Reconstructing human pancreatic differentiation by mapping specific cell populations during development

PubMed Central

Ramond, Cyrille; Glaser, Nicolas; Berthault, Claire; Ameri, Jacqueline; Kirkegaard, Jeannette Schlichting; Hansson, Mattias; Honoré, Christian; Semb, Henrik; Scharfmann, Raphaël

2017-01-01

Information remains scarce on human development compared to animal models. Here, we reconstructed human fetal pancreatic differentiation using cell surface markers. We demonstrate that at 7weeks of development, the glycoprotein 2 (GP2) marks a multipotent cell population that will differentiate into the acinar, ductal or endocrine lineages. Development towards the acinar lineage is paralleled by an increase in GP2 expression. Conversely, a subset of the GP2+ population undergoes endocrine differentiation by down-regulating GP2 and CD142 and turning on NEUROG3, a marker of endocrine differentiation. Endocrine maturation progresses by up-regulating SUSD2 and lowering ECAD levels. Finally, in vitro differentiation of pancreatic endocrine cells derived from human pluripotent stem cells mimics key in vivo events. Our work paves the way to extend our understanding of the origin of mature human pancreatic cell types and how such lineage decisions are regulated. DOI: http://dx.doi.org/10.7554/eLife.27564.001 PMID:28731406

Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells.

PubMed

Lund, Carina; Pulli, Kristiina; Yellapragada, Venkatram; Giacobini, Paolo; Lundin, Karolina; Vuoristo, Sanna; Tuuri, Timo; Noisa, Parinya; Raivio, Taneli

2016-08-09

Gonadotropin-releasing hormone (GnRH) neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs) into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs) by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Quantification of Crypt and Stem Cell Evolution in the Normal and Neoplastic Human Colon

PubMed Central

Baker, Ann-Marie; Cereser, Biancastella; Melton, Samuel; Fletcher, Alexander G.; Rodriguez-Justo, Manuel; Tadrous, Paul J.; Humphries, Adam; Elia, George; McDonald, Stuart A.C.; Wright, Nicholas A.; Simons, Benjamin D.; Jansen, Marnix; Graham, Trevor A.

2014-01-01

Summary Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a “functional” stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC−/+). Furthermore, we show that, in adenomatous crypts (APC−/−), there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics. PMID:25127143

Structural Complexity of Non-acid Glycosphingolipids in Human Embryonic Stem Cells Grown under Feeder-free Conditions*

PubMed Central

Barone, Angela; Benktander, John; Ångström, Jonas; Aspegren, Anders; Björquist, Petter; Teneberg, Susann; Breimer, Michael. E.

2013-01-01

Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 109 cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Lex pentaosylceramide, and the Ley hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated. PMID:23404501

Our Fat Future: Translating Adipose Stem Cell Therapy.

PubMed

Nordberg, Rachel C; Loboa, Elizabeth G

2015-09-01

Human adipose stem cells (hASCs) have the potential to treat patients with a variety of clinical conditions. Recent advancements in translational research, regulatory policy, and industry have positioned hASCs on the threshold of clinical translation. We discuss the progress and challenges of bringing adipose stem cell therapy into mainstream clinical use. This article details the advances made in recent years that have helped move human adipose stem cell therapy toward mainstream clinical use from a translational research, regulatory policy, and industrial standpoint. Four recurrent themes in translational technology as they pertain to human adipose stem cells are discussed: automated closed-system operations, biosensors and real-time monitoring, biomimetics, and rapid manufacturing. In light of recent FDA guidance documents, regulatory concerns about adipose stem cell therapy are discussed. Finally, an update is provided on the current state of clinical trials and the emerging industry that uses human adipose stem cells. This article is expected to stimulate future studies in translational adipose stem cell research. ©AlphaMed Press.

A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

PubMed

Cecchelli, Romeo; Aday, Sezin; Sevin, Emmanuel; Almeida, Catarina; Culot, Maxime; Dehouck, Lucie; Coisne, Caroline; Engelhardt, Britta; Dehouck, Marie-Pierre; Ferreira, Lino

2014-01-01

The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

Effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells investigated by atomic force microscopy.

