CD45RB Is a Novel Molecular Therapeutic Target to Inhibit Aβ Peptide-Induced Microglial MAPK Activation

Background

Microglial activation, characterized by p38 MAPK or p44/42 MAPK pathway signal transduction, occurs in Alzheimer''s disease (AD). Our previous studies demonstrated CD45, a membrane-bound protein tyrosine phosphatase (PTP), opposed β-amyloid (Aβ) peptide-induced microglial activation via inhibition of p44/42 MAPK. Additionally we have shown agonism of the RB isoform of CD45 (CD45RB) abrogates lipopolysaccharide (LPS)-induced microglial activation.

Methodology and Results

In this study, CD45RB modulation of Aβ peptide or LPS-activated primary cultured microglial cells was further investigated. Microglial cells were co-treated with “aged” FITC-Aβ_1–42 and multiple CD45 isoform agonist antibodies. Data revealed cross-linking of CD45, particularly the CD45RB isoform, enhances microglial phagocytosis of Aβ_1–42 peptide and inhibits LPS-induced activation of p44/42 and p38 pathways. Co-treatment of microglial cells with agonist CD45 antibodies results in significant inhibition of LPS-induced microglial TNF-α and IL-6 release through p44/42 and/or p38 pathways. Moreover, inhibition of either of these pathways augmented CD45RB cross-linking induced microglial phagocytosis of Aβ_1–42 peptide. To investigate the mechanism(s) involved, microglial cells were co-treated with a PTP inhibitor (potassium bisperoxo [1,10-phenanthroline oxovanadate; Phen]) and Aβ_1–42 peptides. Data showed synergistic induction of microglial activation as evidenced by TNF-α and IL-6 release; both of which are demonstrated to be dependent on increased p44/42 and/or p38 activation. Finally, it was observed that cross-linking of CD45RB in the presence of Aβ_1–42 peptide, inhibits co-localization of microglial MHC class II and Aβ peptide; suggesting CD45 activation inhibits the antigen presenting phenotype of microglial cells.

Conclusion

In summary, p38 MAPK is another novel signaling pathway, besides p44/42, in which CD45RB cross-linking negatively regulates microglial Aβ phagocytosis while increasing potentially neurotoxic inflammation. Therefore, agonism of CD45RB PTP activity may be an effective therapeutic target for novel agents to treat AD due to its Aβ lowering, and inflammation reducing, properties that are particularly targeted at microglial cells. Such treatments may be more effective with less potential to produce systemic side-effects than therapeutics which induce non-specific, systemic down-regulation of inflammation.     

Hp1404, a New Antimicrobial Peptide from the Scorpion Heterometrus petersii

Antimicrobial peptides have attracted much interest as a novel class of antibiotics against a variety of microbes including antibiotics resistant strains. In this study, a new cationic antimicrobial peptide Hp1404 was identified from the scorpion Heterometrus petersii, which is an amphipathic α-helical peptide and has a specific inhibitory activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus. Hp1404 can penetrate the membrane of S. aureus at low concentration, and disrupts the cellular membrane directly at super high concentration. S. aureus does not develop drug resistance after multiple treatments with Hp1404 at sub MIC concentration, which is possibly associated with the antibacterial mechanism of the peptide. In addition, Hp1404 has low toxicity to both mammalian cells (HC₅₀  =  226.6 µg/mL and CC₅₀ > 100 µg/mL) and balb-c mice (Non-toxicity at 80 mg/Kg by intraperitoneal injection and LD₅₀  =  89.8 mg/Kg by intravenous injection). Interestingly, Hp1404 can improve the survival rate of the MRSA infected balb-c mice in the peritonitis model. Taken together, Hp1404 may have potential applications as an antibacterial agent.     

Chemical Characterization and Antioxidant,Antimicrobial, and Anti-Inflammatory Activities of South Brazilian Organic Propolis

South Brazilian organic propolis (OP), which has never been studied before, was assessed and its chemical composition, scavenging potential of reactive oxygen species, antimicrobial and anti-inflammatory activities are herein presented. Based on the chemical profile obtained using HPLC, OP was grouped into seven variants (OP1–OP7) and all of them exhibited high scavenging activity, mainly against superoxide and hypochlorous acid species. OP1, OP2, and OP3 had the smallest minimal inhibitory concentration (MIC) against Gram-positive bacteria Streptococcus mutans, Streptococcus oralis, and Streptococcus aureus (12.5–100 μg/mL). OP1, OP2, OP3, and OP4 were more effective against Pseudomonas aeruginosa (Gram-negative), with MIC values ranging from 100 to 200 μg/mL. OP6 showed anti-inflammatory activity by decreasing NF-kB activation and TNF-α release in RAW 264.7 macrophages, and expressing the NF-κB-luciferase reporter stable gene. Therefore, south Brazilian OP can be considered an excellent source of bioactive compounds with great potential of application in the pharmaceutical and food industry.     

