Cystic fibrosis (CF) lung disease is characterized by chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in the cystic fibrosis transmembrane conductance regulator, there is still an unmet need to also normalize the inflammatory response. The prolonged and heightened inflammatory response in CF is, in part, mediated by a lack of intrinsic down-regulation of the proinflammatory NF-κB pathway. We have previously identified reduced expression of the NF-κB down-regulator A20 in CF as a key target to normalize the inflammatory response. Here, we have used publicly available gene array expression data together with a statistically significant connections' map (sscMap) to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NF-κB(p65) expression (mRNA) as well as proinflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o−, CFBE41o−) and in primary nasal epithelial cells from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with tnfαip3 (A20) knockdown (siRNA). We also show that the A20-inducing effect of ikarugamycin and quercetin is lower in CF-derived airway epithelial cells than in non-CF cells.A20 | NF-κB | connectivity mapping | drug repositioning | CF airway inflammation
Due to the incidence of diabetes and the related morbidity of diabetic nephropathy, identification of new therapeutic strategies represents a priority. In the last few decades new and growing evidence on the possible role of histamine in diabetes has been provided. In particular, the histamine receptor HR is emerging as a new promising pharmacological target for diabetic nephropathy. The aim of this study was to evaluate the efficacy of selective HR antagonism by JNJ39758979 on the prevention of diabetic nephropathy progression in a murine model of diabetes induced by streptozotocin injection. JNJ39758979 (25, 50, 100 mg/kg/day p.o.) was administered for 15 weeks starting from the onset of diabetes. Functional parameters were monitored throughout the experimental period. JNJ39758979 did not significantly affect glycaemic status or body weight. The urine analysis indicated a dose-dependent inhibitory effect of JNJ39758979 on Albumin-Creatinine-Ratio, the Creatinine Clearance, the 24 h urine volume, and pH urine acidification (P < 0.05). The beneficial effects of JNJ39758979 on renal function paralleled comparable effects on renal morphological integrity. These effects were sustained by a significant immune infiltration and fibrosis reduction. Notably, megalin and sodium-hydrogen-exchanger 3 expression levels were preserved. Our data suggest that the HR participates in diabetic nephropathy progression through both a direct effect on tubular reabsorption and an indirect action on renal tissue architecture via inflammatory cell recruitment. Therefore, HR antagonism emerges as a possible new multi-mechanism therapeutic approach to counteract development of diabetic nephropathy development.
Objective and design The aim of this study is to evaluate the expression of the histamine receptors, particularly focusing on the H 4 R in human renal tubules. MaterialThe ex-vivo evaluation was carried on specimens from human renal cortex. Primary and immortalized tubular epithelial cells (TECs) and the HK-2 cell line were used as in vitro models.Treatment Cells were pretreated for 10 min with chlorfeniramine maleate 10 µM (H 1 R antagonist), ranitidine 10 µM (H 2 R antagonist), GSK189254 1 µM (H 3 R antagonist) or JNJ7777120 10 µM (H 4 R antagonist), and then exposed to histamine (3 pM -10 nM) for 30 min. MethodsThe ex-vivo evaluation on specimens from human renal cortex was performed by immunohistochemistry. The expression of histamine receptors on primary and immortalized TECs and the HK-2 cell line was evaluated at both gene (RT-PCR) and protein (immunocytofluorescence) levels. The pharmacological analysis was performed by TR-FRET measurements of second messenger (IP3 and cAMP) production induced by histamine with or without the selective antagonists.Results Our data revealed the presence of all histamine receptors in human tubules; however, only TECs expressed all the receptors. Indeed, histamine elicited a sigmoid dose-response curve for IP 3 production, shifted to the right by chlorpheniramine maleate, and elicited a double bell-shaped curve for cAMP production, partially suppressed by the selective H 2 R, H 3 R and H 4 R antagonists when each added alone, and completely ablated when combined together.Conclusions Herein, we report the identification of all four histamine receptors in human renal tubules.
Pulmonary fibrosis, a progressive and lethal lung disease characterized by inflammation and accumulation of extracellular matrix components, is a major therapeutic challenge for which new therapeutic strategies are warranted. Cyclooxygenase (COX) inhibitors have been previously utilized to reduce inflammation. Histamine H 4 receptor (H 4 R), largely expressed in hematopoietic cells, has been identified as a novel target for inflammatory and immune disorders. The aim of this study was to evaluate the effect of JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine), a selective H 4 R antagonist, and naproxen, a well known nonsteroidal anti-inflammatory drug, and their combination in a murine model of bleomycin-induced fibrosis. Bleomycin (0.05 IU) was instilled intratracheally to C57BL/6 mice, which were then treated by micro-osmotic pump with vehicle, JNJ7777120 (40 mg/kg b.wt.), naproxen (21 mg/kg b.wt.), or a combination of both. Airway resistance to inflation, an index of lung stiffness, was assessed, and lung specimens were processed for inflammation, oxidative stress, and fibrosis markers. Both drugs alone were able to reduce the airway resistance to inflation induced by bleomycin and the inflammatory response by decreasing COX-2 and myeloperoxidase expression and activity and thiobarbituric acid-reactive substance and 8-hydroxy-29-deoxyguanosine production. Lung fibrosis was inhibited, as demonstrated by the reduction of tissue levels of transforming growth factor-b, collagen deposition, relative goblet cell number, and smooth muscle layer thickness. Our results demonstrate that both JNJ7777120 and naproxen exert an anti-inflammatory and antifibrotic effect that is increased by their combination, which could be an effective therapeutic strategy in the treatment of pulmonary fibrosis.
The adduction of fumaric acid to the sulfhydryl group of certain cysteine (Cys) residues in proteins via a Michaellike reaction leads to the formation of S-(2-succino)cysteine (2SC) sites. Although its role remains to be fully understood, this post-translational Cys modification (protein succination) has been implicated in the pathogenesis of diabetes/obesity and fumarate hydratase-related diseases. In this study, theoretical approaches to address sequence-and 3D-structure-based features possibly underlying the specificity of protein succination have been applied to perform the first analysis of the available data on the succinate proteome. A total of 182 succinated proteins, 205 modifiable, and 1750 non-modifiable sites have been examined. The rate of 2SC sites per protein ranged from 1 to 3, and the overall relative abundance of modifiable sites was 10.8%. Modifiable and nonmodifiable sites were not distinguishable when the hydrophobicity of the Cys-flaking peptides, the acid dissociation constant value of the sulfhydryl groups, and the secondary structure of the Cys-containing segments were compared. By contrast, significant differences were determined when the accessibility of the sulphur atoms and the amino acid composition of the Cys-flaking peptides were analysed. Based on these findings, a sequence-based score function has been evaluated as a descriptor for Cys residues. In conclusion, our results indicate that modifiable and non-modifiable sites form heterogeneous subsets when features often discussed to describe Cys reactivity are examined. However, they also suggest that some differences exist, which may constitute the baseline for further investigations aimed at the development of predictive methods for 2SC sites in proteins.
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