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International audience; Glycosaminoglycan (GAG) chains of proteoglycans (PGs) play important roles in fibrosis through cell-matrix interactions and growth factor binding in the extracellular matrix. We investigated the expression and regulation of PG core protein (versican) and key enzymes (xylosyltransferase [XT]-I, beta 1,3-glucuronosyltransferase [GlcAT]-I, chondroitin-4-sulfotransferase [C4ST]) implicated in synthesis and sulfation of GAGs in bleomycin (BLM) and adenovirus-transforming growth factor (TGF)-beta 1-induced lung fibrosis in rats. We also studied the role of GlcAT-I or TGF-beta 1 and the signaling pathways regulating PG-GAG production in primary lung fibroblasts isolated from saline-or BLM-instilled rats. The mRNA for XT-I, GlcAT-I, C4ST, and versican was increased in the lung 14 days after BLM injury. In vitro studies indicate that fibrotic lung fibroblasts (FLFs) expressed more XT-I, C4ST, and chondroitin sulfate (CS)-GAGs than did normal lung fibroblasts at baseline. TGF-beta 1 enhanced the expression of XT-I, C4ST-I, and versican in normal lung fibroblasts, whereas SB203580 or SB431542, by targeting p38 mitogen-activated protein kinase or TGF-b type-1 receptor/activin receptor-like kinase 5, respectively, attenuated the response to both TGF-beta 1 and FLFs on PG-GAG expression. Neutralizing anti-TGF-beta 1 antibody abrogated FLF-conditioned medium-stimulated expression of XT-I, GlcAT-I, versican, and CS-GAG. Forced expression of TGF-beta 1 in vivo enhanced versican, XT-I, GlcAT-I, and C4ST-I expression and PG-GAG deposition in rat lungs. Finally, induced expression of GlcAT-I gene in rat lung fibroblasts increased GAG synthesis by these cells. Together, our results provide new insights into the basis for increased PG-GAG deposition in lung fibrosis; inhibition of TGF-beta 1-mediated or fibrosis-induced PGGAG production by activin receptor-like kinase 5/p38 inhibitors may contribute to antifibrotic activity.

The asthmatic airway is characterized by alterations in decorin and biglycan and increased airway smooth muscle (ASM). Further, the asthmatic airway may be subjected to abnormal mechanical strain. We hypothesized that ASM cells obtained from ovalbumin (OVA)--and saline (SAL)--challenged rats would respond differently to matrix and mechanical strain. ASMC were seeded on plastic, decorin or biglycan. Additional cells were grown on decorin, biglycan or collagen type 1, and then subjected to mechanical strain (Flexercell). The number of OVA ASMC was significantly greater than SAL ASM when seeded on plastic. A significant decrease was observed for both OVA and SAL ASMC seeded on decorin compared to plastic; the reduction in ASMC number was more modest for OVA. Biglycan decreased SAL ASMC number only. Strain reduced cell number for SAL and OVA ASMC grown on all matrices. Strain affected expression of β1-integrin differently in OVA vs. SAL ASMC. These data suggest that matrix and mechanical strain modulate ASMC number; these effects are differentially observed in OVA ASMC.

Increased proteoglycan (PG) deposition is a feature of airway remodeling in asthma. Glycosaminoglycans (GAGs) mediate many of the biological and mechanical properties of PGs by providing docking sites through their carbohydrate chains to bioactive ligands; therefore, it is imperative to define structural and metabolic changes of GAGs in asthma. Using a Brown Norway (BN) ovalbumin (OVA)-sensitized and -challenged rat model to induce airway remodeling, we found excessive deposition of chondroitin/dermatan (CS/DS)-, heparan (HS), and keratan (KS) sulfate GAGs in the airways and bronchoalveolar lavage cells of OVA-challenged rats. Disaccharide composition of CS/DS of OVA-challenged rats was significantly different compared with saline-treated (SAL) control rats, with increased levels of 0-, 6-, and 4-sulfated disaccharides. Increases in the amount and a change in the proportion of CS/DS versus HS GAGs were noted in OVA-challenged rats. The higher content and sulfation of CS/DS disaccharides was reflected by the increased expression of xylosyltransferase-I, β1,3-glucuronosyltransferase-I, chondroitin-4, and chondroitin-6 sulfotransferase genes and protein expression of xylosyltransferase-I and β1,3-glucuronosyltransferase-I in OVA-challenged rats. Genes encoding the core proteins of the CS/DS and KS-containing PGs, such as versican, biglycan, decorin, and lumican, were overexpressed in OVA-challenged rats. Our results suggest that GAG biosynthetic enzymes may be involved in the altered expression of GAGs in the airways and are potential targets for inhibiting excess PG-GAG deposition and the airway remodeling process in asthma.

The NAD-dependent histone deacetylase silent information regulator 1 (SIRT1) is overexpressed and catalytically activated in a number of human cancers, but recent studies have actually suggested that it may function as a tumor suppressor and metastasis inhibitor in vivo. In breast cancer, SIRT1 stabilization has been suggested to contribute to the oncogenic potential of the estrogen receptor α (ERα), but SIRT1 activity has also been associated with ERα deacetylation and inactivation. In this study, we show that SIRT1 is critical for estrogen to promote breast cancer. ERα physically interacted and functionally cooperated with SIRT1 in breast cancer cells. ERα also bound to the promoter for SIRT1 and increased its transcription. SIRT1 expression induced by ERα was sufficient to activate antioxidant and prosurvival genes in breast cancer cells, such as catalase and glutathione peroxidase, and to inactivate tumor suppressor genes such as cyclin G2 (CCNG2) and p53. Moreover, SIRT1 inactivation eliminated estrogen/ERα-induced cell growth and tumor development, triggering apoptosis. Taken together, these results indicated that SIRT1 is required for estrogen-induced breast cancer growth. Our findings imply that the combination of SIRT1 inhibitors and antiestrogen compounds may offer more effective treatment strategies for breast cancer. Cancer Res; 71(21); 6654–64. ©2011 AACR.

