Corrigendum: Expression of Bioactive Chemerin by Keratinocytes Inhibits Late Stages of Tumor Development in a Chemical Model of Skin Carcinogenesis.

[This corrects the article DOI: 10.3389/fonc.2019.01253.].

Expression of Bioactive Chemerin by Keratinocytes Inhibits Late Stages of Tumor Development in a Chemical Model of Skin Carcinogenesis.

Chemerin is a multifunctional protein acting mainly through the G protein-coupled receptor ChemR23/CMKLR1/Chemerin1. Its expression is frequently downregulated in human tumors, including in melanoma and squamous cell carcinoma of the skin and anti-tumoral properties of chemerin were reported in mouse tumor graft models. In the present study, we report the development of spontaneous skin tumors in aged ChemR23-deficient mice. In order to test the potential therapeutic benefit of chemerin analogs, a transgenic model in which bioactive chemerin is over-expressed by basal keratinocytes was generated. These animals are characterized by increased levels of chemerin immunoreactivity and bioactivity in the skin and the circulation. In a chemical carcinogenesis model, papillomas developed later, were less numerous, and their progression to carcinomas was delayed. Temporal control of chemerin expression by doxycycline allowed to attribute its effects to late stages of carcinogenesis. The protective effects of chemerin were partly abrogated by ChemR23 invalidation. These results demonstrate that chemerin is able to delay very significantly tumor progression in a model that recapitulates closely the evolution of solid cancer types in human and suggest that the chemerin-ChemR23 system might constitute an interesting target for therapeutic intervention in the cancer field.

Implementation of a new health information technology for the management of cancer chemotherapies.

BACKGROUND: Cancer chemotherapy drugs are classified as high-risk molecules. Safety of the cancer chemotherapy process is often achieved with the implementation of a health information technology to each step or to the entire process. However, computerisation could lead to the emergence of new unintended medication errors. The aim of the study was to evaluate the impact of new software designed for the management of anticancer chemotherapies. METHOD: The cartography of the process and the failure modes, effects and criticality analysis were performed by a multidisciplinary team. Criticality indexes were calculated considering or not the implementation of the commercial software (CytoWeb). Quality and satisfactory indicators were measured before the implementation and during the use of the software. RESULTS: Our results demonstrated the complexity of the cancer chemotherapy process in the hospital. Risk analysis highlighted the positive impact of CytoWeb on the process safety but pointed out some steps that were not positively influenced by the software. Although a decrease of 38.6% of error rate was observed with the electronic system, new unintended medication errors emerged. These errors were due to inadequate use of the software (encoding of the wrong drug, the wrong dose, the wrong patient parameters or lab results and lack of prescriber adherence). Our satisfaction survey showed that the hospital pharmacists and doctors were less satisfied by the software than the nurses, mostly in terms of task achievement and time saving. Survey's results highlighted some weaknesses in the user training and in the collaboration between the medical staff. CONCLUSIONS: Our work showed the emergence of unintended medication errors linked to computerisation that were due to an inadequate use of the software. Other issues were highlighted such as the lack of collaboration between the medical staff, the lack of prescriber implication and weaknesses in the user training or in the information related to CytoWeb.

Modular Integrated Secretory System Engineering in Pichia pastoris To Enhance G-Protein Coupled Receptor Expression.

Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies.

Clinical Parameters vs Cytokine Profiles as Predictive Markers of IgE-Mediated Allergy in Young Children.