A Clinical Trial to Evaluate the Efficacy of α-Viniferin in Staphylococcus aureus – Specific Decolonization without Depleting the Normal Microbiota of Nares

Staphylococcus aureus is currently a significant multidrug-resistant bacterium, causing severe healthcare-associated and community-acquired infections worldwide. The current antibiotic regimen against this pathogen is becoming ineffective due to resistance, in addition, they disrupt the normal microbiota. It highlights the urgent need for a pathogen-specific drug with high antibacterial efficacy against S. aureus. α-Viniferin, a bioactive phytochemical compound, has been reported to have excellent anti-Staphylococcus efficacy as a topical agent. However, so far, there were no clinical trials that have been conducted to elucidate its efficacy. The present study aimed to investigate the antibacterial efficacy of α-viniferin against S. aureus in a ten-day clinical trial. Based on the results, α-viniferin showed 50% minimum inhibitory concentrations (MIC₅₀ values) of 7.8 μg/ml in culture broth medium. α-Viniferin was administered in the nares three times a day for ten days using a sterile cotton swab stick. Nasal swab specimens were collected before (0 days) and after finishing the trial (10^th day), and then analyzed. In the culture and RT-PCR-based analysis, S. ureus was reduced significantly: 0.01. In addition, 16S ribosomal RNA-based amplicon sequencing analysis showed that S. aureus reduced from 51.03% to 23.99% at the genus level. RNA-seq analysis was also done to gain insights into molecular mechanisms of α-viniferin against S. aureus, which revealed that some gene groups were reduced in 5-fold FC cutoff at two times MIC conditions. The study results demonstrate α-viniferin as a potential S. aureus-specific drug candidate.     

Oligomeric amyloid β induces IL-1β processing via production of ROS: implication in Alzheimer's disease

Alzheimer''s disease (AD) is a chronic neurodegenerative disease characterized by progressive neuronal loss and cognitive decline. Oligomeric amyloid β (oAβ) is involved in the pathogenesis of AD by affecting synaptic plasticity and inhibiting long-term potentiation. Although several lines of evidence suggests that microglia, the resident immune cells in the central nervous system (CNS), are neurotoxic in the development of AD, the mechanism whether or how oAβ induces microglial neurotoxicity remains unknown. Here, we show that oAβ promotes the processing of pro-interleukin (IL)-1β into mature IL-1β in microglia, which then enhances microglial neurotoxicity. The processing is induced by an increase in activity of caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3) via mitochondrial reactive oxygen species (ROS) and partially via NADPH oxidase-induced ROS. The caspase-1 inhibitor Z-YVAD-FMK inhibits the processing of IL-1β, and attenuates microglial neurotoxicity. Our results indicate that microglia can be activated by oAβ to induce neuroinflammation through processing of IL-1β, a pro-inflammatory cytokine, in AD.     

Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4

The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a critical health issue that has drawn greater attention to the potential use of immunotherapy. Toll-like receptor 2 (TLR2), a pattern recognition receptor, is an essential component in host innate defense system against S. aureus infection. However, little is known about the innate immune response, specifically TLR2 activation, against MRSA infection. Here, we evaluate the protective effect and the mechanism of MRSA murine pneumonia after pretreatment with Pam3CSK4, a TLR2 agonist. We found that the MRSA-pneumonia mouse model, pretreated with Pam3CSK4, had reduced bacteria and mortality in comparison to control mice. As well, lower protein and mRNA levels of TNF-α, IL-1β and IL-6 were observed in lungs and bronchus of the Pam3CSK4 pretreatment group. Conversely, expression of anti-inflammatory cytokine IL-10, but not TGF-β, increased in Pam3CSK4-pretreated mice. Our additional studies showed that CXCL-2 and CXCL1, which are necessary for neutrophil recruitment, were less evident in the Pam3CSK4-pretreated group compared to control group, whereas the expression of Fcγ receptors (FcγⅠ/Ⅲ) and complement receptors (CR1/3) increased in murine lungs. Furthermore, we found that increased survival and improved bacterial clearance were not a result of higher levels of neutrophil infiltration, but rather a result of enhanced phagocytosis and bactericidal activity of neutrophils in vitro and in vivo as well as increased robust oxidative activity and release of lactoferrin. Our cumulative findings suggest that Pam3CSK4 could be a novel immunotherapeutic candidate against MRSA pneumonia.     

