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Purpose: The development of novel and efficient biomarkers for primary bone cancers is of grave importance., Methods: The expression pattern of osteopontin (OPN) was investigated in the 153 patients with benign (n = 72) and malignant (n = 81) primary bone cancers. Both local and circulating OPN mRNA expression levels and their protein concentration in serum and tumor site were assessed using real-time qRT-PCR, ELISA, and immunohistochemistry techniques, respectively. As a control, 29 healthy individuals were considered. The number of 153 tumor tissue specimens and the 153 paired margins were taken on surgical resection from the patients. 153 blood samples were also drained from all participants, then peripheral blood mononuclear cells (PBMC) and sera were separated., Results: The mean mRNA expression was significantly higher in all of the cancerous tissues than the paired margins and the PBMC of the patients than the controls. Consistently, the protein concentrations of OPN in serum and tumor tissues were significantly higher in the patients. Furthermore, the malignant cases had significantly elevated the mRNA levels and the protein compared to the benign cases. OPN could potentially differentiate the patients from the controls with 100% sensitivity and specificity in serum. Moreover, OPN could predict some of the malignant cases' clinicopathological features, including metastasis, recurrence, grade, and response to chemotherapy., Conclusions: In conclusion, OPN might be involved in the pathogenesis of primary bone tumors and can be considered as a potential biomarker to bone cancer diagnosis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier GmbH.)

Chronic inflammation results from excessive pro-inflammatory signaling and the failure to resolve the inflammatory reaction. Lipid mediators orchestrate both the initiation and resolution of inflammation. Switching from pro-inflammatory to pro-resolving lipid mediator biosynthesis is considered as efficient strategy to relieve chronic inflammation, though drug candidates exhibiting such features are unknown. Starting from a library of Vietnamese medical plant extracts, we identified isomers of the biflavanoid 8-methylsocotrin-4'-ol from Dracaena cambodiana , which limit inflammation by targeting 5-lipoxygenase and switching the lipid mediator profile from leukotrienes to specialized pro-resolving mediators (SPM). Elucidation of the absolute configurations of 8-methylsocotrin-4'-ol revealed the 2 S , γS -isomer being most active, and molecular docking studies suggest that the compound binds to an allosteric site between the 5-lipoxygenase subdomains. We identified additional subordinate targets within lipid mediator biosynthesis, including microsomal prostaglandin E 2 synthase-1. Leukotriene production is efficiently suppressed in activated human neutrophils, macrophages, and blood, while the induction of SPM biosynthesis is restricted to M2 macrophages. The shift from leukotrienes to SPM was also evident in mouse peritonitis in vivo and accompanied by a substantial decrease in immune cell infiltration. In summary, we disclose a promising drug candidate that combines potent 5-lipoxygenase inhibition with the favorable reprogramming of lipid mediator profiles., Competing Interests: The authors declare no conflicts of interest. The above mentioned funding sources were neither involved in study design, data collection, analysis, and interpretation nor in writing and submission of the manuscript., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for treating melanoma, gastric cancer (GC) and bladder cancer with clinical benefit. Nevertheless, many patients failed to respond to anti-PD-1/PD-L1 treatment, so it is necessary to seek an alternative strategy for traditional PD-1/PD-L1 targeting immunotherapy. Here with the data from The Cancer Genome Atlas (TCGA) and our in-house tissue library, PD-L1 expression was found to be positively correlated with the expression of ubiquitin-specific processing protease 7 (USP7) in GC. Furthermore, USP7 directly interacted with PD-L1 in order to stabilize it, while abrogation of USP7 attenuated PD-L1/PD-1 interaction and sensitized cancer cells to T cell killing in vitro and in vivo . Besides, USP7 inhibitor suppressed GC cells proliferation by stabilizing P53 in vitro and in vivo . Collectively, our findings indicate that in addition to inhibiting cancer cells proliferation, USP7 inhibitor can also downregulate PD-L1 expression to enhance anti-tumor immune response simultaneously. Hence, these data posit USP7 inhibitor as an anti-proliferation agent as well as a novel therapeutic agent in PD-L1/PD-1 blockade strategy that can promote the immune response of the tumor., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Vascular and cardiovascular inflammation and thrombosis occur in patients with severe coronavirus disease-2019 (COVID-19). Advancing age is the most significant risk factor for severe COVID-19. Using transcriptomic databases, the authors found that: 1) cardiovascular tissues and endothelial cells express putative genes for severe acute respiratory syndrome coronavirus-2 infection, including angiotensin-converting enzyme 2 ( ACE2 ) and basigin ( BSG ); 2) severe acute respiratory syndrome coronavirus-2 receptor pathways ACE2 /transmembrane serine protease 2 and BSG /peptidylprolyl isomerase B(A) polarize to lung/epithelium and vessel/endothelium, respectively; 3) expression of host genes is relatively stable with age; and 4) notable exceptions are ACE2 , which decreases with age in some tissues, and BSG , which increases with age in endothelial cells, suggesting that BSG expression in the vasculature may explain the heightened risk for severe disease with age., Competing Interests: This work was funded by the Wellcome Trust/Imperial College Institutional Support Fellowship for Dr. Ahmetaj-Shala. Dr. Kirkby is a recipient of an Intermediate Research Fellowship from the British Heart Foundation (FS/16/1/31699). Dr. Vaja is a recipient of a Clinical Training Fellowship from the British Heart Foundation (FS/19/6/34129). Drs. Kirkby and Mitchell are holders of a program grant from the British Heart Foundation (RG/18/4/33541). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2020 The Authors.)