Pulmonary fibrosis (PF) is characterized by increased deposition of proteoglycans (PGs), in particular core proteins. Glycosaminoglycans (GAGs) are key players in tissue repair and fibrosis, and we investigated whether PF is associated with changes in the expression and structure of GAGs as well as in the expression of β1,3-glucuronosyltransferase I (GlcAT-I), a rate-limiting enzyme in GAG synthesis. Lung biopsies from idiopathic pulmonary fibrosis (IPF) patients and lung tissue from a rat model of bleomycin (BLM)-induced PF were immunostained for chondroitin sulfated-GAGs and GlcAT-I expression. Alterations in disaccharide composition and sulfation of chondroitin/dermatan sulfate (CS/DS) were evaluated by fluorophore-assisted carbohydrate electrophoresis (FACE) in BLM rats. Lung fibroblasts isolated from control (saline-instilled) or BLM rat lungs were assessed for GAG structure and GlcAT-I expression. Disaccharide analysis showed that 4- and 6-sulfated disaccharides were increased in the lungs and lung fibroblasts obtained from fibrotic rats compared with controls. Fibrotic lung fibroblasts and transforming growth factor-β1 (TGF-β1)-treated normal lung fibroblasts expressed increased amounts of hyaluronan and 4- and 6-sulfated chondroitin, and neutralizing anti-TGF-β1 antibody diminished the same. TGF-β1 upregulated GlcAT-I and versican expression in lung fibroblasts, and signaling through TGF-β type I receptor/p38 MAPK was required for TGF-β1-mediated GlcAT-I and CS-GAG expression in fibroblasts. Our data show for the first time increased expression of CS-GAGs and GlcAT-I in IPF, fibrotic rat lungs, and fibrotic lung fibroblasts. These data suggest that alterations of sulfation isomers of CS/DS and upregulation of GlcAT-I contribute to the pathological PG-GAG accumulation in PF.

Osteoarthritis and rheumatoid arthritis are characterized by loss of proteoglycans (PGs) and their glycosaminoglycan (GAG) chains that are essential for cartilage function. Here, we investigated the role of glycosyltransferases (GTs) responsible for PG-GAG chain assembly during joint cartilage destruction and repair processes. At various times after antigen-induced arthritis (AIA) and papain-induced cartilage repair in rats, PG synthesis and deposition, expression of GTs, and GAG chain composition were analyzed. Our data showed that expression of the GT xylosyltransferase I (XT-I) gene initiating PG-GAG chain synthesis was significantly reduced in AIA rat cartilage and was associated with a decrease in PG synthesis. Interestingly, interleukin-1beta, the main proinflammatory cytokine incriminated in joint diseases, down-regulated the XT-I gene expression with a concomitant decrease in PG synthesis in rat cartilage explants ex vivo. However, cartilage from papain-injected rat knees showed up-regulation of XT-I gene expression and increased PG synthesis at early stages of cartilage repair, a process associated with up-regulation of TGF-beta1 gene expression and mediated by p38 mitogen-activated protein kinase activation. Consistently, silencing of XT-I expression by intraarticular injection of XT-I shRNA in rat knees prevented cartilage repair by decreasing PG synthesis and content. These findings show that GTs play a key role in the loss of PG-GAGs in joint diseases and identify novel targets for stimulating cartilage repair.

This paper describes a novel technique to measure cryogenic fluid flow rate using fiber Bragg grating (FBG) sensors. The proposed design utilizes the drag force caused by fluid flow on the FBG sensor. The strain thus induced in the sensor will cause a Bragg wavelength shift, which can be utilized to estimate the flow rate. Theoretical calculations are done using mathematical models and the design is found to be feasible for a wide range of flow rates and cryogenic temperatures. Calibration experiments are performed for Helium at ambient conditions and the results obtained are regressed to obtain its calibration equation. The sensor shows an overall sensitivity of 20 pm/(g/s) in the range of operation between 0 and 2 g/s and 50 pm/(g/s) in the range of 2 to 5 g/s.

Objectives:We investigated the effect of topical steroids on clinical outcomes and related immune response of chronic rhinosinusitis with nasal polyp (CRSwNP) patients and in eradicating some polyps. We want to explore a new potential mechanism linked to Th-17 cells.Methods:Prospective, double-blind, placebo-controlled studies with 24 allergic and nonallergic patients were randomized to either placebo or fluticasone furoate for 12 weeks. Assessment of clinical response, endoscopic score with biopsies of the inferior turbinate, and polyps before and after treatment were performed. Biopsies were stained for T-cells, eosinophils, neutrophils, and IL-17A/F.Results:Steroid treatment improved the mean symptoms scores from 7.12 to 4.02 ( P < .01) and the polyp score from 5.13 to 3.31 ( P < .05), but the comparison with placebo was not statistically significant in nonallergics due to insufficient study power. Steroid treatment decreased eosinophil counts on allergics but not neutrophils or T-cells. The IL-17A/F expression was higher in nonallergics with high neutrophil counts and was inclined by steroids. Compared to baselines, IL-17 cells were significantly less in allergic individuals and were not observed in allergics and with high neutrophil counts.Conclusion:Topical steroids were more effective on certain nasal polyp phenotypes. Identification of polyp phenotype might be essential to ensure a better therapeutic response to intranasal corticosteroids.

