Search

Supplementary Figures 1-8

Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast, human T cells, a major anticancer effector cell, only rarely express Siglecs. However, here, we report that the majority of intratumoral, but not peripheral blood, cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity, cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement, which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1, but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted, glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset, while confining T-cell activation to the tumor microenvironment.

Background Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance. Methods We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4–11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors. Results Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4–11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4–11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks. Conclusions We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples. Graphical abstract

Intestinal microbiota regulates multiple host metabolic and immunological processes. Consequently, any difference in its qualitative and quantitative composition is susceptible to exert significant effects, in particular along the gut-liver axis. Indeed, recent findings suggest that such changes modulate the severity and the evolution of a wide spectrum of hepatobiliary disorders. However, the mechanisms linking intestinal microbiota and the pathogenesis of liver disease remain largely unknown. In this work, we investigated how a distinct composition of the intestinal microbiota, in comparison with germ-free conditions, may lead to different outcomes in an experimental model of acute cholestasis. Acute cholestasis was induced in germ-free (GF) and altered Schaedler’s flora (ASF) colonized mice by common bile duct ligation (BDL). Studies were performed 5 days after BDL and hepatic histology, gene expression, inflammation, lipids metabolism, and mitochondrial functioning were evaluated in normal and cholestatic mice. Differences in plasma concentration of bile acids (BA) were evaluated by UHPLC-HRMS. The absence of intestinal microbiota was associated with significant aggravation of hepatic bile infarcts after BDL. At baseline, we found the absence of gut microbiota induced altered expression of genes involved in the metabolism of fatty and amino acids. In contrast, acute cholestasis induced altered expression of genes associated with extracellular matrix, cell cycle, autophagy, activation of MAPK, inflammation, metabolism of lipids, and mitochondrial functioning pathways. Ductular reactions, cell proliferation, deposition of collagen 1 and autophagy were increased in the presence of microbiota after BDL whereas GF mice were more susceptible to hepatic inflammation as evidenced by increased gene expression levels of osteopontin, interleukin (IL)-1β and activation of the ERK/MAPK pathway as compared to ASF colonized mice. Additonally, we found that the presence of microbiota provided partial protection to the mitochondrial functioning and impairment in the fatty acid metabolism after BDL. The concentration of the majority of BA markedly increased after BDL in both groups without remarkable differences according to the hygiene status of the mice. In conclusion, acute cholestasis induced more severe liver injury in GF mice compared to mice with limited intestinal bacterial colonization. This protective effect was associated with different hepatic gene expression profiles mostly related to tissue repair, metabolic and immune functions. Our findings suggest that microbial-induced differences may impact the course of cholestasis and modulate liver injury, offering a background for novel therapies based on the modulation of the intestinal microbiota.

Leveraging consumer technology such as smartphone and smartwatch devices to objectively assess people with multiple sclerosis (PwMS) remotely could capture unique aspects of disease progression. This study explores the feasibility of assessing PwMS and Healthy Control's (HC) physical function by characterising gait-related features, which can be modelled using machine learning (ML) techniques to correctly distinguish subgroups of PwMS from healthy controls. A total of 97 subjects (24 HC subjects, 52 mildly disabled (PwMSmild, EDSS [0-3]) and 21 moderately disabled (PwMSmod, EDSS [3.5-5.5]) contributed data which was recorded from a Two-Minute Walk Test (2MWT) performed out-of-clinic and daily over a 24-week period. Signal-based features relating to movement were extracted from sensors in smartphone and smartwatch devices. A large number of features (n = 156) showed fair-to-strong (R 0.3) correlations with clinical outcomes. LASSO feature selection was applied to select and rank subsets of features used for dichotomous classification between subject groups, which were compared using Logistic Regression (LR), Support Vector Machines (SVM) and Random Forest (RF) models. Classifications of subject types were compared using data obtained from smartphone, smartwatch and the fusion of features from both devices. Models built on smartphone features alone achieved the highest classification performance, indicating that accurate and remote measurement of the ambulatory characteristics of HC and PwMS can be achieved with only one device. It was observed however that smartphone-based performance was affected by inconsistent placement location (running belt versus pocket). Results show that PwMSmod could be distinguished from HC subjects (Acc. 82.2 ± 2.9%, Sen. 80.1 ± 3.9%, Spec. 87.2 ± 4.2%, F 1 84.3 ± 3.8), and PwMSmild (Acc. 82.3 ± 1.9%, Sen. 71.6 ± 4.2%, Spec. 87.0 ± 3.2%, F 1 75.1 ± 2.2) using an SVM classifier with a Radial Basis Function (RBF). PwMSmild were shown to exhibit HC-like behaviour and were thus less distinguishable from HC (Acc. 66.4 ± 4.5%, Sen. 67.5 ± 5.7%, Spec. 60.3 ± 6.7%, F 1 58.6 ± 5.8). Finally, it was observed that subjects in this study demonstrated low intra- and high inter-subject variability which was representative of subject-specific gait characteristics. ispartof: Ieee Journal Of Biomedical And Health Informatics vol:25 issue:3 pages:838-849 ispartof: location:United States status: published

