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A tight link has been established between inflammation and cancer. Liver regeneration is a widely used model to study the correlation between inflammation and proliferation. IL-6 is essentially involved in liver regeneration and in cancer. Recently, IL-17A has been shown to regulate not only inflammation, but also cell proliferation. Here, we analyze the role played by IL-17A signaling in liver regeneration by comparing cell proliferation in Wild Type and IL-17RA(−/−) mice. Partial hepatectomy experiments performed in IL-17RA(−/−) mice showed a delay in expression of early-genes to prime the residual hepatocyte to proliferate, with subsequent delay in G(1)/S-phase transition. We demonstrated that IL-17RA regulates, by recruitment of non-parenchymal cell, the expression of IL-6, which in turn triggers the proliferation of residual hepatocytes. Our data indicate an important role played by IL-17RA in liver proliferation via IL-6.

Efficient response to Aspergillusfumigatus requires different mechanisms. Polymorphonuclear neutrophils (PMNs) are the predominant immune cells in the acute stage of most fungal infections and play a crucial role in determining the type of pathology associated with fungal infections in different clinical settings. Dendritic cells (DC) are able to decode the fungus-associated information and translate it into different T helper (Th) and regulatory (Treg) cell responses. Functionally distinct Treg cells are activated after exposure to Aspergillus conidia. Early in infection, inflammation/Th1 reactivity is controlled by Treg cells suppressing PMNs and the immunogenic program of DC. The levels of IFN-γ produced in this phase set the subsequent adaptive stage by conditioning the indoleamine 2, 3-dioxygenase (IDO)-dependent tolerogenic program of DC and the subsequent activation of tolerogenic Treg cells, which inhibit Th2 cells and prevent allergy to the fungus. Knowledge of the immunopathogenesis of Aspergillus infections may pave the way to promising strategies for immunotherapy.

The term liquid biopsy refers to the analysis of biomarkers in any body fluid, including blood, urine and cerebrospinal fluid. In cancer, liquid biopsy testing allows the analysis of tumor-derived DNA, RNA, miRNA and proteins that can be either cell-free or contained in circulating tumor cells (CTC), extracellular vesicles (EVs) or platelets. A number of studies suggest that liquid biopsy testing could have a relevant role in the management of colorectal cancer (CRC) patients at different stages of the disease. Analysis of cell-free DNA (cfDNA), CTC and/or miRNA can provide relevant information for the early diagnosis of CRC and the identification of minimal residual disease and, more generally, the evaluation of the risk of recurrence in early CRC patients. In addition, liquid biopsy testing might allow the assessment of prognostic and predictive biomarkers in metastatic CRC patients, and the monitoring of the response to treatment and of the clonal evolution of the disease. While a number of elegant studies have shown the potential of liquid biopsy in CRC, the possibility to use this approach in the daily clinical practice is still limited. The use of non-standardized methods, the small cohorts of patients analyzed, the lack of demonstration of a clear clinical benefit are the main limitations of the studies with liquid biopsy in CRC reported up to now. The potential of this approach and the steps that need still to be taken to translate these preliminary findings in the clinic are discussed in this review.

Belinsky and colleagues showed the potential utility for predicting tumour recurrence of an 8-gene methylation panel in plasma and sputum from resected Stage I non-small cell lung cancer (NSCLC) patients (1).

Phagocytes fight fungi using canonical and noncanonical, also called LC3-associated phagocytosis (LAP), autophagy pathways. However, the outcomes of autophagy/LAP in shaping host immune responses appear to greatly vary depending on fungal species and cell types. By allowing efficient pathogen clearance and/or degradation of inflammatory mediators, autophagy proteins play a broad role in cellular and immune homeostasis during fungal infections. Indeed, defects in autophagic machinery have been linked with aberrant host defense and inflammatory states. Thus, understanding the molecular mechanisms underlying the relationship between the different forms of autophagy may offer a way to identify drugable molecular signatures discriminating between selective recognition of cargo and host protection. In this regard, IFN-γ and anakinra are teaching examples of successful antifungal agents that target the autophagy machinery. This article provides an overview of the role of autophagy/LAP in response to fungi and in their infections, regulation, and therapeutic exploitation.

// Silvia Vecchiarelli 1, * , Francesco Passiglia 2, * , Armida D’Incecco 3, * , Marianna Gallo 4 , Antonella De Luca 4 , Elisa Rossi 5 , Federica D’Inca 1 , Gabriele Minuti 1 , Lorenza Landi 1 , Chiara Bennati 1 , Michela Spreafico 1 , Manolo D’Arcangelo 1 , Valentina Mazza 1 , Nicola Normanno 4 and Federico Cappuzzo 1 1 Department of Oncology and Hematology, AUSL della Romagna, Ravenna, Italy 2 Department of Surgical, Oncological and Stomatological Disciplines, University of Palermo, Palermo, Italy 3 Medical Oncology and Immunotherapy, University Hospital of Siena, Siena, Italy 4 Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori "Fondazione G Pascale"-IRCCS, Naples, Italy 5 Fondazione Ricerca Traslazionale, Rome, Italy * These authors contributed equally to this work Correspondence to: Federico Cappuzzo, email: federico.cappuzzo@auslromagna.it Keywords: PD-L1; immunotherapy; biomarkers; non-small-cell lung cancer Received: November 28, 2017 Accepted: February 27, 2018 Published: April 03, 2018 ABSTRACT Background: This study aimed at investigating feasibility of programmed death ligand-1 (PD-L1) testing in plasma samples of advanced NSCLC patients receiving first-line treatment, assessing whether circulating (c)PD-L1 levels were modified by the therapy and whether baseline cPD-L1 levels were associated with patients’ clinical responses and survival outcome. Methods: Peripheral blood samples were collected from 16 healthy volunteers and 56 newly diagnosed NSCLC patients before and at 12th week during the course of first-line therapy. The level of PD-L1 was measured in plasma samples using the human (PD-L1/CD274) ELISA kit (CUSABIO, MD, USA). The Mann Whitney test or Fisher’s test were used for comparisons. Survival analysis was performed using Kaplan Meyer method, providing median and p -value. Results: Baseline median cPD-L1 was 42.21 pg/ml (range 12.00-143.49) in NSCLC patients and 37.81 pg/ml (range 9.73-90.21) in healthy control cohort ( p = 0.78). Median cPD-L1 increased in patients treated with first-line chemotherapy (63.20 pg/ml vs 39.34 pg/ml; p = 0.002), with no changes in patients exposed to non-chemotherapy drugs (42.39 pg/ml vs 50.67 pg/ml; p = 0.398). Time to progression and overall survival were 4.4 vs 6.9 months ( p = 0.062) and 8.8 vs 9.3 months ( p = 0.216) in cPD-L1 positive vs cPD-L1 negative patients. Baseline cPD-L1 levels increased with the ascending number of metastatic sites, even if the association was not statistically significant ( p = 0.063). Conclusions: This study showed that cPD-L1 testing is feasible, with chemotherapy influencing PD-L1 plasma levels. The possibility of using such test for predicting or monitoring the effect of immunotherapy or combination of chemotherapy and immunotherapy warrant further investigations.