PubMed

Li, Mi; Liu, LianQing; Xi, Ning; Wang, YueChao; Xiao, XiuBin; Zhang, WeiJing

2015-09-01

Cell mechanics plays an important role in cellular physiological activities. Recent studies have shown that cellular mechanical properties are novel biomarkers for indicating the cell states. In this article, temperature-controllable atomic force microscopy (AFM) was applied to quantitatively investigate the effects of temperature and cellular interactions on the mechanics and morphology of human cancer cells. First, AFM indenting experiments were performed on six types of human cells to investigate the changes of cellular Young's modulus at different temperatures and the results showed that the mechanical responses to the changes of temperature were variable for different types of cancer cells. Second, AFM imaging experiments were performed to observe the morphological changes in living cells at different temperatures and the results showed the significant changes of cell morphology caused by the alterations of temperature. Finally, by co-culturing human cancer cells with human immune cells, the mechanical and morphological changes in cancer cells were investigated. The results showed that the co-culture of cancer cells and immune cells could cause the distinct mechanical changes in cancer cells, but no significant morphological differences were observed. The experimental results improved our understanding of the effects of temperature and cellular interactions on the mechanics and morphology of cancer cells.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

PubMed

Dussault, Nathalie; Ducas, Eric; Racine, Claudia; Jacques, Annie; Paré, Isabelle; Côté, Serge; Néron, Sonia

2008-11-01

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Generation of functional podocytes from human induced pluripotent stem cells.

PubMed

Ciampi, Osele; Iacone, Roberto; Longaretti, Lorena; Benedetti, Valentina; Graf, Martin; Magnone, Maria Chiara; Patsch, Christoph; Xinaris, Christodoulos; Remuzzi, Giuseppe; Benigni, Ariela; Tomasoni, Susanna

2016-07-01

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro. Copyright © 2016. Published by Elsevier B.V.

Fermentation product of soybean, black bean, and green bean mixture induces apoptosis in a wide variety of cancer cells.

PubMed

Chia, Jean-San; Du, Jia-Ling; Wu, Ming-Shiou; Hsu, Wei-Bin; Chiang, Chun-Pin; Sun, Andy; Lu, John Jenn-Yenn; Wang, Won-Bo

2013-05-01

Previous studies have shown that soybean fermentation products can act as cancer chemoprevention or therapeutic agents. In this study, the anticancer activities of a fermentation product of soybean, black bean, and green bean mixture (BN999) were investigated. We found that BN999 inhibited the growth of human breast cancer AU565 cells and prostate adenocarcinoma PC-3 cells but not that of normal human cells. BN999 induced apoptosis in various human cancer cells but not in normal human cells. BN999 treatment of AU565 cancer cells resulted in activation of calpain and caspase-8, -9, and -3, suggesting that BN999 induces apoptosis via receptor-, mitochondria-, and endoplasmic reticulum-mediated pathways. Finally, we showed that BN999 inhibited the growth of mouse CT-26 colon cancer xenografts in syngenic BALB/c mice without causing obvious side effects. Together, these data suggest that BN999 has potential to be used as a cancer chemoprevention or therapeutic agent.

Mitosis can drive cell cannibalism through entosis

PubMed Central

Durgan, Joanne; Tseng, Yun-Yu; Hamann, Jens C; Domart, Marie-Charlotte; Collinson, Lucy; Overholtzer, Michael; Florey, Oliver

2017-01-01

Entosis is a form of epithelial cell cannibalism that is prevalent in human cancer, typically triggered by loss of matrix adhesion. Here, we report an alternative mechanism for entosis in human epithelial cells, driven by mitosis. Mitotic entosis is regulated by Cdc42, which controls mitotic morphology. Cdc42 depletion enhances mitotic deadhesion and rounding, and these biophysical changes, which depend on RhoA activation and are phenocopied by Rap1 inhibition, permit subsequent entosis. Mitotic entosis occurs constitutively in some human cancer cell lines and mitotic index correlates with cell cannibalism in primary human breast tumours. Adherent, wild-type cells can act efficiently as entotic hosts, suggesting that normal epithelia may engulf and kill aberrantly dividing neighbours. Finally, we report that Paclitaxel/taxol promotes mitotic rounding and subsequent entosis, revealing an unconventional activity of this drug. Together, our data uncover an intriguing link between cell division and cannibalism, of significance to both cancer and chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.27134.001 PMID:28693721

A green approach toward quinoxalines and bis-quinoxalines and their biological evaluation against A431, human skin cancer cell lines.

