Showing papers on "Receptor expression published in 2015"

Non-Classical monocytes display inflammatory features: Validation in Sepsis and Systemic Lupus Erythematous

Abstract: Given the importance of monocytes in pathogenesis of infectious and other inflammatory disorders, delineating functional and phenotypic characterization of monocyte subsets has emerged as a critical requirement. Although human monocytes have been subdivided into three different populations based on surface expression of CD14 and CD16, published reports suffer from contradictions with respect to subset phenotypes and function. This has been attributed to discrepancies in reliable gating strategies for flow cytometric characterization and purification protocols contributing to significant changes in receptor expression. By using a combination of multicolour flow cytometry and a high-dimensional automated clustering algorithm to confirm robustness of gating strategy and analysis of ex-vivo activation of whole blood with LPS we demonstrate the following: a. ‘Classical’ monocytes are phagocytic with no inflammatory attributes, b. ‘Non-classical’ subtype display ‘inflammatory’ characteristics on activation and display properties for antigen presentation and c. ‘Intermediate’ subtype that constitutes a very small percentage in circulation (under physiological conditions) appear to be transitional monocytes that display both phagocytic and inflammatory function. Analysis of blood from patients with Sepsis, a pathogen driven acute inflammatory disease and Systemic Lupus Erythmatosus (SLE), a chronic inflammatory disorder validated the broad conclusions drawn in the study.

Adenosine Receptor 2A Blockade Increases the Efficacy of Anti–PD-1 through Enhanced Antitumor T-cell Responses

Abstract: Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti-PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A2A receptors could enhance the efficacy of anti-PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti-PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A2A adenosine receptor antagonist. The blockade of PD-1 enhanced A2A receptor expression on tumor-infiltrating CD8(+) T cells, making them more susceptible to A2A-mediated suppression. Thus, dual blockade of PD-1 and A2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8(+) T cells and, accordingly, increased growth inhibition of CD73(+) tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti-PD-1 mAb in patients with cancer and that the efficacy of anti-PD-1 mAb can be significantly enhanced by A2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti-PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer.

Generation of a Transgenic Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease

Abstract: The emergence of Middle East respiratory syndrome-coronavirus (MERS-CoV) in the Middle East since 2012 has caused more than 900 human infections with ∼40% mortality to date. Animal models are needed for studying pathogenesis and for development of preventive and therapeutic agents against MERS-CoV infection. Nonhuman primates (rhesus macaques and marmosets) are expensive models of limited availability. Although a mouse lung infection model has been described using adenovirus vectors expressing human CD26/dipeptidyl peptidase 4 (DPP4), it is believed that a transgenic mouse model is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. We show that transgenic mice globally expressing hCD26/DPP4 were fully permissive to MERS-CoV infection, resulting in relentless weight loss and death within days postinfection. High infectious virus titers were recovered primarily from the lungs and brains of mice at 2 and 4 days postinfection, respectively, whereas viral RNAs were also detected in the heart, spleen, and intestine, indicating a disseminating viral infection. Infected Tg + mice developed a progressive pneumonia, characterized by extensive inflammatory infiltration. In contrast, an inconsistent mild perivascular cuffing was the only pathological change associated with the infected brains. Moreover, infected Tg + mice were able to activate genes encoding for many antiviral and inflammatory mediators within the lungs and brains, coinciding with the high levels of viral replication. This new and unique transgenic mouse model will be useful for furthering knowledge of MERS pathogenesis and for the development of vaccine and treatments against MERS-CoV infection. IMPORTANCE Small and economical animal models are required for the controlled and extensive studies needed for elucidating pathogenesis and development of vaccines and antivirals against MERS. Mice are the most desirable small-animal species for this purpose because of availability and the existence of a thorough knowledge base, particularly of genetics and immunology. The standard small animals, mice, hamsters, and ferrets, all lack the functional MERS-CoV receptor and are not susceptible to infection. So, initial studies were done with nonhuman primates, expensive models of limited availability. A mouse lung infection model was described where a mouse adenovirus was used to transfect lung cells for receptor expression. Nevertheless, all generally agree that a transgenic mouse model expressing the DPP4 receptor is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. This new and unique transgenic mouse model will be useful for furthering MERS research.