Weight-bearing foot structure may influence postural control by either decreasing the base of support (BOS) or increasing the passive instability of the joints of the foot. Poor postural control has been implicated as the main causative factor for foot and ankle injuries. The purpose of this study was to examine the influence of forefoot postures on postural stability during single limb stance.Sixty healthy individuals between the ages of 18 to 31 were selected using a purposive sampling procedure based on forefoot angle measurements and categorized into three groups; high forefoot varus (≥8°) (n=20), neutral forefoot varus (1°-8°) (n=20) and low forefoot varus group (≤1°) (n=20). Static foot measurements, including relaxed rearfoot angle and navicular drop, and foot dimentsions were performed. Height and weight were also recorded for all the subjects. Center of Pressure (COP) excursion in Anterior-posterior (AP) and Medial-lateral (ML) planes and Stability Index (SI) with eyes open and eyes closed conditions were also measured using the force platform.Strong correlations were found between forefoot angle and rearfoot angle (r=0.71, p0.01), forefoot angle and navicular drop (r=0.58, p0.01), and between rearfoot angle and navicular drop (r=0.661, p0.01). There were no correlations (p0.05) between the forefoot angle and all the five COP measures, except between forefoot angle and SI with eyes closed (r= -0.25 p0.01).There is a significant positive correlation between forefoot angle and rearfoot angle and between forefoot angle and navicular drop. Forefoot angles did not affect the maximum AP COP and ML COP excursions or SI in healthy subjects.3.

(1) We have studied whether curcumin prevents amiodarone-induced lung fibrosis in rats. Intratracheal instillation of amiodarone (6.25 mg kg(-1) on days 0 and 2, and then killed on day 3, day 5, week 1, week 3 and week 5 after amiodarone administration) induced increases in total protein and lactate dehydrogenase (LDH) activity on days 3 and 5 in bronchoalveolar lavage fluid (BALF). Total cell counts, alveolar macrophages, neutrophils and eosinophils recovered by BAL, and lung myeloperoxidase (MPO) activity were significantly higher in amiodarone rats. (2) Tumor necrosis factor-alpha (TNF-alpha) release after lipopolysaccharide (LPS) stimulation and superoxide anion generation after phorbol myristate acetate (PMA) stimulation were higher in the alveolar macrophages of amiodarone rats at 3 and 5 weeks postamiodarone instillation than in controls. Amiodarone also induced increases in transforming growth factor-beta1 (TGF-beta1) expression, collagen deposition, type I collagen expression and c-Jun protein in lungs. (3) Curcumin (200 mg kg(-1) body weight after first amiodarone instillation and daily thereafter for 5 weeks)-treated amiodarone rats had reduced levels of protein, LDH activity, total cell numbers and differential cell counts in BALF. LPS-stimulated TNF-alpha release and PMA-stimulated superoxide generation were significantly suppressed by curcumin. Furthermore, curcumin inhibited the increases in lung MPO activity, TGF-beta1 expression, lung hydroxyproline content, expression of type I collagen and c-Jun protein in amiodarone rats. Our results have important implications for the treatment of amiodarone-induced lung fibrosis.

Bleomycin (BM)-induced pulmonary fibrosis involves excess production of proteoglycans (PGs). Because transforming growth factor-β1(TGF-β1) promotes fibrosis, and interferon-γ (IFN-γ) inhibits it, we hypothesized that TGF-β1treatment would upregulate PG production in fibrotic lung fibroblasts, and IFN-γ would abrogate this effect. Primary lung fibroblast cultures were established from rats 14 days after intratracheal instillation of saline (control) or BM (1.5 units). PGs were extracted and subjected to Western blot analysis. Bleomycin-exposed lung fibroblasts (BLF) exhibited increased production of versican (VS), heparan sulfate proteoglycan (HSPG), and biglycan (BG) compared with normal lung fibroblasts (NLF). Compared with NLF, BLF released significantly increased amounts of TGF-β1. TGF-β1(5 ng/ml for 48 h) upregulated PG expression in both BLF and NLF. Incubation of BLF with anti-TGF-β antibody (1, 5, and 10 μg/ml) inhibited PG expression in a dose-dependent manner. Treatment of BLF with IFN-γ (500 U · ml−1× 48 h) reduced VS, HSPG, and BG expression. Furthermore, IFN-γ inhibited TGF-β1-induced increases in PG expression by these fibroblasts. Activation of fibroblasts by TGF-β1promotes abnormal deposition of PGs in fibrotic lungs; downregulation of TGF-β1by IFN-γ may have potential therapeutic benefits in this disease.

Background Alpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown. We investigated the expression, accumulation, and secretion of Z-alpha-1 antitrypsin and its polymers in cultures of transfected cells and in cells originating from alpha-1 antitrypsin-deficient patients. Methods Experiments using a conformation-specific antibody were carried out on M- and Z-variant–transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann–Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of

Bleomycin-induced lung fibrosis results in changes in tissue mechanical properties due to alterations in the extracellular matrix (ECM). How oscillatory mechanics and changes in the matrix evolve over time has not been addressed. Sprague-Dawley rats were instilled with bleomycin sulfate (BM) (1.5 U) intratracheally; control animals (C) received saline. At 7, 14, and 28 d after BM, parenchymal strips (7 x 2 x 2 mm) were obtained and strips suspended in a Krebs-filled organ bath. One end of the strip was attached to a force (F) transducer and the other to a lever arm that effected sinusoidal length (L) oscillations. Strips were oscillated at varying amplitudes (1, 3, and 10% of resting L) and frequencies (f = 0.3, 1, 3, and 10 Hz) at an operating stress of 2 kPa. Resistance (R) and elastance (E) were estimated by fitting changes in F and L to the equation of motion. Hysteresivity (eta) was calculated as eta = (R/E) 2pif. Strips were then fixed for morphological study of collagen, elastic fibers, and the small proteoglycans (PGs), biglycan and fibromodulin (FM). R and E were significantly greater and eta significantly less in BM versus C strips (p < 0.001). The increase in R and E peaked at 14 d after BM; the decrement in eta was maximal at Day 7. Biglycan was increased in BM lung strips at all time points, FM and elastic fibers were increased at 14 and 28 d, and collagen was increased at 28 d only. Hence, changes in mechanics were maximal before collagen content had increased. In addition, we demonstrated a significant correlation between biglycan and all mechanical parameters. These data suggest that changes in PGs may be critical in determining changes in lung tissue viscoelastic behavior in this fibrosis model