PubMed

Bandyopadhyay, Debasish; Cruz, Jessica; Morales, Liza D; Arman, Hadi D; Cuate, Erica; Lee, Young S; Banik, Bimal K; Kim, Dae J

2013-08-01

The objective of this study was to develop a practical green procedure to synthesize quinoxalines and bis-quinoxalines and evaluate their inhibitory effects on the viability of A431 human epidermoid carcinoma cells. A series of quinoxaline and bis-quinoxaline derivatives have been designed and synthesized following a microwave-assisted and bismuth nitrate-catalyzed eco-friendly route. A detailed comparison has been made between microwave-induced protocol with the reactions occurred at room temperature. The structure of the compounds have been elucidated by various spectroscopic methods and finally confirmed by x-ray crystallographic analyses. Two quinoxaline derivatives, compounds 6 and 12 have demonstrated inhibitory effects on the viability of A431 human epidermoid carcinoma cells when compared with HaCaT nontumorigenic human keratinocyte cells. Notably, compound 6 inhibits Stat3 phosphorylation/activation in A431 skin cancer cells.

4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin

PubMed Central

1984-01-01

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120- kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts. PMID:6538202

Investigation of Influenza Virus Polymerase Activity in Pig Cells

PubMed Central

Moncorgé, Olivier; Long, Jason S.; Cauldwell, Anna V.; Zhou, Hongbo; Lycett, Samantha J.

2013-01-01

Reassortant influenza viruses with combinations of avian, human, and/or swine genomic segments have been detected frequently in pigs. As a consequence, pigs have been accused of being a “mixing vessel” for influenza viruses. This implies that pig cells support transcription and replication of avian influenza viruses, in contrast to human cells, in which most avian influenza virus polymerases display limited activity. Although influenza virus polymerase activity has been studied in human and avian cells for many years by use of a minigenome assay, similar investigations in pig cells have not been reported. We developed the first minigenome assay for pig cells and compared the activities of polymerases of avian or human influenza virus origin in pig, human, and avian cells. We also investigated in pig cells the consequences of some known mammalian host range determinants that enhance influenza virus polymerase activity in human cells, such as PB2 mutations E627K, D701N, G590S/Q591R, and T271A. The two typical avian influenza virus polymerases used in this study were poorly active in pig cells, similar to what is seen in human cells, and mutations that adapt the avian influenza virus polymerase for human cells also increased activity in pig cells. In contrast, a different pattern was observed in avian cells. Finally, highly pathogenic avian influenza virus H5N1 polymerase activity was tested because this subtype has been reported to replicate only poorly in pigs. H5N1 polymerase was active in swine cells, suggesting that other barriers restrict these viruses from becoming endemic in pigs. PMID:23077313

Hydrophobic chalcogenide fibers for cell-based bio-optical sensors

NASA Astrophysics Data System (ADS)

Lucas, Pierre; Riley, Mark R.; Solis, Michelle A.; Juncker, Christophe; Collier, Jayne; Boesewetter, Dianne E.

2005-03-01

Chalcogenide fibers are shown to exhibit a hydrophobic surface behavior which results in detection enhancement for organic species in aqueous solutions. We use these fibers to monitor the infrared signature of human lung cells and detect the presence of toxic agents in the cell surrounding media. The signal is collected using a fiber evanescent wave spectroscopy set up with live human cells acting as a sensitizer for detection of minute quantities of toxicant. A monolayer of human alveolar epithelial cells form strong attachment at the surface of the fiber sensing zone and live in contact with the fiber while their IR spectra is collected remotely. Biochemical change in the living cells are detected during exposure to toxic agents. Variations in the spectroscopic features of the cells are observed in different spectral regions. Finally, the toxicity of Te2As3Se5 fibers is investigated.

