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proliferation - Top 30 Publications

Regulation of c-MYC transcriptional activity by transforming growth factor-beta 1-stimulated clone 22.

C-MYC stimulates cell proliferation through the suppression of cyclin-dependent kinase (CDK) inhibitors including P15 (CDKN2B) and P21 (CDKN1A). It also activates E-box-mediated transcription of various target genes including telomerase reverse transcriptase (TERT) that is involved in cellular immortality and tumorigenesis. Transforming growth factor-beta 1 (TGF-β1)-stimulated clone 22 (TSC-22/TSC22D1) encodes a highly conserved leucine zipper protein that is induced by various stimuli, including TGF-β. TSC-22 inhibits cell growth in mammalian cells and in Xenopus embryos. However, underlying mechanisms of growth inhibition by TSC-22 remain unclear. Here, we show that TSC-22 physically interacts with c-MYC to inhibit the recruitment of c-MYC on the P15 (CDKN2B) and P21 (CDKN1A) promoters, effectively inhibiting c-MYC-mediated suppression of P15 (CDKN2B) and also P21 (CDKN1A) promoter activities. On the other hand, TSC-22 enhances c-MYC-mediated activation of the TERT promoter. Additionally, the expression of TSC-22 in embryonic stem cells inhibits cell growth without affecting its pluripotency-related gene expression. These results indicate that TSC-22 differentially regulates c-MYC-mediated transcriptional activity to regulate cell proliferation. This article is protected by copyright. All rights reserved.

The Role of Estrogens in Pancreatic Islet Physiopathology.

In rodent models of insulin-deficient diabetes, 17β-estradiol (E2) protects pancreatic insulin-producing β-cells against oxidative stress, amyloid polypeptide toxicity, gluco-lipotoxicity, and apoptosis. Three estrogen receptors (ERs)-ERα, ERβ, and the G protein-coupled ER (GPER)-have been identified in rodent and human β-cells. This chapter describes recent advances in our understanding of the role of ERs in islet β-cell function, nutrient homeostasis, survival from pro-apoptotic stimuli, and proliferation. We discuss why and how ERs represent potential therapeutic targets for the maintenance of functional β-cell mass.

Cellular Mechanisms Driving Sex Differences in Adipose Tissue Biology and Body Shape in Humans and Mouse Models.

Sex differences in adipose tissue distribution and the metabolic, endocrine, and immune functions of different anatomical fat depots have been described, but they are incompletely documented in the literature. It is becoming increasingly clear that adipose depots serve distinct functions in males and females and have specific physiological roles. However, the mechanisms that regulate the size and function of specific adipose tissues in men and women remain poorly understood. New insights from mouse models have advanced our understanding of depot differences in adipose growth and remodeling via the proliferation and differentiation of adipose progenitors that can expand adipocyte number in the tissue or simply replace dysfunctional older and larger adipocytes. A limited ability of a depot to expand or remodel can lead to excessive adipocyte hypertrophy, which is often correlated with metabolic dysfunction. However, the relationship of adipocyte size and function varies by depot and sex. For example, femoral adipose tissues of premenopausal women appear to have a greater capacity for adipose expansion via hyperplasia and hypertrophy; although larger, these gluteal-femoral adipocytes remain insulin sensitive. The microenvironment of specific depots, including the composition of the extracellular matrix and cellular composition, as well as cell-autonomous genetic differences, influences sex- and depot-dependent metabolic and growth properties. Although there are some species differences, studies of the molecular and physiological determinants of sex differences in adipocyte growth and function in humans and rodents are both needed for understanding sex differences in health and disease.

The IL-2/Anti-IL-2 Complex Attenuates Cardiac Ischaemia-Reperfusion Injury Through Expansion of Regulatory T Cells.

Regulatory T cells (Tregs) can suppress immunologic damage in myocardial ischaemia/reperfusion injury (MIRI), however, the isolation and ex vivo expansion of these cells for clinical application remains challenging. Here, we investigated whether the IL-2/anti-IL-2 complex (IL-2C), a mediator of Treg expansion, can attenuate MIRI in mice.