Curcumin, an anti-inflammatory, antioxidant, was evaluated for its ability to suppress bleomycin (BLM)-induced pulmonary fibrosis in rats. A single intratracheal instillation of BLM (0.75 U 100−1 g, sacrificed 3, 5, 7, 14 and 28 days post-BLM) resulted in significant increases in total cell numbers, total protein, and angiotensin-converting enzyme (ACE), and alkaline phosphatase (AKP) activities in bronchoalveolar lavage fluid. Animals with fibrosis had a significant increase in lung hydroxyproline content. Alveolar macrophages from BLM-administered rats elaborated significant increases in tumour necrosis factor (TNF)-α release, and superoxide and nitric oxide production in culture medium. Interestingly, oral administration of curcumin (300 mg kg−1 10 days before and daily thereafter throughout the experimental time period) inhibited BLM-induced increases in total cell counts and biomarkers of inflammatory responses in BALF. In addition, curcumin significantly reduced the total lung hydroxyproline in BLM rats. Furthermore, curcumin remarkably suppressed the BLM-induced alveolar macrophage production of TNF-α, superoxide and nitric oxide. These findings suggest curcumin as a potent anti-inflammatory and anti-fibrotic agent against BLM-induced pulmonary fibrosis in rats. British Journal of Pharmacology (2000) 131, 169–172; doi:10.1038/sj.bjp.0703578

The present study investigated the effect of curcumin on adriamycin (ADR) nephrosis in rats. The results indicate that ADR-induced kidney injury was remarkably prevented by treatment with curcumin. Treatment with curcumin markedly protected against ADR-induced proteinuria, albuminuria, hypoalbuminaemia and hyperlipidaemia. Similarly, curcumin inhibited ADR-induced increase in urinary excretion of N-acetyl-beta-D-glucosaminidase (a marker of renal tubular injury), fibronectin and glycosaminoglycan and plasma cholesterol. Curcumin restored renal function in ADR rats, as judged by the increase in GFR. The data also demonstrated that curcumin protected against ADR-induced renal injury by suppressing oxidative stress and increasing kidney glutathione content and glutathione peroxidase activity. In like manner, curcumin abolished ADR-stimulated kidney microsomal and mitochondrial lipid peroxidation. These data suggest that administration of curcumin is a promising approach in the treatment of nephrosis caused by ADR.

The present investigation was designed to characterize the biochemical and connective tissue components and to correlate the significance of morphological and biochemical perturbations in cyclophosphamide (CP)-induced lung fibrosis in rats. Lung fibrosis was induced in male Wistar rats by intraperitoneal injection of 20 mg/100 g body weight of CP, and their pneumotoxic derangements were characterized during an early destructive phase followed by a proliferative and synthetic phase. Serum angiotensin-converting enzyme (ACE) activity was higher in CP-treated rats at days 2, 3, 5, 7, and 11, but there was a significant decrease in lung ACE activity during the same time period. Elevated levels of beta-glucuronidase activity were observed in the lung lavage fluid of CP-administered rats days 2, 3, 5, and 7. Lung myeloperoxidase activity was higher in CP rats. Of significance was the presence of collagenase and collagenolytic cathepsin in the lavage fluid of CP rats, when compared with the barely detectable levels in controls. A similar increase in these enzyme activities was also noticed in the lung tissue of CP rats during the same experimental period. Lavage fluid hydroxyproline content was higher in CP rats when compared with controls. Similarly, lung protein and DNA levels were elevated significantly after treatment with CP. The pulmonary histamine and serotonin contents were significantly higher in CP rats. The incorporation of [3H]thymidine into lung total DNA, [3H]proline into lung hydroxyproline, and [35S]sulphate into lung glycosaminoglycan, measured as indicators of lung DNA, collagen, and glycosaminoglycan synthesis, respectively, was also higher in CP groups. Increased levels of hydroxyproline, elastin, hexosamine, total hexose, fucose, sialic acid, and uronic acid in the lungs of rats 14, 28, and 42 days after CP insult were characterized as biomarkers of CP-induced interstitial changes. These findings indicate that CP-induced lung fibrosis results in alterations not only in collagen synthesis and accumulation, but also in glycosaminoglycan and glycoprotein content.

The importance of heparan- and chondroitin-sulfate proteoglycans in physiological and pathological processes led to the investigation of the regulation of beta1,3-glucuronosyltransferase I (GlcAT-I), responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide, a key step prior to polymerization of chondroitin- and heparan-sulfate chains. We have cloned and functionally characterized GlcAT-I 5'-flanking regulatory region. Mutation analysis and electrophoretic mobility shift assays demonstrated the importance of Sp1 motif located at -65/-56 position in promoter activity. Furthermore, we found that elevation of intracellular calcium concentration by the calcium ionophore ionomycin stimulated GlcAT-I gene expression as well as glycosaminoglycan chain synthesis in HeLa cells. Bisanthracycline, an anti-Sp1 compound, inhibited GlcAT-I basal promoter activity and suppressed ionomycin induction, suggesting the importance of Sp1 in calcium induction of GlcAT-I gene expression. Nuclear protein extracts from ionomycin-induced cells exhibited an increased DNA binding of Sp1 factor to the consensus sequence at position -65/-56. Signaling pathway analysis and MEK inhibition studies revealed the important role of p42/p44 MAPK in the stimulation of GlcAT-I promoter activity by ionomycin. The present study identifies, for the first time, GlcAT-I as a target of calcium-dependent signaling pathway and evidences the critical role of Sp1 transcription factor in the activation of GlcAT-I expression.