Membrane oxidation in cell delivery and cell killing applications

PubMed Central

Wang, Ting-Yi; Libardo, M. Daben J.; Angeles-Boza, Alfredo M.; Pellois, Jean-Philippe

2018-01-01

Cell delivery or cell killing processes often involve the crossing or disruption of cellular membranes. We review how, by modifying the composition and properties of membranes, membrane oxidation can be exploited to enhance the delivery of macromolecular cargos into live human cells. We also describe how membrane oxidation can be utilized to achieve efficient killing of bacteria by antimicrobial peptides. Finally, we present recent evidence highlighting how membrane oxidation is intimately engaged in natural biological processes such as antigen delivery in dendritic cells and in the killing of bacteria by human macrophages. Overall, the insights that have been recently gained in this area should facilitate the development of more effective delivery technologies and antimicrobial therapeutic approaches. PMID:28355059

Human melanoma immunotherapy using tumor antigen-specific T cells generated in humanized mice

PubMed Central

Hu, Zheng; Xia, Jinxing; Fan, Wei; Wargo, Jennifer; Yang, Yong-Guang

2016-01-01

A major factor hindering the exploration of adoptive immunotherapy in preclinical settings is the limited availability of tumor-reactive human T cells. Here we developed a humanized mouse model that permits large-scale production of human T cells expressing the engineered melanoma antigen MART-1-specific TCR. Humanized mice, made by transplantation of human fetal thymic tissue and CD34+ cells virally-transduced with HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene, showed efficient development of MART-1-TCR+ human T cells with predominantly CD8+ cells. Importantly, MART-1-TCR+CD8+ T cells developing in these mice were capable of mounting antigen-specific responses in vivo, as evidenced by their proliferation, phenotypic conversion and IFN-γ production following MART-1 peptide immunization. Moreover, these MART-1-TCR+CD8+ T cells mediated efficient killing of melanoma cells in an HLA/antigen-dependent manner. Adoptive transfer of in vitro expanded MART-1-TCR+CD8+ T cells induced potent antitumor responses that were further enhanced by IL-15 treatment in melanoma-bearing recipients. Finally, a short incubation of MART-1-specific T cells with rapamycin acted synergistically with IL-15, leading to significantly improved tumor-free survival in recipients with metastatic melanoma. These data demonstrate the practicality of using humanized mice to produce potentially unlimited source of tumor-specific human T cells for experimental and preclinical exploration of cancer immunotherapy. This study also suggests that pretreatment of tumor-reactive T cells with rapamycin in combination with IL-15 administration may be a novel strategy to improve the efficacy of adoptive T cell therapy. PMID:26824989

Acute cardiovascular toxicity of sterilizers, PHMG, and PGH: severe inflammation in human cells and heart failure in zebrafish.

PubMed

Kim, Jae-Yong; Kim, Hak Hyeon; Cho, Kyung-Hyun

2013-06-01

In 2011, dozens of children and pregnant women in Korea died by exposure to sterilizer for household humidifier, such as Oxy(®) and Cefu(®). Until now, however, it remains unknown how the sterilizer affect the human health to cause the acute deaths. To find its toxicity for organ, we investigated the putative toxicity of the sterilizer in the cardiovascular system. The sterilizers, polyhexamethylene guanidine phosphate (PHMG, Cefu(®)), and oligo-[2-(2-ethoxy)-ethoxyethyl)-guanidinium-chloride (PGH, Oxy(®)) were treated to human lipoproteins, macrophages, and dermal fibroblast cells. The PGH and PHMG at normal dosages caused severe atherogenic process in human macrophages, cytotoxic effect, and aging in human dermal cell. Zebrafish embryos, which were exposed to the sterilizer, showed early death with acute inflammation and attenuated developmental speed. All zebrafish exposed to the working concentration of PHMG (final 0.3 %) and PGH (final 10 mM) died within 70 min and displayed acute increases in serum triacylglycerol level and fatty liver induction. The dead zebrafish showed severe accumulation of fibrous collagen in the bulbous artery of the heart with elevation of reactive oxygen species. In conclusion, the sterilizers showed acute toxic effect in blood circulation system, causing by severe inflammation, atherogenesis, and aging, with embryo toxicity.

Immunohistochemical Analysis of Human Vallate Taste Buds.

PubMed

Tizzano, Marco; Grigereit, Laura; Shultz, Nicole; Clary, Matthew S; Finger, Thomas E

