Facile one-pot in-situ synthesis of novel graphene oxide-cellulose nanocomposite for enhanced azo dye adsorption at optimized conditions.

This is the first report on utilization of modified Hummers' method for in-situ synthesis of novel graphene oxide-cellulose nanocrystals nanocomposite in a single reaction vessel. Cellulose used for nanocomposite preparation was extracted from waste jute. The synthesized nanocomposite was characterized with FTIR, XRD, SEM, EDX, DLS, and Zeta potential analyzer. It was applied as an adsorbent for the removal of toxic dye methylene blue from aqueous solutions. Around 98 % MB removal was achieved in 135 min. Under optimum experimental conditions recommended by response surface methodology, adsorption capacity of the nanocomposite was found to be 334.19 mg g-1 while the maximum adsorption capacity as determined by Langmuir isotherm 751.88 mg g-1. Further analysis revealed that the process was guided by both Langmuir and Freundlich isotherm and followed pseudo-second-order kinetics. This cost-effective synthesis route and efficient adsorption capacity of the nanocomposite indicate its immense potential for large-scale application in wastewater treatment.

Sustained release of ketorolac tromethamine from poloxamer 407/cellulose nanofibrils graft nanocollagen based ophthalmic formulations.

There has been extensive utilization of poloxamer 407 (PM) for the delivery of various ophthalmic drugs aimed at efficient ophthalmic drug delivery approach for longer precorneal residence time along with acceptable bioavailability of drugs. We have studied the effect of nanocellulose grafted collagen (CGC) on the performance of in situ gels based on PM for the controlled in vitro release of Ketorolac Tromethamine (KT). CGC has shown great influence evident by the reduction in PM critical gelation concentration, increased gel strength, and prolonged the release of loaded drugs compared with the virgin PM gel. The engineered nanocomposite formulations established an anomalous diffusion mechanism along with a Fickian diffusion controlled drug release for 1.5 & 1.75 w/v% CGC reinforced PM. Hence, the synthesized in situ nanocomposites are potential candidates for ophthalmic drug delivery system.

Toll-like receptor 2 (TLR2), transforming growth factor-ß, hyaluronan (HA), and receptor for HA-mediated motility (RHAMM) are required for surfactant protein A-stimulated macrophage chemotaxis.

The innate immune system protects the host from bacterial and viral invasion. Surfactant protein A (SPA), a lung-specific collectin, stimulates macrophage chemotaxis. However, the mechanisms regulating this function are unknown. Hyaluronan (HA) and its receptors RHAMM (receptor for HA-mediated motility, CD168) and CD44 also regulate cell migration and inflammation. We therefore examined the role of HA, RHAMM, and CD44 in SPA-stimulated macrophage chemotaxis. Using antibody blockade and murine macrophages, SPA-stimulated macrophage chemotaxis was dependent on TLR2 but not the other SPA receptors examined. Anti-TLR2 blocked SPA-induced production of TGFß. In turn, TGFß1-stimulated chemotaxis was inhibited by HA-binding peptide and anti-RHAMM antibody but not anti-TLR2 antibody. Macrophages from TLR2(-/-) mice failed to migrate in response to SPA but responded normally to TGFß1 and HA, effects that were blocked by anti-RHAMM antibody. Macrophages from WT and CD44(-/-) mice had similar responses to SPA, whereas those from RHAMM(-/-) mice had decreased chemotaxis to SPA, TGFß1, and HA. In primary macrophages, SPA-stimulated TGFß production was dependent on TLR2, JNK, and ERK but not p38. Pam3Cys, a specific TLR2 agonist, stimulated phosphorylation of JNK, ERK, and p38, but only JNK and ERK inhibition blocked Pam3Cys-stimulated chemotaxis. We have uncovered a novel pathway for SPA-stimulated macrophage chemotaxis where SPA stimulation via TLR2 drives JNK- and ERK-dependent TGFß production. TGFß1, in turn, stimulates macrophage chemotaxis in a RHAMM and HA-dependent manner. These findings are highly relevant to the regulation of innate immune responses by SPA with key roles for specific components of the extracellular matrix.

Ciliary beating is depressed in nasal cilia from chronic obstructive pulmonary disease subjects.

UNLABELLED: COPD is characterized by increased cough, mucus production, and airway inflammation. Beating epithelial cell cilia contribute to mucociliary clearance with ciliary beat frequency (CBF) an important measure of cilia function. However, whether CBF varies with COPD severity is unknown. AIMS: 1) to compare nasal cilia samples and their CBF from healthy non-smokers (Control), COPD and At Risk (cough and sputum production) subjects. 2) to determine the effect of pharmacologic agents that modulate mediators implicated in the pathogenesis of COPD on nasal CBF. Nasal brushings of ciliated cells were obtained from Control, At Risk and COPD subjects. Using high speed digital imaging, we measured baseline CBF ex vivo. Then, CBF was re-measured after 30 min perfusion with pharmacologic agents that modulate mediators implicated in COPD (salmeterol xinafoate, tiotropium bromide, licofelone, luteolin, YM976, Defensin HNP-1) and again after 30 min washout. CBF was significantly depressed in moderate and severe COPD compared to At Risk and Control subjects. There was an evident and persistent rise in CBF with all agents tested in COPD cilia except that YM976 effects persisted only in severe COPD. Only YM976 and tiotropium caused a persistent increase in CBF in At Risk cilia. The reduction of nasal CBF in moderate and severe COPD implies that impaired ciliary function may impact mucociliary clearance in COPD, potentially contributing to retention of secretions and infection. Pharmacologic agents with different mechanisms of action can increase CBF of COPD cilia. Further investigation of the signalling pathways influencing CBF of COPD cilia is needed.