Tryptophan (trp) metabolism is an important regulatory component of gut mucosal homeostasis and the microbiome. Metabolic pathways targeting the trp can lead to a myriad of metabolites, of both host and microbial origins, some of which act as endogenous low-affinity ligands for the aryl hydrocarbon receptor (AhR), a cytosolic, ligand-operated transcription factor that is involved in many biological processes, including development, cellular differentiation and proliferation, xenobiotic metabolism, and the immune response. Low-level activation of AhR by endogenous ligands is beneficial in the maintenance of immune health and intestinal homeostasis. We have defined a functional node whereby certain bacteria species contribute to host/microbial symbiosis and mucosal homeostasis. A microbial trp metabolic pathway leading to the production of indole-3-aldehyde (3-IAld) by lactobacilli provided epithelial protection while inducing antifungal resistance via the AhR/IL-22 axis. In this review, we highlight the role of AhR in inflammatory lung diseases and discuss the possible therapeutic use of AhR ligands in cystic fibrosis.

The stability of ferric nitrilotriacetate chelates (Fe(III)-NTA) was studied under thermal, oxidative and photochemical stress. The knowledge of chelate stability is fundamental to correctly implement the management system of wastewater treatment plant for application of chelates as catalyst in photo-Fenton process at neutral pH. Fe(III)-NTA solution stability was monitored under different temperature conditions (T = 10–30 °C), in presence and absence of UV-A irradiation and by adding three different concentrations of H2O2. The additional effect on chelate stability caused by different irradiation source (UV-A, UV-C and Xenon lamps) was also evaluated. Although the complexes were stable under the temperature test range, temperature control is crucial when stressing the solution by irradiation or by adding hydrogen peroxide. The solution was kept stable during two hours of reaction under UV-A irradiation only when temperature was set at 10–15 °C while in presence of hydroxyl radicals (HO ) the temperature control could only reduce the chelates decomposition. Fe(III)-NTA solution mineralization could be obtained under irradiation and radical strain. Thus, the production of organic radical from free ligands was also demonstrated. Finally, the suitableness of UV-C lamps as light source for the process application was questioned since they caused a strong degradation of the chelate solution. Indeed, only 30 min of UV-C irradiation by adding 0.59 mM of hydrogen peroxide caused almost 90% reduction of the chelate content in the solution.

Vascular endothelial growth factor A (VEGFA) is one of the main mediators of angiogenesis in non-small cell lung cancer (NSCLC). Recently, it has been described an autocrine feed-forward loop in NSCLC cells in which tumor-derived VEGFA promoted the secretion of VEGFA itself, amplifying the proangiogenic signal. In order to investigate the role of VEGFA in lung cancer progression, we assessed the effects of recombinant VEGFA on proliferation, migration, and secretion of other angiogenic factors in A549, H1975, and HCC827 NSCLC cell lines. We found that VEGFA did not affect NSCLC cell proliferation and migration. On the other hand, we demonstrated that VEGFA not only produced a strong and persistent increase of VEGFA itself but also significantly induced the secretion of a variety of angiogenic factors, including follistatin (FST), hepatocyte growth factor (HGF), angiopoietin-2 (ANGPT2), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-8, leptin (LEP), platelet/endothelial cell adhesion molecule 1 (PECAM-1), and platelet-derived growth factor bb (PDGF-BB). PI3K/AKT, RAS/ERK, and STAT3 signalling pathways were found to mediate the effects of VEGFA in NSCLC cell lines. We also observed that VEGFA regulation mainly occurred at post-transcriptional level and that NSCLC cells expressed different isoforms of VEGFA. Collectively, our data suggested that VEGFA contributes to lung cancer progression by inducing a network of angiogenic factors, which might offer potential for therapeutic intervention.

Although evidence suggests that the RAF/MEK/ERK pathway plays an important role in triple negative breast cancer (TNBC), resistance to MEK inhibitors has been observed in TNBC cells. Different mechanisms have been hypothesized to be involved in this phenomenon, including receptor tyrosine kinase-dependent activation of the PI3K/AKT pathway. In this study, we analyzed the effects of the MEK1/2 inhibitor selumetinib in combination with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in a panel of TNBC cell lines that showed different levels of sensitivity to single-agent selumetinib: SUM-149 and MDA-MB-231 cells resulted to be sensitive, whereas SUM-159, MDA-MB-468 and HCC70 cells were relatively resistant to the drug. Treatment of TNBC cells with selumetinib produced an increase of the phosphorylation of the EGFR both in selumetinib-sensitive SUM-149, MDA-MB-231 and in selumetinib-resistant MDA-MB-468 TNBC cells. The combination of selumetinib and gefitinib resulted in a synergistic growth inhibitory effect in all the TNBC cell lines, although the IC50 was not reached in SUM-159 and MDA-MB-468 cells. This effect was associated with an almost complete suppression of ERK1/2 activation and a reduction of selumetinib-induced AKT phosphorylation. In addition, in selumetinib-sensitive TNBC cells the combination of selumetinib and gefitinib induced a significant G0/G1 cell cycle arrest and apoptosis. Taken together, our data demonstrated that blockade of the EGFR might efficiently increase the antitumor activity of selumetinib in a subgroup of TNBC and that this phenomenon might be related to the effects of such combination on both ERK1/2 and AKT activation.

The Parkinson’s disease-associated protein, Leucine-rich repeat kinase 2 (LRRK2), a known negative regulator of nuclear factor of activated T cells (NFAT), is expressed in myeloid cells such as macrophages and dendritic cells (DCs) and is involved in the host immune response against pathogens. Since, the Ca2+/NFAT/IL-2 axis has been previously found to regulate DC response to the fungus Aspergillus, we have investigated the role played by the kinase LRRK2 during fungal infection. Mechanistically, we found that in the early stages of the non-canonical autophagic response of DCs to the germinated spores of Aspergillus, LRRK2 undergoes progressive degradation and regulates NFAT translocation from the cytoplasm to the nucleus. Our results shed new light on the complexity of the Ca2+/NFAT/IL-2 pathway, where LRRK2 plays a role in controlling the immune response of DCs to Aspergillus.

Introduction The vascular endothelial growth factor A (VEGF) is the main mediator of angiogenesis. In addition, VEGF contributes to cancer growth and metastasis directly targeting tumor cells. VEGF overexpression and/or high VEGF serum levels have been reported in lung cancer. Areas covered We searched Pubmed for relevant preclinical studies with the terms 'lung cancer' 'VEGF' and 'in vivo'. We also searched the Clinicaltrials.gov database, the FDA and the EMA websites for the most recent updates on clinical development of anti-VEGF agents. Expert opinion VEGF plays an important role in sustaining the development and progression of lung cancer and it might represent an attractive target for therapeutic strategies. Nevertheless, clinical trials failed to attend the promising expectations deriving from preclinical studies with anti-VEGF agents. To improve the efficacy of anti-VEGF therapies in lung cancer, potential strategies might be the employment of combinatory therapies with immune checkpoint inhibitors or agents that inhibit signaling pathways and proangiogenic factors activated in response to VEGF blockade, and the identification of novel targets in the VEGF cascade. Finally, the identification of predictive markers might help to select patients who are more likely to respond to anti-angiogenic drugs.

Several different studies have addressed the role of the circulating tumor cells (CTC) in non-small-cell lung cancer (NSCLC). In particular, the potential of CTC analysis in the early diagnosis of NSCLC and in the prediction of the outcome of patients with early and advanced NSCLC have been explored. A major limit of these studies is that they used different techniques for CTC isolation and enumeration, they employed different thresholds to discriminate between high- and low-risk patients, and they enrolled heterogeneous and often small cohort of patients. Nevertheless, the results of many studies are concordant in indicating a correlation between high CTC count and poor prognosis in both early and advanced NSCLC. The reduction of CTC number following treatment might also represent an important indicator of sensitivity to therapy in patients with metastatic disease. Preliminary data also suggest the potential for CTC analysis in the early diagnosis of NSCLC in high-risk individuals. However, these findings need to be confirmed in large prospective trials in order to be transferred to the clinical practice. The molecular profiling of single CTC in NSCLC might provide important information on tumor biology and on the mechanisms involved in tumor dissemination and in acquired resistance to targeted therapies. In this respect, xenografts derived from CTC might represent a valuable tool to investigate these phenomena and to develop novel therapeutic strategies.