Iron regulatory proteins and their role in controlling iron metabolism

Abstract: Cellular iron homeostasis is regulated by post-transcriptional feedback mechanisms, which control the expression of proteins involved in iron uptake, release and storage. Two cytoplasmic proteins with mRNA-binding properties, iron regulatory proteins 1 and 2 (IRP1 and IRP2) play a central role in this regulation. Foremost, IRPs regulate ferritin H and ferritin L translation and thus iron storage, as well as transferrin receptor 1 (TfR1) mRNA stability, thereby adjusting receptor expression and iron uptake via receptor-mediated endocytosis of iron-loaded transferrin. In addition splice variants of iron transporters for import and export at the plasma-membrane, divalent metal transporter 1 (DMT1) and ferroportin are regulated by IRPs. These mechanisms have probably evolved to maintain the cytoplasmic labile iron pool (LIP) at an appropriate level. In certain tissues, the regulation exerted by IRPs influences iron homeostasis and utilization of the entire organism. In intestine, the control of ferritin expression limits intestinal iron absorption and, thus, whole body iron levels. In bone marrow, erythroid heme biosynthesis is coordinated with iron availability through IRP-mediated translational control of erythroid 5-aminolevulinate synthase mRNA. Moreover, the translational control of HIF2α mRNA in kidney by IRP1 coordinates erythropoietin synthesis with iron and oxygen supply. Besides IRPs, body iron absorption is negatively regulated by hepcidin. This peptide hormone, synthesized and secreted by the liver in response to high serum iron, downregulates ferroportin at the protein level and thereby limits iron absorption from the diet. Hepcidin will not be discussed in further detail here.

Trailing TRAIL Resistance: Novel Targets for TRAIL Sensitization in Cancer Cells

Abstract: Resistance to chemotherapeutic drugs is the major hindrance in the successful cancer therapy. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands, which initiates apoptosis in cancer cells through interaction with the death receptors DR4 and DR5. TRAIL is perceived as an attractive chemotherapeutic agent as it specifically targets cancer cells while sparing the normal cells. However, TRAIL therapy has a major limitation as a large number of the cancer develop resistance toward TRAIL and escape from the destruction by the immune system. Therefore, elucidation of the molecular targets and signaling pathways responsible for TRAIL resistance is imperative for devising effective therapeutic strategies for TRAIL resistant cancers. Although, various molecular targets leading to TRAIL resistance are well-studied, recent studies have implicated that the contribution of some key cellular processes toward TRAIL resistance need to be fully elucidated. These processes primarily include aberrant protein synthesis, protein misfolding, ubiquitin regulated death receptor expression, metabolic pathways, epigenetic deregulation, and metastasis. Novel synthetic/natural compounds that could inhibit these defective cellular processes may restore the TRAIL sensitivity and combination therapies with such compounds may resensitize TRAIL resistant cancer cells toward TRAIL-induced apoptosis. In this review, we have summarized the key cellular processes associated with TRAIL resistance and their status as therapeutic targets for novel TRAIL-sensitizing agents.

Hyaluronan, Inflammation, and Breast Cancer Progression

Abstract: Breast cancer induced inflammation in the tumor reactive stroma supports invasion and malignant progression and is contributed to by a variety of host cells including macrophages and fibroblasts. Inflammation appears to be initiated by tumor cells and surrounding host fibroblasts that secrete pro-inflammatory cytokines and chemokines and remodel the extracellular matrix (ECM) to create a pro-inflammatory “cancerized” or tumor reactive microenvironment that supports tumor expansion and invasion. The tissue polysaccharide hyaluronan (HA) is an example of an ECM component within the cancerized microenvironment that promotes breast cancer progression. Like many ECM molecules the function of native high molecular weight HA is altered by fragmentation, which is promoted by oxygen/nitrogen free radicals and release of hyaluronidases within the tumor microenvironment. HA fragments are pro-inflammatory and activate signaling pathways that promote survival, migration and invasion within both tumor and host cells through binding to HA receptors such as CD44 and RHAMM/HMMR. In breast cancer, elevated HA in the peri-tumor stroma and increased HA receptor expression are prognostic for poor outcome and are associated with disease recurrence. This review addresses the critical issues regarding tumor-induced inflammation and its role in breast cancer progression focusing specifically on the changes in HA metabolism within tumor reactive stroma as a key factor in malignant progression.