The formyl peptide receptor 2 (FPR2) belongs to the family of seven-transmembrane G protein-coupled receptors (GPCR) and are expressed by many different cells but mainly studied in immune cells. FPR2 is involved in host defense against bacterial infections and clearance of damaged cells through the oxidative burst and chemotaxis of neutrophils. In addition, FPR2 has also been implicated as an immunomodulator in sterile inflammations, e.g. inflammatory joint diseases. Here we present data regarding FPR2 expression in human articular chondrocytes, isolated from healthy individuals and osteoarthritic patients, on both mRNA and protein level using qPCR and Imagestream flow cytometry. We also present data after receptor stimulation and monitoring of production of nitric oxide, reactive oxygen species, IL-6, IL-8 and MMP-3. The presented data show that human articular chondrocytes from patients with osteoarthritis as well as from healthy individuals express FPR2 both at mRNA and protein level. The biological relevance of FPR2 expression in chondrocytes needs to be further investigated., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 The Authors.)

There is mounting evidence of systemic inflammation in post-traumatic stress disorder (PTSD) and Parkinson's disease (PD), yet inconsistency and a lack of replicability in findings of putative biological markers have delayed progress in this space. Variability in performance between platforms may contribute to the lack of consensus in the biomarker literature, as has been seen for a number of psychiatric disorders, including PTSD. Thus, there is a need for high-performance, scalable, and validated platforms for the discovery and development of biomarkers of inflammation for use in drug development and as clinical diagnostics. To identify the best platform for use in future biomarker discovery efforts, we conducted a comprehensive cross-platform and cross-assay evaluation across five leading platform technologies. This initial assessment focused on four cytokines that have been implicated PTSD - interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ. To assess platform performance and understand likely measurements in individuals with brain disorders, serum and plasma samples were obtained from individuals with PTSD (n = 13) or Parkinson's Disease (n = 14) as well as healthy controls (n = 5). We compared platform performance across a number of common analytic parameters, including assay precision, sensitivity, frequency of endogenous analyte detection (FEAD), correlation between platforms, and parallelism in measurement of cytokines using a serial dilution series. The single molecule array (Simoa™) ultra-sensitive platform (Quanterix), MESO V-Plex (Mesoscale Discovery), and Luminex xMAP® (Myriad) were conducted by their respective vendors, while Luminex® and Quantikine® high-sensitivity ELISA assays were evaluated by R&D System's Biomarker Testing Services. The assay with the highest sensitivity in detecting endogenous analytes across all analytes and clinical populations (i.e. the highest FEAD), was the Simoa™ platform. In contrast, more variable performance was observed for MESO V-plex, R&D Luminex® and Quantikine®, while Myriad's Luminex xMAP® exhibited low FEAD across all analytes and samples. Simoa™ also demonstrated