MicroRNA-195 Suppresses the Progression of Pancreatic Cancer by Targeting DCLK1.

Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor-specific stem cell marker in pancreatic cancer (PC). MicroRNA-195 (miR-195) plays an important role in many types of tumors. However, the roles of DCLK1 in cancer and miRNAs that directly regulate DCLK1 have not been elucidated. The goal of this study is to assess the effects of miR-195 on inhibiting DCLK1 and to clarify the regulating mechanism of miR-195-DCLK1 in PC cells.

MiRNAs Mediate GDNF-Induced Proliferation and Migration of Glioma Cells.

Glial cell line-derived neurotrophic factor (GDNF) is an important factor promoting invasive glioma growth. This study was performed to reveal a unique mechanism of glioma cell proliferation and migration.

Human Leukocyte Antigen-G Inhibits the Anti-Tumor Effect of Natural Killer Cells via Immunoglobulin-Like Transcript 2 in Gastric Cancer.

Human leukocyte antigen-G (HLA-G) plays an important role in inhibiting natural killer (NK) cell function and promoting immune escape. However, the specific mechanism of HLA-G on NK in gastric cancer (GC) remains not well understood. This study investigated the expression of HLA-G in GC and the role of HLA-G-effected NK cells in GC progression.

The Protective Effects of Κ-Opioid Receptor Stimulation in Hypoxic Pulmonary Hypertension Involve Inhibition of Autophagy Through the AMPK-MTOR Pathway.

In a previous study, we showed that κ-opioid receptor stimulation with the selective agonist U50,488H ameliorated hypoxic pulmonary hypertension (HPH). However, the roles that pulmonary arterial smooth muscle cell (PASMC) proliferation, apoptosis, and autophagy play in κ-opioid receptor-mediated protection against HPH are still unknown. The goal of the present study was to investigate the role of autophagy in U50,488H-induced HPH protection and the underlying mechanisms.

Increased ADAM10 expression in patients with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disease, which is characterized by abnormal of T immunity. A disintegrin and metalloproteinase (ADAM) 10, a member of proteinase family, has been demonstrated to regulate T cell proliferation and effector function. Considering the closely association of dysregulation of T cell function with ITP, whether ADAM10 involves in the pathogenesis of ITP remains unclear. In this study, 54 active ITP patients, 18 ITP in remission and 24 age and gender matched healthy control were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and control for isolation of RNA and plasma which were used to measure mRNA level of ADAM10 and tissue inhibitor of metalloproteinase 3 (TIMP3) by quantitative real-time PCR and soluble level of FasL and lymphocyte activation gene-3 (LAG-3) in plasma by ELISA. Meanwhile, T cell activation was measured by flow cytometry. Our results showed significantly higher expression of ADAM10 and lower expression of TIMP3 in active ITP patients compared with control, which were all restored into normal level in remission patients. Consistent with the expression profile of ADAM10, increased soluble plasma level of FasL and LAG-3 were observed in active ITP patients and reduced to normal level in patients in remission. Furthermore, increased T cell activation as demonstrated by higher expression of HLA-DR and CD69 were found in active ITP patients. In conclusion, elevated expression of ADAM10 was associated with the pathogenesis and development of ITP and therapeutically targeting it might be a novel approach for the treatment of ITP.

Exposure to acrylamide induces cardiac developmental toxicity in zebrafish during cardiogenesis.