BACKGROUND Remote monitoring of Huntington disease (HD) signs and symptoms using digital technologies may enhance early clinical diagnosis and tracking of disease progression, guide treatment decisions, and monitor response to disease-modifying agents. Several recent studies in neurodegenerative diseases have demonstrated the feasibility of digital symptom monitoring. OBJECTIVE The aim of this study was to evaluate a novel smartwatch- and smartphone-based digital monitoring platform to remotely monitor signs and symptoms of HD. METHODS This analysis aimed to determine the feasibility and reliability of the Roche HD Digital Monitoring Platform over a 4-week period and cross-sectional validity over a 2-week interval. Key criteria assessed were feasibility, evaluated by adherence and quality control failure rates; test-retest reliability; known-groups validity; and convergent validity of sensor-based measures with existing clinical measures. Data from 3 studies were used: the predrug screening phase of an open-label extension study evaluating tominersen (NCT03342053) and 2 untreated cohorts—the HD Natural History Study (NCT03664804) and the Digital-HD study. Across these studies, controls (n=20) and individuals with premanifest (n=20) or manifest (n=179) HD completed 6 motor and 2 cognitive tests at home and in the clinic. RESULTS Participants in the open-label extension study, the HD Natural History Study, and the Digital-HD study completed 89.95% (1164/1294), 72.01% (2025/2812), and 68.98% (1454/2108) of the active tests, respectively. All sensor-based features showed good to excellent test-retest reliability (intraclass correlation coefficient 0.89-0.98) and generally low quality control failure rates. Good overall convergent validity of sensor-derived features to Unified HD Rating Scale outcomes and good overall known-groups validity among controls, premanifest, and manifest participants were observed. Among participants with manifest HD, the digital cognitive tests demonstrated the strongest correlations with analogous in-clinic tests (Pearson correlation coefficient 0.79-0.90). CONCLUSIONS These results show the potential of the HD Digital Monitoring Platform to provide reliable, valid, continuous remote monitoring of HD symptoms, facilitating the evaluation of novel treatments and enhanced clinical monitoring and care for individuals with HD.

The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is

INTRODUCTION Preterm birth is a major cause of neonatal morbidity and mortality. There is an urgent need to accurately predict imminent delivery to enable necessary interventions such as tocolytic, glucocorticoid, and magnesium sulfate administration. We aimed to evaluate placental α-macroglobulin-1 as a new diagnostic marker in the prediction of preterm birth. MATERIAL AND METHODS We performed a prospective observational trial in women with intact membranes between 24+0 and 36+6 weeks of gestation. We included both women with and without threatened preterm labor symptoms. We evaluated the test performance of placental α-macroglobulin-1 measurements in cervicovaginal fluid regarding three different presentation-to-delivery intervals: ≤2, ≤7, ≤14 days. In addition, we calculated placental α-macroglobulin-1 performance in combination with other prognostic factors such as ultrasonographic cervical length measurements. RESULTS We included 126 women in the study. We detected high specificity (97%-98%) and negative predictive value (89%-97%) for placental α-macroglobulin-1 at all time intervals. We assessed placental α-macroglobulin-1 in combination with cervical length measurements (≤15 mm) in the sub-group of women presenting with threatened preterm labor symptoms (n = 63) and detected high positive predictive values (100%) for 7- and 14-day presentation-to-delivery intervals. CONCLUSIONS Our study provides evidence that placental α-macroglobulin-1 testing in cervicovaginal fluid, in combination with cervical length measurements, accurately predicts preterm birth in women with preterm labor symptoms. This novel test combination may be used clinically to triage women presenting with threatened preterm labor, avoiding overtreatment and unnecessary hospitalizations.

Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT, and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling, suggesting that cell-extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK inhibition. PDGF-BB maintained ERK activation in the presence of ruxolitinib, consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.