JAK Inhibition Impairs NK Cell Function in Myeloproliferative Neoplasms

Abstract: Ruxolitinib is a small-molecule inhibitor of the JAK kinases, which has been approved for the treatment of myelofibrosis, a rare myeloproliferative neoplasm (MPN), but clinical trials are also being conducted in inflammatory-driven solid tumors. Increased infection rates have been reported in ruxolitinib-treated patients, and natural killer (NK) cells are immune effector cells known to eliminate both virus-infected and malignant cells. On this basis, we sought to compare the effects of JAK inhibition on human NK cells in a cohort of 28 MPN patients with or without ruxolitinib treatment and 24 healthy individuals. NK cell analyses included cell frequency, receptor expression, proliferation, immune synapse formation, and cytokine signaling. We found a reduction in NK cell numbers in ruxolitinib-treated patients that was linked to the appearance of clinically relevant infections. This reduction was likely due to impaired maturation of NK cells, as reflected by an increased ratio in immature to mature NK cells. Notably, the endogenous functional defect of NK cells in MPN was further aggravated by ruxolitinib treatment. In vitro data paralleled these in vivo results, showing a reduction in cytokine-induced NK cell activation. Further, reduced killing activity was associated with an impaired capacity to form lytic synapses with NK target cells. Taken together, our findings offer compelling evidence that ruxolitinib impairs NK cell function in MPN patients, offering an explanation for increased infection rates and possible long-term side effects associated with ruxolitinib treatment.

T-bet– and STAT4–dependent IL-33 receptor expression directly promotes antiviral Th1 cell responses

Abstract: During infection, the release of damage-associated molecular patterns, so-called "alarmins," orchestrates the immune response. The alarmin IL-33 plays a role in a wide range of pathologies. Upon release, IL-33 signals through its receptor ST2, which reportedly is expressed only on CD4(+) T cells of the Th2 and regulatory subsets. Here we show that Th1 effector cells also express ST2 upon differentiation in vitro and in vivo during lymphocytic choriomeningitis virus (LCMV) infection. The expression of ST2 on Th1 cells was transient, in contrast to constitutive ST2 expression on Th2 cells, and marked highly activated effector cells. ST2 expression on virus-specific Th1 cells depended on the Th1-associated transcription factors T-bet and STAT4. ST2 deficiency resulted in a T-cell-intrinsic impairment of LCMV-specific Th1 effector responses in both mixed bone marrow-chimeric mice and adoptive cell transfer experiments. ST2-deficient virus-specific CD4(+) T cells showed impaired expansion, Th1 effector differentiation, and antiviral cytokine production. Consequently, these cells mediated little virus-induced immunopathology. Thus, IL-33 acts as a critical and direct cofactor to drive antiviral Th1 effector cell activation, with implications for vaccination strategies and immunotherapeutic approaches.

Evaluating the evidence for targeting FOXO3a in breast cancer: a systematic review

Abstract: Background: Tumour cells show greater dependency on glycolysis so providing a sufficient and rapid energy supply for fast growth. In many breast cancers, estrogen, progesterone and epidermal growth factor receptor-positive cells proliferate in response to growth factors and growth factor antagonists are a mainstay of treatment. However, triple negative breast cancer (TNBC) cells lack receptor expression, are frequently more aggressive and are resistant to growth factor inhibition. Downstream of growth factor receptors, signal transduction proceeds via phosphatidylinositol 3-kinase (PI3k), Akt and FOXO3a inhibition, the latter being partly responsible for coordinated increases in glycolysis and apoptosis resistance. FOXO3a may be an attractive therapeutic target for TNBC. Therefore we have undertaken a systematic review of FOXO3a as a target for breast cancer therapeutics. Methods: Articles from NCBI were retrieved systematically when reporting primary data about FOXO3a expression in breast cancer cells after cytotoxic drug treatment. Results: Increased FOXO3a expression is common following cytotoxic drug treatment and is associated with apoptosis and cell cycle arrest. There is some evidence that metabolic enzyme expression is also altered and that this effect is also elicited in TNBC cells. FOXO3a expression serves as a positive prognostic marker, especially in estrogen (ER) receptor positive cells. Discussion: FOXO3a is upregulated by a number of receptor-dependent and -independent anti-cancer drugs and associates with apoptosis. The identification of microRNA that regulate FOXO3a directly suggest that it offers a tangible therapeutic target that merits wider evaluation.