Acrylamide (AA), an environmental pollutant, has been linked to neurotoxicity, genotoxicity and carcinogenicity. AA is widely used to synthesize polymers for industrial applications, is widely found in Western-style carbohydrate-rich foods and cigarette smoke, and can also be detected in human umbilical cord blood and breast milk. This is the first study that demonstrated the cardiac developmental toxicity of AA in zebrafish embryos. Post-fertilization exposure to AA caused a clearly deficient cardiovascular system with a shrunken heart and abortive morphogenesis and function. Disordered expression of the cardiac genes, myl7, vmhc, myh6, bmp4, tbx2b and notch1b, as well as reduced number of myocardial cells and endocardial cells, indicated the collapsed development of ventricle and atrium and failed differentiation of atrioventricular canal (AVC). Although cell apoptosis was not affected, the capacity of cardiomyocyte proliferation was significantly reduced by AA exposure after fertilization. Further investigation showed that treatment with AA specifically reduced the expressions of nkx2.5, myl7 and vmhc in the anterior lateral plate mesoderm (ALPM) during the early cardiogenesis. In addition, AA exposure disturbed the restricted expressions of bmp4, tbx2b and notch1b during atrioventricular (AV) valve development and cardiac chambers maturation. Our results showed that AA-induced cardiotoxicity was related to decreased cardiac progenitor genes expression, reduced myocardium growth, abnormal cardiac chambers morphogenesis and disordered AVC differentiation. Our study demonstrates that AA exposure during a time point analogous to the first trimester in humans has a detrimental effect on early heart development in zebrafish. A high ingestion rate of AA-containing products may be an underlying risk factor for cardiogenesis in fetuses.

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus.

Down the local: A qualitative case study of daytime drinking spaces in the London Borough of Islington.

Recognising the lack of research on daytime drinking practices in areas with managed night-time economies (NTEs), this qualitative study explores the phenomena in the London Borough of Islington; a rapidly gentrifying area with a highly regulated night-time economy (NTE). The objectives were to (i) Characterise the daytime drinking spaces of the local alcohol environment and (ii) Theorise the ways in which these spaces, and the practices and performativities within them, are situated within broader social and economic trends.

Buparlisib plus fulvestrant in postmenopausal women with hormone-receptor-positive, HER2-negative, advanced breast cancer progressing on or after mTOR inhibition (BELLE-3): a randomised, double-blind, placebo-controlled, phase 3 trial.

Activation of the PI3K/AKT/mTOR pathway occurs frequently in breast cancer that is resistant to endocrine therapy. Approved mTOR inhibitors effectively inhibit cell growth and proliferation but elicit AKT phosphorylation via a feedback activation pathway, potentially leading to resistance to mTOR inhibitors. We evaluated the efficacy and safety of buparlisib plus fulvestrant in patients with advanced breast cancer who were pretreated with endocrine therapy and mTOR inhibitors.

Functional hepatocyte clusters on bioactive blend silk matrices towards generating bioartificial liver constructs.

The creation of in vitro functional hepatic tissue simulating micro-environmental niche of native liver is a keen area of research due to its demand in bioartificial liver (BAL) and cell-based tissue engineering. Here, we investigated the potential of novel blend (BA) silk scaffold fabricated by blending mulberry (Bombyx mori, BM) silk fibroin with cell adhesion motif (RGD) rich non-mulberry (Antheraea assamensis, AA) silk fibroin in generating a functional liver construct. Three-dimensional (3D) porous silk scaffolds (BM, AA and BA) were physico-chemically characterized and functionally evaluated using human hepatocarcinoma cells (HepG2) and primary neonatal rat hepatocytes. The growth and distribution of hepatocytes within the scaffolds were tracked by FESEM, alamar blue proliferation assay and live/dead staining. Hemocompatible BA scaffolds supported the formation of high density hepatocyte clusters, facilitating cell-matrix and cell-cell interactions. Blend scaffolds evinced enhanced liver-specific functions of cultured hepatocytes in terms of albumin synthesis, urea synthesis and cytochrome P450 enzyme activity over 21 days. Subcutaneous implantation of scaffolds demonstrated minimal macrophage infiltration in blend scaffolds. These findings substantiate that the integral property of blend (BA) scaffold offers a befitting environment by influencing spheroidal growth of hepatocytes with enhanced biological activity. Collectively, the present study provides a new 3D bio-matrix niche for growing functional liver cells that would have future prospects in BAL as well as regenerative medicine.