World Health Organization estimates that almost 1 billion people in the world have no access to potable water and that 2.6 billion people lack access to adequate sanitation (World Health Organization (WHO), in Meeting the MDG drinking water and sanitation target: a mid-term assessment of progress/WHO/UNICEF Joint Monitoring Programme, 2004). The high level of quality required to make possible the utilization of reclaimed water to fill this lack of resource leads into the necessity of adapting conventional treatment schemes to more restrictive requirements. Nanotechnology processes could represent one of the most useful options for the achievement of this goal. National Nanotechnology Initiative (NNI) defined nanotechnology as “the understanding and control of matter at dimension between approximately 1 and 100 nanometers (nm), where unique phenomena enable novel application not feasible when working with bulk materials or even single atoms or molecules”. The knowledge achieved in this field offers the possibility of using novel material in the treatment of surface water, groundwater and wastewater especially for the removal of heavy metals, organic and inorganic solutes and microorganism. Due to the generally new ambit of research, the scientific community is actively involved in the development of new strategies and novel nanomaterials synthesis. Thousands of scientific articles related to nanotechnology applications in water sanitation have been published in the last decade. On the basis of their function, nanomaterials can be easily classified as: nanosorbents, nanocatalysts and redox active nanoparticles, nanostructured and reactive membranes, bioactive nanoparticles. Literature about current research on nanomaterials, methods of synthesis and characterization are reviewed in this chapter. Moreover, possible environmental impacts related with their application are also commented.

The interleukin 17 (IL-17) cytokine and receptor family is central to antimicrobial resistance and inflammation in the lung. Mice lacking IL-17A, IL-17F, or the IL-17RA subunit were compared with wild-type mice for susceptibility to airway inflammation in models of infection and allergy. Signaling through IL-17RA was required for efficient microbial clearance and prevention of allergy; in the absence of IL-17RA, signaling through IL-17RC on epithelial cells, predominantly by IL-17F, significantly exacerbated lower airway Aspergillus or Pseudomonas infection and allergic airway inflammation. In contrast, following infection with the upper respiratory pathogen Staphylococcus aureus, the IL-17F/IL-17RC axis mediated protection. Thus, IL-17A and IL-17F exert distinct biological effects during pulmonary infection; the IL-17F/IL-17RC signaling axis has the potential to significantly worsen pathogen-associated inflammation of the lower respiratory tract in particular, and should be investigated further as a therapeutic target for treating pathological inflammation in the lung.

The mechanisms how environmental compounds influence the human immune system are unknown. The environmentally sensitive transcription factor aryl hydrocarbon receptor (AhR) has immune-modulating functions and responds to a wide variety of small molecules. Since AhR is highly expressed in cells at body surfaces, such as skin, gut mucosa and particularly in mucosal-associated lymphocytes, this molecule is perfectly positioned to be a sensor of external environmental signals. The role of AhR in the balance of immunity and tolerance and in the control of local homeostasis has been clearly demonstrated in recent years [Kiss et al., Science (New York, NY) 334(6062):1561–1565, 2011; Li et al., Cell 147(3):629–640, 2011]. Deletion of AhR in mice resulted in altered composition of gut microbiota, impaired function and inflammatory immune activation of gut epithelium. In addition to xenobiotics, AhR ligands now include endogenous metabolites, dietary derivatives and bacterial metabolites (Denison and Nagy. Annu Rev Pharmacol Toxicol 43:309–334, 2003). Xenobiotics such as dietary components, products of microbiota, and ubiquitous environmental pollutants may have shaped the AhR system in intestinal epithelia or other body surfaces during millions of years of evolution. Thus, the crosstalk among the dietary components/xenobiotics, AhR and the gut microbiome appears to be an important factor in the maintenance of the mucosal immunity and immune homeostasis. These exciting discoveries provide a novel perspective for the biological role of AhR, which has been originally studied only as a sensor of toxicants, but which has been now implicated in a wide range of human conditions, including autoimmune and allergic disorders.

An increased understanding of the importance of microbiota in shaping the host's immune and metabolic activities has rendered fungal interactions with their hosts more complex than previously appreciated. The aryl hydrocarbon receptor (AhR) has a pivotal role in connecting tryptophan catabolism by microbial communities and the host's own pathway of tryptophan metabolite production with the orchestration of T-cell function. AhR activation by a Lactobacillus-derived AhR ligand leads to the production of IL-22 to the benefit of mucosal defense mechanisms, an activity upregulated in the absence of the host tryptophan catabolic enzyme, indoleamine 2,3-dioxygenase 1 (IDO1), which is required for protection from fungal diseases ("disease tolerance"). As AhR activation in turn leads to the activation-in a feedback fashion-of IDO1, the regulatory loop involving AhR and IDO1 may have driven the coevolution of commensal fungi with the mammalian immune system and the microbiota, to the benefit of host survival and fungal commensalism. This review will discuss the essential help the microbiota provides in controlling the balance between the dual nature of the fungal-host relationship, namely, commensalism vs. infection.

In this study, homogeneous photo-Fenton like at neutral pH was applied to remove sulfamethoxazole from water. The process was performed using different chelating agents in order to solubilize iron in a neutral water solution. The chelating agents tested were: ethylenediaminetetraacetic acid (EDTA); nitrilotriacetic acid (NTA); oxalic acid (OA) and tartaric acid (TA). The iron leaching was monitored over reaction time to evaluate the chelates stability and their resistance to HO· and UV-A radiation. Chelates of EDTA and NTA presented more stability than OA and TA, which also confirmed their higher efficiency. Total Organic Carbon (TOC) analyses were also performed to evaluate the contribution in terms of solution contamination related to the use of chelating agents. The better properties of biodegradability in respect of EDTA combined with better efficiency in terms of microcontaminant removal and the smallest TOC contribution indicate that NTA could represent a useful option to perform photo-Fenton processes at neutral pH.

The VEGF A /VEGF receptor (VEGFR) network actively contributes to breast cancer pathogenesis and progression, playing a pivotal role in promoting tumour-associated angiogenesis. Vandetanib is a multitargeted tyrosine kinase inhibitor that selectively blocks VEGFR-2, the EGFR and RET tyrosine kinases. The drug has shown promising anti-tumour activity in preclinical models of breast cancer.The authors summarise the data on pharmacodynamics, pharmacokinetics, preclinical and clinical studies of vandetanib up to April 2014, using: the PubMed and the clinicaltrials.gov databases; the FDA and EMA websites and the ASCO proceedings.Vandetanib has demonstrated a modest efficacy in patients with metastatic breast cancer. In this respect, the increased number of angiogenic pathways activated during tumour progression might partially explain the intrinsic and acquired resistance to the drug in advanced breast cancer. The activity of vandetanib in early phases of the disease, and in combination with other anti-angiogenic factors or metronomic therapy, should be explored in order to improve the clinical efficacy of the drug. Finally, the identification of predictive markers might help to select patients who are more likely to respond to anti-angiogenic drugs.