SummaryBackground Infiltration of fibrocytes (FC) in the airway smooth muscle is a feature of asthma, but the pathological significance is unknown. Objective We sought to explore whether FC modulate the phenotype of airway smooth muscle cells (ASMC) in asthmatic vs. control subjects. Methods Fibrocytes were isolated from CD14+ monocytes from asthmatic and normal subjects. Proliferation of ASMC of asthmatic or normal subjects was analysed by 3H-thymidine incorporation, cell number counting and Ki-67 expression after treatment of ASMC with FC-conditioned medium (FCCM) or co-culture with FC. ASMC-associated cytokines/chemokines implicated in asthma (TGF-β1, eotaxin, IL-6 and IL-8) were measured in co-culture or transwell culture of ASMC + FC by ELISA. Immunofluorescence staining was performed to localize these cytokines in ASMC. Cytokine secretion was measured in the transwell culture of ASMC + FC, where NF-κB-p65 or ERK1/2 in ASMC was silenced by siRNA. Contractile phenotype of ASMC in transwell culture was assessed by immunoblotting of α-smooth muscle actin (α-SMA) and myosin light chain kinase (MLCK). Results Fibrocytes did not affect ASMC proliferation and expression of TGF-β1, eotaxin, α-SMA and MLCK; however, ASMC production of IL-8 and IL-6 was increased in the co-culture and transwell culture by FC. ASMC treated with FCCM were immunopositive for IL-8/IL-6 and produced more IL-8/IL-6. Furthermore, siRNA silencing of NF-κB-p65 or ERK1/2 in transwell cultures of asthmatic ASMC with normal subject FC decreased IL-8 and IL-6 production. Conclusions and Clinical Relevance Fibrocytes promoted IL-8 and IL-6 production by ASMC, demonstrating a proinflammatory role for FC and a possible mechanism of the inflammatory phenotype in asthma.

Purpose of review The activation and regulation of lymphocytes play a central role in asthmatic inflammation. It is increasingly recognized that diverse panels of lymphocyte lineages and cytokine profiles are involved in the asthmatic phenotypes. In this review, we discuss the advances in the gene variants associated with the regulation of lymphocytes and relevant cytokines underlying asthma and allergic diseases. We also discuss the current evidence about the epigenetic regulation of lymphocyte differentiation and the interaction with environment. Recent findings Many genetic variants in asthma are functionally associated with lymphocytes and relevant cytokines. Interleukin (IL)-2RB is important in the homeostasis of T regulatory cells (Tregs) through effects from IL-2. IL-18R1 and ST2/IL-1RL1 drive the T helper 1 and 2 inflammation via the ligands of their encoding receptors. Novel genes, like orosomucoid 1-like 3/gasdermin-like gene and taste receptor type 2 members are being explored for their roles in T-cell activation. T-cell lineages are epigenetically regulated by de novo methyltransferases, histone methylase, CD44 and microRNA. Environmental factors such as second-hand smoke and ambient air pollution modify Tregs differentiation significantly. Summary Plenty of genetic loci of lymphocyte regulation provide us a deeper insight into the asthma pathogenesis. Future challenge is to define genetic drivers in asthma phenotypes to provide therapeutic targets.

Cyclophosphamide causes lung injury in rats through its ability to generate free radicals with subsequent endothelial and epithelial cell damage. In order to observe the protective effects of a potent anti-inflammatory antioxidant, curcumin (diferuloyl methane) on cyclophosphamide-induced early lung injury, healthy pathogen free male Wistar rats were exposed to 20 mg/100 g body weight of cyclophosphamide, intraperitoneally as a single injection. Prior to cyclophosphamide intoxication oral administration of curcumin was performed daily for 7 days. At various time intervals (2, 3, 5 and 7 days post insult) serum and lung samples were analyzed for angiotensin converting enzyme, lipid peroxidation, reduced glutathione and ascorbic acid. Bronchoalveolar lavage fluid was analyzed for biochemical constituents. The lavage cells were examined for lipid peroxidation and glutathione content. Excised lungs were analyzed for antioxidant enzyme levels. Biochemical analyses revealed time course increases in lavage fluid total protein, albumin, angiotensin converting enzyme (ACE), lactate dehydrogenase, N-acetyl-β-D-glucosaminidase, alkaline phosphatase, acid phosphatase, lipid peroxide levels and decreased levels of glutathione (GSH) and ascorbic acid 2, 3, 5 and 7 days after cyclophosphamide intoxication. Increased levels of lipid peroxidation and decreased levels of glutathione and ascorbic acid were seen in serum, lung tissue and lavage cells of cyclophosphamide groups. Serum angiotensin converting enzyme activity increased which coincided with the decrease in lung tissue levels. Activities of antioxidant enzymes were reduced with time in the lungs of cyclophosphamide groups. However, a significant reduction in lavage fluid biochemical constituents, lipid peroxidation products in serum, lung and lavage cells with concomitant increase in antioxidant defense mechanisms occurred in curcumin fed cyclophosphamide rats. Therefore, our results suggest that curcumin is effective in moderating the cyclophosphamide induced early lung injury and the oxidant-antioxidant imbalance was partly abolished by restoring the glutathione (GSH) with decreased levels of lipid peroxidation.

Repair of damaged articular cartilage in osteoarthritis (OA) is a clinical challenge. Because cartilage is an avascular and aneural tissue, normal mechanisms of tissue repair through recruitment of cells to the site of tissue destruction are not feasible. Proteoglycan (PG) depletion induced by the proinflammatory cytokine interleukin-1β, a principal mediator in OA, is a major factor in the onset and progression of joint destruction. Current symptomatic treatments of OA by anti-inflammatory drugs do not alter the progression of the disease. Various therapeutic strategies have been developed to antagonize the effect of proinflammatory cytokines. However, relatively few studies were conducted to stimulate anabolic activity, in an attempt to enhance cartilage repair. To this aim, a nonviral gene transfer strategy of glycosyltransferases responsible for PG synthesis has been developed and tested for its capacity to promote cartilage PG synthesis and deposition. Transfection of chondrocytes or cartilage explants by the expression vector for the glycosyltransferase β-1,3-glucuronosyltransferase-I (GlcAT-I) enhanced PG synthesis and deposition in the ECM by promoting the synthesis of chondroitin sulfate GAG chains of the cartilage matrix. This indicates that therapy mediated through GT gene delivery may constitute a new strategy for the treatment of OA.