Antimicrobial eugenol-loaded electrospun membranes of poly(ε-caprolactone)/gelatin incorporated with REDV for vascular graft applications.

Confronted with the risk of stenosis and bacterial infection, vascular grafts are necessarily required with rapid endothelialization and antibacterial activity. In this work, dual-functional electrospun membranes of poly(ε-caprolactone) (PCL)/gelatin were developed. A short active peptide, REDV, was covalently conjugated with a low molecular weight PCL to obtain REDV-PCL-REDV, which was introduced into the electrospun fibers to improve the adhesion and proliferation of vascular endothelial cells (VECs) on the electrospun membranes. Additionally, a plant-extracted antibacterial agent, eugenol, was loaded for the antibacterial purpose. Results suggested that the electrospun membranes demonstrated acceptable mechanical properties and release profiles. The electrospun membrane containing 30% of eugenol could inhibit Escherichia coli and Staphylococcus aureus with 71.6±3.3% and 78.6±2.5% of growth inhibition rates, respectively. Further results showed all the electrospun membranes exhibited lower cytotoxicity towards L929 fibroblasts with more than 80% of relative cell viability. The VEC culture assays indicated that the REDV-modified electrospun membranes by the incorporation of REDV-PCL-REDV could significantly promote VEC adhesion and proliferation. Therefore, the dual-functional electrospun membranes with endothelialization and antibacterial abilities by incorporating REDV and eugenol could be potentially applied as promising vascular grafts.

(-)-9'-O-(α-l-Rhamnopyranosyl)lyoniresinol from Lespedeza cuneata suppresses ovarian cancer cell proliferation through induction of apoptosis.

Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae), known as Chinese bushclover or sericea lespedeza, has been used in traditional medicine to treat diabetes, hematuria, and insomnia, and it has been reported that bioactive compounds from L. cuneata possess various pharmacological properties. However, there has been no study to determine the active compounds from L. cuneata with potential activity against ovarian cancer. This study aimed to isolate cytotoxic compounds from L. cuneata and identify the molecular mechanisms underlying the apoptosis pathway in ovarian cancer cells. Based on cytotoxic activity identified in the screening test, chemical investigation of the active fraction of L. cuneata led to the isolation of nine compounds including four lignanosides (1-4), three flavonoid glycosides (5-7), and two phenolics (8-9). Cytotoxicity and the molecular mechanism were examined by methyl thiazolyl tetrazolium (MTT) assay and Western blot analysis. Of the isolated compounds, (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol (3) demonstrated the strongest effect in suppressing A2780 human ovarian carcinoma cell proliferation in a dose-dependent manner, with an IC50 value of 35.40 ± 2.78 μM. Control A2780 cells had normal morphology, whereas cell blebbing, shrinkage, and condensation were observed after treatment with compound 3. Western blotting analysis showed that compound 3 inhibited A2780 human ovarian cancer cell viability by activating caspase-8, caspase-3, and PARP, which contributed to apoptotic cell death. These results suggest that (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol (3) has potent anticancer activities against A2780 human ovarian carcinoma cells through the extrinsic apoptotic pathway. Therefore, (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol is an excellent candidate for the development of novel chemotherapeutics.

Emerging roles of circular RNA hsa_circ_0000064 in the proliferation and metastasis of lung cancer.