BACKGROUND: Allergy afflicts one third of children, negatively impacting their quality of life and generating a significant socio-economic burden. To this day, this disorder remains difficult to diagnose early in young patients, with no predictive test available. OBJECTIVE: This study was designed to correlate cytokine profiles with clinical phenotypes of allergy development. METHODS: Three hundred patients were recruited and followed from birth to 18 months of age. They were given a clinical exam at birth and at 2, 6, 12, and 18 months of age, with skin prick tests at 6, and 18 months, in order to have a record of their medical history and determine their allergic status. In addition, mononuclear cells from 131 patients were isolated from cord blood and from peripheral blood samples at 2, 6 and 18 months of age, to analyse their cytokine and chemokine production. RESULTS: Cord blood mononuclear cells (CBMCs) from future Immunoglobulin (Ig) E-mediated allergic children produced significantly less Interleukin (IL)-12p70 and IL-15 than cells from the rest of the cohort. Multivariate analyses revealed that the best predictive model of allergy was built on cytokine data, whereas the best predictive model of IgE-mediated allergy was built on clinical parameters. CONCLUSIONS AND CLINICAL RELEVANCE: Although univariate analyses can yield interesting information regarding the immune responses of allergic children, finding predictive markers of the disorder will likely rely on monitoring multiple parameters. Nonetheless these analyses suggest a potential key role for IL-15 in the development of atopic disease. In addition, the study highlights the importance of clinical parameters in predicting the development of IgE-mediated allergy.

PET/CT with 18F-FDG- and 18F-FBEM-labeled leukocytes for metabolic activity and leukocyte recruitment monitoring in a mouse model of pulmonary fibrosis.

UNLABELLED: Idiopathic pulmonary fibrosis is characterized by a progressive and irreversible respiratory failure. Validated noninvasive methods able to assess disease activity are essential for prognostic purposes as well as for the evaluation of emerging antifibrotic treatments. METHODS: C57BL/6 mice were used in a murine model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin (control mice were instilled with a saline solution). At different times after instillation, PET/CT with (18)F-FDG- or (18)F-4-fluorobenzamido-N-ethylamino-maleimide ((18)F-FBEM)-labeled leukocytes was performed to assess metabolic activity and leukocyte recruitment, respectively. RESULTS: In bleomycin-treated mice, a higher metabolic activity was measured on (18)F-FDG PET/CT scans from day 7 to day 24 after instillation, with a peak of activity measured at day 14. Of note, lung mean standardized uptake values correlated with bleomycin doses, histologic score of fibrosis, lung hydroxyproline content, and weight loss. Moreover, during the inflammatory phase of the model (day 7), but not the fibrotic phase (day 23), bleomycin-treated mice presented with an enhanced leukocyte recruitment as assessed by (18)F-FBEM-labeled leukocyte PET/CT. Autoradiographic analysis of lung sections and CD45 immunostaining confirm the higher and early recruitment of leukocytes in bleomycin-treated mice, compared with control mice. CONCLUSION: (18)F-FDG- and (18)F-FBEM-labeled leukocyte PET/CT enable monitoring of metabolic activity and leukocyte recruitment in a mouse model of pulmonary fibrosis. Implications for preclinical evaluation of antifibrotic therapy are expected.

Adult human hepatocytes promote CD4(+) T-cell hyporesponsiveness via interleukin-10-producing allogeneic dendritic cells.

The success of liver cell therapy remains closely dependent on how well the infused cells can be accepted after transplantation and is directly related to their degree of immunogenicity. In this study, we investigated the in vitro immunogenic properties of isolated human hepatocytes (hHeps) and adult-derived human liver progenitor cells (ADHLPCs), an alternative cell candidate for liver cell transplantation (LCT). The constitutive expression of immune markers was first analyzed on these liver-derived cells by flow cytometry. Human liver-derived cells were then cocultured with allogeneic human adult peripheral blood mononuclear cells (PBMCs), and the resulting activation and proliferation of PBMCs was evaluated, as well as the cytokine levels in the coculture supernatant. The effect of liver-derived cells on monocyte-derived dendritic cell (MoDC) properties was further analyzed in a secondary coculture with naive CD4(+) T-cells. We report that hHeps and ADHLPCs expressed human leukocyte antigen (HLA) class I and Fas but did not express HLA-DR, Fas ligand, and costimulatory molecules. hHeps and ADHLPCs did not induce T-cell activation or proliferation. Moreover, hHeps induced a cell contact-dependent production of interleukin (IL)-10 that was not observed with ADHLPCs. The IL-10 was produced by a myeloid DC subset characterized by an incomplete mature state. Furthermore, hHep-primed MoDCs induced an antigen-independent hyporesponsiveness of naive CD4(+) T lymphocytes that was partially reversed by blocking IL-10, whereas nonprimed MoDCs (i.e., those cultured alone) did not. hHeps and ADHLPCs present a low immunogenic phenotype in vitro. Allogeneic hHeps, but not ADHLPCs, promote a cell contact-dependent production of IL-10 by myeloid DCs, which induces naive CD4(+) T-cells antigen-independent hyporesponsiveness.