The antiinflammatory, antioxidant activity of taurine and niacin against cyclophosphamide-induced early lung injury in rats was investigated. A single intraperitoneal injection of cyclophosphamide markedly altered the levels of several biomarkers in bronchoalveolar lavage fluid: total protein, albumin, angiotensin converting enzyme, lactate dehydrogenase, lactate, N-acetyl-beta-D-glucosaminidase, alkaline phosphatase, acid phosphatase and lipid peroxidation product were significantly elevated. In contrast, decreased levels of total reduced glutathione (GSH) and ascorbic acid were observed. Cyclophosphamide significantly increased malondialdehyde levels in serum and lung. Significant increases in lung content of lipid hydroperoxides were seen that paralleled the decreased levels of total reduced glutathione and total sulfhydryl groups. Pretreatment of rats with daily intraperitoneal injection of taurine plus niacin 7 days prior to and 2 days after cyclophosphamide insult significantly inhibited the development of lung injury, prevented the alterations in lavage fluid biomarkers associated with inflammatory reactions, with less lipid peroxidation and restoration of antioxidants. In conclusion, our results suggest that taurine and niacin in combination is efficient in blunting cyclophosphamide-induced pulmonary damage.

The present investigation evaluated the effectiveness of taurine in attenuating the acute nephrosis in rats following intraperitoneal administration of puromycin aminonucleoside. Animals given a single injection of aminonucleoside (100mg/kg) developed nephrotic syndrome at the end of 10 days, as evidenced by heavy proteinuria, albuminuria, and increased urinary excretion of N-acetyl-β-D-glucos-aminidase when compared with the controls. Significant increases in kidney lipid peroxides, hydroxyl radicals, and hydroperoxides were also evident. Aminonucleoside significantly reduced the levels of glutathione, total thiol, ascorbic acid, and vitamin E in the kidney tissue. Significant reduction in the activities of antioxidant enzymes such as glucose-6-phosphate dehydrogenase, superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase were also observed in the nephrotic rats. Neither saline nor saline plus taurine treatment caused detectable changes in the above biochemical variables. Daily taurine administration to nephrotic rats for 10 days, however, reduced the severity of aminonucleoside-associated renal injury with significant reduction in urinary protein constituents, decreased lipid peroxides, and increased levels of antioxidants and antioxidant enzymes. Collectively, the results of this study demonstrate a protective role of taurine in ameliorating the acute nephrosis in response to puromycin aminonucleoside administration.

Repair of damaged articular cartilage in osteoarthritis (OA) is a clinical challenge. Because cartilage is an avascular and aneural tissue, normal mechanisms of tissue repair through recruitment of cells to the site of tissue destruction are not feasible. Proteoglycan (PG) depletion induced by the proinflammatory cytokine interleukin-1β, a principal mediator in OA, is a major factor in the onset and progression of joint destruction. Current symptomatic treatments of OA by anti-inflammatory drugs do not alter the progression of the disease. Various therapeutic strategies have been developed to antagonize the effect of proinflammatory cytokines. However, relatively few studies were conducted to stimulate anabolic activity, in an attempt to enhance cartilage repair. To this aim, a nonviral gene transfer strategy of glycosyltransferases responsible for PG synthesis has been developed and tested for its capacity to promote cartilage PG synthesis and deposition. Transfection of chondrocytes or cartilage explants by the expression vector for the glycosyltransferase β-1,3-glucuronosyltransferase-I (GlcAT-I) enhanced PG synthesis and deposition in the ECM by promoting the synthesis of chondroitin sulfate GAG chains of the cartilage matrix. This indicates that therapy mediated through GT gene delivery may constitute a new strategy for the treatment of OA.

Activity of the dipeptidyl hydrolase angiotensin converting enzyme (ACE) has been observed to be altered by treatment with adriamycin (ADR). We used an animal model of ADR nephrotoxicity to study the effects on ACE in serum, urine and tissues on days 5, 10, 15, 20, 25 and 30 after ADR administration. Both glomerular and tubular injury occurred as evidenced by heavy proteinuria, albuminuria and increased urine N -acetyl glucosaminidase (NAG) excretion. Serum ACE was signifcaantly elevated on days 20, 25 and 30. Of great interest was the excretion of ACE in urine of treated rats which ran parallel with the total protein excretion above the barely detectable levels found in controls. ACE activity increased in kidney, adrenal gland and liver on days 15, 20, 25 and 30. Heart and brain ACE levels increased on days 25 and 30. Increased ACE activity in aorta and lungs occurred on days 20, 25 and 30. ACE activity decreased in kidney, aorta, heart and brain on days 5 and 10. These observations strongly suggest a contribution of various tissues to elevate the serum ACE level. Urinary ACE may be of potential use as an index for renal glomerular and tubular damage.

The protective effects of taurine on hyperlipidemia associated with puromycin aminonucleoside-induced nephrotis were assessed in male Wistar rats. Administration of puromycin aminonucleoside to rats resulted in hyperlipidemia at the end of 10 days, as indicated by significant elevation of cholesterol, triglycerides, and phospholipids in plasma and liver. A significant increase in plasma lipid peroxides associated with lipoprotein fractions was observed in nephrotic rats. Liver lipids and lipid peroxides were also higher in these animals. On the other hand, reduced lipoprotein lipase activity was noted in liver of the puromycin-treated rats. In addition, urinary excretion of cholesterol was also high in them. However, treatment of the nephrotic rats with taurine modified hyperlipidemia with reduction in lipid peroxide and all major lipoprotein fractions. Taurine significantly prevented the decrease in lipoprotein lipase activity induced by puromycin. Therefore, our results suggest that prevention of hyperlipidemia by taurine contributes to its therapeutic effects in attenuating the puromycin-induced nephrotic syndrome.