Galectin-1 stimulates motility of human umbilical cord blood-derived mesenchymal stem cells by downregulation of smad2/3-dependent collagen 3/5 and upregulation of NF-κB-dependent fibronectin/laminin 5 expression

Galectin-1 (Gal-1) belongs to a family of endogenous lectins with conserved carbohydrate recognition domains binding β-galactosidase sugars and plays a vital role in regulating stem cell functions including determination of cell fate. However, our understanding of the functional roles of Gal-1 in human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) is still fragmentary and incomplete. Gal-1 significantly increased motility after a 24-h incubation, and this effect was inhibited by β-lactose. We analyzed 17 extracellular matrix (ECM) genes in UCB-MSCs. Gal-1 decreased the expression of collagen genes COL3A1 (COL-3) and COL5A1 (COL-5) but increased the expression of fibronectin (FN) and laminin 5 (LM-5), that were reversed by β-lactose. Gal-1 increased protein kinase C (PKC), c-Src, and caveolin-1 (Cav-1) phosphorylation that was attenuated by β-lactose and the Src inhibitor PP2. In addition, pretreatment with the lipid raft disruptor Mβ-CD and the PKC inhibitors inhibited Gal-1-induced UCB-MSC motility. In addition, Gal-1 reduced smad2/3 phosphorylation and induced nuclear factor (NF)-κB phosphorylation. Pretreatment with Mβ-CD attenuated Gal-1-reduced smad2/3 phosphorylation, COL-3, and COL-5 expression but did not affect NF-κB phosphorylation, FN, or LM-5 expression. In contrast, PKC inhibitors only attenuated NF-κB phosphorylation, FN, and LM-5 expression. Reconstructing Gal-1-induced genetic changes by replacing it with siRNA specific for COL-3 or COL-5, or treatment of the cells with FN and LM-5 proteins, increased motility and its related proteins such as focal adhesion kinase, Akt, Erk, integrins, and matrix metalloproteinase-2. A combined treatment with COL-3/COL-5 siRNA or FN/LM-5 compared with that of single treatments was synergistic. However, a single Gal-1 treatment maximally stimulated motility and related protein phosphorylation/expression. These results demonstrate that Gal-1 stimulated human UCB-MSC motility by decreasing COL-3/COL-5 expression and increasing FN/LM-5 expression through a PKC-dependent NF-κB and c-Src/Cav-1-dependent smad2/3 pathway that was critical for governing the activation of FAK, Akt, Erk, integrins, and MMP2.     

The Antibacterial Properties of 4, 8, 4′, 8′-Tetramethoxy (1,1′-biphenanthrene) -2,7,2′,7′-Tetrol from Fibrous Roots of Bletilla striata

Biphenanthrene compound, 4, 8, 4′, 8′-tetramethoxy (1, 1′-biphenanthrene)—2, 7, 2′, 7′-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10 min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.     

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α_S1-casein (α_S1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α_S1-CN f1-6 and α_S2-CN f182-185 and f186-188; caprine β-CN f58-65 and α_S2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC₅₀) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC₅₀s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

Mechanism of Action of Recombinant Acc-Royalisin from Royal Jelly of Asian Honeybee against Gram-Positive Bacteria

The antibacterial activity of royalisin, an antimicrobial peptide from the royal jelly produced by honeybees, has been addressed extensively. However, its mechanism of action remains unclear. In this study, a recombinant royalisin, RAcc-royalisin from the royal jelly of Asian honeybee Apis cerana cerana, was expressed by fusing with glutathione S-transferase (GST) in Escherichia coli BL21, isolated and purified. The agar dilution assays with inhibition zone showed that RAcc-royalisin, similar to nisin, inhibits the growth of Gram-positive bacteria. The antibacterial activity of RAcc-royalisin was associated with its concentration, and was weakened by heat treatment ranging from 55°C to 85°C for 15 min. Both RAcc-royalisin and nisin exhibited the minimum inhibitory concentrations (MIC) of 62.5 µg/ml, 125 µg/ml, and 250 µg/ml against Gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. However, RAcc-royalisin did not show antimicrobial activity against tested Gram-negative bacterial and fungal strains. The antibacterial activity of RAcc-royalisin agrees well with the decrease in bacterial cell hydrophobicity, the leakage of 260-nm absorbing materials, and the observation by transmission electron microscopy, all indicating that RAcc-royalisin induced the disruption and dysfunction of cell walls and membranes. This is the first report detailing the antibacterial mechanism of royalisin against Gram-positive bacteria, and provides insight into the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent.     