Interleukin-17 produced by tumor microenvironment promotes self-renewal of CD133 + cancer stem-like cells in ovarian cancer

Abstract: Inflammatory cytokines, components of cancer stem cells (CSCs) niche, could affect the characteristics of CSCs such as self-renewal and metastasis. Interleukin-17 (IL-17) is a new pro-inflammatory cytokine mainly produced by T-helper (Th17) cells and macrophages. The effects of IL-17 on the characteristics of CSCs remain to be explored. Here we first demonstrated a role of IL-17 in promoting the self-renewal of ovarian CD133(+) cancer stem-like cells (CSLCs). We detected IL-17-producing cells (CD4(+) cells and CD68(+) macrophages) in the niche of CD133(+)CSLCs. Meanwhile, there was IL-17 receptor expression on CD133(+)CSLCs derived from A2780 cell line and primary ovarian cancer tissues. By recombinant human IL-17 stimulation and IL-17 transfection, the growth and sphere formation capacities of ovarian CD133(+)CSLCs were significantly enhanced in a dose-dependent manner. Moreover, ovarian CD133(+)CSLCs transfected with IL-17 showed greater tumorigenesis capacity in nude mice. These data suggest that IL-17 promoted the self-renewal of ovarian CD133(+)CSLCs. Further investigation through gene profiling revealed that the stimulation function of IL-17 on self-renewal of ovarian CD133(+)CSLCs might be mediated by the nuclear factor (NF)-κB and p38 mitogen-activated protein kinases (MAPK) signaling pathway. NF-κB and p38 MAPK were activated by IL-17. More importantly, IL-17-promoted self-renewal was inhibited by specific inhibitors of NF-κB and p38 MAPK. Taken together, our data indicate that IL-17 contributed to ovarian cancer malignancy through promoting the self-renewal of CD133(+)CSLCs and that IL-17 and its signaling pathway might serve as therapeutic targets for the treatment of ovarian cancer.

Defining Breast Cancer Intrinsic Subtypes by Quantitative Receptor Expression

Abstract: Purpose. To determine intrinsic breast cancer subtypes represented within categories defined by quantitative hormone receptor (HR) and HER2 expression.

Role of IGF1R in Breast Cancer Subtypes, Stemness, and Lineage Differentiation

Abstract: Insulin-like growth factor (IGF) signaling is fundamental for growth and survival. A large body of evidence (laboratory, epidemiological, and clinical) implicates the exploitation of this pathway in cancer. Up to 50% of breast tumors express the activated form of the type 1 insulin-like growth factor receptor (IGF1R). Breast cancers are categorized into subtypes based upon hormone and ERRB2 receptor expression and/or gene expression profiling. Even though IGF1R influences tumorigenic phenotypes and drug resistance across all breast cancer subtypes, it has specific expression and function in each. In some subtypes, IGF1R levels correlate with a favorable prognosis, while in others it is associated with recurrence and poor prognosis, suggesting different actions based upon cellular and molecular contexts. In this review, we examine IGF1R expression and function as it relates to breast cancer subtype and therapy-acquired resistance. Additionally, we discuss the role of IGF1R in stem cell maintenance and lineage differentiation and how these cell fate influences may alter the differentiation potential and cellular composition of breast tumors.

Role and species-specific expression of colon T cell homing receptor GPR15 in colitis

Abstract: Lymphocyte recruitment maintains intestinal immune homeostasis but also contributes to inflammation. The orphan chemoattractant receptor GPR15 mediates regulatory T cell homing and immunosuppression in the mouse colon. We show that GPR15 is also expressed by mouse TH17 and TH1 effector cells and is required for colitis in a model that depends on the trafficking of these cells to the colon. In humans GPR15 is expressed by effector cells, including pathogenic TH2 cells in ulcerative colitis, but is expressed poorly or not at all by colon regulatory T (Treg) cells. The TH2 transcriptional activator GATA-3 and the Treg-associated transcriptional repressor FOXP3 robustly bind human, but not mouse, GPR15 enhancer sequences, correlating with receptor expression. Our results highlight species differences in GPR15 regulation and suggest it as a potential therapeutic target for colitis.