The present study evaluated the efficacy of taurine and niacin alone and in combination, in ameliorating the early lung injury found in paraquat-administered rats. At 24h after paraquat insult several biochemical indicators in bronchoalveolar lavage fluid were analyzed. Increases in total protein, albumin, lactate, lactate dehydrogenase, Nacetyl-β-D-glucosaminidase, alkaline phosphatase, acid phosphatase, and angiotensin-converting enzyme were evident in bronchoalveolar lavage fluid of paraquat-intoxicated rats. Serum and lung lipid peroxidation products were also found to be elevated in the paraquat rats. Pretreatment with taurine or niacin for 7 days before paraquat intoxication reduced the incidence of lung injury; however, far more dramatic protective effects was observed in combined treatment with taurine and niacin. A significant reduction in biochemical constituents in bronchoalveolar lavage fluid and lipid peroxidation occurred in paraquat groups pretreated with taurine and niacin. We conclude that taurine and niacin administration has a protective effect against paraquat-induced early lung injury through the beneficial effects of their antioxidant and membrane-stabilizing properties.

The aim of this review has been to describe the current state of the therapeutic potential of curcumin in acute and chronic lung injuries. Occupational and environmental exposures to mineral dusts, airborne pollutants, cigarette smoke, chemotherapy, and radiotherapy injure the lungs, resulting in acute and chronic inflammatory lung diseases. Despite major advances in treating lung diseases, until now disease-modifying efficacy has not been demonstrated for any of the existing drugs. Current medical therapy offers only marginal benefit; therefore, there is an essential need to develop new drugs that might be of effective benefit in clinical settings. Over the years, there has been increasing evidence that curcumin, a phytochemical present in turmeric (Curcuma longa), has a wide spectrum of therapeutic properties and a remarkable range of protective effects in various diseases. Several experimental animal models have tested curcumin on lung fibrosis and these studies demonstrate that curcumin attenuates lung injury and fibrosis caused by radiation, chemotherapeutic drugs, and toxicants. The growing amount of data from pharmacological and animal studies also supports the notion that curcumin plays a protective role in chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, and allergic asthma, its therapeutic action being on the prevention or modulation of inflammation and oxidative stress. These findings give substance to the possibility of testing curcumin in patients with lung diseases.

The aim of this review has been to describe the current state of the therapeutic potential of curcumin in acute and chronic lung injuries. Occupational and environmental exposures to mineral dusts, airborne pollutants, cigarette smoke, chemotherapy, and radiotherapy injure the lungs, resulting in acute and chronic inflammatory lung diseases. Despite major advances in treating lung diseases, until now disease-modifying efficacy has not been demonstrated for any of the existing drugs. Current medical therapy offers only marginal benefit; therefore, there is an essential need to develop new drugs that might be of effective benefit in clinical settings. Over the years, there has been increasing evidence that curcumin, a phytochemical present in turmeric (Curcuma longa), has a wide spectrum of therapeutic properties and a remarkable range of protective effects in various diseases. Several experimental animal models have tested curcumin on lung fibrosis and these studies demonstrate that curcumin attenuates lung injury and fibrosis caused by radiation, chemotherapeutic drugs, and toxicants. The growing amount of data from pharmacological and animal studies also supports the notion that curcumin plays a protective role in chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, and allergic asthma, its therapeutic action being on the prevention or modulation of inflammation and oxidative stress. These findings give substance to the possibility of testing curcumin in patients with lung diseases.

The protective effect of curcumin on acute adriamycin (ADR) myocardial toxicity was analysed in rats. ADR toxicity, induced by a single intraperitoneal injection (30 mg kg(-1)), was revealed by elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH). The level of the lipid peroxidation products, conjugated dienes and malondialdehyde, was markedly elevated by ADR. ADR caused a decrease in myocardial glutathione content and glutathione peroxidase activity. In contrast, cardiac catalase activity was increased in ADR rats. Curcumin treatment (200 mg kg(-1), seven days before and two days following ADR) significantly ameliorated the early manifestation of cardiotoxicity (ST segment elevation and an increase in heart rate) and prevented the rise in serum CK and LDH exerted by ADR. ADR rats that received curcumin displayed a significant inhibition of lipid peroxidation and augmentation of endogenous antioxidants. These results suggest that curcumin inhibits ADR cardiotoxicity and might serve as novel combination chemotherapeutic agent with ADR to limit free radical-mediated organ injury.

Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, β1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1β down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by 35 S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1β-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1β-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1β. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.

Administration of bleomycin (BM) produces inflammation and fibrosis of the lung in humans and experimental animals. The molecular defects by which BM induces these pathological effects have not been studied in detail. We studied the expression of Smad family proteins, key molecules involved in mediating transforming growth factor (TGF)-β signaling from the cell membrane to the nucleus, during the early and late phases of BM-induced fibrogenesis. Pulmonary fibrosis was induced in male Sprague-Dawley rats by a single intratracheal injection (1.5 units) of BM. Control rats received saline. Rats were killed at 3, 5, 7, 14, and 28 days after BM, cytosolic and nuclear proteins were extracted and isolated from lung tissues, and Smad proteins were probed with specific antibodies. In BM-exposed lung tissue, compared with control, Smad3 decreased persistently in the cytosol and increased transiently in the nucleus. There was a persistent increase in phosphorylation and nuclear accumulation of Smad2/3. Smad4 was increased transiently in both the cytosol and nucleus. A significant and progressive decrease in the expression of Smad7, the endogenous inhibitor of TGF-β/Smad signaling, was observed after BM instillation. Collectively, our results indicate that an imbalance between agonistic Smads2–4 and antagonistic Smad7 may result in the unchecked activation of an autocrine TGF-β loop, which contributes to the pathogenesis of BM-induced pulmonary fibrosis.