2015-11-01

The morphology of the vallate papillae from postmortem human samples was investigated with immunohistochemistry. Microscopically, taste buds were present along the inner wall of the papilla, and in some cases in the outer wall as well. The typical taste cell markers PLCβ2, GNAT3 (gustducin) and the T1R3 receptor stain elongated cells in human taste buds consistent with the Type II cells in rodents. In the human tissue, taste bud cells that stain with Type II cell markers, PLCβ2 and GNAT3, also stain with villin antibody. Two typical immunochemical markers for Type III taste cells in rodents, PGP9.5 and SNAP25, fail to stain any taste bud cells in the human postmortem tissue, although these antibodies do stain numerous nerve fibers throughout the specimen. Car4, another Type III cell marker, reacted with only a few taste cells in our samples. Finally, human vallate papillae have a general network of innervation similar to rodents and antibodies directed against SNAP25, PGP9.5, acetylated tubulin and P2X3 all stain free perigemmal nerve endings as well as intragemmal taste fibers. We conclude that with the exception of certain molecular features of Type III cells, human vallate papillae share the structural, morphological, and molecular features observed in rodents. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Functional Characterization of the Human Mariner Transposon Hsmar2

PubMed Central

Gil, Estel; Bosch, Assumpcio; Lampe, David; Lizcano, Jose M.; Perales, Jose C.; Danos, Olivier; Chillon, Miguel

2013-01-01

DNA transposons are mobile elements with the ability to mobilize and transport genetic information between different chromosomal loci. Unfortunately, most transposons copies are currently inactivated, little is known about mariner elements in humans despite their role in the evolution of the human genome, even though the Hsmar2 transposon is associated to hotspots for homologous recombination involved in human genetic disorders as Charcot–Marie–Tooth, Prader-Willi/Angelman, and Williams syndromes. This manuscript describes the functional characterization of the human HSMAR2 transposase generated from fossil sequences and shows that the native HSMAR2 is active in human cells, but also in bacteria, with an efficiency similar to other mariner elements. We observe that the sub-cellular localization of HSMAR2 is dependent on the host cell type, and is cytotoxic when overexpressed in HeLa cells. Finally, we also demonstrate that the binding of HSMAR2 to its own ITRs is specific, and that the excision reaction leaves non-canonical footprints both in bacteria and eukaryotic cells. PMID:24039890

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells.

PubMed

Shi, Cheng; Shen, Huan; Jiang, Wei; Song, Zhi-Hua; Wang, Cheng-Yan; Wei, Li-Hui

2011-04-01

Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability. Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propagate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells. We generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line. This newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo development research, along with clinical treatments for diabetes and some hepatic diseases.

Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.

PubMed

Rodrigues, Gonçalo M C; Fernandes, Tiago G; Rodrigues, Carlos A V; Cabral, Joaquim M S; Diogo, Maria Margarida

2015-01-01

Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

Sulforaphane inhibits hypoxia-induced HIF-1α and VEGF expression and migration of human colon cancer cells.

PubMed

Kim, Dong Hwan; Sung, Bokyung; Kang, Yong Jung; Hwang, Seong Yeon; Kim, Min Jeong; Yoon, Jeong-Hyun; Im, Eunok; Kim, Nam Deuk

2015-12-01

The effects of sulforaphane (a natural product commonly found in broccoli) was investigated on hypoxia inducible factor-1α (HIF-1α) expression in HCT116 human colon cancer cells and AGS human gastric cancer cells. We found that hypoxia-induced HIF-1α protein expression in HCT116 and AGS cells, while treatment with sulforaphane markedly and concentration-dependently inhibited HIF-1α expression in both cell lines. Treatment with sulforaphane inhibited hypoxia-induced vascular endothelial growth factor (VEGF) expression in HCT116 cells. Treatment with sulforaphane modulated the effect of hypoxia on HIF-1α stability. However, degradation of HIF-1α by sulforaphane was not mediated through the 26S proteasome pathway. We also found that the inhibition of HIF-1α by sulforaphane was not mediated through AKT and extracellular signal-regulated kinase phosphorylation under hypoxic conditions. Finally, hypoxia-induced HCT116 cell migration was inhibited by sulforaphane. These data suggest that sulforaphane may inhibit human colon cancer progression and cancer cell angiogenesis by inhibiting HIF-1α and VEGF expression. Taken together, these results indicate that sulforaphane is a new and potent chemopreventive drug candidate for treating patients with human colon cancer.

Tumorigenicity studies for human pluripotent stem cell-derived products.