Tumour necrosis factor-alpha participates on the endothelin-1/nitric oxide imbalance in small arteries from obese patients: role of perivascular adipose tissue

Abstract: Aims We assessed the impact of vascular and perivascular tumour necrosis factor-alpha (TNF-α) on the endothelin (ET)-1/nitric oxide (NO) system and the molecular pathways involved in small arteries from visceral fat of obese patients (Obese) and Controls. Methods and results Isolated small arteries from 16 Obese and 14 Controls were evaluated on a pressurized micromyograph. Endogenous ET-1 activity was assessed by the ETA blocker BQ-123. TNF-α and NO were tested by anti-TNF-α infliximab (IFX) and N ω-nitro-l-arginine methylester (L-NAME). Gene and protein expression of TNF-α, ET-1, ETA, and ETB receptors were determined by RT-PCR and IHC on arterial wall and in isolated adipocytes. Obese showed a blunted L-NAME-induced vasoconstriction, which was potentiated by IFX, and an increased relaxation to BQ-123, unaffected by L-NAME but attenuated by IFX. Perivascular adipose tissue (PVAT) removal reversed these effects. Obese showed intravascular superoxide excess, which was decreased by apocynin (NAD(P)H oxidase inhibitor), L-NAME, and BQ-123 incubations, and abolished by IFX. An increased vascular expression of ET-1, ETA, and ETB receptors, and higher vascular/perivascular TNF-α and TNF-α receptor expression were also detected. The arterial expression and phosphorylation of c-Jun N-terminal kinase (JNK) were higher in Obese vs. Controls, and downregulated by IFX. Conclusions In small arteries of Obese, PVAT-derived TNF-α excess, and an increased vascular expression of ET-1 and ETA receptor, contribute to the ET-1/NO system imbalance, by impairing tonic NO release. Reactive oxygen species excess, via NAD(P)H oxidase activation, induces the endothelial nitric oxide synthase uncoupling, which in turn generates superoxide and impairs NO production. The up-regulated JNK pathway represents a crucial molecular signalling involved in this process.

Interaction of thymic stromal lymphopoietin, IL-33, and their receptors in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps

Abstract: Background Thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 system contribute to the initiation and development of Th2 responses. This study aimed to explore the involvement of TSLP, IL-25, IL-33, and their receptors in type 2 T-helper (Th) responses in chronic rhinosinusitis with nasal polyps (CRSwNPs) and their cross-regulation in human nasal epithelial cells (HNECs). Methods Immunohistochemistry, quantitative RT-PCR, ELISA, Bio-Plex assay, and flow cytometry were used to detect the expression of TSLP/common γ-like TSLP receptor (TSLPR)/IL-7 receptor α (IL-7Rα), IL-25/IL-17B receptor (IL-17RB), and IL-33/membrane-bound ST2 (ST2L)/soluble ST2 (sST2) in sinonasal mucosa and HNECs. HNECs cultured at an air–liquid interface were used to explore the expression in regulation of these cytokine systems. Results Compared with controls and noneosinophilic CRSwNP, the expression of TSLP/TSLPR/IL-7Rα and ST2L/sST2 was significantly increased in eosinophilic CRSwNP, predominantly in epithelial cells. In contrast, the expression of IL-33 and IL-25/IL-17RB was enhanced in epithelial cells in both eosinophilic and noneosinophilic CRSwNP compared to controls. The expression of TSLP, TSLPR, and ST2L was positively correlated with symptom and computer tomography scan scores in eosinophilic CRSwNP and with Th2 cytokine expression in sinonasal mucosa. The expression of ST2L was correlated with TSLP and its receptor expression. TSLP could induce ST2L expression that promoted IL-33-induced TSLP expression in HNECs. In addition, TSLP/TSLPR/IL-7Rα and ST2L could be induced by Th2 cytokines, while IL-25/IL-17RB and IL-33 could be upregulated by Th1/Th17 cytokines, in HNECs. Conclusions The positive feedback loop between TSLP, IL-33 and their receptors, and Th2 cytokines may facilitate Th2-skewed inflammation in eosinophilic CRSwNP.

A pH-Responsive Drug-Delivery Platform Based on Glycol Chitosan-Coated Liposomes.