The chemerin/ChemR23 system does not affect the pro-inflammatory response of mouse and human macrophages ex vivo.

Macrophages constitute a major component of innate immunity and play an essential role in defense mechanisms against external aggressions and in inflammatory responses. Chemerin, a chemoattractant protein, is generated in inflammatory conditions, and recruits cells expressing the G protein-coupled receptor ChemR23, including macrophages. Chemerin was initially expected to behave as a pro-inflammatory agent. However, recent data described more complex activities that are either pro- or anti-inflammatory, according to the disease model investigated. In the present study, peritoneal macrophages were generated from WT or ChemR23(-/-) mice, stimulated with lipopolyssaccharide in combination or not with IFN-γ and the production of pro- (TNF-α, IL-1ß and IL-6) and anti-inflammatory (IL-10) cytokines was evaluated using qRT-PCR and ELISA. Human macrophages generated from peripheral blood monocytes were also tested in parallel. Peritoneal macrophages from WT mice, recruited by thioglycolate or polyacrylamide beads, functionally expressed ChemR23, as assessed by flow cytometry, binding and chemotaxis assays. However, chemerin had no effect on the strong upregulation of cytokine release by these cells upon stimulation by LPS or LPS/IFN-γ, whatever the concentration tested. Similar data were obtained with human macrophages. In conclusion, our results rule out the direct anti-inflammatory effect of chemerin on macrophages ex vivo, described previously in the literature, despite the expression of a functional ChemR23 receptor in these cells.

ChemR23 dampens lung inflammation and enhances anti-viral immunity in a mouse model of acute viral pneumonia.

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23(-/-) mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23(-/-) mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.

AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity.

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-kappaB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4(+) T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

N-acetylcysteine derivative inhibits CD40-dependent proinflammatory properties of human pancreatic duct cells.

OBJECTIVES: We recently observed that duct cells constitutively express CD40, a membrane molecule whose engagement results in duct cell activation and proinflammatory cytokine secretion. This observation suggests a potential role of this pathway in the pathogenesis of type 1 diabetes, islet graft rejection, or acute pancreatitis. In this article, we investigated whether a salt derivative of N-acetyl-L-cysteine, Nacystelyn, could modulate CD40 expression on duct cells and the response of activated duct cells to CD40 engagement. METHODS: We assessed the effects of Nacystelyn on CD40 expression and function in human caucasian pancreatic adenocarcinoma, ATCC n degrees THB-80 (CAPAN-2) cells, a human pancreatic duct cell line. CD40 expression was analyzed by flow cytometry. To assess CAPAN-2 cell responses to CD40 engagement, we looked at nuclear factor-kappaB transcription factor activation using enzyme-linked immunosorbent assay and electrophoretic mobility shift assay and cytokine mRNA levels by quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: We observed that Nacystelyn dose-dependently inhibited CD40 expression on CAPAN-2 cells as well as CD40-induced nuclear factor kappaB activation and proinflammatory cytokines up-regulation. CONCLUSIONS: Our data suggest that Nacystelyn could be considered as a useful tool to prevent immune and inflammatory responses in pancreatic disorders by interfering with the CD40 pathway in pancreatic duct cells.

Tissue factor-dependent procoagulant activity of isolated human hepatocytes: relevance to liver cell transplantation.