high precision in detecting endogenous cytokines, as reflected in < 20 percent coefficient of variance (%CV) across replicate runs for samples from the healthy controls, PTSD patients, and PD patients. In contrast, MESO V-Plex, R&D Luminex® and Quantikine® had variable performance in terms of precision across cytokines. Myriad Luminex xMAP® could not be included in precision estimates because the vendor did not run samples in duplicate. For cross-platform performance comparisons, the highest cross-platform correlations were observed for IL-6 such that all platforms - except for Myriad's Luminex xMAP® - had strong correlations with one another in measurements of IL-6 (r range = 0.59 - 0.86). For the other cytokines, there was low to no correlation across platforms, such that reported measurements of IL-1β, TNF-α, and IFN-γ varied across assays. Taken together, these findings provide novel evidence that the choice of immunoassay could greatly impact reported cytokine findings. The current study provides crucial information on the variability in performance between platforms and across immunoassays that may help inform the selection of assay in future research studies. Further, the results emphasize the need for performing comparative evaluations of immunoassays as new technologies emerge over time, particularly given the lack of reference standards for the quantitative assessments of cytokines., Competing Interests: AJ has been an advisor to Quanterix, Inc and holds stock options. The other authors (HCL, ACP, LC, NPD, and MH) declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Among Plant Protection Products (PPP), a new emerging category of pesticides act by stimulating plant defense in order to improve plant resistance against microbial pathogens. Given that these compounds, the so-called Plant Defense Stimulators (PDS) act on innate immunity, we tested, using an in vitro approach on human mononuclear leucocytes (PBMC), the potential toxicity (XTT assay) and inflammatory effects (production of IL-1β) of 4 PPP belonging to different chemical families. We found that two products (LBG-01F34® and Regalis®) did not induce any cytotoxicity or IL-1 β production. The product BION-50 WG®, that contains Acibenzolar-S-methyl (ASM) and silica particles did not present any cytotoxicity but induced a significant increase in the production of the inflammatory cytokine IL-1 β. Finally, Vacciplant® that contains laminarin, was highly cytotoxic and pro-inflammatory. It induced a strong production of IL-1 β when used at a concentration in the culture medium, as low as 0.02 mg/mL. We also tested the potential toxic effect of these 4 PPP on 4 days old zebra fish larvae. After 24 h of exposure, our results indicate that Vacciplant® induced zebra fish larvae mortality at concentration of 20 μg/mL. LBG did not induced significant mortality at concentrations up to 1 mg/mL whereas Regalis was lethal for 0,3 mg/mL concentrations and BION-50 WG began to induce mortality at 2,5 mg/mL. Our results indicate possible effects of PDS on IL-1β production in human cells and fish survival, calling for more studies on the potential noxious side effects of these compounds., Competing Interests: There are no conflicts of interest to declare., (© 2020 The Authors. Published by Elsevier B.V.)