Abstract: Currently, a substantial amount of effort is focused on developing actively targeted, receptor-specific nanoparticles for the delivery of anticancer drugs and diagnostic imaging contrast agents.[1–7] Receptor-targeted nanoparticles hold much promise and are often shown to provide nanoparticle delivery beyond that seen with the enhanced permeability and retention (EPR) effect alone. However, these nanoparticles still face considerable challenges as a result of the significant heterogeneity in receptor expression, not only between patients and tumor types, but also within individual tumors.[8] In many cases, the overexpressed receptor may also be present on normal tissues leading to detrimental off-target effects. Perhaps even more troubling is that several recent studies have shown that cancer stem cells may not even possess any known up-regulated receptor.[9] Therefore, targeting strategies that are more generalizable across a broad range of tumors than receptor-specific targeting are highly desirable.

Cisplatin Induces Overactivation of the Dormant Primordial Follicle through PTEN/AKT/FOXO3a Pathway which Leads to Loss of Ovarian Reserve in Mice

Abstract: Cisplatin is a first-line chemotherapeutic agent for ovarian cancer that acts by promoting DNA cross links and adduct. However drug resistance and considerable side effects including reproductive toxicity remain a significant challenge. PTEN is well known as a tumor suppressor function which plays a fundamental role in the regulation of the cell cycle, apoptosis and development of cancer. At the same time PTEN has been revealed to be critically important for the maintenance of the primordial follicle pool. In this study, we investigated the role of PTEN/Akt/FOXO3 pathway in cisplatin-induced primordial follicle depletion. Cisplatin induced ovarian failure mouse model was used to evaluate how this pathway involves. In vitro maturation was used for oocyte rescue after cisplatin damage. We found that cisplatin treatment decreased PTEN levels, leading to a subsequent increase in the phosphorylation of key molecules in the pathway. The activation of the PTEN/Akt/FOXO3 pathway cascade increased cytoplasmic translocation of FOXO3a in cisplatin-treated follicles, which in turn increased the pool size of growing follicles, and rapidly depleted the number of dormant follicles. Once activated, the follicles were more prone to apoptosis, and their cumulus cells showed a loss of luteinizing hormone (LH) receptor expression, which leads to failure during final maturation and ovulation. In vitro maturation to rescue oocytes in a cisplatin-treated mouse model resulted in successful maturation and fertilization. This study is the first to show the involvement of the PTEN/Akt/FOXO3 pathway in premature ovarian failure after cisplatin treatment and the possibility of rescue through in vitro maturation.

Contact-dependent carcinoma aggregate dispersion by M2a macrophages via ICAM-1 and β2 integrin interactions

Abstract: // Jing Bai 1, 2, * , Giulia Adriani 1, * , Truong-Minh Dang 3 , Ting-Yuan Tu 1 , Hwei-Xian Leong Penny 3 , Siew-Cheng Wong 3 , Roger D. Kamm 1, 2 , Jean-Paul Thiery 1, 4, 5 1 BioSystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 138602, Singapore 2 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA 3 SIgN (Singapore Immunology Network), A*STAR (Agency for Science, Technology and Research), Biopolis, 138648, Singapore 4 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore 5 Institute of Molecular and Cell Biology, Proteos, 138673, Singapore * These authors have contributed equally to this work Correspondence to: Jean-Paul Thiery, e-mail: bchtjp@nus.edu.sg Keywords: epithelial-mesenchymal transition, microfluidics, macrophage phenotypes, macrophage polarization, cancer microenvironment Received: June 25, 2015 Accepted: July 17, 2015 Published: July 30, 2015 ABSTRACT Tumor-associated macrophages (TAMs) can constitute up to 50% of the tumor mass and have strong implications in tumor progression and metastasis. Macrophages are plastic and can polarize to various subtypes that differ in terms of surface receptor expression as well as cytokine and chemokine production and effector function. Conventionally, macrophages are grouped into two major subtypes: the classically activated M1 macrophages and the alternatively activated M2 macrophages. M1 macrophages are pro-inflammatory, promote T helper (Th) 1 responses, and show tumoricidal activity, whereas M2 macrophages contribute to tissue repair and promote Th2 responses. Herein, we present a microfluidic system integrating tumor cell aggregates and subtypes of human monocyte-derived macrophages in a three-dimensional hydrogel scaffold, in close co-culture with an endothelial monolayer to create an in vitro tumor microenvironment. This platform was utilized to study the role of individual subtypes of macrophages (M0, M1, M2a, M2b and M2c) in human lung adenocarcinoma (A549) aggregate dispersion, as a representation of epithelial-mesenchymal transition (EMT). A significant difference was observed when M2a macrophages were in direct contact with or separated from A549 aggregates, suggesting a possible mechanism for proximity-induced, contact-dependent dissemination via ICAM-1 and integrin β2 interactions. Indeed, M2a macrophages tended to infiltrate and release cells from carcinoma cell aggregates. These findings may help in the development of immunotherapies based on enhancing the tumor-suppressive properties of TAMs.