OBJECTIVES Stem cells secrete significant amounts of bioactive factors in their secretome that can be immunosuppressive. We studied the effect of the secretome obtained from bone marrow-derived mesenchymal stem cells (BMSC-sec) in combination with cyclosporine A following acute rejection of lung allografts in the rat. METHODS Lung allotransplants were performed from male Brown Norway donor rats to recipient male Fisher 344 rats. Rat BMSC-sec was introduced intratracheally in the recipient every day after the transplant until the day the animal was sacrificed. Group A (n = 5) received control medium and cyclosporine A (2.5 mg/kg body weight intraperitoneally) for 5 days post-transplant and group B (n = 5) received BMSC-sec and cyclosporine A. Blood gas analysis was performed to assess graft function at day 5 only from the graft, and the tissue was sampled for measurement of the wet/dry ratio and histological grading of rejection. RESULTS All control animals (group A) showed severe signs of rejection. At day 5 grafts in group B showed improved gas exchange (i.e. mean PaO2 mmHg 237.9 ± 130 mmHg vs 24.9 ± 7.8 mmHg in group A). Histological examination according to the International Society of Heart and Lung Transplantation (ISHLT) revealed moderate to severe rejection in all animals in group A (III B) and a significant improvement in group B (I-IIA). The wet/dry ratio was also reduced in group B to 6.19 ± 0.6 compared to 9.36 ± 2 in group A. Furthermore, in vitro T-cell proliferation was reduced after treatment with BMSC-sec for CD 3 cells (69.55 ± 07 vs 73 ± 0.84), for CD 4 (24.95 ± 1.2 vs 27.75 ± 0.21) and for CD 8 cells (3.75 ± 0.2 vs 5.68 ± 0.02). CONCLUSIONS The BMSC-sec is a promising novel cell-based therapeutic option for acute rejection in a rat lung allograft model.

Simple Summary Macrophages play essential role in repair, regeneration and tissue remodeling. Role of macrophages in progression of lung fibrosis is established. Secretome of Induced pluripotent stem cells (iPSC-CM) has shown to reduce lung fibrosis and regulate macrophage phenotype, however exact mechanism is not known. Using advanced bioinformatics analysis by gene network analysis in this study we identified two components AAP and ELAVL-1 present in the iPSC-CM playing important role in regulation of macrophage phenotype. In this invitro study we confirmed experimentally that AAP and ELAVL1 play essential role by changing the profibrotic phenotype of the macrophages to pro resolution macrophages. We demonstrate reduction in gene expression and cytokine secretion of profibrotic macrophages after iPSC-CM treatment. Our study confirms antifibrotic and regenerative potential of iPSC-CM. Abstract Induced pluripotent stem cell secretome (iPSC-CM) mitigate organ injury and help in repair. Macrophages play a critical role in tissue repair and regeneration and can be directed to promote tissue repair by iPSC-CM, although the exact mechanisms are not known. In the current investigative study, we evaluated the possible mechanism by which iPSC-CM regulates the phenotype and secretory pattern of macrophages in vitro. Macrophages were obtained from human peripheral blood mononuclear cells and differentiated to various subpopulations and treated with either iPSC-CM or control media in vitro. Macrophage phenotype was assessed by flow cytometry, gene expression changes by qRT PCR and secretory pattern by multiplex protein analysis. The protein and gene interaction network revealed the involvement of Amyloid precursor protein (APP) and ELAV-like protein 1 (ELAVL-1) both present in the iPSC-CM to play an important role in regulating the macrophage phenotype and their secretory pattern. This exploratory study reveals, in part, the possible mechanism and identifies two potential targets by which iPSC-CM regulate macrophages and help in repair and regeneration.

Recent data suggest that Pancreatic Neuroendocrine Tumours (PanNETs) originate from α- or β-cells of the islets of Langerhans. The majority of PanNETs are non-functional and do not express cell-type specific hormones. In the current study we examine whether tumour DNA methylation (DNAme) profiling combined with genomic data is able to identify cell of origin and to reveal pathways involved in PanNET progression. We analyse genome-wide DNAme data of 125 PanNETs and sorted α- and β-cells. To confirm cell identity, we investigate ARX and PDX1 expression. Based on epigenetic similarities, PanNETs cluster in α-like, β-like and intermediate tumours. The epigenetic similarity to α-cells progressively decreases in the intermediate tumours, which present unclear differentiation. Specific transcription factor methylation and expression vary in the respective α/β-tumour groups. Depending on DNAme similarity to α/β-cells, PanNETs have different mutational spectra, stage of the disease and prognosis, indicating potential means of PanNET progression., Di Domenico et al., provide a comprehensive assessment of DNA methylation in a large cohort of pancreatic neuroendocrine tumors. This work provides new insights into the epigenetic heterogeneity of the disease and cellular basis.