Aerosols of Mycoplasmas, L Forms, and Bacteria: Comparison of Particle Size, Viability, and Lethality of Ultraviolet Radiation

Aerosols of microorganisms were tested for particle size by use of an Andersen sampler. Mycoplasma aerosols had an average count median diameter (CMD) of 2.1 ± 0.5 μ. Staphylococcus aureus L forms gave an average CMD of 4.6 ± 1.7 μ; the diphtheroid L form, a CMD of 3.4 ± 0.3 μ. Escherichia coli had a CMD of 5.4 ± 2.5 μ; Neisseria sicca, 3.3 ± 0.5 μ; N. meningitidis, 3.4 ± 0.2 μ. S. aureus ATCC 6538, the parent strain of the L form, yielded a CMD of 3.9 ± 1.2 μ. Candida albicans gave an average CMD of 5.9 ± 1.4 μ. All organisms tested survived aerosolizing and could be recovered in viable form for at least 1 hr. Ultraviolet radiation at 2,537 A destroyed the bacteria and mycoplasmas instantaneously, and destroyed 87% of the L forms of S. aureus, 69% of the diphtheroid L form, and 98% of the C. albicans cells. After irradiation, viable particles of the L form and C. albicans aerosols were consistently larger, indicating that clumping led to survival. Submicron size particles were found in aerosols of all species tested except C. albicans.     

Lactococcin Q, a Novel Two-Peptide Bacteriocin Produced by Lactococcus lactis QU 4

A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, α and β, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qα plus Qβ) and lactococcin G (Gα plus Gβ) clarified that hybrid combinations (Qα plus Gβ and Gα plus Qβ) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qα plus Gβ was 32 times higher than that of Qα plus Qβ, suggesting that the difference in β peptides was important for the intensity of antibacterial activity.     

Two-Component Anti-Staphylococcus aureus Lantibiotic Activity Produced by Staphylococcus aureus C55

Staphylococcus aureus C55 was shown to produce bacteriocin activity comprising three distinct peptide components, termed staphylococcins C55α, C55β, and C55γ. The three peptides were purified to homogeneity by a simple four-step purification procedure that consisted of ammonium sulfate precipitation followed by XAD-2 and reversed-phase (C₈ and C₁₈) chromatography. The yield following C₈ chromatography was about 86%, with a more-than-300-fold increase in specific activity. When combined in approximately equimolar amounts, staphylococcins C55α and C55β acted synergistically to kill S. aureus or Micrococcus luteus but not S. epidermidis strains. The N-terminal amino acid sequences of all three peptides were obtained and staphylococcins C55α and C55β were shown to be lanthionine-containing (lantibiotic) molecules with molecular weights of 3,339 and 2,993, respectively. The C55γ peptide did not appear to be a lantibiotic, nor did it augment the inhibitory activities of staphylococcin C55α and/or C55β. Plasmids of 2.5 and 32.0 kb are present in strain C55, and following growth of this strain at elevated temperature (42°C), a large proportion of the progeny failed to produce strong bacteriocin activity and also lost the 32.0-kb plasmid. Protoplast transformation of these bacteria with purified 32-kb plasmid DNA regenerates the ability to produce the strong bacteriocin activity.     

Rational Design of Berberine-Based FtsZ Inhibitors with Broad-Spectrum Antibacterial Activity

Inhibition of the functional activity of Filamenting temperature-sensitive mutant Z (FtsZ) protein, an essential and highly conserved bacterial cytokinesis protein, is a promising approach for the development of a new class of antibacterial agents. Berberine, a benzylisoquinoline alkaloid widely used in traditional Chinese and native American medicines for its antimicrobial properties, has been recently reported to inhibit FtsZ. Using a combination of in silico structure-based design and in vitro biological assays, 9-phenoxyalkyl berberine derivatives were identified as potent FtsZ inhibitors. Compared to the parent compound berberine, the derivatives showed a significant enhancement of antibacterial activity against clinically relevant bacteria, and an improved potency against the GTPase activity and polymerization of FtsZ. The most potent compound 2 strongly inhibited the proliferation of Gram-positive bacteria, including methicillin-resistant S. aureus and vancomycin-resistant E. faecium, with MIC values between 2 and 4 µg/mL, and was active against the Gram-negative E. coli and K. pneumoniae, with MIC values of 32 and 64 µg/mL respectively. The compound perturbed the formation of cytokinetic Z-ring in E. coli. Also, the compound interfered with in vitro polymerization of S. aureus FtsZ. Taken together, the chemical modification of berberine with 9-phenoxyalkyl substituent groups greatly improved the antibacterial activity via targeting FtsZ.     