Background & Aims: C-C motif chemokine ligand 24 (CCL24) is a chemokine that regulates inflammatory and fibrotic activities through its receptor, C-C motif chemokine receptor (CCR3). The aim of the study was to evaluate the involvement of the CCL24-CCR3 axis in liver fibrosis and inflammation and to assess the potential of its blockade, by a monoclonal anti-CCL24 antibody, as a therapeutic strategy for non-alcoholic steatohepatitis (NASH) and liver fibrosis., Methods: Expression of CCL24 and CCR3 was evaluated in liver biopsies and blood samples. CCL24 involvement in NAFLD/NASH pathogenesis was assessed in Ccl24 knockout mouse using the methionine-choline deficient (MCD) diet experimental model. Antifibrotic and anti-inflammatory effects of CM-101 were tested in the MCD and STAM mouse models and in the thioacetamide (TAA) model in rats. Liver enzymes, liver morphology, histology and collagen deposition, as well as fibrosis- and inflammation-related protein expression were assessed. Activation of hepatic stellate cells (HSCs) was evaluated in the human LX2 cell line., Results: Patients with NASH and advanced NAFLD exhibited significant expression of both CCL24 and CCR3 in liver and blood samples. In the experimental MCD-diet model, Ccl24 knockout mice showed an attenuated liver damage response compared to wild-type mice, exhibiting reduced histological NAFLD activity scores and fibrosis, as well as lower levels of liver enzymes. Blocking CCL24 using CM-101 robustly reduced liver damage in 3 experimental animal models (MCD, STAM and TAA), as demonstrated by attenuation of liver fibrosis and NAFLD activity score. Furthermore, blocking CCL24 by CM-101 significantly inhibited CCL24-induced HSC motility, α-SMA expression and pro-collagen I secretion., Conclusion: Our results reveal that blocking CCL24 significantly attenuates liver fibrosis and inflammation and may have a potential therapeutic effect in patients with NASH and/or liver fibrosis., Lay Summary: CCL24 is a chemokine that regulates inflammation and fibrosis. It was found to be significantly expressed in patients with non-alcoholic steatohepatitis, in whom it regulates profibrotic processes in the liver. Herein, we show that blockade of CCL24 using a monoclonal antibody robustly attenuated liver fibrosis and inflammation in animal models, thus suggesting a potential therapeutic role for an anti-CCL24 agent., (© 2019 The Author(s).)

Atherosclerosis involves phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs). Data are given in tabular or figure format that illustrate genome-wide DNA methylation alterations in atherosclerotic vs. control aorta (athero DMRs). Data based upon publicly available chromatin state profiles are also shown for normal aorta, monocyte, and skeletal muscle tissue-specific DMRs and for aorta-specific chromatin features (enhancer chromatin, promoter chromatin, repressed chromatin, actively transcribed chromatin). Athero hypomethylated and hypermethylated DMRs as well as epigenetic and transcription profiles are described for the following genes: ACTA2, MYH10, MYH11 (SMC-associated genes); SMAD3 (a signaling gene for SMCs and other cell types); CD79B and SH3BP2 (leukocyte-associated genes); and TBX20 and genes in the HOXA, HOXB, HOXC , and HOXD clusters (T-box and homeobox developmental genes). The data reveal strong correlations between athero hypermethylated DMRs and regions of enhancer chromatin in aorta, which are discussed in the linked research article "Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers" (M. Lacey et al., 2019).

This toxicogenomic study was conducted to predict (post)menopausal human health effects of commercial soy supplementation using ovariectomized rats as a model. Different target tissues (i.e. breast, uterus and sternum) and non-target tissues (i.e. peripheral blood mononuclear cells (PBMC), adipose and liver) of ovariectomized F344 rats exposed to a commercially available soy supplement for eight weeks, were investigated. Changes in gene expression in these tissues were analysed using whole-genome microarray analysis. No correlation in changes in gene expression were observed among different tissues, indicating tissue specific effects of soy isoflavone supplementation. Out of 87 well-established estrogen responsive genes (ERGs), only 19 were found to be significantly regulated (p < 0.05) in different tissues, particularly in liver, adipose and uterus tissues. Surprisingly, no ERGs were significantly regulated in estrogen sensitive breast and sternum tissues. The changes in gene expression in PBMC and adipose tissue in rats were compared with those in (post)menopausal female volunteers who received the same supplement in a similar oral dose and exposure duration in human intervention studies. No correlation in changes in gene expression between rats and humans was observed. Although receiving a similar dose, in humans the plasma levels expressed as total free aglycones were several folds higher than in the rat. Therefore, the overall results in young ovariectomized female F344 rats indicated that using rat transcriptomic data does not provide a suitable model for human risk or benefit analysis of soy isoflavone supplementation.