Lung injury in mice induces mobilization of discrete subsets of epithelial progenitor cells to promote new airway and alveolar structures. However, whether similar cell types exist in human lung remains unresolved. Using flow cytometry, we identified a distinct cluster of cells expressing the epithelial cell adhesion molecule (EpCAM), a cell surface marker expressed on epithelial progenitor cells, enriched in the ecto-5′-nucleotidase CD73 in unaffected postnatal human lungs resected from pediatric patients with congenital lung lesions. Within the EpCAM+CD73+ population, a small subset coexpresses integrin β4 and HTII-280. This population remained stable with age. Spatially, EpCAM+CD73+ cells were positioned along the basal membrane of respiratory epithelium and alveolus next to CD73+ cells lacking EpCAM. Expanded EpCAM+CD73+ cells give rise to a pseudostratified epithelium in a two-dimensional air–liquid interface or a clonal three-dimensional organoid assay. Organoids generated under alveolar differentiation conditions were cystic-like and lacked robust alveolar mature cell types. Compared with unaffected postnatal lung, congenital lung lesions were marked by clusters of EpCAM+CD73+ cells in airway and cystic distal lung structures lined by simple epithelium composed of EpCAM+SCGB1A1+ cells and hyperplastic EpCAM+proSPC+ cells. In non-small-cell lung cancer (NSCLC), there was a marked increase in EpCAM+CD73+ tumor cells enriched in inhibitory immune checkpoint molecules CD47 and programmed death-ligand 1 (PD-L1), which was associated with poor survival in lung adenocarcinoma (LUAD). In conclusion, EpCAM+CD73+ cells are rare novel epithelial progenitor cells in the human lung. Importantly, reemergence of CD73 in lung adenocarcinoma enriched in negative immune checkpoint molecules may serve as a novel therapeutic target.

Lung injury in mice induces mobilization of discrete subsets of epithelial progenitor cells to promote new airway and alveolar structures. However, whether similar cell types exist in human lung remains unresolved. Using flow cytometry, we identified a distinct cluster of cells expressing the epithelial cell adhesion molecule (EpCAM), a cell surface marker expressed on epithelial progenitor cells, enriched in the ecto-5'-nucleotidase CD73 in unaffected postnatal human lungs resected from pediatric patients with congenital lung lesions. Within the EpCAM

Objective: Smartphone devices may enable out-of-clinic assessments in chronic neurological diseases. We describe the Draw a Shape (DaS) Test, a smartphone-based and remotely administered test of Upper Extremity (UE) function developed for people with multiple sclerosis (PwMS). This work introduces DaS-related features that characterise UE function and impairment, and aims to demonstrate how multivariate modelling of these metrics can reliably predict the 9-Hole Peg Test (9HPT), a clinician-administered UE assessment in PwMS. Approach: The DaS Test instructed PwMS and healthy controls (HC) to trace predefined shapes on a smartphone screen. A total of 93 subjects (HC, n = 22; PwMS, n = 71) contributed both dominant and non-dominant handed DaS tests. PwMS subjects were characterised as those with normal (nPwMS, n = 50) and abnormal UE function (aPwMS, n = 21) with respect to their average 9HPT time (≤ or > 22.7 (s), respectively). L 1-regularization techniques, combined with linear least squares (OLS, IRLS), or non-linear support vector (SVR) or random forest (RFR) regression were investigated as functions to map relevant DaS features to 9HPT times. Main results: It was observed that average non-dominant handed 9HPT times were more accurately predicted by DaS features (r 2 = 0.41, P < 0.05; MAE: 2.08 ± 0.34 (s)) than average dominant handed 9HPTs (r 2 = 0.39, P < 0.05; MAE: 2.32 ± 0.43 (s)), using simple linear IRLS ( P < 0.01). Moreover, it was found that the Mean absolute error (MAE) in predicted 9HPTs was comparable to the variability of actual 9HPT times within HC, nPwMS and aPwMS groups respectively. The 9HPT however exhibited large heteroscedasticity resulting in less stable predictions of longer 9HPT times. Significance: This study demonstrates the potential of the smartphone-based DaS Test to reliably predict 9HPT times and remotely monitor UE function in PwMS.

Background Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The macrolide Azithromycin (AZT) has anti-inflammatory and immunomodulatory properties. Recently anti-fibrotic effects have been described. However, the anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects. Methods Primary normal human lung and IPF-FB were exposed to TGF-β (5 ng/ml), Azithromycin (50 μM) alone or in combination prior to gene expression analysis. Pro-collagen Iα1 secretion was assessed by ELISA and protein expression by western blot (αSMA, Fibronectin, ATP6V1B2, LC3 AB (II/I), p62, Bcl-xL). Microarray analysis was performed to screen involved genes and pathways after Azithromycin treatment in control-FB. Apoptosis and intraluminal lysosomal pH were analyzed by flow cytometry. Results AZT significantly reduced collagen secretion in TGF-β treated IPF-FB compared to TGF-β treatment alone, but not in control-FB. Pro-fibrotic gene expression was similarly reduced after AZT treatment in IPF and control-FB. P62 and LC3II/I western blot revealed impaired autophagic flux after AZT in both control and IPF-FB with significant increase of LC3II/I after AZT in control and IPF-FB, indicating enhanced autophagy inhibition. Early apoptosis was significantly higher in TGF-β treated IPF-FB compared to controls after AZT. Microarray analysis of control-FB treated with AZT revealed impaired lysosomal pathways. The ATPase and lysosomal pH regulator ATP6V0D2 was significantly less increased after additional AZT in IPF-FB compared to controls. Lysosomal function was impaired in both IPF and control FB, but pH was significantly more increased in TGF-β treated IPF-FB. Conclusion We report different treatment responses after AZT with enhanced anti-fibrotic and pro-apoptotic effects in IPF compared to control-FB. Possibly impaired lysosomal function contributes towards these effects. In summary, different baseline cell phenotype and behavior of IPF and control cells contribute to enhanced anti-fibrotic and pro-apoptotic effects in IPF-FB after AZT treatment and strengthen its role as a new potential anti-fibrotic compound, that should further be evaluated in clinical studies.