The Wound Healing and Antibacterial Activity of Five Ethnomedical Calophyllum inophyllum Oils: An Alternative Therapeutic Strategy to Treat Infected Wounds

Background

Calophyllum inophyllum L. (Calophyllaceae) is an evergreen tree ethno-medically used along the seashores and islands of the Indian and Pacific Oceans, especially in Polynesia. Oil extracted from the seeds is traditionally used topically to treat a wide range of skin injuries from burn, scar and infected wounds to skin diseases such as dermatosis, urticaria and eczema. However, very few scientific studies reported and quantified the therapeutic properties of Calophyllum inophyllum oil (CIO). In this work, five CIO from Indonesia (CIO1), Tahiti (CIO2, 3), Fiji islands (CIO4) and New Caledonia (CIO5) were studied and their cytotoxic, wound healing, and antibacterial properties were presented in order to provide a scientific support to their traditional use and verify their safety.

Methods

The safety of the five CIO was ascertained using the Alamar blue assay on human keratinocyte cells. CIO wound healing properties were determined using the scratch test assay on human keratinocyte cells. CIO-stimulated antibacterial innate immune response was evaluated using ELISA by measuring β defensin-2 release in human derivative macrophage cells. CIO antibacterial activity was tested using oilogramme against twenty aerobic Gram- bacteria species, twenty aerobic Gram+ bacteria species, including a multi-drug resistant Staphylococcus aureus strain and two anaerobic Gram+ bacteria species e.g. Propionibacterium acnes and Propionibacterium granulosum. To detect polarity profile of the components responsible of the antibacterial activity, we performed bioautography against a Staphylococcus aureus strain.

Results

Based on Alamar Blue assay, we showed that CIO can be safely used on keratinocyte cells between 2.7% and 11.2% depending on CIO origin. Concerning the healing activity, all the CIO tested accelerated in vitro wound closure, the healing factor being 1.3 to 2.1 higher compared to control when keratinocytes were incubated after scratch with CIO at 0.1%. Furthermore, our results showed that CIO exhibit two distinct antibacterial effects: one against Gram+ bacteria by direct inhibition of mitotic growth and another potent effect against Gram- bacteria due to increased release of β-defensin 2 peptide by macrophages. Interestingly, the needed concentrations of CIO to inhibit bacteria growth and to promote wound healing are lower than concentrations exhibiting cytotoxic effects on keratinocyte cells. Finally, we performed bioautography assay against Staphylococcus aureus to determine polarity profile of the components responsible for CIO antibacterial activity. Our results showed for the five tested CIO that components responsible of the bacterial growth inhibition are the more polar one on the TLC chromatographic profile and are contained in the resinous fraction of the oil.

Conclusions

This study was conducted to evaluate cytotoxicity, wound healing and antibacterial properties of five CIO traditionally used to treat infected wounds. Using cell and bacteria cultures, we confirmed the pharmacological effects of CIO as wound healing and antimicrobial agent. Moreover, we showed that concentration of CIO needed to exhibit therapeutic effects are lower than concentrations exhibiting cytotoxic effects in vitro. For the first time, this study provides support for traditional uses of CIO. These wound healing and antibiotic properties make CIO a valuable candidate to treat infected wounds especially in tropical areas.     

Moxifloxacin modulates inflammation during murine pneumonia

Background

Moxifloxacin is a synthetic antibacterial agent belonging to the fluoroquinolone family. The antimicrobial activity of quinolones against Gram-positive and Gram-negative bacteria is based on their ability to inhibit topoisomerases. Quinolones are described to have immunomodulatory features in addition to their antimicrobial activities. It was the goal of this study to examine whether a short term treatment with moxifloxacin modulates the inflammation during a subsequently induced bacterial infection in an animal model.

Methods

Mice were treated with moxifloxacin or saline for two consecutive days and were subsequently intranasally infected with viable or heat-inactivated bacterial pathogens (Streptococcus pneumoniae, Pseudomonas aeruginosa) for 6 and 24 hours. Measurements of cytokines in the lungs and plasma were performed. Alveolar cells were determined in bronchoalveolar lavage fluits.