Circular RNAs (circRNAs), a novel class of widespread and diverse endogenous RNAs, can regulate gene expression in mammals. CircRNAs have recently been identified as microRNA sponges and involved in the development of some human diseases. However, the role of circRNAs in the process of tumorigenesis and development of lung cancer remains vague. The purpose of this study is to investigate the role of circRNAs in the lung cancer. In this study, we chose hsa_circ_0000064 as a targeted circRNA to investigate its clinical significances in lung cancer patients. The result indicated that hsa_circ_0000064 was up-regulated in lung cancer tissues and lung cancer cell lines (A549 and H1229). Moreover, its aberrant expression was correlated with several clinical characteristics, including T stage, lymphatic metastasis, and TNM stage. Fluorescence in situ hybridization detected that hsa_circ_0000064 was mostly located in the cytoplasm in A549 and H1229 cells. In addition, knockdown of hsa_circ_0000064 with siRNA dramatically attenuated the proliferation, blocked cell cycle progression, and promoted cell apoptosis. Western blot analysis showed that the protein levels of caspase-3, caspase-9, bax, p21, CDK6 and cyclin D1 significantly restrained by si-hsa_circ_0000064, while the expression of bcl-2 notably increased in A549 and H1229 cells. Further, si-hsa_circ_0000064 also abated migration and invasion activities of A549 and H1229 cells, which may be associated with reduced expressions of MMP-2 and MMP-9. In general, our data suggest that hsa_circ_0000064 represents a novel potential biomarker and therapeutic target of lung cancer.

Interval exercise training increases LIF expression and prevents myocardial infarction-induced skeletal muscle atrophy in rats.

Myocardial infarction (MI) is commonly associated with body weight loss and skeletal muscle atrophy. Studies have shown that exercise training could give beneficial effects on skeletal muscle growth. Leukemia inhibitory factor (LIF) is a key regulator of muscle growth and regeneration. The aim of this study was to investigate the effects of interval exercise training (IET) on the expression of LIF and the MI-induced skeletal muscle atrophy.

Functional role of lncRNA LOC101927497 in N-methyl-N'-nitro-N-nitrosoguanidine-induced malignantly transformed human gastric epithelial cells.

Evidence shows that aberrant expression of long non-coding RNA (lncRNA) is closely associated with tumor development and progression. However, the role of lncRNA in environmental carcinogen induced gastric tumorigenesis remains largely unknown. This study aimed at investigating the function role of lncRNA in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induce malignantly transformed human gastric epithelial cells.

Evidence Suggests Sphingosine 1-Phosphate Might Be Actively Generated, Degraded, and Transported to Extracellular Spaces With Increased S1P2 and S1P3 Expression in Colon Cancer.

A pivotal role of sphingosine 1-phosphate (S1P) in cancer has been suggested based on the ceramide-S1P rheostat theory that the intracellular balance between prosurvival S1P and proapoptotic ceramide determines cell fate. Upregulation of S1P-generating sphingosine kinases (SKs) and downregulation of S1P-degrading S1P lyase (SPL) might increase intracellular S1P levels to exert a prosurvival effect in cancer in general, such as colon cancer. However, we recently observed a distinct S1P metabolism in hepatocellular carcinoma tissues that increased SPL mRNA levels with reduced S1P levels. Thus, we investigated S1P metabolism in colon cancer.

Protein Poly(ADP-ribosyl)ation System: Changes in Development and Aging as well as due to Restriction of Cell Proliferation.

It is well known that the number of dividing cells in an organism decreases with age. The average rate of cell division in tissues and organs of a mature organism sharply decreases, which is probably a trigger for accumulation of damage leading to disturbance of genome integrity. This can be a cause for the development of many age-related diseases and appearance of phenotypic and physiological signs of aging. In this connection, the protein poly(ADP-ribosyl)ation system, which is activated in response to appearance of various DNA damage, attracts great interest. This review summarizes and analyzes data on changes in the poly(ADP-ribosyl)ation system during development and aging in vivo and in vitro, and due to restriction of cell proliferation. Special attention is given to methodological aspects of determination of activity of poly(ADP-ribose) polymerases (PARPs). Analysis of relevant publications and our own data has led us to the conclusion that PARP activity upon the addition of free DNA ends (in this review referred to as stimulated PARP activity) is steadily decreasing with age. However, the dynamics of PARP activity measured without additional activation of the enzyme (in this review referred to as unstimulated activity) does not have such a clear trend: in many studies, the presented differences are statistically non-significant, although it is well known that the number of unrepaired DNA lesions steadily increases with aging. Apparently, the cell has additional regulatory systems that limit its own capability of reacting to DNA damage. Special attention is given to the influence of the cell proliferative status on PARP activity. We have systematized and analyzed data on changes in PARP activity during development and aging of an organism, as well as data on differences in the dynamics of this activity in the presence/absence of additional stimulation and on cellular processes that are associated with activation of these enzymes. Moreover, data obtained in different models of cellular aging are compared.