Idiopathic pulmonary fibrosis (IPF) is a complex disease involving various cell types. Macrophages are essential in maintenance of physiological homeostasis, wound repair and fibrosis in the lung. Macrophages play a crucial role in repair and remodeling by altering their phenotype and secretory pattern in response to injury. The secretome of induced pluripotent stem cells (iPSC-cm) attenuates injury and fibrosis in bleomycin injured rat lungs. In the current study, we evaluate the effect of iPSC-cm on gene expression and phenotype of interstitial macrophage in bleomycin injured rat lungs in vivo. iPSC-cm was intratracheally instilled 7 days after bleomycin induced lung injury and assessed 7 days later and single cell isolation was performed. Macrophages were FACS sorted and microarray analysis was performed. We characterized changes in the rat lung interstitial macrophages using transcriptional profiling. iPSC-cm reduced the total collagen content of the lung and reduced different macrophage populations. Gene set enrichment analysis revealed involvement of three essential pathways (a) immune modulation, (b) branching morphogenesis and (c) canonical Wnt signaling. This study demonstrates that iPSC-cm reduces fibrosis in bleomycin injured rat lung by partially altering the macrophages and regulating their gene expression. Electronic supplementary material The online version of this article (10.1007/s12015-017-9790-9) contains supplementary material, which is available to authorized users.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune responses. Increasing evidence suggests a role for TREM-1 not only in acute pathogen-induced reactions but also in chronic and non-infectious inflammatory disorders, including various types of cancer. Here, we demonstrate that genetic deficiency in Trem1 protects from colorectal cancer. In particular, Trem1−/− mice exhibited reduced tumor numbers and load in an experimental model of inflammation-driven tumorigenesis. Gene expression analysis of Trem1−/− versus Trem1+/+ tumor tissue demonstrated distinct immune signatures. Whereas Trem1−/− tumors showed an increased abundance of transcripts linked to adaptive immunity, Trem1+/+ tumors were characterized by overexpression of innate pro-inflammatory genes associated with tumorigenesis. Compared to adjacent tumor-free colonic mucosa, expression of Trem1 was increased in murine and human colorectal tumors. Unexpectedly, TREM-1 was not detected on tumor-associated Ly6C− MHC class II+ macrophages. In contrast, TREM-1 was highly expressed by tumor-infiltrating neutrophils which represented the predominant myeloid population in Trem1+/+ but not in Trem1−/− tumors. Collectively, our findings demonstrate a clear role of TREM-1 for intestinal tumorigenesis and indicate TREM-1-expressing neutrophils as critical players in colorectal tumor development.

Diagnostic markers are needed for accidental or deliberate radiation exposure that could cause acute and chronic radiation toxicity. Biomarkers of temporal, dose-dependent, aging-attenuated and multiple radiation exposures have been previously described by others. However, the physiological origin and biochemical networks that generate these biomarkers and their association at the molecular level have yet to be explored. Hence, the discovery and identification of total-body-irradiation-induced tissue specific biomarkers remains an enormous challenge within radiation biodosimetry research. To determine the tissue level response of total-body exposure (6 Gy), metabolomics analysis was carried out on radiosensitive tissues bone marrow, ileum, liver, muscle and lung as well as serum and on urine within 12 h postirradiation. Differences in the metabolic signatures between the sham and gamma-irradiated groups were analyzed by hydrophilic interaction liquid chromatography (HILIC)-based ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS). A panel of 67 biomarkers identified in radiosensitive tissues and biofluids (serum and urine) at a 6 Gy dose. Among the identified biomarkers, 3-methylglutarylcarnitine (3-MGC) was found to be a novel metabolite in liver, serum and urine that could potentially be an early radiation response marker. The degree of metabolic changes among different tissues showed perturbations in pathways including DNA methylation, energy, nucleic acid, amino acid, glutathione and bile acid metabolism. These results highlight metabolomics as a potential novel approach to understand functional alterations in the metabolome that could be adapted for use in the rapid assessment of radiation exposure and triage protocols in the case of nuclear incidents.

