Monocytes from infliximab-resistant patients with Crohn's disease exhibit a disordered cytokine profile

Crohn's disease (CD) is a chronic inflammatory disorder characterized by immune response dysregulation. Tumor necrosis factor-alpha (TNF alpha) is a key cytokine in the pathogenesis of CD, as indicated by the efficacy of anti-TNF-alpha therapy with infliximab (IFX). However, approximately 30-40% of CD patients fail to respond to IFX with still unclear underlying mechanisms. This study compares the inflammatory phenotype of monocytes from CD patients, who respond or non-respond to IFX. Under basal conditions, the mRNA for the cytokines TNF alpha, IL-23, IL-1 beta and the chemokines CXCL8/IL-8, CCL5/RANTES and CCL2/MCP-1 was up-regulated in monocytes from non-responders than responders. The expression of the same cytokines and CCL2/MCP-1 was higher in non-responders also upon LPS treatment. Moreover, higher secretion of TNF alpha, IL-1 beta, IFN gamma and IL-2 proteins occurred in the supernatants of LPS-treated non-responders cells. Resistance to IFX in CD may result from a transcriptional dysregulation of circulating monocytes, leading to hyperactivation of pro-inflammatory pathways. Monocytes' cytokine profile may thus represent a predictive marker of response to IFX. Monocytes were isolated from blood samples of 19 CD patients (11 responders, 8 non-responders) and incubated with or without LPS. Cytokine profiles were assessed by RT-qPCR and, in the supernatants, by ELISA assay

Glutamine depletion by crisantaspase hinders the growth of human hepatocellular carcinoma xenografts

Background: A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO). Methods: Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1. Results: Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro. Conclusions: The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth

ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger-for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg2+ and/or H+ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP4- in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed

Changes in neutral amino acid efflux and membrane potential associated with the expression of CFTR protein

The expression of wild type CFTR facilitates the efflux of neutral amino acids; as a result, after an extensive depletion of intracellular amino acid pool obtained through an incubation in saline solution, the intracellular leucine levels were lower in murine C127 cells transfected with the wild type CF gene (C127 CFTRw/t) than in cells transfected with either mutant CF (C127 CFTR\u394F508 cells) or mock vector only. No change in amino acid efflux was detected when C127 CFTRw/t and C127 CFTR\u394F508 cells were studied under conditions known to activate protein kinase A. Upon an incubation in Cl- free medium, a permeant analogue of cAMP caused a marked cell depolarization of C127 CFTRw/t cells but not of C127 CFTR\u394F508 cells, thus showing a functional expression of CFTR protein in the former cell line. However, we found that, upon a Cl- free incubation and in the absence of exogenous cAMP, C127 CFTRw/t cells developed a marked hyperpolarization that was not detected in C127 CFTR\u394F508 cells. It is concluded that the expression of normal CFTR accelerates amino acid efflux and enhances cell hyperpolarization in Cl- free media; both these effects appear to be independent from PKA stimulation of CFTR

Alterations of arginine in Lysinuric Protein Intolerance (LPI) macrophages

Lysinuric Protein Intolerance (LPI) is an inherited transport defect of cationic amino acids (arginine, lysine and ornithine) transport at the basolateral membrane of intestinal and renal tubular cells caused by mutations in SLC7A7 encoding for System y+L-related y+LAT1 protein. The severe clinical course of this disorder suggests that LPI should be considered a severe multisystem disease with features proper of 'metabolic' disorders. In particular, immune dysfunction could be attributed to an altered metabolism of nitric oxide (NO) secondary to abnormal arginine intracellular metabolism. To address this hypothesis we measured arginine transport/metabolism in monocyte-derived macrophages (MDM), obtained from peripheral blood monocytes isolated from 5 LPI patients. In these cells the efflux of 3H-arginine through System y+L was markedly lower than in normal MDM, with a decrease that ranged from 60 to 75%. Moreover, SLC7A7 silencing in a human monocytic cell line (THP-1 cells) caused a significant increase of intracellular arginine content. These results indicate that arginine entrapping takes place in LPI monocytes-macrophages, supporting the hypothesis that NO overproduction observed in vivo may be due to an abnormal intracellular metabolism of arginine. Supported by CLIMB (Children living with inherited metabolic diseases, Crewe, UK

The transport of L-arginine in Chinese hamster ovary cells.

The transport of L-arginine has been characterized in Chinese hamster ovary cells (CHO). In the absence of Na+ the influx of the amino acid decreased. Both in the presence and in the absence of Na+ L-arginine influx was trans-stimulated and cis-inhibited by cationic amino acids. The amino acid entered CHO cells through an apparently non saturable mechanism and a single saturable agency whose Km increased in the absence of Na+. These results indicate that the agency devoted to transport cationic amino acids in CHO cells resembles system y+, the Na+-independent route that transports cationic amino acids in a number of mammalian models, although its activity is lowered by the replacement of extracellular sodium

CFTR protein is involved in the efflux of neutral amino acids

Trans-membrane fluxes of leucine were measured in mouse C127i cells transfected with the wild type (C127 CFTRw/t) or the \u394F508 CF gene (C127 CFTR\u394F508). Leucine efflux was significantly faster in C127 CFTRw/t cells. On the contrary, leucine influx was comparable in the two cell lines and referable to a 'L-type' transport system. No significant differences in leucine content were detected among the two cell lines when maintained in complete growth medium; in contrast, after prolonged incubation in amino-acid-free saline solution, the amount of intracellular leucine was significantly smaller in C127 CFTRw/t than in C127 CFTR\u394F508 cells. Leucine behavior was shared by other neutral amino acids with non polar side chains. These results suggest that the expression of normal CFTR increases the efflux of a subgroup of neutral amino acids