Objectives Circulating tumor cells (CTCs) have been hypothesized to be a prognostic factor in small-cell lung cancer (SCLC), and different cutoffs have been proposed to identify patients at high risk. We assessed the prognostic value of CTCs in patients with extensive SCLC. Materials and methods CTCs were assessed with the CellSearch system in 60 extensive SCLC patients. CTC count at baseline or after one cycle of chemotherapy (cycle-1) or as change after chemotherapy were analyzed separately. Primary outcome was overall survival. The accuracy of prognostic role was assessed by Harrell's c-index. “Optimal” cutoffs were derived by bootstrap resampling to reduce the overfitting bias; accuracy improvement was estimated by calculating the difference of c-indexes of models including clinical variables with or without CTCs. Results CTCs were identified in 90% (54/60) of patients at baseline, in which CTC count ranged from 0 to 24,281. CTC count was strongly associated with the number of organs involved. The prognostic accuracy was only marginally increased by the addition to clinical information of “optimal” CTC cutoffs at baseline and after cycle-1. Conversely, a reduction of CTC count higher than 89% following chemotherapy significantly improved prognostic accuracy (bootstrap p -value = 0.009) and was associated with a lower risk of death (HR 0.24, 95% CI 0.09–0.61). When previously proposed cutoffs were applied to our cohort, they showed only marginal improvement of the prognostic accuracy. Conclusion CTCs have useful prognostic role in extensive SCLC, but only the change of CTC count after the first cycle of chemotherapy provides clinically relevant information. Previously reported CTC cutoffs were not prognostic in our cohort of patients.

Introduction. We previously demonstrated that the activation of the epidermal growth factor receptor (EGFR) in mesenchymal stem cells (MSCs) modulates the expression of a number of genes coding for secreted proteins that promote breast cancer cell progression. As different microRNAs (miRNAs) have been shown to be involved in the cross-talk between MSCs and tumor cells, we hypothesized that the EGFR might regulate in MSCs the expression of miRNAs that might affect breast cancer progression through paracrine circuits. To this aim, we analysed the whole small RNA transcriptome of MSCs stimulated or not with transforming growth factor α (TGFα), one of the main ligands of the EGFR. Materials and methods. Small RNA sequencing was performed using the SOLiD 5500xl System. The DAVID (Database for Annotation, Visualization and Integrated Discovery) database was used for pathway analysis. Exosomes containing miRNAs were isolated from conditioned media with the ExoQuick reagent. MiRNA expression levels in conditioned medium were analyzed by Real Time PCR. Anchorage-dependent growth assays and Boyden-chamber based colorimetric migration assays were used to assess the proliferation and the migration of breast cancer cells, respectively. Results. Following small RNA sequencing, we identified 36 miRNAs differentially expressed between untreated and TGFα-treated MSCs with a fold change (FC) Conclusion. Collectively, our data suggest that the EGFR signaling regulate in MSCs a wide number of miRNAs that might be involved in breast cancer progression, providing novel information on the mechanisms that regulate the MSC-tumor cross-talk. Citation Format: Marianna Gallo, Marianeve Carotenuto, Cristin Roma, Francesca Bergantino, Pasqualino de Antonellis, Nicola Normanno, Antonella De Luca. The EGFR signaling modulates in mesenchymal stem cells the expression of microRNAs involved in the interaction with breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 142.

Rationale: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator affect the innate epithelial immune function of the lung, resulting in exaggerated and ineffective airway inflammation that fails to eradicate pathogenic fungi. The appreciation of whether such fungi are primarily responsible for or a consequence of ineffective airway inflammation is important for future therapeutics development.Objectives: To characterize the impact of the tryptophan/kynurenine pathway on pathogenic airway inflammation preventing effective fungal clearance in CF.Methods: We studied the expression of indoleamine 2,3-dioxygenase (IDO), the first enzyme in the kynurenine pathway of tryptophan degradation, in human and murine CF, the impact of IDO on lung inflammation and immunity in murine CF, and the potential role of tryptophan catabolism in pathogenesis and therapy of fungus-associated lung inflammation.Measurements and Main Results: IDO was defective in murine and human CF. Genetic and transcriptional regulatory mechanisms contributed to dysfunctional IDO activity that, in turn, correlated with imbalanced Th17/Treg-cell responses to Aspergillus fumigatus in murine CF. Treatments enhancing IDO function or preventing pathogenic Th17-cell activation restored protective immunity to the fungus and improved lung inflammation in murine CF.Conclusions: This study provides a link between tryptophan catabolism and lung immune homeostasis in murine CF, representing a proof-of-concept that targeting pathogenic inflammation via IDO-mimetic drugs may benefit patients with CF., Supported by a grant from the Italian Cystic Fibrosis Research Foundation (Research Project no. FFC#21/2010 to L. Romani) with the contribution of Francesca Guadagnin, Coca Cola Light Tribute to Fashion, and Delegazione FFC di Belluno; and the Specific Targeted Research Project "ALLFUN" (FP7-HEALTH-2009 contract no. 260338 to L. Romani). R.G.I. gratefully acknowledges a fellowship from the Italian Cystic Fibrosis Research Foundation. A. Carvalho and C. Cunha were financially supported by fellowships from the Fundacao para a Ciencia e Tecnologia, Portugal (contracts SFRH/BPD/46292/2008 and SFRH/BD/65962/2009, respectively).

The presence of activating mutations of the epidermal growth factor receptor (EGFR) is predictive of response to first- and second-generation tyrosine kinase inhibitors (TKIs) in patients with advanced non-small-cell lung cancer (NSCLC). However, patients that initially respond to these drugs inexorably become resistant. The T790M mutation in the exon 20 of the EGFR is the main mechanism of resistance to EGFR TKIs occurring in over 50% of the cases. Third generation EGFR TKIs have been shown to be active in patients who progressed after TKI treatment and carry the T790M mutation. Areas covered: This review is focused on the implications of tumor heterogeneity for targeting the T790M in patients with NSCLC. Expert opinion: Pre-clinical and clinical data suggest that the T790M is heterogeneously expressed in tumors that become resistant to first- and second-generation EGFR TKIs. These findings have important implications for the molecular diagnostic of the T790M mutation. Indeed, the analysis of both the circulating free tumor DNA (ctDNA) isolated from plasma and the tumor tissue might provide complimentary information to identify patients carrying the T790M mutation. However, further studies are needed to better understand the influence of tumor heterogeneity on the activity of drugs targeting the T790M.

The wide use of organophosphorous flame retardants (OPFR) and plasticizers causes a continuous release of large quantities into natural waters. One of the main contributors to micropollutants depletion in surface water is sunlight induced phototransformations. This study aims to elucidate whether alkyl, chloroalkyl and aryl organophosphorus flame retardants undergo phototransformations in river water. To perform the experiments, nine OPFR were subjected to natural sunlight, Xe lamp (simulated sunlight) and UV-C irradiations in ultra-pure Milli-Q water, Milli-Q water with humic acid and river water. Experiments demonstrated that OPFR achieve an important degree of photodegradation noticeable at long irradiation time, although direct photolysis did not account as the main photodegration mechanism. Results indicated that sunlight absorbing OPFR exhibited photosensitizing activity. The presence of azide in ultra- pure water inhibited some OPFR photodegration by singlet oxygen (1O2) scavenging, and the absence of dissolved oxygen significantly depleted most of OPFR removal. In the conditions studied, humic acid inhibited OPFR phototransformations, while river water enhanced their removal. Results from this study point out the need to further investigate the role of some OPFR as photosensitizers, which are important for fate and ecological risk assessment of flame retardants and other micropollutants in water.