Practical NK cell phenotyping and variability in healthy adults.

Abstract: Human natural killer (NK) cells display a wide array of surface and intracellular markers that indicate various states of differentiation and/or levels of effector function. These NK cell subsets exist simultaneously in peripheral blood and may vary among individuals. We examined variety among selected NK cell receptors expressed by NK cells from normal donors, as well as the distribution of select NK cell subsets and NK cell receptor expression over time in several individual donors. Peripheral blood mononuclear cells were evaluated using flow cytometry via fluorochrome-conjugated antibodies against a number of NK cell receptors. Results were analyzed for both mean fluorescence intensity (MFI) and the percent positive cells for each receptor. CD56(bright) and CD56(dim) NK cell subsets were also considered separately, as was variation in receptor expression in NK cell subsets over time in selected individuals. Through this effort, we provide ranges of NK cell surface receptor expression for a local adult population as well as provide insight into intra-individual variation.

Curcumin inhibits cancer-associated fibroblast-driven prostate cancer invasion through MAOA/mTOR/HIF-1α signaling.

Abstract: Cancer-associated fibroblasts (CAFs) are key determinants in the malignant progression of cancer, supporting tumorigenesis and metastasis. CAFs also mediate epithelial to mesenchymal transition (EMT) in tumor cells and their achievement of stem cell traits. Curcumin has recently been found to possess anticancer activities via its effect on a variety of biological pathways involved in cancer progression. In this study, we found that CAFs could induce prostate cancer cell EMT and invasion through a monoamine oxidase A (MAOA)/mammalian target of rapamycin (mTOR)/hypoxia-inducible factor-1α (HIF-1α) signaling pathway, which exploits reactive oxygen species (ROS) to drive a migratory and aggressive phenotype of prostate carcinoma cells. Moreover, CAFs was able to increase CXC chemokine receptor 4 (CXCR4) and interleukin-6 (IL-6) receptor expression in prostate cancer cells. However, curcumin abrogated CAF-induced invasion and EMT, and inhibited ROS production and CXCR4 and IL-6 receptor expression in prostate cancer cells through inhibiting MAOA/mTOR/HIF-1α signaling, thereby supporting the therapeutic effect of curcumin in prostate cancer.

Quantitative in vivo cell-surface receptor imaging in oncology: kinetic modeling and paired-agent principles from nuclear medicine and optical imaging.

Abstract: The development of methods to accurately quantify cell-surface receptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surface receptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surface receptor concentrations present in tissue, although each likely has a different clinical niche.

Contribution of MINCLE–SYK Signaling to Activation of Primary Human APCs by Mycobacterial Cord Factor and the Novel Adjuvant TDB

Abstract: Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk-Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF-driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA-mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK-CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE.

Adolescent Intermittent Alcohol Exposure: Persistence of Structural and Functional Hippocampal Abnormalities into Adulthood