The direct effect of hyperosmolar agents on ciliary beating of human bronchial epithelial cells.

BACKGROUND: Inhalation of hypertonic saline and mannitol improve mucociliary clearance in patients with bronchiectasis, but little is known about how the relative osmotic strengths of these compounds affect ciliary beat frequency (CBF) of ciliated human bronchial epithelial cells (HBEC). Our aim was to compare in vitro the direct effects of osmotically equivalent solutions on CBF of HBEC. METHODS: HBEC were acutely (10, 30 min) exposed to comparable osmolar solutions of saline (0.03-0.48%), mannitol (0.19-3%) and dextran (10%-39.39%). Effects of higher % solutions, reversibility of responses, and prolonged treatments (15-20 h) were also compared. CBF was measured using digital videomicroscopy at baseline and at all time points. RESULTS: CBF of HBEC increased significantly after acute exposure to mannitol (0.19%, 0.38%), decreased with dextran and remained unchanged with saline. Prolonged exposure to mannitol at high (3%, 6%) osmolar concentrations reversibly suppressed CBF. In comparison, acute and prolonged treatment with 39.39% dextran (equivalent to 3% mannitol) reduced CBF irreversibly. Furthermore, acute and prolonged treatment with 1% saline (equivalent to 6% mannitol) suppressed CBF with only the acute effect being reversible. CONCLUSIONS: Mannitol had a direct osmolarity-independent cilio-stimulatory effect at lower % solutions and a reversible cilio-inhibitory effect at higher % solutions, and prolonged exposure to mannitol inhibited CBF reversibly. Both the acute and prolonged effects of mannitol compared to dextran and saline on CBF of HBEC in vitro, imply a unique mechanism of action for mannitol on ciliary beating and might contribute to the improved clearance observed in mannitol-treated patients.

Primary human bronchial epithelial cells grown from explants.

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and < or =1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm(3) pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 microg/ml), fibronectin (10 microg/ml), and BSA (10 microg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37 degrees C in 5% CO(2) humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

S-nitrosylation of surfactant protein-D controls inflammatory function.

The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional "bunch of flowers" arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NO's role in innate immunity.

Loss of PECAM-1 function impairs alveolarization.

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.

Expression and role of the hyaluronan receptor RHAMM in inflammation after bleomycin injury.

Lung injury is associated with increased concentrations of hyaluronan (hyaluronic acid, HA). HA modifies cell behavior through interaction with cell-associated receptors such as receptor for HA-mediated motility (RHAMM, CD168). Using a function blocking anti-RHAMM antibody (R36), we investigated the expression and role of RHAMM in the inflammatory response to intratracheal bleomycin in rats. Immunostaining showed increased expression of RHAMM in macrophages 4-7 d after injury. Surface biotin labeling of cells isolated by lavage confirmed increased surface expression of a 70-kD RHAMM after lung injury, and in situ hybridization demonstrated increased RHAMM mRNA in macrophages responding to injury. Time-lapse cinemicrography demonstrated a 5-fold increase in motility of alveolar macrophages from bleomycin-treated animals that was completely blocked by R36 in vitro. Further, HA-stimulated macrophage chemotaxis was also inhibited by R36. Daily administration of R36 to injured animals resulted in a 40% decrease in macrophage accumulation 7 d after injury. Further, H&E staining of tissue sections showed that bleomycin-mediated changes in lung architecture were improved with R36 treatment. Taken together with previous results showing the inhibitory effects of HA-binding peptide on inflammation and fibrosis, we conclude that the interaction of RHAMM with HA is a critical component of the recruitment of inflammatory cells to the lung after injury.

Human surfactant protein B promoter in transgenic mice: temporal, spatial, and stimulus-responsive regulation.

Surfactant protein B (SP-B) is a developmentally and hormonally regulated lung protein that is required for normal surfactant function. We generated transgenic mice carrying the human SP-B promoter (-1,039/+431 bp) linked to chloramphenicol acetyltransferase (CAT). CAT activity was high in lung and immunoreactive protein localized to alveolar type II and bronchiolar epithelial cells. In addition, thyroid, trachea, and intestine demonstrated CAT activity, and each of these tissues also expressed low levels of SP-B mRNA. Developmental expression of CAT activity and SP-B mRNA in fetal lung were similar and both increased during explant culture. SP-B mRNA but not CAT activity decreased during culture of adult lung, and both were reduced by transforming growth factor (TGF)-beta(1). Treatment of adult mice with intratracheal bleomycin caused similar time-dependent decreases in lung SP-B mRNA and CAT activity. These findings indicate that the human SP-B promoter fragment directs tissue- and lung cell-specific transgene expression and contains cis-acting elements involved in regulated expression during development, fetal lung explant culture, and responsiveness to TGF-beta and bleomycin-induced lung injury.