Background: Although the divergent male and female differentiation depends on key genes, many biological differences seen in men and women are driven by relative differences in estrogen and testosterone levels. Gender dysphoria denotes the distress that gender incongruence with the assigned sex at birth may cause. Gender-affirming treatment includes medical intervention such as inhibition of endogenous sex hormones and subsequent replacement with cross-sex hormones. The aim of this study is to investigate consequences of an altered sex hormone profile on different tissues and metabolic risk factors. By studying subjects undergoing gender-affirming medical intervention with sex hormones, we have the unique opportunity to distinguish between genetic and hormonal effects., Methods: The study is a single center observational cohort study conducted in Stockholm, Sweden. The subjects are examined at four time points; before initiation of treatment, after endogenous sex hormone inhibition, and three and eleven months following sex hormone treatment. Examinations include blood samples, skeletal muscle-, adipose- and skin tissue biopsies, arteriography, echocardiography, carotid Doppler examination, whole body MRI, CT of muscle and measurements of muscle strength., Results: The primary outcome measure is transcriptomic and epigenomic changes in skeletal muscle. Secondary outcome measures include transcriptomic and epigenomic changes associated with metabolism in adipose and skin, muscle strength, fat cell size and ability to release fatty acids from adipose tissue, cardiovascular function, and body composition., Conclusions: This study will provide novel information on the role of sex hormone treatment in skeletal muscle, adipose and skin, and its relation to cardiovascular and metabolic disease.

Marine n -3 (omega-3) fatty acids alter gene expression by regulating the activity of transcription factors. Krill oil is a source of marine n -3 fatty acids that has been shown to modulate gene expression in animal studies; however, the effect in humans is not known. Hence, we aimed to compare the effect of intake of krill oil, lean and fatty fish with a similar content of n -3 fatty acids, and high-oleic sunflower oil (HOSO) with added astaxanthin on the expression of genes involved in glucose and lipid metabolism and inflammation in peripheral blood mononuclear cells (PBMC) as well as circulating inflammatory markers. In an 8-week trial, healthy men and women aged 18-70 years with fasting TAG of 1·3-4·0 mmol/l were randomised to receive krill oil capsules ( n 12), HOSO capsules ( n 12) or lean and fatty fish ( n 12). The weekly intakes of marine n -3 fatty acids from the interventions were 4654, 0 and 4103 mg, respectively. The mRNA expression of four genes, PPAR γ coactivator 1A ( PPARGC1A ), steaoryl-CoA desaturase ( SCD ), ATP binding cassette A1 ( ABCA1 ) and cluster of differentiation 40 ( CD40 ), were differently altered by the interventions. Furthermore, within-group analyses revealed that krill oil down-regulated the mRNA expression of thirteen genes, including genes involved in glucose and cholesterol metabolism and β-oxidation. Fish altered the mRNA expression of four genes and HOSO down-regulated sixteen genes, including several inflammation-related genes. There were no differences between the groups in circulating inflammatory markers after the intervention. In conclusion, the intake of krill oil and HOSO with added astaxanthin alter the PBMC mRNA expression of more genes than the intake of fish.

Arylamine N -acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The in situ activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. In situ NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

Programmed death-1 (PD-1), upon engagement by its ligands, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), provides signals that attenuate adaptive immune responses. Here we describe the identification of the Pekin duck PD-L2 (duPD-L2) and its gene structure. The duPD-L2 cDNA encodes a 321 amino acid protein that has an amino acid identity of 76% and 35% with chicken and human PD-L2, respectively. Mapping of the duPD-L2 cDNA with duck genomic sequences revealed an exonic structure similar to that of the human P dcd1lg2 gene. Homology modelling of the duPD-L2 protein was compatible with the murine PD-L2 ectodomain structure. Residues known to be important for PD-1 receptor binding of murine PD-L2 were mostly conserved in duPD-L2 within sheets A and G and partially conserved within sheets C and F. DuPD-L2 mRNA was constitutively expressed in all tissues examined with highest expression levels in lung, spleen, cloaca, bursa, cecal tonsil, duodenum and very low levels of expression in muscle, kidney and brain. Lipopolysaccharide treatment of adherent duck PBMC upregulated duPD-L2 mRNA expression. Our work shows evolutionary conservation of the PD-L2 ectodomain structure and residues important for PD-1 binding in vertebrates including fish. The information provided will be useful for further investigation of the role of duPD-L2 in the regulation of duck adaptive immunity and exploration of PD-1-targeted immunotherapies in the duck hepatitis B infection model.

