Destruction of Pseudomonas aeruginosa pre-formed biofilms by cationic polymer micelles bearing silver nanoparticles

Abstract

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen often associated with biofilm infections. This study evaluated the capacity for biofilm destruction of a novel combination of cationic polymer micelles formed from poly(2-(dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA-PCL-PDMAEMA) triblock copolymer either alone, or loaded with silver nanoparticles (M_AgNPs). Pre-formed P. aeruginosa biofilms were incubated with either blank micelles, AgNO₃, or M_AgNPs. Biofilm biomass (crystal violet assay), metabolic activity (Alamar blue reduction), structure (SEM) and viability (CLSM after Live/Dead staining, or plating for CFU) were checked. The results showed that the micelles alone loosened the biofilm matrix, and caused some alterations in the bacterial surface. AgNO₃ killed the bacteria in situ leaving dead biofilm bacteria on the surface. M_AgNPs combined the two types of activities causing significant biofilm reduction, and alteration and death of biofilm bacteria. Therefore, the applied PDMAEMA-based micelles appear to be a successful candidate for the treatment of P. aeruginosa biofilm infections.     

Synthesis and in vitro biological evaluation of aryl boronic acids as potential inhibitors of factor XIa

A series of functionalized aryl boronic acids were synthesized and evaluated as potential inhibitors of factor XIa. Crystal structures of the protein-inhibitor complexes led to the design and synthesis of second generation compounds showing single digit micromolar inhibition against FXIa and selectivity against thrombin, trypsin, and FXa.     

Vasoactive intestinal peptide induces IL-8 production in human colonic epithelial cells via MAP kinase-dependent and PKA-independent pathways

Vasoactive intestinal peptide (VIP) has been shown to be a key regulator of intestinal epithelial functions such as mucus and chloride secretion, paracellular permeability, and cell proliferation. However, its regulatory role in intestinal epithelial chemokine production remains unknown. The aim of this study was (1) to determine whether VIP can modulate intestinal epithelial interleukin-8 (IL-8) production and (2) to identify intracellular mediators responsible for this effect. In the human colonic epithelial cell line HT29-Cl.16E, VIP stimulates IL-8 secretion dose-dependently and IL-8 mRNA level at 10(-9) M. The protein kinase A (PKA) inhibitor PKI did not abolish the effect of VIP. However, inhibition of the ERK1/2 and p38 MAPK pathways reduced the VIP-stimulated IL-8 secretion and mRNA level. Together, our results showed that VIP stimulates IL-8 production in intestinal epithelial cells via PKA-independent and MAPK-dependent pathways. These data suggest that VIPergic pathways can play an immunomodulatory role in intestinal epithelial cells, by regulating epithelial IL-8 secretion.     

Visualization and phenotyping of proinflammatory antigen-specific T cells during collagen-induced arthritis in a mouse with a fixed collagen type II-specific transgenic T-cell receptor β-chain

Introduction  

The Vβ12-transgenic mouse was previously generated to investigate the role of antigen-specific T cells in collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. This mouse expresses a transgenic collagen type II (CII)-specific T-cell receptor (TCR) β-chain and consequently displays an increased immunity to CII and increased susceptibility to CIA. However, while the transgenic Vβ12 chain recombines with endogenous α-chains, the frequency and distribution of CII-specific T cells in the Vβ12-transgenic mouse has not been determined. The aim of the present report was to establish a system enabling identification of CII-specific T cells in the Vβ12-transgenic mouse in order to determine to what extent the transgenic expression of the CII-specific β-chain would skew the response towards the immunodominant galactosylated T-cell epitope and to use this system to monitor these cells throughout development of CIA.     