In childhood acute lymphoblastic leukemia, treatment failure is associated with resistance to glucocorticoid agents. Resistance to this class of drugs represents one of the strongest indicators of poor clinical outcome. We show that leukemic cells, which are resistant to the glucocorticoid drug methylprednisolone, display a higher demand of glucose associated with a deregulation of metabolic pathways, in comparison to sensitive cells. Interestingly, a combinatorial treatment of glucocorticoid and the glucose analog 2-deoxy-D-glucose displayed a synergistic effect in methylprednisolone-resistant cells, in an oxygen tension-independent manner. Unlike solid tumors, where 2-deoxy-D-glucose promotes inhibition of glycolysis by hexokinase II exclusively under hypoxic conditions, we were able to show that the antileukemic effects of 2-deoxy-D-glucose are far more complex in leukemia. We demonstrate a hexokinase II-independent cell viability decrease and apoptosis induction of the glucose analog in leukemia. Additionally, due to the structural similarity of 2-deoxy-D-glucose with mannose, we could confirm that the mechanism by which 2-deoxy-D-glucose predominantly acts in leukemia is via modification in N-linked glycosylation, leading to endoplasmic reticulum stress and consequently induction of the unfolded protein response., Oxidative Medicine and Cellular Longevity, 2017, ISSN:1942-0994, ISSN:1942-0900

The liver has an intrinsic capacity to regenerate in response to injury or surgical resection. Nevertheless, circumstances in which hepatocytes are unresponsive to proliferative signals result in impaired regeneration and hepatic failure. As the Hippo pathway has a canonical role in the maintenance of liver size, we investigated whether it could serve as a therapeutic target to support regeneration. Using a standard two‐thirds partial hepatectomy (PH) model in young and aged mice, we demonstrate that the Hippo pathway is modulated across the phases of liver regeneration. The activity of the core kinases MST1 and LATS1 increased during the early hypertrophic phase and returned to steady state levels in the proliferative phase, coinciding with activation of YAP1 target genes and hepatocyte proliferation. Moreover, following PH in aged mice, we demonstrate that Hippo signaling is anomalous in non‐regenerating livers. We provide pre‐clinical evidence that silencing the Hippo core kinases MST1 and MST2 with siRNA provokes hepatocyte proliferation in quiescent livers and rescues liver regeneration in aged mice following PH. Our data suggest that targeting the Hippo core kinases MST1/2 has therapeutic potential to improve regeneration in non‐regenerative disorders.

CD8+ T cell immunosurveillance is crucial in solid tumors and T cell dysfunction leads to tumor progression. In contrast, the role of CD8+ T cells in the control of leukemia is less clear. We characterized the molecular signature of leukemia stem/progenitor cells (LSPCs) and paired CD8+ T cells in patients with acute myeloid leukemia (AML). Epigenetic alterations via histone deacetylation reduced the expression of immune-related genes in bone marrow (BM)-infiltrating CD8+ T cells. Surprisingly, a silenced gene expression pattern in CD8+ T cells significantly correlated with an improved prognosis. To define interactions between CD8+ T cells and LSPCs, we performed comprehensive correlative network modeling. This analysis indicated that CD8+ T cells contribute to the maintenance/expansion of LSPCs, particularly in favorable risk AML. Functionally, CD8+ T cells in favorable AML induced the expansion of LSPCs by stimulating the autocrine production of important hematopoietic cytokines such as interleukin (IL)-3. In contrast, LSPCs in aggressive AML were characterized by a higher activation of stemness/proliferation-related pathways and develop independent of BM CD8+ T cells. Overall, our study indicates that CD8+ T cells support and expand LSPCs in favorable risk AML whereas intermediate and adverse risk AML possess the intrinsic molecular abnormalities to develop independently.

The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is

Five percent of papillary thyroid carcinomas (PTC) show an adverse clinical outcome (ACO). The tall cell variant of papillary thyroid carcinomas (TCV) is a good predictor of an ACO, however, the identification of tall-cells is subjective. Micro RNAs are short non-coding ribonucleic acids (miRNA). Their expression in PTC could be a powerful, more objective predictor of prognosis. Methods: Forty-four PTC underwent miRNA profiling, twenty-four of them were TCV. The miRNA dataset was validated by analysis of expression of known target proteins (vascular endothelial growth factor (VEGF) and phosphatase and tensin homolog (PTEN)) in 125 patients including 48 TCV and 57 with an ACO. Results: One hundred and forty-nine miRNAs were significantly associated with an ACO, seventy-one of them with TC-morphology. Twenty-two miRNAs were identified as targets for VEGF and thirty-two as targets for PTEN. In univariate and multivariable analysis, reduced expression of PTEN and an increased expression of VEGF were associated with shorter relapse free survival. A classifier, including TC-morphology, pT-stage, VEGF, and PTEN, predicted relapse with an 80% accuracy. Conclusions: Some miRNAs predict outcome in PTC and are involved in TC-morphology in PTC. These miRNAs may serve as more objective indicators of an ACO than tall cell morphology. PTEN and VEGF protein expression are prognostically relevant and are at least partially regulated by miRNAs.