Background & Aims: Systemic cellular immunity elicited by the Ty21a oral typhoid vaccine has been extensively characterized. However, very limited data are available in humans regarding mucosal immunity at the site of infection (terminal ileum [TI]). Here we investigated the host immunity elicited by Ty21a immunization on terminal ileum-lamina propria mononuclear cells (LPMC) and peripheral blood in volunteers undergoing routine colonoscopy., Methods: We characterized LPMC-T memory (T M ) subsets and assessed Salmonella enterica serovar Typhi ( S Typhi)-specific responses by multichromatic flow cytometry., Results: No differences were observed in cell yields and phenotypes in LPMC CD8 + -T M subsets following Ty21a immunization. However, Ty21a immunization elicited LPMC CD8 + T cells exhibiting significant S Typhi-specific responses (interferon-γ, tumor necrosis factor-α, interleukin-17A, and/or CD107a) in all major T M subsets (T-effector/memory [T EM ], T-central/memory, and T EM -CD45RA + ), although each T M subset exhibited unique characteristics. We also investigated whether Ty21a immunization elicited S Typhi-specific multifunctional effectors in LPMC CD8 + T EM . We observed that LPMC CD8 + T EM responses were mostly multifunctional, except for those cells exhibiting the characteristics associated with cytotoxic responses. Finally, we compared mucosal with systemic responses and made the important observation that LPMC CD8 + S Typhi-specific responses were unique and distinct from their systemic counterparts., Conclusions: This study provides the first demonstration of S Typhi-specific responses in the human terminal ileum mucosa and provides novel insights into the generation of mucosal immune responses following oral Ty21a immunization.

Systems biology is an interdisciplinary research field in life sciences, which involves a comprehensive and quantitative analysis of the interactions between all of the components of biological systems over time. For the past 50 years the discipline of virology has overly focused on the pathogen itself. However, we now know that the host response is equally or more important in defining the eventual pathological outcome of infection. Systems biology has in recent years been increasingly recognised for its importance to infectious disease research. Host-virus interactions can be better understood by taking into account the dynamical molecular networks that constitute a biological system. To decipher the pathobiological mechanisms of any disease requires a deep knowledge of how multiple and concurrent signal-transduction pathways operate and are deregulated. Hence the intricacies of signalling pathways can be dissected only by system level approaches., (© 2016 Elsevier Inc. All rights reserved.)

9 kDa granulysin is a protein present in the granules of human CTL and NK cells, with cytolytic activity against microbes and tumors. Previous work from our group demonstrated that this granulysin isoform induced apoptosisin vitroon hematological tumor cells and on primary tumor cells from B-CLL patients. In the present work, recombinant 9 kDa granulysin was used as an anti-tumoral agent to study itsin vivoeffect on tumor development in athymic “nude” mice models bearing human breast adenocarcinoma MDA-MB-231 or multiple myeloma NCI-H929–derived xenografts. Granulysin prevented thein vivodevelopment of detectable MDA-MB-231-derived tumors. In addition, recombinant granulysin was able to completely eradicate NCI-H929-derived tumors. All granulysin-treated tumors exhibited signs of apoptosis induction and an increased NK cell infiltration inside the tumor tissue comparing to control ones. Moreover, noin vivodeleterious effects of the recombinant 9 kDa granulysin doses used in this study were observed on the skin or on the internal organs of the animals. In conclusion, granulysin was able to inhibit the progression of MDA-MB-231-derived xenografts and also to eradicate multiple myeloma NCI-H929-derived xenografts. This work opens the door to the initiation of preclinical and possibly clinical studies for the use of 9 kDa granulysin as a new anti-tumoral treatment. [ABSTRACT FROM AUTHOR]