PubMed

Kuroda, Takuya; Yasuda, Satoshi; Sato, Yoji

2013-01-01

Human pluripotent stem cells (hPSCs), i.e. human embryonic stem cells and human induced pluripotent stem cells, are able to self-renew and differentiate into multiple cell types. Because of these abilities, numerous attempts have been made to utilize hPSCs in regenerative medicine/cell therapy. hPSCs are, however, also tumorigenic, that is, they can give rise to the progressive growth of tumor nodules in immunologically unresponsive animals. Therefore, assessing and managing the tumorigenicity of all final products is essential in order to prevent ectopic tissue formation, tumor development, and/or malignant transformation elicited by residual pluripotent stem cells after implantation. No detailed guideline for the tumorigenicity testing of hPSC-derived products has yet been issued for regenerative medicine/cell therapy, despite the urgent necessity. Here, we describe the current situations and issues related to the tumorigenicity testing of hPSC-derived products and we review the advantages and disadvantages of several types of tumorigenicity-associated tests. We also refer to important considerations in the execution and design of specific studies to monitor the tumorigenicity of hPSC-derived products.

Towards a global human embryonic stem cell bank.

PubMed

Lott, Jason P; Savulescu, Julian

2007-08-01

An increasingly unbridgeable gap exists between the supply and demand of transplantable organs. Human embryonic stem cell technology could solve the organ shortage problem by restoring diseased or damaged tissue across a range of common conditions. However, such technology faces several largely ignored immunological challenges in delivering cell lines to large populations. We address some of these challenges and argue in favor of encouraging contribution or intentional creation of embryos from which widely immunocompatible stem cell lines could be derived. Further, we argue that current immunological constraints in tissue transplantation demand the creation of a global stem cell bank, which may hold particular promise for minority populations and other sub-groups currently marginalized from organ procurement and allocation systems. Finally, we conclude by offering a number of practical and ethically oriented recommendations for constructing a human embryonic stem cell bank that we hope will help solve the ongoing organ shortage problem.

Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes.

PubMed

Limat, A; Hunziker, T; Boillat, C; Bayreuther, K; Noser, F

1989-05-01

For growth at low seeding densities, keratinocytes isolated from human tissues like epidermis or hair follicles are dependent on mesenchyme-derived feeder cells such as the 3T3-cell employed so far. As an alternative method, the present study describes the use of post-mitotic human dermal fibroblasts sublethally irradiated or mitomycin C-treated. Special emphasis was put on efficient growth of primary keratinocyte cultures plated at very low seeding densities. Thus, outer root sheath cells isolated from two anagen human hair follicles and plated in a 35-mm culture dish (3 - 6 X 10(2) attached cells) grew to confluence within 3 weeks (6 - 8 X 10(5) cells). Similar results were obtained for interfollicular keratinocytes. A crucial point for the function of these fibroblast feeder cells is plating at appropriate densities, considering their tremendous increase in cell size at the post-mitotic state. Plating densities of 4 - 5 X 10(3/cm2 allow full spreading of the feeder cells and do not impede the settling and expansion of the keratinocytes. Major advantages of this system include easier handling and better reproducibility than using 3T3-cells. Moreover, homologous fibroblast feeders mimic more closely the physiologic situation and therefore might provide a valuable tool for studying interactions between human mesenchymal and epithelial cells. Finally, potential hazards of using transformed feeder cells from a different species in keratinocyte cultures raised for wound covering in humans could be thus avoided.

Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

PubMed

Winge, Stefan; Yderland, Louise; Kannicht, Christoph; Hermans, Pim; Adema, Simon; Schmidt, Torben; Gilljam, Gustav; Linhult, Martin; Tiemeyer, Maya; Belyanskaya, Larisa; Walter, Olaf

2015-11-01

Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

GMP-Compliant Expansion of Clinical-Grade Human Mesenchymal Stromal/Stem Cells Using a Closed Hollow Fiber Bioreactor.

PubMed

Barckhausen, Christina; Rice, Brent; Baila, Stefano; Sensebé, Luc; Schrezenmeier, Hubert; Nold, Philipp; Hackstein, Holger; Rojewski, Markus Thomas

2016-01-01

This chapter describes a method for GMP-compliant expansion of human mesenchymal stromal/stem cells (hMSC) from bone marrow aspirates, using the Quantum(®) Cell Expansion System from Terumo BCT. The Quantum system is a functionally closed, automated hollow fiber bioreactor system designed to reproducibly grow cells in either GMP or research laboratory environments. The chapter includes protocols for preparation of media, setup of the Quantum system, coating of the hollow fiber bioreactor, as well as loading, feeding, and harvesting of cells. We suggest a panel of quality controls for the starting material, the interim product, as well as the final product.