The composition of immune infiltrates strongly affects the prognosis of patients with colorectal cancer (CRC). Interleukin (IL)-33 and regulatory T cells (Tregs) in the tumor microenvironment have been separately implicated in CRC; however their contribution to intestinal carcinogenesis is still controversial. Here, we reveal that IL-33 signaling promotes CRC by changing the phenotype of Tregs. In mice with CRC, tumor-infiltrating Tregs preferentially upregulate IL-33 receptor (ST2), and IL-33/ST2 signaling positively correlates with tumor number and size. Transcriptomic and flow cytometry analyses demonstrate that ST2 expression induces a more activated and migratory phenotype in FOXP3+ Tregs, which favors their accumulation in the tumor environment. Consequently, genetic ablation of St2 reduces Treg infiltration and concomitantly enhances the frequencies of effector CD8+ T cells, thereby restraining CRC. Mechanistically, IL-33 curtails IL-17 production by FOXP3+ Tregs and inhibits Th17 differentiation. In humans, numbers of activated ST2-expressing Tregs are increased in blood and tumor lesions of CRC patients, suggesting a similar mode of regulation. Together, these data indicate a central role of IL-33/ST2 signaling in shaping an immunosuppressive environment during intestinal tumorigenesis. Blockade of this pathway may provide a strategy to modulate the composition of CRC immune infiltrates.

Although TH1, TH2, and TH17 cells are well-defined TH cell lineages in humans, it remains debated whether IL-9-producing TH cells represent a bona fide "TH9" lineage. Our understanding of the cellular characteristics and functions of IL-9-producing TH cells in humans is still nascent. Here, we report that human IL-9-producing TH cells express the chemokine receptors CCR4 and CCR8, produce high levels of IL-5 and IL-13, and express TH2 lineage-associated transcription factors. In these cells, IL-9 production is activation dependent, transient, and accompanied by down-regulation of TH2 cytokines, leading to an apparent "TH9" phenotype. IL-9+ TH2 cells can be distinguished from "conventional" TH2 cells based on their expression of the transcription factor PPAR-γ. Accordingly, PPAR-γ is induced in naive TH cells by priming with IL-4 and TGF-β ("TH9" priming) and is required for IL-9 production. In line with their identity as early activated TH2 cells, IL-9+ TH2 cells are found in acute allergic skin inflammation in humans. We propose that IL-9-producing TH cells are a phenotypically and functionally distinct subpopulation of TH2 cells that depend on PPAR-γ for full effector functions.

Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast, human T cells, a major anticancer effector cell, only rarely express Siglecs. However, here, we report that the majority of intratumoral, but not peripheral blood, cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity, cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement, which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1, but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted, glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset, while confining T-cell activation to the tumor microenvironment.

Interferon-γ (IFN-γ) plays an important role in innate and adaptive immunity against intracellular infections and is used clinically for the prevention and control of infections in chronic granulomatous disease (CGD) and inborn defects in the IFN-γ/interleukin (IL)-12 axis. Using transcriptome profiling (RNA-seq), we sought to identify differentially expressed genes, transcripts and exons in Epstein-Barr virus-transformed B lymphocytes (B-EBV) cells from CGD patients, IFN-γ receptor deficiency patients, and normal controls, treated in vitro with IFN-γ for 48 hours. Our results show that IFN-γ increased the expression of a diverse array of genes related to different cellular programs. In cells from normal controls and CGD patients, IFN-γ-induced expression of genes relevant to oxidative killing, nitric oxide synthase pathway, proteasome-mediated degradation, antigen presentation, chemoattraction, and cell adhesion. IFN-γ also upregulated genes involved in diverse stages of messenger RNA (mRNA) processing including pre-mRNA splicing, as well as others implicated in the folding, transport, and assembly of proteins. In particular, differential exon expression of WARS (encoding tryptophanyl-transfer RNA synthetase, which has an essential function in protein synthesis) induced by IFN-γ in normal and CGD cells suggests that this gene may have an important contribution to the benefits of IFN-γ treatment for CGD. Upregulation of mRNA and protein processing related genes in CGD and IFNRD cells could mediate some of the effects of IFN-γ treatment. These data support the concept that IFN-γ treatment may contribute to increased immune responses against pathogens through regulation of genes important for mRNA and protein processing.