Cathelicidin LL37 Promotes Epithelial and Smooth-Muscle-Like Differentiation of Adipose-Derived Stem Cells through the Wnt/β-Catenin and NF-κB Pathways.

Ureter reconstruction is a difficult procedure in urology. Adipose-derived stem cells (ADSCs), along with multipotency and self-renewal capacity, are a preferred choice for tissue engineering-based ureteral reconstruction. We explored the synergic role of cathelicidin LL37 (LL37) in epithelial and smooth-muscle-like differentiation. ADSCs were separated from adipose tissues of mouse and characterized by flow cytometry. The ADSCs were then stably transfected with pGC-FU-GFP (pGC) or pGC containing full-length LL37 (pGC-LL37), respectively. Cell viability and apoptosis were respectively estimated in the stably transfected cells and non-transfected cells. Then, qRT-PCR and Western blot analysis were used for determinations of epithelial marker expressions after induction by all-trans retinoic acid as well as smooth-muscle-like marker expressions after induction by transforming growth factor-β1. Then, possibly involved signaling pathways and extracellular expression of LL37 were detected. Cell viability and apoptosis were not changed after LL37 overexpression. Expression levels of epithelial and smooth-muscle-like markers were significantly upregulated by LL37 overexpression. Moreover, expressions of key kinases involved in the Wnt/β-catenin pathway as well as epithelial marker were upregulated by the LL37 overexpression, while it was reversed by Wnt/β-catenin inhibitor. Likewise, expressions of key kinases involved in the nuclear factor κB (NF-κB) pathway as well as smooth-muscle-like markers were upregulated by LL37 overexpression, which was reversed by NF-κB inhibitor. LL37 was found in the culture medium. LL37, which could be released into the medium, had no impact on cell proliferation and apoptosis of ADSCs. However, LL37 promoted epithelial and smooth-muscle-like differentiation through activating the Wnt/β-catenin and NF-κB pathways, respectively.

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi-Pasteur Denvaxia vaccine does not protect children under the age of nine and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant-expression to produce a humanised and highly immunogenic Poly-Immunoglobulin G Scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralising antibodies and evidence of cell-mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen-specific CD4+ and CD8+ T cell proliferation, IFN-γ and antibody production. The purified polymeric fraction of dengue PIGS (D-PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low affinity Fcγ receptors on antigen-presenting cells. These results show that the plant-expressed D-PIGS have the potential for translation towards a safe and easily scalable single antigen based tetravalent dengue vaccine. This article is protected by copyright. All rights reserved.

Stemazole Promotes Survival and Preserves Stemness in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) are extremely delicate, and survive poorly under suboptimal culture conditions, severely restricting long-term studies and practical applications. Thus, a protective agent that promotes stem cell survival is urgently needed. In this study, we evaluated the protective effects of stemazole in single-cell and starved hESC cultures. Colony formation was quantified by alkaline phosphatase and immunofluorescence staining, while apoptosis was assessed by flow cytometry and TUNEL assay. Expression of hESC and other stem cell markers was evaluated by western blot, RT-PCR, and QPCR. We found that stemazole enhanced clonal expansion from single cells in dose-dependent fashion, and clearly decreased apoptosis from 54.1% to 25.2%. Furthermore, the drug reduced apoptosis from 43.6% to 8.4% over 15 hours of starvation, with 66% of stemazole-treated cells remaining viable after two weeks of starvation. Importantly, starved cells protected with stemazole retained the same proliferation and differentiation properties as cells in normal culture. In conclusion, stemazole significantly promotes survival of stem cells in single-cell or starvation cultures without compromising stemness and pluripotency. This article is protected by copyright. All rights reserved.

Elevated levels of the antimicrobial peptide LL-37 in hidradenitis suppurativa are associated with a Th1/Th17 immune response.