Autologous dendritic cell (DC) therapy is an experimental cellular immunotherapy that is safe and immunogenic in patients with advanced melanoma. In an attempt to further improve the therapeutic responses, we treated 15 patients with melanoma, with autologous monocyte-derived immature DC electroporated with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively active TLR4 (caTLR4) together with mRNA encoding a tumor-associated antigen (TAA; respectively gp100 or tyrosinase). In addition, DC were pulsed with keyhole limpet hemocyanin (KLH) that served as a control antigen. Production of this DC vaccine with high cellular viability, high expression of co-stimulatory molecules and MHC class I and II and production of IL-12p70, was feasible in all patients. A vaccination cycle consisting of three vaccinations with up to 15×106DC per vaccination at a biweekly interval, was repeated after 6 and 12 months in the absence of disease progression. mRNA-optimized DC were injected intranodally, because of low CCR7 expression on the DC, and induced de novo immune responses against control antigen. T cell responses against tyrosinase were detected in the skin-test infiltrating lymphocytes (SKIL) of two patients. One mixed tumor response and two durable tumor stabilizations were observed among 8 patients with evaluable disease at baseline. In conclusion, autologous mRNA-optimized DC can be safely administered intranodally to patients with metastatic melanoma but showed limited immunological responses against tyrosinase and gp100. [ABSTRACT FROM PUBLISHER]

Chronic pancreatitis (CP) is a risk factor of pancreatic ductal adenocarcinoma (PDAC) and characterized by a pronounced desmoplastic reaction with CD4+T cells accounting for the majority of the stromal T cell infiltrate. Epithelial-mesenchymal-transition (EMT) is a critical process for metastasis by which epithelial/carcinoma cells become enabled to disseminate probably prior to tumor formation. To investigate whether CD4+T cells induce EMT in human pancreatic ductal epithelial cells, premalignant H6c7 cells were mono- or co-cultured with human CD4+CD25+CD127−CD49d−regulatory T cells (T-regs) or CD4+CD25−T-effector cells (T-effs) being isolated by negative magnetic bead separation from blood of healthy donors. Particularly in the presence of activated T-effs, H6c7 cells acquired a spindle-shaped morphology, reduced E-cadherin expression, and elevated expression of the mesenchymal proteins vimentin, L1CAM, and ZEB-1. This was accompanied by an increased invasive behavior. Moreover, activated T-effs exerted similar effects in the PDAC cell line T3M4. Blocking of TNF-α and IL-6 being released at greater amounts into supernatants during co-cultures with activated T-effs attenuated the EMT-associated alterations in H6c7 cells. Supporting these findings, EMT-associated alterations (exemplified by reduced E-cadherin expression and enhanced expression of vimentin and L1CAM) were predominantly detected in ductal epithelium of CP tissues surrounded by a dense stroma enriched with CD4+T cells. Overall this study points to a novel role of CD4+T cells beyond their immune function in pancreatic tumorigenesis and underscores the view that EMT induction in pancreatic ductal epithelial cells represents an early event in PDAC development being essentially promoted by inflammatory processes. [ABSTRACT FROM PUBLISHER]

Indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO) represent some of the key immune regulators. Their increased activity has been demonstrated in a number of human malignancies but not yet in chronic myeloid leukemia (CML). In the present study, the activity of these enzymes was tested in 29 CML patients and 28 healthy subjects by monitoring the kynurenine (KYN)/tryptophan ratio. Serum samples taken prior to the therapy displayed a highly significant difference in KYN levels between the patient and control groups. However, increased KYN levels were detected in only 13 (44.8%) of these CML patients. The KYN levels in pretreatment sera of the patients correlated with the tumor burden. There was also a strong correlation between KYN levels and uric acid levels (UA). This suggests but does not prove the possible involvement of UA in activating IDO family of enzymes. Whenever tested, the increased KYN levels normalized in the course of the therapy. Patients with normal KYN levels in their pretreatment sera and subsequently treated with interferon-α, showed a transitory increase in their KYN levels. The present data indicate that CML should be added to the malignancies with an increased activity of the IDO family of enzymes and suggest that IDO inhibitors may be used in the treatment of CML patients. [ABSTRACT FROM PUBLISHER]