Abstract: Adolescence is a critical period for cognitive, emotional, and social maturation (Choudhury et al., 2006) that is accompanied by the pruning of synapses, refinement of neural circuitry, and changes in receptor expression and sensitivity (Kilb, 2012). These processes contribute to the normal maturation of cognitive processes crucial for successful adult function, including planning, inhibitory control, and working memory (Paus, 2005). Adolescence is also a period during which alcohol consumption is often initiated and sustained at high levels (Squeglia et al., 2012). While it has become clear that adolescents respond differently than adults to the acute effects of ethanol (EtOH) on learning, sedation, and motor function (Little et al., 1996;Markwiese et al., 1998; Spear, 2000), the enduring consequences of repeated EtOH exposure during this developmental period have only recently begun to be addressed. In humans, chronic excessive alcohol use during adolescence has been associated with cognitive deficits manifesting in adulthood, particularly in the domain of memory function (Brown et al., 2000; Hanson et al., 2011). In animal models used to reflect human levels of consumption, behavioral studies have shown that adolescent intermittent EtOH (AIE) exposure in rats results in long-lasting changes in EtOH sensitivity, consumption, and aversion (Diaz-Granados and Graham, 2007; Matthews et al., 2008; Risher et al., 2013). With respect to learning and memory in particular, it has been reported that AIE, but not chronic intermittent EtOH (CIE) exposure in adulthood, results in greater sensitivity to the spatial memory-impairing effects of acute EtOH in the radial arm maze, in the absence of an effect on baseline learning ability (Risher et al., 2013; White et al., 2000). Interestingly, AIE has also been shown to reduce the efficacy of EtOH to impair spatial learning in the Morris water maze 24 hours after the last EtOH dose (Silvers et al., 2003, 2006), although it is noteworthy that the effect may have been driven by withdrawal, tolerance, or both, given the brief delay between repeated EtOH exposure and testing. Aside from the subsequent responsiveness to EtOH challenge, Sircar and Sircar (2005) reported that AIE caused deficits in Morris water maze performance up to 25 days after the last EtOH treatment, and fear retention deficits have also been observed 25 days following AIE exposure (Broadwater and Spear, 2013). At the neuronal level in similar AIE rodent models (which achieve equivalent blood EtOH concentrations [BECs]), enduring functional effects are produced that are not apparent when comparable EtOH exposure is administered in adulthood. For example, AIE has been shown to reduce A-type potassium current (IA) in GABAergic hippocampal interneurons (Li et al., 2013), GABAA receptor-mediated tonic current in dentate granule cells (Fleming et al., 2012, 2013) in adulthood, and protein levels of delta and alpha-4 GABA receptors in whole hippocampus (Centanni et al., 2014), while comparable EtOH exposure during adulthood did not produce equivalent long-lasting changes in these cellular functions or receptor proteins. Thus, not only does adolescent EtOH exposure promote long-lasting changes in hippocampal cellular function, but that adolescence is also a period of distinctive vulnerability to the long-term effects of EtOH, enduring even after an extended period of abstinence. While these findings support heightened vulnerability to the long-term consequences of repeated alcohol exposure during adolescence, the extent to which AIE alters subsequent hippocampal neuronal, synaptic, and behavioral processes is unclear. We used hippocampal slices to assess the long-term effects of AIE (an established model of intermittent EtOH exposure) on CA1 structure and function. The CA1 area of the hippocampus was selected because of its role in learning/memory processes and because it has been a focus of studies in the emerging literature on the enduring effects of AIE. Here, we assessed the effects of AIE on long-term potentiation (LTP), dendritic spine morphology (to determine whether AIE alters synaptic structure that in turn can influence synaptic function), and postsynaptic density proteins; the latter of which has been found to influence dendritic spine morphology and play a critical role in the recruitment and stabilization of excitatory synapses and synaptic function (Beique et al., 2006; El-Husseini et al., 2000; Zheng et al., 2012). This approach represents a comprehensive analysis combining physiological, biochemical, and morphological assessments that support the hypothesis that AIE results in neuronal changes that persist into adulthood, emphasizing the vulnerability of this critical developmental period to insults that can potentially alter the trajectory of brain development.

Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis

Abstract: Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts. FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed. Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects. Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

Serotonergic Regulation of Prefrontal Cortical Circuitries Involved in Cognitive Processing: A Review of Individual 5-HT Receptor Mechanisms and Concerted Effects of 5-HT Receptors Exemplified by the Multimodal Antidepressant Vortioxetine.

Abstract: It has been known for several decades that serotonergic neurotransmission is a key regulator of cognitive function, mood, and sleep. Yet with the relatively recent discoveries of novel serotonin (5-HT) receptor subtypes, as well as an expanding knowledge of their expression level in certain brain regions and localization on certain cell types, their involvement in cognitive processes is still emerging. Of particular interest are cognitive processes impacted in neuropsychiatric and neurodegenerative disorders. The prefrontal cortex (PFC) is critical to normal cognitive processes, including attention, impulsivity, planning, decision-making, working memory, and learning or recall of learned memories. Furthermore, serotonergic dysregulation within the PFC is implicated in many neuropsychiatric disorders associated with prominent symptoms of cognitive dysfunction. Thus, it is important to better understand the overall makeup of serotonergic receptors in the PFC and on which cell types these receptors mediate t...