In bleomycin (BM)-induced lung fibrosis, alterations have been shown in the expression and deposition of small proteoglycans (PGs). Less, however, is known about changes in large PGs. We investigated changes in large aggregating (versican [VS]), basement membrane (heparan sulfate proteoglycan [HSPG]), and small (biglycan and fibromodulin) PGs during the development of BM-induced pulmonary fibrosis. BM (1.5 U) was instilled intratracheally into male Sprague-Dawley rats. Control rats received saline. At 7, 14, and 28 d after administration of BM, lungs were excised; one lung was fixed in formalin and 5-microm sections were cut and stained with hematoxylin-eosin. The other lung was used for PG extraction. PGs were extracted with guanidine and were separated through composite gel polyacrylamide gel electrophoresis (PAGE) (large PGs) and sodium dodecylsulfate-PAGE (small PGs). Gels were either stained or electrophoretically transferred and probed with antibodies to VS, HSPG, biglycan, and fibromodulin. Histologic samples showed prominent inflammation, with abundant proteinaceous material, most evident in the samples obtained at 7 and 14 d after administration of BM. By 28 d after BM, much of the inflammatory response had resolved, and heterogeneous distribution of fibrosis was observed. Immunoblots showed a relative abundance of VS at 7 and 14 d. Control lungs stained minimally for VS. Levels of HSPG, biglycan, and fibromodulin were increased maximally at 14 d after administration of BM. Immunocytochemistry showed intense immunostaining of biglycan and fibromodulin in the areas of injured lung tissue from rats 14 and 28 d after BM administration. Control lungs revealed minimal staining for small PGs. Our findings indicate that changes in all subclasses of PGs occur during the development of BM-induced pulmonary fibrosis.

An early feature of paraquat (PQ) toxicity is the influx of inflammatory cells, releasing proteolytic enzymes and oxygen free radicals, which can destroy the lung epithelium and result in pulmonary fibrosis. Therefore, the ability to suppress early lung injury seems to be an appropriate therapy of pulmonary damage before the development of irreversible fibrosis. Here I show curcumin confers remarkable protection against PQ lung injury. A single intraperitoneal injection of PQ (50 mg/kg) resulted in a significant rise in the levels of protein, angiotensin converting enzyme (ACE), alkaline phosphatase (AKP), N-acetyl-beta-D-glucosaminidase (NAG) and thiobarbituric acid reactive substances (TBARS), and neutrophils in the bronchoalveolar lavage fluid (BALF), while a decrease in glutathione levels. In paraquat rats bronchoalveolar lavage (BAL) cell TBARS concentration was increased with a simultaneous decrease in glutathione content. In addition, the data also demonstrated that PQ caused a decrease in ACE and glutathione levels and an increase in levels of TBARS and myeloperoxidase (MPO) activity in the lung. Interestingly, curcumin prevented the general toxicity and mortality induced by PQ and blocked the rise in BALF protein, ACE, AKP, NAG TBARS and neutrophils. Similarly, curcumin prevented the rise in TBARS content in both BAL cell and lung tissue and MPO activity of the lung. In addition, PQ induced reduction in lung ACE and BAL cell and lung glutathione levels was abolished by curcumin treatment. These findings indicate that curcumin has important therapeutic implications in facilitating the early suppression of PQ lung injury.

Taurine was used in the present study to evaluate its beneficial effects against proteinuria and hyperlipidemia associated with nephrotic syndrome. Rats made nephrotic with adriamycin had a high excretion of protein, albumin, and N-acetyl-beta-D-glucosaminidase compared with nonnephrotic rats. Nephrotic rats manifested hyperlipidemia with significant elevation in all major lipoprotein fractions. Treatment with taurine significantly suppressed adriamycin-induced proteinuria, albuminuria, and urinary excretion of N-acetyl-beta-D-glucosaminidase. Treatment of rats wit taurine for 7 days before adriamycin, and daily thereafter, significantly lowered plasma cholesterol, triglycerides, phospholipids, lipid peroxides, and malondialdehyde associated with lipoprotein fractions. Similarly, total lipids, cholesterol, triglycerides, lipid peroxides, hydroperoxides, and hydroxyl radicals in the liver and kidneys of taurine-treated adriamycin rats were decreased significantly compared with adriamycin alone. Lecithin cholesterol acyl transferase activity and free fatty acid levels in plasma, lipoprotein lipase activity, glutathione, total thiol, and ascorbic acid in the liver and the kidneys of taurine-treated adriamycin groups were significantly elevated compared with adriamycin alone. These results suggest that taurine might be applicable as a protective agent for proteinuria and hyperlipidemia associated with nephrotic syndrome.

The present study was designed to determine the protective effects of curcumin against bleomycin (BLM)-induced inflammatory and oxidant lung injury. The data indicate that BLM-mediated lung injury resulted in increases in lung lavage fluid biomarkers such as total protein, angiotensin-converting enzyme (ACE), lactate dehydrogenase (LDH), N-acetyl-β-D-glucosaminidase (NAG), lipid peroxidation (LPO) products, Superoxide dismutase (SOD) and catalase. Bleomycin administration also resulted in increased levels of malondialdehyde (MDA) in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage (BAL) cells and greater amounts of alveolar macrophage (AM) Superoxide dismutase activity. By contrast, lower levels of reduced glutamione (GSH) were observed in lung lavage fluid, BAL cells and AM. Stimulated Superoxide anion and hydrogen peroxide release by AM from BLM rats were found to be higher. Curcumin treatment resulted in a significant reduction in lavage fluid biomarkers. In addition, curcumin treatment resulted in the restoration of antioxidant status in BLM rats. These data suggest that curcumin treatment reduces the development of BLM-induced inflammatory and oxidant activity. Therefore, curcumin offers the potential for a novel pharmacological approach in the suppression of drug or chemical-induced lung injury.