Aspergillosis includes a spectrum of diseases caused by different Aspergillus spp. New insights into the cellular and molecular mechanisms of resistance and immune tolerance to the fungus in infection and allergy have been obtained in experimental settings. The fact that virulence factors, traditionally viewed as fungal attributes, are contingent upon microbial adaptation to various environmental stresses encountered in the human host implies that the host and fungus are jointly responsible for pathogenicity. Ultimately, despite the occurrence of severe aspergillosis in immunocompromised patients, clinical evidence indicates that aspergillosis also occurs in the setting of a heightened inflammatory response, in which immunity occurs at the expense of host damage and pathogen eradication. Thus, targeting pathogenicity rather than microbial growth, tolerance rather than resistance mechanisms of defense may pave the way to targeted anti-inflammatory strategies in difficult-to-treat patients. The challenge now is to translate promising results from experimental models to the clinic.

Short synthetic oligodeoxynucleotides (ODNs) rich in CpG or GpG motifs have been considered as potential modulators of immunity in clinical settings. In this study, we show that a synthetic GpC-ODN conferred highly suppressive activity on mouse splenic plasmacytoid dendritic cells, demonstrable in vivo in a skin test assay. The underlying mechanism involved signaling by noncanonical NF-κB family members and TGF-β–dependent expression of the immunoregulatory enzyme IDO. Unlike CpG-ODNs, the effects of GpC-ODN required TLR7/TRIF-mediated but not TLR9/MyD88-mediated events, as do sensing of viral ssRNA and the drug imiquimod. Induction of IDO by a GpC-containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3+ T cell generation in vitro. Among potentially therapeutic ODNs, this study identifies GpC-rich sequences as novel activators of TLR7-mediated, IDO-dependent regulatory responses.

Molecular diagnosis of invasive aspergillosis (IA) is a potentially life-saving tool in the care of at-risk individuals. To date, the development of PCR-based diagnostic tests has been hampered by the lack of standardization in the methods for such critical activities. In this study, we used both spiked volunteer blood samples and a murine model of IA to test the utility of the PAXgene and Tempus systems for stabilization and isolation of fungal RNA from blood as part of an evaluation of a new diagnostic strategy. In spiking experiments, RNA isolation followed by RT-qPCR that targeted the 18S gene was compared to a standard DNA isolation and qPCR assay that targeted the ITS ribosomal region. We demonstrated that both PAXgene and Tempus RNA stabilization and extraction systems followed by RT-qPCR had similar performance in detecting fungal RNA in blood samples from Aspergillus fumigates-infected mice. In spiked samples, the Tempus system performed better than the PAXgene system as it detected 100% of all samples spiked with 10 or 20 germinated Aspergillus conidia/ml blood sample as compared to the PAXgene system which detected 33% and 56% of the samples spiked with 10 or 20 conidia/ml, respectively. The stabilization of fungal nucleic acids in blood samples and its efficient isolation by a commercial method is an important step in the development of standardized molecular diagnostic tools that are needed to improve the outcomes for individuals with IA.

We thank Dr. Cristina Massi Benedetti for digital art and editing, Recognition of β-glucans by dectin-1 has been shown to mediate cell activation, cytokine production and a variety of antifungal responses. Here, we report that the functional activity of dectin-1 in mucosal immunity to Candida albicans is influenced by the genetic background of the host. Dectin-1 was required for the proper control of gastrointestinal and vaginal candidiasis in C57BL/6 but not BALB/c mice, the latter actually showing increased resistance in the absence of dectin-1. Susceptibility of dectin-1-deficient C57BL/6 mice to infection was associated with defective IL-17A, aryl hydrocarbon receptor-dependent IL-22 production as well as adaptive Th1 responses. In contrast, resistance of dectin-1-deficient BALB/c mice was associated with increased IL-17A and IL-22 production, and the skewing towards Th1/Treg immune responses that provide immunological memory. Disparate canonical/noncanonical NF-κB signaling pathways downstream dectin-1were activated in the two different mouse strains. Thus, the net activity of dectin-1 in antifungal mucosal immunity is dependent on the host’s genetic background that affects both the innate cytokine production as well as the adaptive Th1/Th17 cell activation upon dectin-1 signaling., The studies were supported by the Specific Targeted Research Project “ALLFUN” (FP7−HEALTH−2009 contract number 260338 to LR) and the Italian Project AIDS 2010 by ISS (Istituto Superiore di Sanità - contract number 40H40 to LR) and Fondazione Cassa di Risparmio di Perugia Project n. 2011.0124.021. AC and CC were financially supported by fellowships from Fundação para a Ciência e Tecnologia, Portugal (contracts SFRH/BPD/46292/2008 and SFRH/BD/65962/2009, respectively).

The RAS/RAF/MAP kinase-ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) (MAPK) and the PI3K/AKT/mammalian target of rapamycin (mTOR) (PI3K) pathways are frequently deregulated in human cancer as a result of genetic alterations in their components or upstream activation of cell-surface receptors. These signalling cascades are regulated by complex feedback and cross-talk mechanisms.In this review the key components of the MAPK and AKT pathways and their molecular alterations are described. The complex interactions between these signalling cascades are also analysed.The observation that the MAPK and the PI3K pathways are often deregulated in human cancer makes the components of these signalling cascades interesting targets for therapeutic intervention. Recently, the presence of compensatory loops that activate one pathway following the blockade of the other signalling cascade has been demonstrated. Therefore, the blockade of both pathways with combinations of signalling inhibitors might result in a more efficient anti-tumor effect as compared with a single agent. In addition, the MAPK and PI3K pathways are activated by mutations that coexist or can be mutually exclusive. In this regard, a large-scale characterization of the cancer genome might offer personalized cancer genomic information, which may improve the anti-tumor efficacy of signalling inhibitors.

Aspergillus fumigatus is a model fungal pathogen and a common cause of severe infections and diseases. CD8+ T cells are present in the human and murine T-cell repertoire to the fungus. However, CD8+ T-cell function in infection and the molecular mechanisms that control their priming and differentiation into effector and memory cells in vivo remain elusive. In the present study, we report that both CD4+ and CD8+ T cells mediate protective memory responses to the fungus contingent on the nature of the fungal vaccine. Mechanistically, class I MHC-restricted, CD8+ memory T cells were activated through TLR3 sensing of fungal RNA by cross-presenting dendritic cells. Genetic deficiency of TLR3 was associated with susceptibility to aspergillosis and concomitant failure to activate memory-protective CD8+ T cells both in mice and in patients receiving stem-cell transplantations. Therefore, TLR3 essentially promotes antifungal memory CD8+ T-cell responses and its deficiency is a novel susceptibility factor for aspergillosis in high-risk patients., These studies were supported by the Specific Targeted Research Project ALLFUN (FP7-HEALTH-2009 contract number 260338 to L.R.), by SYBARIS (FP7-HEALTH-2009 contract number 242220 to L.R.), and by the Italian Project AIDS 2010 by the Istituto Superiore di Sanita (contract number 40H40 to L.R.). A.C. and C.C. were supported by fellowships from Fundacao para a Ciencia e Tecnologia, Portugal (contracts SFRH/BPD/46292/2008 and SFRH/BD/65962/2009, respectively).

Lapatinib, an oral, small-molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity. Here, we found that quercetin-3-methyl ether caused a significant growth inhibition of lapatinib-sensitive and -resistant breast cancer cells. Western blot data showed that quercetin-3-methyl ether had no effect on Akt or ERKs signaling in resistant cells. However, quercetin-3-methyl ether caused a pronounced G(2)/M block mainly through the Chk1-Cdc25c-cyclin B1/Cdk1 pathway in lapatinib-sensitive and -resistant cells. In contrast, lapatinib produced an accumulation of cells in the G(1) phase mediated through cyclin D1, but only in lapatinib-sensitive cells. Moreover, quercetin-3-methyl ether induced significant apoptosis, accompanied with increased levels of cleaved caspase 3, caspase 7, and poly(ADP-ribose) polymerase (PARP) in both cell lines. Overall, these results suggested that quercetin-3-methyl ether might be a novel and promising therapeutic agent in lapatinib-sensitive or -resistant breast cancer patients.