In melanoma, both the induction of immunosuppression by tumor cells and the inflammatory antitumor response can induce an upregulation of counter-regulatory mechanisms such as indoleamine 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1) and CTLA-4+regulatory T-cells (Tregs) in the tumor microenvironment. Even though these immunosuppressive mediators are targets for immunotherapy, research investigating their expression in the peripheral blood is lacking. We therefore, performed flow cytometry on PBMCs of stage I–IV melanoma patients. IDO expression was detected in plasmacytoid dendritic cells (pDC) and monocytic myeloid-derived suppressor cells (mMDSC), and increased in advanced disease stage (p= 0.027). Tryptophan breakdown confirmed the functional activity of IDO and was linked with increased PD-L1+ cytotoxic T-cells (p= 0.009), relative lymphopenia (p= 0.036), and a higher mDC/pDC ratio (p= 0.002). High levels of circulating PD-L1+ cytotoxic T-cells were associated with increasedCTLA-4expression by Tregs (p= 0.005) and MDSC levels (p= 0.033). This illustrates that counter-regulatory immune mechanisms in melanoma should be considered as one interrelated signaling network. Moreover, both increased PD-L1+ T-cells andCTLA-4expression in Tregs conferred a negative prognosis, indicating theirin vivorelevance. Remarkably, circulatingCTLA-4, IDO, and pDC levels were altered according to prior invasion of the sentinel lymph node and IDO expression in the sentinel was associated with more IDO+ PBMCs. We conclude that the expression of IDO, PD-L1, andCTLA-4in the peripheral blood of melanoma patients is strongly interconnected, associated with advanced disease and negative outcome, independent of disease stage. Combination treatments targeting several of these markers are therefore likely to exert a synergistic response. [ABSTRACT FROM PUBLISHER]

Both IL-17 and Th17 cells have been ascribed tumor promoting as well as tumor suppressing functions. We reviewed the literature on correlations between IL-17 versus Th17 cells and survival in human cancer, following the PRISMA guidelines. Serum, formalin-fixed, paraffin-embedded (FFPE) tissue and peripheral blood samples were most frequently studied. High IL-17 quantities were correlated with poor prognosis, whereas high Th17 cell frequencies were correlated with improved prognosis. Since Th17 cells are a subpopulation of IL-17+cells and had a different correlation with prognosis than total IL-17, we substantiate that a distinction should be made between Th17 and other IL-17+cells. [ABSTRACT FROM PUBLISHER]

Background.Glioblastoma (GBM) is the most common malignant brain tumor in adults and is nearly always fatal. Emerging evidence suggests that humanCytomegalovirus(HCMV) is present in 90–100% of GBMs and that add-on antiviral treatment for HCMV show promise to improve survival. Methods.In a randomized, placebo-controlled trial of valganciclovir in 42 GBM patients, blood samples were collected for analyses of HCMV DNA, RNA, reactivity against HCMV peptides, IgG, and IgM at baseline and at 3, 12, and 24 weeks of treatment. Results.All 42 tumors were positive for HCMV protein. All patients examined had at least one blood sample positive for HCMV DNA, 63% were HCMV RNA positive, and 21% were IgM positive. However, 29% of GBM patients were IgG negative for HCMV. Five of these samples were positive in an enzyme-linked immunosorbent assay (ELISA) that used antigens derived from a clinical isolate. Blood T cells from 11 of 13 (85%) HCMV IgG-negative GBM patients reacted against HCMV peptides. Valganciclovir did not affect IgG titers, DNA, or RNA levels of theHCMV immediate early (HCMV IE)gene in blood. Conclusion.In GBM patients, HCMV activity is higher than in healthy controls and serology is a poor test to define previous or active HCMV infection in these patients. [ABSTRACT FROM PUBLISHER]

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme. Remarkably, we discovered IDO-specific T cells that can influence adaptive immune reactions in patients with cancer. Further, a recent phase I clinical trial demonstrated long-lasting disease stabilization without toxicity in patients with non-small-cell lung cancer (NSCLC) who were vaccinated with an IDO-derived HLA-A2-restricted epitope. [ABSTRACT FROM PUBLISHER]