Hidradenitis suppurativa (HS) is an inflammatory skin disease with poorly understood immunopathogenic mechanisms. LL-37 is an antimicrobial peptide, which is transcribed from the CAMP (cathelicidin antimicrobial peptide) gene. Previous reports showed upregulated levels of CAMP and LL-37 in HS lesions and therefore the aim of this study was to compare levels of LL-37 in HS to other inflammatory skin diseases and to establish immunomodulatory functions of LL-37 in HS. We confirm an upregulation of the LL-37 peptide in lesional HS skin with comparable levels as in psoriasis patients and are able to positively correlate the presence of LL-37 in HS with the presence of T cells, macrophages, neutrophils, IFNγ, IL-17, IL-23, TNFα, IL-32 and IL-1β. Mechanistically, LL-37 boosts the proliferation of unspecifically activated CD4+ T cells via an increased calcium signalling independent of antigen presenting cells. Targeting LL-37 may therefore represent a new therapeutic option for the treatment of this recalcitrant disease, but it has to be kept in mind that LL-37 also has an antimicrobial function. This article is protected by copyright. All rights reserved.

Dominant role of microglial and macrophage innate immune responses in human ischemic infarcts.

Inflammatory mechanisms, involving granulocytes, T-cells, B-cells, macrophages and activated microglia, have been suggested to play a pathogenic role in experimental models of stroke and may be targets for therapeutic intervention. However, knowledge on the inflammatory response in human stroke lesions is limited. Here, we performed a quantitative study on the inflammatory reaction in human ischemic infarct lesions. We found increased numbers of T-lymphocytes, mainly CD8+ cells, but not of B-lymphocytes. Their number was very low in comparison to that seen in inflammatory diseases of the central nervous system and they did not show signs of activation. Polymorphonuclear leukocytes were present in meninges and less prominently in the perivascular space in early lesions, but their infiltration into the lesioned tissue was sparse with the exception of a single case. Microglia were lost in the necrotic core of fresh lesions, their number was increased in the surrounding penumbra, apparently due to proliferation. Using TMEM119 as a marker for the resident microglia pool, macrophages in lesions were in part derived from the original microglia pool, depending upon the lesion stage. Most microglia and macrophages revealed a pro-inflammatory activation pattern, expressing molecules involved in phagocytosis, oxidative injury, antigen presentation and iron metabolism and had partially lost the expression of P2RY12, an antigen expressed on homeostatic ("resting") microglia in rodents. At later lesion stages, the majority of macrophages showed intermediate activation patterns, expressing pro-inflammatory and anti-inflammatory markers. Microglia in the normal white matter of controls and stroke patients were already partly activated towards a pro-inflammatory phenotype. Our data suggest that the direct contribution of lymphocytes and granulocytes to active tissue injury in human ischemic infarct lesions is limited and that stroke therapy that targets pro-inflammatory microglia and macrophage activation may be effective. This article is protected by copyright. All rights reserved.

RELN signaling modulates glioblastoma growth and substrate-dependent migration.

Glioblastoma (GBM) represents the most common and most malignant type of primary brain tumor and significantly contributes to cancer morbidity and mortality. Invasion into the healthy brain parenchyma is a major feature of glioblastoma aggressiveness. Reelin (RELN) is a large secreted extracellular matrix glycoprotein that regulates neuronal migration and positioning in the developing brain and sustains functionality in the adult brain. We here show that both RELN and its main downstream effector DAB1 are silenced in glioblastoma as compared to non-neoplastic tissue and mRNA expression is inversely correlated with malignancy grade. Furthermore, RELN expression is positively correlated with patient survival in two large, independent clinically annotated datasets. RELN silencing occurs via promoter hypermethylation as shown by both database mining and bisulfite sequencing of the RELN promoter. Consequently, treatment with 5'-Azacytidine and trichostatin A induced RELN expression in vitro. On the functional level, we found RELN to regulate glioblastoma cell migration both in a DAB1 (tyrosine phosphorylation)-dependent and -independent fashion, depending on the substrate provided. Moreover, stimulation of RELN signaling strongly reduced proliferation in glioblastoma cells. This phenotype depends on DAB1 stimulation by RELN, as a mutant that lacks all RELN induced tyrosine phosphorylation sites (DAB1-5F) failed to induce a growth arrest. Proteomic analyzes revealed that these effects are mediated by a reduction in E2F targets and dephosphorylation of ERK1/2. Taken together, our data establish a relevance of RELN signaling in glioblastoma pathology and thereby might unearth novel, yet unrecognized treatment options. This article is protected by copyright. All rights reserved.