Deciphering cellular and molecular mechanisms that maintain host immune homeostasis with fungi and the breakdown of this homeostatic tolerance during fungal infections disease is a challenge in medical mycology. In fact, the virulence of fungi may be determined by the interaction between fungi and the host immune status and its classification as a commensal microorganism or a pathogen may shift depending on the balance. In addition to the central role of the IL-12/IFN-γ-dependent Th1 responses in cell-mediated immune protection against fungi, Th17 cells provide protection and inflammation at mucosal surfaces, and Tregs fine-tune immune responses to prevent damage to the host. Recent evidence indicates that IL-22-producing cells, employing primitive antifungal effector mechanisms, contribute to antifungal resistance at mucosal surfaces under conditions of defective adaptive immunity. The fact that IL-22 production is driven by commensals points to the need of an integrated, systems biology approach to improve our understanding of the inherent and intimate mechanisms underlying multilevel host-fungus interactions.

Innate responses combine with adaptive immunity to generate the most effective form of anti-Aspergillus immune resistance. Whereas the pivotal role of dendritic cells in determining the balance between immunopathology and protective immunity to the fungus is well established, we determined that epithelial cells (ECs) also contributes to this balance. Mechanistically, EC-mediated protection occurred through a Toll-like receptor 3/Toll/IL-1 receptor domain-containing adaptor-inducing interferon (TLR3/TRIF)-dependent pathway converging on indoleamine 2,3-dioxygenase (IDO) via non-canonical nuclear factor-?B activation. Consistent with the high susceptibility of TRIF-deficient mice to pulmonary aspergillosis, bone marrow chimeric mice with TRIF unresponsive ECs exhibited higher fungal burdens and inflammatory pathology than control mice, underexpressed the IDO-dependent T helper 1/regulatory T cell (Th1/Treg) pathway and overexpressed the Th17 pathway with massive neutrophilic inflammation in the lungs. Further studies with interferon (IFN)-?, IDO or IL-17R unresponsive cells confirmed the dependency of immune tolerance to the fungus on the IFN-?/IDO/Treg pathway and of immune resistance on the MyD88 pathway controlling the fungal growth. Thus, distinct immune pathways contribute to resistance and tolerance to the fungus, to which the hematopoietic/non-hematopoietic compartments contribute through distinct, yet complementary, roles., We thank Cristina Massi Benedetti for digital art and editing. This work was supported by the Specific Targeted Research Project 'Sybaris' (LSHE-CT-2006), contract number 037899 (FP7) and by the Italian Projects PRIN 2007KLCKP8_004 (to LR) and 2007XYB9T9_001 (to SB). CC and AC were financially supported by fellowships from Fundacao para a Ciencia e Tecnologia, Portugal (contracts SFRH/BD/65962/2009 and SFRH/BPD/46292/2008, respectively).

Introduction: The Cetuximab After Progression in KRAS wild type colorectal cancer patients (CAPRI) study enrolled KRAS exon 2 wild-type (wt) metastatic colorectal cancer (mCRC) patients who received first-line FOLFIRI-cetuximab. Retrospective analysis showed that mCRC patients with KRAS/NRAS/BRAF/PIK3CA wt (quadruple-wt) tumors had an excellent prognosis when treated with anti-EGFR agents. In order to identify additional mechanisms that might allow to better select patients who benefit of anti-EGFR monoclonal antibodies, we analyzed quadruple-wt tumors from the CAPRI clinical trial with a large targeted sequencing panel and correlated results with patients' outcome. Materials and methods: NGS analysis was performed using the Oncomine Comprehensive v.2 panel from Thermofisher on Ion Torrent PGM. The panel covers 143 cancer genes providing information on hotspot mutations of 73 oncogenes, copy number variation (CNV) of 49 genes, full-length sequence of 26 tumor suppressor genes, and sequence of 22 fusion driver genes. Data analysis was carried out using Ion Reporter™ Software v5.0. Results: Complete data of the first cohort of 17 patients are summarized. These patients had a median progression-free survival (mPFS) of 12,3 months (m) and median overall survival (mOS) of 34,03 mos. The analysis revealed in all 17 tumors the presence of at least one mutation, and in 9/17 showed the presence of at least one CNV. Variants were detected in the following genes: TP53 (17 mutations), APC (16), FBXW7 (2), MAP2K1 (1), PTPN11 (1), PTCH1 (1), ATM (1), CTNNB1 (1), PIK3R1 (1), PTEN (1), CDKN2A (1), KRAS (1). CNVs were found in APC (2 deletions), TP53 (1 deletion), PIK3R1 (1 deletion), BCL2L1 (4 amplifications), GAS6 (3 amplifications), MYC (2 amplifications), ZNF217 (2 amplifications), FLT3 (1 amplification), ERBB2 (1 amplification), APEX 1 (1 amplification). The 3 patients with the shortest PFS and OS had peculiar genetic alterations that might be associated with resistance to EGFR targeting drugs. Case #980 (PFS 3,93 m; OS 9,2 m) carried a KRAS p.Gln61His variant at a low allelic frequency (0,38%) that was confirmed by plasma testing with BEAMing. Case #4120 (PFS 5,77 m; OS 25,33 m) had a loss of function PTCH1 variant (p.Tyr1316fs) leading to activation of the sonic hedgehog (SHH) pathway. Case #1491 (PFS 6,63 m; OS 19,03 m) carried a p.Lys57Glu mutations in the MAP2K1 gene that causes constitutive activation of the corresponding protein. Conclusions: These preliminary data show that quadruple-wt CRC carry genetic alterations that potentially can drive resistance to anti-EGFR monoclonal antibodies. While the significance of the identified variants needs to be explored in experimental models, these findings do suggest that wide genetic profiling of CRC might improve our ability to select patients who are highly sensitive to EGFR inhibition. Citation Format: Anna Maria Rachiglio, Matilde Lambiase, Francesca Fenizia, Alessia Iannaccone, Cristin Roma, Claudia Cardone, Antonella De Luca, Erika Martinelli, Evaristo Maiello, Fortunato Ciardiello, Nicola Normanno. Genetic landscape of KRAS-NRAS-BRAF-PIK3CA wild type metastatic colorectal cancer patients enrolled in the CAPRI clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2619.

Patients affected by haemophilia A require treatment with factor VIII (FVIII) protein. The most relevant complication is the development of neutralizing FVIII-specific antibodies or “inihibitors”. We reported that the inhibitor-positive status was associated with reduced activity of the immune-regulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1), that promotes regulatory effects via the production of tryptophan catabolites, known as kynurenines. Some of those tryptophan derivatives are endogenous ligands for the Aryl hydrocarbon receptor (AhR). In this study we tested the potential of tryptophan-related AhR ligands for inhibiting the development of anti-FVIII antibodies in hemophilic (F8 KO) mice. To this aim, F8 KO mice were treated with recombinant human FVIII (rhFVIII) alone or in combination with selected AhR ligands once weekly for four weeks. Antibody titers were tested by specific ELISA and Bethesda test. All mice treated with rhFVIII developed high-titer anti-FVIII antibodies after 4 weeks of treatment. Administration of a specific tryptophan metabolite prevented the generation of anti-FVIII antibodies in almost 80% of F8 KO mice. The protective effect of these AhR ligands was negated by co-administration of the AhR antagonist CH-223191 or in AhR KO mice. Similar results were obtained by administration of engineered gold nanoparticles loaded with the same tryptophan metabolite and rhFVIII. These results suggest that the engagement of AhR, by specific tryptophan derivatives, may be a possible new strategy to control the immune response to rhFVIII. Our findings might lead to the development of novel immunomodulatory interventions for preventing or eradicating inhibitors in hemophilia A patients.