Results

The inflammation was increased after the inoculation of viable bacteria compared to inactivated bacteria. Numbers of total immune cells and neutrophils and concentrations of inflammatory mediators (e.g. KC, IL-1β, IL-17A) were significantly reduced in lungs of moxifloxacin-treated mice infected with inactivated and viable bacterial pathogens as compared to infected control mice. Plasma concentrations of inflammatory mediators were significantly reduced in moxifloxacin-treated mice. Immunohistochemistry showed a stronger infiltrate of TNF-α-expressing cells into lungs of saline-treated mice infected with viable P. aeruginosa as compared to moxifloxacin-treated mice.

Conclusions

These data show that in this pneumonia model moxifloxacin has anti-inflammatory properties beyond its antibacterial activity.     

An Important Role of α-Hemolysin in Extracellular Vesicles on the Development of Atopic Dermatitis Induced by Staphylococcus aureus

Skin barrier disruption and dermal inflammation are key phenotypes of atopic dermatitis (AD). Staphylococcus aureus secretes extracellular vesicles (EVs), which are involved in AD pathogenesis. Here, we evaluated the role of EVs-associated α-hemolysin derived from S. aureus in AD pathogenesis. α-hemolysin production from S. aureus was detected using western blot analyses. The cytotoxic activity of α-hemolysin on HaCaT keratinocytes was evaluated by measuring cell viability after treating cells with soluble and EVs-associated α-hemolysin. To determine the type of cell death, HaCaT keratinocytes were stained with annexin V and 7-AAD. The in vivo effects of α-hemolysin were evaluated by application of soluble and EV-associated α-hemolysin on the mouse skin. The present study showed that increased α-hemolysin was produced by S. aureus colonized on AD patients compared to healthy subjects. α-hemolysin production was also related to AD severity. In addition, EV-associated α-hemolysin was more cytotoxic to HaCaT keratinocytes than soluble α-hemolysin, and α-hemolysin-negative EVs did not induce keratinocyte death. EV-associated α-hemolysin induced necrosis, but soluble α-hemolysin induced apoptosis of keratinocytes. In vivo, skin barrier disruption and epidermal hyperplasia were induced by soluble and EV-associated α-hemolysin. However, AD-like dermal inflammation was only caused by EV-associated α-hemolysin. Moreover, neither skin barrier disruption nor AD-like skin inflammation was induced by α-hemolysin-negative EVs. Taken together, α-Hemolysin secreted from S. aureus, particularly the EV-associated form, induces both skin barrier disruption and AD-like skin inflammation, suggesting that EV-associated α-hemolysin is a novel diagnostic and therapeutic target for the control of AD.     

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.     

Evidence of the Innate Antiviral and Neuroprotective Properties of Progranulin

Background

Compelling data exist that show that normal levels of progranulin (PGRN) are required for successful CNS aging. PGRN production is also modulated by inflammation and infection, but no data are available on the production and role of PGRN during CNS HIV infection.

Methods

To determine the relationships between PGRN and HIV disease, neurocognition, and inflammation, we analyzed 107 matched CSF and plasma samples from CHARTER, a well-characterized HIV cohort. Levels of PGRN were determined by ELISA and compared to levels of several inflammatory mediators (IFNγ, IL-6, IL-10, IP-10, MCP-1, TNFα, IL-1β, IL-4 and IL-13), as well as clinical, virologic and demographic parameters. The relationship between HIV infection and PGRN was also examined in HIV-infected primary human microglial cultures.

Results

In plasma, PGRN levels correlated with the viral load (VL, p<0.001). In the CSF of subjects with undetectable VL, lower PGRN was associated with neurocognitive impairment (p = 0.046). CSF PGRN correlated with CSF IP-10, TNFα and IL-10, and plasma PGRN correlated with plasma IP-10. In vitro, microglial HIV infection increased PGRN production and PGRN knockdown increased HIV replication, demonstrating that PGRN is an innate antiviral protein.

Conclusions

We propose that PGRN plays dual roles in people living with HIV disease. With active HIV replication, PGRN is induced in infected macrophages and microglia and functions as an antiviral protein. In individuals without active viral replication, decreased PGRN production contributes to neurocognitive dysfunction, probably through a diminution of its neurotrophic functions. Our results have implications for the pathogenesis, biomarker studies and therapy for HIV diseases including HIV-associated neurocognitive dysfunction (HAND).