// Reut Shainer 1 , Osnat Almogi-Hazan 1 , Arye Berger 1 , Liad Hinden 1 , Martin Mueller 2, 3 , Chaya Brodie 4 , Cedric Simillion 5 , Michael Paidas 2 , Eytan R. Barnea 6, 7, * , Reuven Or 1, * 1 Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah-Hebrew University Medical Center, Jerusalem, 91120, Israel 2 Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06510, USA 3 Department of Obstetrics and Gynecology, University Hospital Bern, Bern, 3003, Switzerland 4 Henry Ford Hospital, Detroit, MI 48202, USA 5 Department of Clinical Research, University of Bern, Bern, 3003, Switzerland 6 The Society for The Investigation of Early Pregnancy (SIEP), Cherry Hill, NJ 08003, USA 7 BioIncept, LLC (PreImplantation Factor* Proprietary), Cherry Hill, NJ 08003, USA * These authors contributed equally to this work Correspondence to: Eytan R. Barnea, email: barnea@earlypregnancy.org Keywords: ARS, PreImplantation Factor (PIF), oxidative stress, local & systemic protection Received: May 03, 2016 Accepted: June 30, 2016 Published: July 16, 2016 ABSTRACT Acute Radiation Syndrome (ARS) may lead to cancer and death and has few effective countermeasures. Efficacy of synthetic PIF treatment was demonstrated in preclinical autoimmune and transplantation models. PIF protected against inflammation and mortality following lethal irradiation in allogeneic bone marrow transplant (BMT) model. Herein, we demonstrate that PIF imparts comprehensive local and systemic protection against lethal and sub-lethal ARS in murine models. PIF treatment 2 h after lethal irradiation led to 100% survival and global hematopoietic recovery at 2 weeks after therapy. At 24 h after irradiation PIF restored hematopoiesis in a semi-allogeneic BMT model. PIF-preconditioning provided improved long-term engraftment. The direct effect of PIF on bone marrow cells was also demonstrated in vitro : PIF promoted pre-B cell differentiation and increased immunoregulatory properties of BM-derived mesenchymal stromal cells. PIF treatment also improved hematopoietic recovery and reduced systemic inflammatory cytokine production after sub-lethal radiation exposure. Here, PIF also prevented colonic crypt and basal membrane damage coupled with reduced nitric oxide synthetase ( iNOS ) and increased (B7h1) expression. Global upper GI gene pathway analysis revealed PIF’s involvement in protein-RNA interactions, mitochondrial oxidative pathways, and responses to cellular stress. Some effects may be attributed to PIF’s influence on macrophage differentiation and function. PIF demonstrated a regulatory effect on irradiated macrophages and on classically activated M1 macrophages, reducing inflammatory gene expression ( iNOS, Cox2 ), promoting protective ( Arg1 ) gene expression and inducing pro-tolerance cytokine secretion. Notably, synthetic PIF is stable for long-term field use. Overall, clinical investigation of PIF for comprehensive ARS protection is warranted.

CD8

Regular physical exercise has many beneficial effects, including antitumor properties, and is associated with a reduced risk of developing hepatocellular carcinoma (HCC). Less is known about the impact of exercise on HCC growth and progression. Here, we investigated the effects of exercise on HCC progression and assessed whether any beneficial effects would be evident under sorafenib treatment and could be mimicked by metformin. American Cancer Institute rats with orthotopic syngeneic HCC derived from Morris Hepatoma-3924A cells were randomly assigned to exercise (Exe) and sedentary groups, or sorafenib±Exe groups or sorafenib±metformin groups. The Exe groups ran on a motorized treadmill for 60 minutes/day, 5 days/week for 4 weeks. Tumor viable area was decreased by exercise, while cell proliferation and vascular density were reduced. Exercise increased the expression of phosphatase and tensin homolog deleted from chromosome 10 and increased the phosphorylation of adenosine monophosphate-activated protein kinase, while the phosphorylation of protein kinase B, S6 ribosomal protein, and signal transducer and activator of transcription 3 were decreased. Transcriptomic analysis suggested major effects of exercise were on nontumoral liver rather than tumor tissue. Exercise demonstrated similar effects when combined with sorafenib. Moreover, similar effects were observed in the group treated with sorafenib+metformin, revealing an exercise-mimicking effect of metformin. Conclusion: Exercise attenuates HCC progression associated with alterations in key signaling pathways, cellular proliferation, tumor vascularization, and necrosis. These beneficial effects are maintained when combined with sorafenib and can be mimicked by metformin. (Hepatology Communications 2018;2:607-620).