Innate-like behavior of human invariant natural killer T cells during herpes simplex virus infection.

PubMed

Novakova, Lucie; Nevoralova, Zuzana; Novak, Jan

2012-01-01

Invariant natural killer T (iNKT) cells, CD1d restricted T cells, are involved in the immune responses against various infection agents. Here we describe their behavior during reactivation of human herpes simplex virus (HSV). iNKT cells exhibit only discrete changes, which however, reached statistically significant level due to the relatively large patient group. Higher percentage of iNKT cells express NKG2D. iNKT cells down-regulate NKG2A in a subset of patients. Finally, iNKT cells enhance their capacity to produce TNF-α. Our data suggests that iNKT cells are involved in the immune response against HSV and contribute mainly to its early, innate phase. Copyright © 2012 Elsevier Inc. All rights reserved.

AMPK/p53 Axis Is Essential for α-Lipoic Acid-Regulated Metastasis in Human and Mouse Colon Cancer Cells.

PubMed

Park, Sunmi; Choi, Seung Kug; Choi, Yura; Moon, Hyun-Seuk

2015-10-01

α-Lipoic acid (ALA) has an anticancer property of lung, cervix, and prostate cancer cells. However, direct evidence that ALA contributes to the development of colon cancer has not been fully elucidated. In addition, no previous studies have evaluated whether ALA may regulate malignant potential, such as adhesion, invasion, and colony formation of colon cancer cells. To address the aforementioned questions, we conducted in vitro ALA signaling studies using human (HT29) and mouse (MCA38) colon cancer cell lines. We observed that cell proliferation is reduced by ALA administration in a dose-dependent manner in human and mouse colon cancer cell lines. Specifically, 0.5 to 1 mM concentration of ALA significantly decreased cell proliferation when compared with control. Similarly, we found that ALA downregulates adhesion, invasion, and colony formation. Finally, we observed that ALA activates p53 and AMPK signaling pathways in human and mouse colon cancer cells. We found for the first time that ALA suppresses cell proliferation and malignant potential via p53 and AMPK signaling pathways in human and mouse colon cancer cells. These new and early mechanistic studies provide a causal role of ALA in colon cancer, suggesting that ALA might be a useful agent in the management or chemoprevention of colon cancer.

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

PubMed

Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

2017-01-01

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017. © 2016 American Institute of Chemical Engineers.

Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao

Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression ofmore » Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target. - Highlights: • GGCT expression is up-regulated in human glioma tissues and cell lines. • GGCT promotes glioma cell growth and colony formation. • GGCT promotes the activation of Notch-Akt signaling in glioma cells and tissues. • Notch inhibition blocks the role of GGCT in human glioma cells.« less

Bioreactor expansion of human mesenchymal stem cells according to GMP requirements.

PubMed

Elseberg, Christiane L; Salzig, Denise; Czermak, Peter

2015-01-01

In cell therapy, the use of autologous and allogenic human mesenchymal stem cells is rising. Accordingly, the supply of cells for clinical applications in highest quality is required. As hMSCs are considered as an advanced therapy medicinal products (ATMP), they underlie the requirements of GMP and PAT according to the authorities (FDA and EMA). The production process of these cells must therefore be documented according to GMP, which is usually performed via a GMP protocol based on standard operating procedures. This chapter provides an example of such a GMP protocol for hMSC, here a genetically modified allogenic cell line, based on a production process in a microcarrier-based stirred tank reactor including process monitoring according to PAT and final product quality assurance.

Reconstruction of genome-scale human metabolic models using omics data.

PubMed

Ryu, Jae Yong; Kim, Hyun Uk; Lee, Sang Yup

2015-08-01

The impact of genome-scale human metabolic models on human systems biology and medical sciences is becoming greater, thanks to increasing volumes of model building platforms and publicly available omics data. The genome-scale human metabolic models started with Recon 1 in 2007, and have since been used to describe metabolic phenotypes of healthy and diseased human tissues and cells, and to predict therapeutic targets. Here we review recent trends in genome-scale human metabolic modeling, including various generic and tissue/cell type-specific human metabolic models developed to date, and methods, databases and platforms used to construct them. For generic human metabolic models, we pay attention to Recon 2 and HMR 2.0 with emphasis on data sources used to construct them. Draft and high-quality tissue/cell type-specific human metabolic models have been generated using these generic human metabolic models. Integration of tissue/cell type-specific omics data with the generic human metabolic models is the key step, and we discuss omics data and their integration methods to achieve this task. The initial version of the tissue/cell type-specific human metabolic models can further be computationally refined through gap filling, reaction directionality assignment and the subcellular localization of metabolic reactions. We review relevant tools for this model refinement procedure as well. Finally, we suggest the direction of further studies on reconstructing an improved human metabolic model.

