monocytic cells, iron supplementation to EA.hy926 cells resulted in promotion of C. pneumoniae proliferation and differentiation while IFN-gamma had an inhibitory effect. Furthermore, the number of infected endothelial cells was significantly decreased upon 1-MT treatment. C. pneumoniae infection induced a pro-inflammatory immune response as evidenced by increased IDO activity, neopterin formation or TNF-alpha production in THP-1 but not in endothelial cells. These pathways were superinduced upon IFN-gamma treatment and partly modulated by iron supplementation. Our results demonstrate that the infectious cycle of C. pneumoniae behaves differently between monocytic and endothelial cells. While the intracellular pathogen remains in a persistent form within monocytes, it can differentiate and proliferate within endothelial cells indicating that endothelial cells are a preferred environment for Chlamydia. Nutritive factors such as iron have subtle effects on C. pneumoniae biology in endothelial, but not monocytic cells. Our results contribute to a better understanding of C. pneumoniae infection and its role in chronic inflammatory diseases such as atherosclerosis.

Screening a mushroom extract library for activity against Acinetobacter baumannii and Burkholderia cepacia and the identification of a compound with anti-Burkholderia activity.

BACKGROUND: Acinetobacter baumannii and species within the Burkholderia cepacia complex (BCC) are significant opportunistic bacterial pathogens of humans. These species exhibit a high degree of antibiotic resistance, and some clinical isolates are resistant to all currently available antimicrobial drugs used for treatment. Thus, new drugs are needed to treat infections by these species. Mushrooms could be a potential source for new drugs to treat A. baumannii and BCC infections. METHODS: The aim of this study was to screen a library of crude extracts from 330 wild mushrooms by disk diffusion assays for antibacterial activity against A. baumannii and Burkholderia cepacia in the hope of identifying a novel natural drug that could be used to treat infections caused by these species. Once positive hits were identified, the extracts were subjected to bioassay-guided separations to isolate and identify the active drug molecules. MICs were performed to gauge the in vitro activity of the purified compounds. RESULTS: Only three crude extracts (0.9%) had activity against A. baumannii and B. cepacia. Compounds from two of these extracts had MICs greater than 128 microg/ml, and further analyses were not performed. From the third extract, prepared from Leucopaxillus albissimus, 2-aminoquinoline (2-AQ) was isolated. This compound exhibited a modest MIC in vitro against strains from nine different BCC species, including multi-drug resistant clinical isolates (MIC = 8-64 microg/ml), and a weak MIC (128 microg/ml) against A baumannii. The IC50 against a murine monocyte line was 1.5 mg/ml. CONCLUSION: The small number of positive hits in this study suggests that finding a new drug from mushrooms to treat Gram-negative bacterial infections may be difficult. Although 2-AQ was identified in one mushroom, and it was shown to inhibit the growth of multi-drug resistant BCC isolates, the relatively high MICs (8-128 microg/ml) for both A. baumannii and BCC strains suggests that 2-AQ is not suitable for further drug development in its current form.

Expression of inflammatory host genes in Chlamydia trachomatis-infected human monocytes.

The aim of this study was to perform a comprehensive gene expression analysis of cytokines, chemokines, and their receptors in Chlamydia trachomatis-infected human monocytes in order to elucidate molecular aspects of their involvement in the host response. Peripheral blood mononuclear cells from three healthy donors were separated and infected with C. trachomatis elementary bodies serovar K (UW/31/Cx) at a multiplicity of infection of 5:1. Three time points of infection were studied by gene expression analysis using microarray: 4 hours (active infection), 1 day (transition), and 7 days (persistent infection). Expression levels of selected genes were confirmed by quantitative real-time reverse transcription-polymerase chain reaction. Transcripts encoding 10 cytokines, chemokines, and receptors were found to be upregulated exclusively in the early, active phase of the infection as compared to four genes in the late, persistent state of the infection. Apart from receptors, both the level and the number of transcripts encoding inflammatory products decreased with ongoing infection. Four genes (interferon-gamma, macrophage inflammatory protein [MIP]-1-alpha, MIP-1-beta, and interleukin-2 receptor-gamma) were constantly expressed over a period of 7 days. The current study provides data on the induction of mRNA encoding cytokines, chemokines, and their receptors in C. trachomatis-infected human monocytes. This pro-inflammatory gene expression profile of the monocytic host cell showed several differences between active and persistent chlamydial infections.

Hairpin extensions enhance the efficacy of mycolyl transferase-specific antisense oligonucleotides targeting Mycobacterium tuberculosis.

We have investigated the efficacy of modifying gene-specific antisense phosphorothioate oligodeoxyribonucleotides (PS-ODNs) by the addition of 5' and 3' hairpin extensions. As a model system, we have targeted the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase protein complex genes encoding three highly related enzymes (antigens 85 A, B, and C). Whereas the addition of a hairpin extension at only one end of the PS-ODNs did not improve their inhibitory capacity, the addition of hairpin extensions at both ends enhanced their capacity to inhibit M. tuberculosis multiplication in comparison with unmodified PS-ODNs. A combination of three 5'-, 3'-hairpin-modified PS-ODNs (HPS-ODNs) targeting each of the three mycolyl transferase transcripts inhibited bacterial growth in broth culture by approximately 1.75 log units (P < 0.0001) and in human THP-1 macrophages by approximately 0.4 log units (P < 0.0001), which to our knowledge has not previously been demonstrated for any PS-ODN; reduced target gene transcription by > or =90%; caused approximately 90% reduction in mycolyl transferase expression; and increased bacterial sensitivity to isoniazid by 8-fold. The growth-inhibitory effect of the HPS-ODNs was gene-specific. Mismatched HPS-ODNs had no growth-inhibitory capacity. This study demonstrates that 5'- and 3'-HPS-ODNs are highly efficacious against M. tuberculosis and supports the further development of antisense technology as a therapeutic modality against tuberculosis.