Culture of Human Primary Bone Cells and Phenotype Assessment.

Bone engineering scaffolds and biomimetic substitutes are currently the leading techniques to repair and regenerate bone defects. Ideally, bone grafts should imitate the structure and properties of bone extracellular matrix, house osteoprogenitor cells, and provide all the necessary environmental cues to orchestrate the functions of osteoblast and osteoclast cells. Consequently, there is an increasing demand for preclinical models based on in vitro bone-derived cell cultures for screening of novel biomaterials. In this chapter, we provide the protocols for culture of primary human bone cells from explants of cortical mandible bone and for characterization of cell behavior on biomimetic surfaces in terms of strength of adhesion, proliferation, differentiation, and matrix mineralization.

Nerve Growth Factor-Induced Angiogenesis: 1. Endothelial Cell Tube Formation Assay.

Nerve growth factor (NGF) is a neurotrophin promoting survival, proliferation, differentiation, and neuroprotection in the embryonal and adult nervous system. NGF also induces angiogenic effects in the cardiovascular system, which may be beneficial in engineering new blood vessels and for developing novel anti-angiogenesis therapies for cancer. Angiogenesis is a cellular process characterized by a number of events, including endothelial cell migration, invasion, and assembly into capillaries. In vitro endothelial tube formation assays are performed using primary human umbilical vein endothelial cells, human aortic endothelial cells, and other human or rodent primary endothelial cells isolated from the vasculature of both tumors and normal tissues. Immortalized endothelial cell lines are also used for these assays. When seeded onto Matrigel, these cells reorganize to create tubelike structure, which may be used as models for studying some aspects of in vitro angiogenesis. Image acquisition by light and fluorescence microscopy and/or quantification of fluorescently labeled cells can be carried out manually or digitally, using commercial software and automated image processing. Here we detail materials, procedure, assay conditions, and cell labeling for quantification of endothelial cell tube formation. This model can be applied to study cellular and molecular mechanisms by which NGF or other neurotrophins promote angiogenesis. This model may also be useful for the development of potential angiogenic and/or anti-angiogenic drugs targeting NGF receptors.

Hypoxia-inducible factor 1α is Essential for Macrophage-mediated Erythroblast Proliferation in Acute Friend Retrovirus Infection.

Macrophages are the frontline of defence against foreign microorganisms, including bacteria, parasites, and viruses. During acute viral infection, macrophages must invade the inflamed tissue toward low oxygen concentrations, where genetic cellular responses depend on hypoxia-inducible factors (HIF). In the study reported here we investigated the role of HIF-1α in macrophage function during acute retroviral infection. Wild-type and myeloid cell-specific HIF-1α knockout mice were infected with Friend retrovirus (FV), and immune response was analysed 7 and 10 days after infection. FV infection led to increased spleen weight in wild-type and knockout mice, whereas a profound proliferation of erythroblasts was seen only in wild-type mice. The number of spleen-infiltrating macrophages was also significantly lower in knockout animals. Macrophage invasion after FV infection in wild-type mice led to elevated amounts of activated macrophage-stimulating 1 protein that resulted in massive proliferation of erythrocyte precursor cells. This proliferation was absent from knockout mice because of impaired invasion capabilities of HIF-1α-deficient macrophages. Our study elucidated a novel mechanism of FV-induced erythrocyte precursor cell proliferation.