Liver cell transplantation (LCT) aims to correct inborn liver function defects by infusing metabolically active cells into the diseased liver. Further improvement in LCT might depend on the prevention of early loss of transplanted cells. As tissue factor (TF)-dependent activation of coagulation was found to contribute to a low rate of beta cell engraftment in islet transplantation, we investigated the potential procoagulant activity (PCA) of hepatocyte preparations. TF expression on hepatocyte preparations was assessed by flow cytometry, reverse-transcription polymerase chain reaction and immunofluorescence. PCA depending on TF was evaluated in human plasma and in whole blood systems. Coagulation parameters were followed by routine techniques in a LCT recipient Crigler-Najjar patient. We determined that hepatocytes express soluble and membrane-bound forms of TF. We showed that hepatocytes exert a TF-dependent PCA. In parallel, delayed increase in D-dimer levels was observed following the hepatocyte infusions in the Crigler-Najjar patient. Furthermore, in vitro experiments demonstrated that TF-dependent PCA of hepatocytes is inhibited by N-acetyl-L-cysteine. In conclusion, hepatocytes exert TF-dependent PCA, which may contribute to early loss of infused cells. Addition of N-acetyl-L-cysteine to the suspensions of hepatocytes might be beneficial in LCT by inhibiting activation of coagulation.

Rottlerin inhibits human T cell responses.

Rottlerin is a pharmacological inhibitor of protein kinase C (PKC) theta, a novel PKC selectively expressed in T lymphocytes. PKC theta is known to regulate T cell receptor (TCR)/CD28 signalling pathways in T lymphocytes, but the impact of PKC theta inhibition on human T cell responses remains undefined. In this work, we describe the effects of rottlerin on the responses of CD4+ and CD8+ human T lymphocytes upon polyclonal activation. We observed a dose-dependent inhibition of CD4+ and CD8+ T cell proliferation in response to anti-CD3/anti-CD28 antibodies stimulation in the presence of rottlerin. This inhibition was associated with impaired CD25 expression and decreased interleukin (IL)-2 production in activated T cells. In contrast, rottlerin did not alter IL-2-induced T cell proliferation. Furthermore, we demonstrated that rottlerin blocked interferon (IFN) gamma, IL-10 and IL-13 mRNA expression in TCR/CD28 activated CD4+ T cells. These findings place rottlerin as a potent immunosuppressive agent for the development of novel therapies in T cell mediated immune disorders.

Human pancreatic duct cells exert tissue factor-dependent procoagulant activity: relevance to islet transplantation.

Activation of the coagulation cascade contributes to early graft loss and intraportal thrombotic events in clinical islet transplantation. Although these complications were shown to be related to the presence of tissue factor in human islet preparations, the contribution of duct cells, which represent a major contaminant of clinical islet isolates, has not been specified so far. Herein, we used flow cytometry, immunohistochemistry, RT-PCR, and functional coagulation assays to demonstrate that duct cells exert a potent factor VII-dependent procoagulant activity related to their expression of tissue factor. Both the classical membrane-bound and the recently described soluble form of tissue factor were shown to be synthesized by duct cells. We conclude that contaminating duct cells contribute to early beta-cell damage after islet transplantation through their involvement in tissue factor-mediated thrombotic and inflammatory events.

Induction of FOXP3-expressing regulatory CD4pos T cells by human mature autologous dendritic cells.