The peripheral immune system can promote either immunity or tolerance when presented with new antigens. Current knowledge withholds that populations of suppressor or regulatory T cells (T(reg) cells) constitute a pivotal mechanism of immunological tolerance. The potential role of malfunctioning T(reg) cells in chronic inflammatory immune and auto-immune diseases is well-documented. Learning how to successfully manipulate T(reg) responses could result in more effective vaccines and immunomodulators. We have already shown that Thymosin alpha1 (Talpha1), a naturally occurring thymic peptide first described and characterized by Allan Goldstein in 1972, by modulating signals delivered through innate immune receptors on dendritic cells, affects adaptive immune responses via modulation of Th cell effector and regulatory functions. We will discuss recent molecular mechanisms underlying the ability of Talpha1 to activate or inhibit immune responses.

EGFR regulates cancer-cell proliferation, apoptosis and tumor-induced neoangiogenesis, and has been validated as a relevant therapeutic target in several human cancers, including metastatic colorectal cancer (mCRC). The anti-EGFR monoclonal antibodies cetuximab and panitumumab are available for the treatment of patients with mCRC. Although EGFR is expressed in approximately 85% of patients with mCRC, the clinical efficacy of treatment with anti-EGFR antibodies is limited to a subset of patients. A series of potential biomarkers that could be useful in predicting response to EGFR inhibitors has been investigated. In patients with mCRC, activating mutations within KRAS can predict resistance to anti-EGFR monoclonal antibodies. Activating mutations in KRAS, which could result in EGFR-independent intracellular signal transduction activation, are found in approximately 35-40% of patients with mCRC. These mutations are almost exclusively detected in codons 12 and 13 of exon 2. KRAS mutations have been significantly associated with lack of response to cetuximab or panitumumab therapy in patients with mCRC, which suggests that EGFR-independent, constitutive activation of the RAS signaling pathway could impair response to anti-EGFR drugs. We summarize the experimental and clinical evidence supporting the use of KRAS testing for the optimal selection of patients with mCRC to be treated with anti-EGFR monoclonal antibodies.

How the immune system tailors protective responses to suit the infectious challenge while limiting damage to the host is an emerging theme in T-cell biology. Although many studies have focused on the pathological aspects of IL-17-producing T cells in many autoimmune diseases, their role in protective anti-microbial immunity has also been increasingly recognized. This increased recognition also applies to their role in anti-fungal immunity; however, the role of IL-17-producing T cells in protection versus pathology in fungal infections is still controversial. Although both positive and negative effects on immune resistance have been attributed to the IL-23/Th17 axis in experimental models of fungal infections, defective Th17 cell differentiation has been linked to recurrent pneumonia by filamentous fungi and the occurrence of mucocutaneous candidiasis in patients with primary immunodeficiencies. Here we discuss how recent findings in experimental candidiasis and aspergillosis shed new lights on the contribution of Th17 cells to resistance and pathology to fungi.

Renal-cell cancer (RCC) is an heterogeneous disease consisting of different subtypes that show peculiar histological features and genetic alterations. Although inherited or familial predisposition occurs in less than 4% of renal cancers, most of the available information on the genetic alterations involved in the pathogenesis of RCC derives from the study of the inherited forms of kidney cancer: von Hippel-Lindau ( VHL gene), hereditary papillary renal carcinoma ( MET proto-oncogene), hereditary leiomyomatosis and renal-cell cancer ( fumarate hydratase gene), and Birt–Hogg–Dube ( BHD gene) syndromes. Such genetic alterations have also been detected in sporadic RCCs. In particular, inactivation of VHL gene by mutation or hypermethylation has been found in up to 70% of sporadic clear-cell RCC, and it has been associated with increased hypoxia-inducible factor (HIF) activity. The knowledge of these deregulated genes and their downstream pathways provides the rationale for the development of target-based approaches for RCC.

The ability of regulatory T (Treg) cells to inhibit aspects of innate and adaptive immunity is central to their protective function in fungal infections. In murine candidiasis, CD4+CD25+ Treg cells prevent excessive inflammation but enable fungal persistence in the gastrointestinal tract, which underlies the onset of durable antifungal protection. In this study, we show that fungal growth, inflammatory immunity, and tolerance to the fungus were all controlled by the coordinate activation of naturally occurring Treg cells, which limited early inflammation at the sites of infection, and pathogen-induced Treg cells (that regulated the expression of adaptive Th immunity in secondary lymphoid organs). Naturally occurring Treg cells required the TRIF pathway for migration to inflamed sites, where the MyD88 pathway would then restrain their suppressive function. Subsequent inflammatory Th1-type immunity was modulated by induced Treg cells, which required the TRIF pathway as well, and acted through activation of IDO in dendritic cells and Th17 cell antagonism. In vitro, using naive CD4+ cells from TRIF-deficient mice, tryptophan metabolites were capable of inducing the Foxp3-encoding gene transcriptionally and suppressing the gene encoding RORγt, Th17 lineage specification factor. This is the first study to show that the same tryptophan catabolites can foster dendritic cell-supported generation of Foxp3+ cells and mediate, at the same time, inhibition of RORγt-expressing T cells.

Dysregulated inflammasome activation contributes to respiratory infections and pathologic airway inflammation. Through basic and translational approaches involving murine models and human genetic epidemiology, we show here the importance of the different inflammasomes in regulating inflammatory responses in mice and humans with cystic fibrosis (CF), a life-threatening disorder of the lungs and digestive system. While both contributing to pathogen clearance, NLRP3 more than NLRC4 contributes to deleterious inflammatory responses in CF and correlates with defective NLRC4-dependent IL-1Ra production. Disease susceptibility in mice and microbial colonization in humans occurrs in conditions of genetic deficiency of NLRC4 or IL-1Ra and can be rescued by administration of the recombinant IL-1Ra, anakinra. These results indicate that pathogenic NLRP3 activity in CF could be negatively regulated by IL-1Ra and provide a proof-of-concept evidence that inflammasomes are potential targets to limit the pathological consequences of microbial colonization in CF., IL-1-mediated inflammation contributes to the pathogenesis of cystic fibrosis. Here the authors show that this is largely due to NLRP3 activation, whereas NLRP4 induces IL-1Ra, limiting the overall inflammasome activity and providing a therapeutic angle to ameliorate the disease.

Although evidence suggests that the RAF/MEK/ERK pathway plays an important role in triple negative breast cancer (TNBC), resistance to MEK inhibitors has been observed in TNBC cells. Different mechanisms have been hypothesized to be involved in this phenomenon, including receptor tyrosine kinase-dependent activation of the PI3K/AKT pathway. In this study, we analyzed the effects of the MEK1/2 inhibitor selumetinib in combination with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in a panel of TNBC cell lines that showed different levels of sensitivity to single-agent selumetinib: SUM-149 and MDA-MB-231 cells resulted to be sensitive, whereas SUM-159, MDA-MB-468 and HCC70 cells were relatively resistant to the drug. Treatment of TNBC cells with selumetinib produced an increase of the phosphorylation of the EGFR both in selumetinib-sensitive SUM-149, MDA-MB-231 and in selumetinib-resistant MDA-MB-468 TNBC cells. The combination of selumetinib and gefitinib resulted in a synergistic growth inhibitory effect in all the TNBC cell lines, although the IC50 was not reached in SUM-159 and MDA-MB-468 cells. This effect was associated with an almost complete suppression of ERK1/2 activation and a reduction of selumetinib-induced AKT phosphorylation. In addition, in selumetinib-sensitive TNBC cells the combination of selumetinib and gefitinib induced a significant G0/G1 cell cycle arrest and apoptosis. Taken together, our data demonstrated that blockade of the EGFR might efficiently increase the antitumor activity of selumetinib in a subgroup of TNBC and that this phenomenon might be related to the effects of such combination on both ERK1/2 and AKT activation.