Effect of Cytokinin and Auxin Treatments on Morphogenesis,Terpenoid Biosynthesis,Photosystem Structural Organization,and Endogenous Isoprenoid Cytokinin Profile in <Emphasis Type="Italic">Artemisia alba</Emphasis> Turra In Vitro

Developmental pattern modification in essential oil bearing Artemisia alba Turra was obtained by exogenous plant growth regulator (PGRs) treatments in vitro. Enhanced rooting (in PGR-free and auxin-treated plants) led to elevation of the monoterpenoid/sesquiterpenoid ratio in the essential oils of aerials. On the contrary, root inhibition and intensive callusogenesis [combined cytokinin (CK) and auxin treatments] reduced this ratio more than twice, significantly enhancing sesquiterpenoid production. Both morphogenic types displayed sesquiterpenoid domination in the underground tissues, which however differed qualitatively from the sesquiterpenoids of the aerials, excluding the hypothesis of their shoot-to-root translocation and implying the possible role of another signaling factor, affecting terpenoid biosynthesis. Inhibited rooting also resulted in a significant drop of endogenous isoprenoid CK bioactive-free bases and ribosides as well as CK N-glycoconjugates and in decreased trans-zeatin (transZ):cis-zeatin (cisZ) ratio in the aerials. Marked impairment of the structural organization of the photosynthetic apparatus and chloroplast architecture were also observed in samples with suppressed rooting. It is well known that in the plant cell monoterpenoid and transZ-type CKs biogenesis are spatially bound to plastids, while sesquiterpenoid and cisZ production are compartmented in the cytosol. In the present work, interplay between the biosynthesis of terpenoids and CK bioactive free bases and ribosides in A. alba in vitro via possible moderation of chloroplast structure has been hypothesized.     

Human ENS regulates the intestinal epithelial barrier permeability and a tight junction-associated protein ZO-1 via VIPergic pathways

Although the enteric nervous system (ENS) has been shown to regulate various mucosal functions, its role in the physiological control of the human intestinal epithelial barrier is unknown. The aim of this study was to investigate whether the ENS is able to modulate epithelial barrier permeability and a key tight junction-associated protein, zonula occludens-1 (ZO-1). Therefore, we developed a co-culture model, consisting of human submucosa containing the submucosal neuronal network and human polarized colonic epithelial monolayers (HT29-Cl.16E or Caco-2). Submucosal neurons were activated by electrical field stimulation (EFS). Permeability was assessed by measuring the flux of paracellular permeability markers (FITC-dextran or FITC-inulin) across epithelial monolayers. Expression of ZO-1 was determined by immunofluorescence, quantitative immunoblot analysis, and real time RT-PCR. Using the coculture model, we showed that EFS of submucosal neurons resulted in a reduction in FITC-dextran or FITC-inulin fluxes, which was blocked by TTX. In HT29-Cl.16E, the effect of submucosal neuron activation was blocked by a VIP receptor antagonist (VIPra) and reproduced by VIP. Furthermore, ZO-1 expression (mRNA, protein) assessed in HT29-Cl.16E, was significantly increased after submucosal neuron activation by EFS. These effects on ZO-1 expression were blocked by TTX and VIPra and reproduced by VIP. In conclusion, our results strongly suggest a modulatory role of VIPergic submucosal neuronal pathways on intestinal epithelial barrier permeability and ZO-1 expression.     

Cells Isolated from Human Glioblastoma Multiforme Express Progesterone-Induced Blocking Factor (PIBF)

Glioblastoma multiforme (GBM) is the most common and malignant tumor in the central nervous system. One of the contemporary hypotheses postulates that its pathogenesis is associated with the cancer stem cells (CSCs) which originate from mutations in the normal neural stem cells residing in their specific “niches.” Simultaneously with its aggressive development the tumor suppresses the local immune system by different secreted and/or cell expressed factors. Progesterone-induced blocking factor (PIBF) is an immunomodulatory protein with known role in the regulation of the immune response in the reproductive system. Expression of PIBF has been described in some tumors as one of the factors suppressing the anti-tumor immunity. The aim of the present study was to check for the expression of PIBF from cells isolated from six GBMs. To characterize the cultured cells and to study the PIBF expression confocal microscopy, flow cytometry, ELISA, and real-time PCR were used. The results obtained showed expression of markers typical for cancer CSCs and secretion of interleukin 6 by the GBM-derived cultured cells. The results convincingly prove that PIBF is intracellularly expressed by the cultured cells from the all six GBM samples, and this fact is confirmed by three different methods—flow cytometry, confocal microscopy, and real-time PCR. This paper reports for the first time the expression of PIBF by GBM-derived cells cultured in vitro and reveals a new aspect of the immunosuppressive mechanism used by GBM in escaping the immune control.