Current literature suggests that T cells recognizing antigen on mature dendritic cells (DC) differentiate into effector T cells whereas tolerance is induced when antigen is presented by immature DC. We investigated the consequences of the interactions between immature or lipopolysaccharide-matured DC and CD4(pos) T lymphocytes in absence of foreign antigen. While immature DC did not induce significant CD4(pos) T cell activation, we observed that a significant fraction of CD4(pos) T cells cultured with mature autologous DC displayed phenotypic features of activation and produced IL-2, IFN-gamma, IL-10 and TGF-beta. Furthermore, CD4(pos) T lymphocytes primed by mature, but not immature, autologous DC acquired regulatory properties. Indeed, when added to an allogeneic mixed leukocyte reaction, they suppressed the response of alloreactive T lymphocytes to the priming DC while responses to third-party stimulators were spared. The generation of CD4(pos) T cells with regulatory function by autologous stimulation did not require the presence of natural CD4(pos)CD25(pos) regulatory T cells. In addition, the acquisition of regulatory function by CD4(pos)CD25(neg) T cells stimulated by autologous mature DC was accompanied by the induction of FOXP3 expression. Our data suggest that during inflammatory conditions, presentation of self antigens by mature DC to autologous T lymphocytes could contribute to the generation of regulatory mechanisms.

Dendritic cells exposed to nacystelyn are refractory to maturation and promote the emergence of alloreactive regulatory t cells.

BACKGROUND: Drugs blocking dendritic cell (DC) maturation might be useful in transplantation by inhibiting the induction of primary alloimmune responses and promoting the emergence of regulatory T lymphocytes (Treg). We investigated the effects of Nacystelyn (NAL), an N-acetyl-L-cysteine derivative, on human DCs, paying attention to the T-cell responses elicited by NAL-treated DCs in vitro. METHODS: Lipopolysaccharide (LPS) was used to induce the maturation of DCs naturally present in blood or generated from human monocytes cultured in interleukin-4 and granulocyte-macrophage colony-stimulating activity. We first analyzed the consequences of NAL on cytokine production and expression of major histocompatibility complex class II and costimulatory molecules. Monocyte-derived DCs were then used as stimulators in mixed leukocyte cultures with naive CD4 T cells. Cytokine levels were measured in culture supernatants; the phenotype of T cells and their capacity to inhibit the proliferation of third-party T-cell responders was determined at the end of the culture. RESULTS: NAL proved to be a potent inhibitor of DC maturation in whole blood experiments and on monocyte-derived DCs. Alloreactive T cells stimulated with DCs pretreated with LPS in the presence of NAL produced much less interferon-gamma but similar levels of interleukin-13 compared with DCs treated with LPS alone. Immature DCs induced Treg, which was not observed with mature DCs. DCs cultured with LPS in the presence of NAL were as efficient as immature DCs to generate alloreactive T cells with regulatory activity. CONCLUSIONS: NAL is a potent inhibitor of DC maturation, which might be useful to promote allograft acceptance by inducing the differentiation of allospecific Treg.

Inhibitory effects of thiol-containing drugs on erythrocyte oxidative damages investigated with an improved assay system.

The involvement of oxygen-derived free radicals and other oxidant species in numerous physiopathological processes makes it necessary to develop suitable analytical systems to study their effects and also to assess the antioxidant activity of endogenous and exogenous compounds. In this respect, the properties of three selected thiol-containing drugs (Captopril, N-acetylcysteine and its lysine salt Nacystelyn, a newly developed mucoactive agent) and of reference compounds were examined in a lipid peroxidation model using human red blood cells (RBC) as biological substrate. The thermolabile azo-compound 2,2'-azobis-2-amidinopropane dihydrochloride (AAPH) served for the generation of an oxidative stress and the determination of the extent of RBC haemolysis was recorded. Experimental conditions were developed and optimised to ensure the stability and reproducibility of the system and to establish complete dose-response relationships in order to determine relevant pharmacological parameters. Actually, the AAPH/haemolysis system shrewdly combined with a procedure to measure the extent of haemolysis in which all common haemoglobin derivatives released following haemolysis are converted to cyanomethaemoglobin, allowed the assessment of the antioxidant activity of most investigated drugs. Precision was also improved by considering readings at 50% haemolysis (T(50)). The following sequence was obtained for the antioxidant properties of investigated drugs: uric acid>Trolox>ascorbic acid>N-acetylcysteine approximately Nacystelyn>Captopril>>l-lysine.