Item does not contain fulltext Candida albicans is a well-tolerated resident of human mucosal tissues. This implies that host defense mechanisms cooperate to limit inflammation while controlling fungal burden. The cytokine IL-22 and inflammasomes are essential components of the mucosal responses to C. albicans. How these components cooperate to mediate the balance of inflammation and host defense is not explored. We find that NLRP3 inflammasome activation promotes neutrophil recruitment and inflammation during infection and that this activity is counteracted by IL-22. Mechanistically, IL-22 activated NLRC4 for sustained production of the IL-1 receptor antagonist IL-1Ra, which restrained NLRP3 activity. Symptomatic infection in mice and humans occurred under conditions of IL-1Ra deficiency and was rescued in mice by replacement therapy with the recombinant IL-1Ra anakinra. Thus, pathogenic inflammasome activity during Candida infection is negatively regulated by the IL-22/NLRC4/IL-1Ra axis. Our findings offer insights into the pathogenesis of C. albicans and suggest therapeutic avenues for candidiasis.

Aim: We assessed the ability of the Therascreen® kit (plasma-Therascreen) and of a peptide nucleic acids (PNA)-clamp approach to detect EGFR mutations in plasma-derived circulating-free tumor DNA (cftDNA) from non-small-cell lung cancer patients. Materials & methods: cftDNA from 96 patients was analyzed for exon 19 deletions and the p.L858R mutation, using both plasma-Therascreen and PNA-clamp-based assays. Results: None of the 70 EGFR wild-type patients showed EGFR mutations in cftDNA with both techniques (specificity: 100%). In 17/26 EGFR-mutant patients, plasma-Therascreen analysis confirmed the mutation identified in the primary tumor (analytical sensitivity: 65.4%). Similar results were obtained with the PNA-clamp method. Conclusion: Both approaches were specific and sensitive for EGFR mutational analysis of cftDNA in non-small-cell lung cancer patients.

ABSTRACT The presence of EGFR mutations predicts the sensitivity to EGF receptor (EGFR)-tyrosine kinase inhibitors in a molecularly defined subset of non-small-cell lung carcinoma (NSCLC) patients. For this reason, EGFR testing of NSCLC is required to provide personalized treatment options and better outcomes for NSCLC patients. As surgery specimens are not available in the majority of NSCLC, other currently available DNA sources are small biopsies and cytological samples, providing however limited and low-quality material. In order to address this issue, the use of surrogate sources of DNA, such as blood, serum and plasma samples, which often contains circulating free tumor DNA or circulating tumor cells, is emerging as a new strategy for tumor genotyping.

AKT and MAPK signaling are involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. RAS proteins are upstream mediators that transfer messages from surface receptors to intracellular signal transducers including MAPK and AKT pathways. AZD3409 is a novel prenyl inhibitor that has shown activity against both farnesyl transferase and geranylgeranyl transferase in isolated enzyme studies. We explored the activity of AZD3409 on breast cancer cell lines with high (SK-Br-3), intermediate (MDA-MB-361) or low (MDA-MB-468) sensitivity to gefitinib. We found that AZD3409 inhibits the growth of breast cancer cells in a dose-dependent manner, with the MDA-MB-468 and MDA-MB-361 cell lines showing higher sensitivity as compared with SK-Br-3 cells. Treatment with AZD3409 produced a significant reduction in the levels of activation of AKT in the three cell lines. AZD3409 also induced an increase in the expression of p27kip-1 and of hypophosphorylated forms of pRb2 in MDA-MB-468 cells that was associated with accumulation of cells in G0/G1 and the appearance of a sub-G1 peak suggestive of apoptosis. In contrast, AZD3409 produced a G2 arrest associated with reduced expression of pRb2 in MDA-MB-361 cells. A synergistic anti-tumor effect was observed when MDA-MB-468 or MDA-MB-361 cells were treated with both AZD3409 and gefitinib, whereas this combination was only additive in SK-Br-3 cells. However, treatment of breast cancer cells with AZD3409 and gefitinib did not produce a more significant blockade of AKT signaling as compared with gefitinib alone. These data suggest that AZD3409 might be active in gefitinib-resistant breast carcinoma.

Purpose: Human Cripto-1 (CR-1), a cell membrane glycosylphosphatidylinositol-anchored glycoprotein that can also be cleaved from the membrane, is expressed at high levels in several different types of human tumors. We evaluated whether CR-1 is present in the plasma of patients with breast and colon cancer, and if it can represent a new biomarker for these malignancies. Experimental Design: We determined CR-1 plasma levels using a sandwich-type ELISA in 21 healthy volunteers, 54 patients with breast cancer, 33 patients with colon carcinoma, and 21 patients with benign breast lesions. Immunohistochemical analysis was also used to assess CR-1 expression in cancerous tissues. Results: Very low levels of CR-1 (mean ± SD) were detected in the plasma of healthy volunteers (0.32 ± 0.19 ng/mL). A statistically significant increase in the levels of plasma CR-1 was found in patients with colon carcinoma (4.68 ± 3.5 ng/mL) and in patients with breast carcinoma (2.97 ± 1.48 ng/mL; P < 0.001). Although moderate levels of plasma CR-1 were found in women with benign lesions of the breast (1.7 ± 0.99 ng/mL), these levels were significantly lower than in patients with breast cancer (P < 0.001). Finally, immunohistochemical analysis and real-time reverse transcription-PCR confirmed strong positivity for CR-1 in colon and/or breast tumor tissues. Conclusion: This study suggests that plasma CR-1 might represent a novel biomarker for the detection of breast and colon carcinomas.

The structural gene for glutathione transferase (XlGSTS1-1) in the amphibia Xenopus laevis has been cloned from an embryo library and its nucleotide sequence has been determined. Open reading frame analysis indicated that xlgsts1 gene encodes the smallest protein of sigma class GST so far identified as being composed of only 194 amino acid residues. The recombinant XlGSTS1-1 shows a narrow range of substrate specificity as well as a significantly lower 1-chloro-2,4-dinitrobenzene conjugation capacity than that of squid sigma class GST. To compare the structural and functional differences between the squid and amphibian enzymes, several site-specific mutations were introduced in XlGSTS1-1, i.e., Ser100Asn, Phe102Tyr, Trp143Leu, Phe146Leu, and Trp148Cys. The results obtained indicate that Trp143 and Trp148 are more important determinants for the structural stability of XlGSTS1-1 rather than for its substrate specificity.

The expression of glutathione transferase isoenzymes has been investigated in embryo and adult liver of the frog Xenopus laevis. By analysing the GST isoenzymes recovered from GSH-affinity chromatography in terms of electrophoretic mobility, HPLC elution profile, immunological reactivity, N-terminal amino acid sequence and mass spectrometry molecular mass no significant difference in the GST subunit composition between embryos and liver was found. In both tissues the same three subunits, showing similarity to mu, alpha and sigma class GSTs, are present. These results, together with those previously reported for toad (Bufo bufo), strongly support the notion that the transition from an aquatic environment to a terrestrial atmosphere containing high oxygen concentration has accompanied specific GST gene expression.