BETA-ADRENOCEPTOR SIGNALING AND ITS CONTROL OF CELL REPLICATION IN MDA-MB-231 HUMAN BREAST CANCER CELLS. (U915722)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation.

PubMed

Ishikawa, Takanori; Nishida, Takashi; Ono, Mitsuaki; Takarada, Takeshi; Nguyen, Ha Thi; Kurihara, Shinnosuke; Furumatsu, Takayuki; Murase, Yurika; Takigawa, Masaharu; Oohashi, Toshitaka; Kamioka, Hiroshi; Kubota, Satoshi

2018-06-01

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates. © 2017 Wiley Periodicals, Inc.

The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent.

PubMed

Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

2016-01-01

Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species.

The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent

PubMed Central

Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

2016-01-01

Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species. PMID:26901159

Cell sources for in vitro human liver cell culture models.

PubMed

Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

2016-09-01

In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. © 2016 by the Society for Experimental Biology and Medicine.

Cell sources for in vitro human liver cell culture models

PubMed Central

Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

2016-01-01

In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

[Unresolved issues in the evaluation of research projects involving induced pluripotent stem cells (iPS)].

PubMed

Casado, María; de Lecuona, Itziar

2013-01-01

This paper identifies problems and analyzes those conflicts posed by the evaluation of research projects involving the collection and use of human induced pluripotent stem cells (iPS) in Spain. Current legislation is causing problems of interpretation, circular and unnecessary referrals, legal uncertainty and undue delays. Actually, this situation may cause a lack of control and monitoring, and even some paralysis in regenerative medicine and cell therapy research, that is a priority nowadays. The analysis of the current legislation and its bioethical implications, led us to conclude that the review of iPS research projects cannot be assimilated to the evaluation of research projects that involve human embryonic stem cell (hESC). In this context, our proposal is based on the review by the Research Ethics Committees and the checkout by the Spanish Comission of Guarantees for Donation and Use of Human Cells and Tissues (CGDUCTH) of human iPS cells research projects. Moreover, this article claims for a more transparent research system, by effectively articulating the Registry on Research Projects. Finally, a model of verification protocol (checklist) for checking out biomedical research projects involving human iPS cells is suggested.

Direct cytotoxicity evaluation of 63S bioactive glass and bone-derived hydroxyapatite particles using yeast model and human chondrocyte cells by microcalorimetry.

PubMed

Doostmohammadi, A; Monshi, A; Fathi, M H; Karbasi, S; Braissant, O; Daniels, A U

2011-10-01

In this study, the cytotoxicity evaluation of prepared 63S bioactive glass and bone-derived hydroxyapatite particles with yeast and human chondrocyte cells was carried out using isothermal micro-nano calorimetry (IMNC), which is a new method for studying cell/biomaterial interactions. Bioactive glass particles were made via sol-gel method and hydroxyapatite was obtained from bovine bone. Elemental analysis was carried out by XRF and EDXRF. Amorphous structure of the glass and completely crystalline structure of HA were detected by XRD analysis. Finally, the cytotoxicity of bioactive glass and bone-derived HA particles with yeast and cultured human chondrocyte cells was evaluated using IMNC. The results confirmed the viability, growth and proliferation of human chondrocyte cells in contact with 63S bioactive glass, and bone-derived HA particles. Also the results indicated that yeast model which is much easier to handle, can be considered as a good proxy and can provide a rapid primary estimate of the ranges to be used in assays involving human cells. All of these results confirmed that IMNC is a convenient method which caters to measuring the cell-biomaterial interactions alongside the current methods.

Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

NASA Astrophysics Data System (ADS)

Taik Lim, Yong; Cho, Mi Young; Noh, Young-Woock; Chung, Jin Woong; Chung, Bong Hyun

2009-11-01

This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

HIV/AIDS

MedlinePlus

HIV stands for human immunodeficiency virus. It harms your immune system by destroying the white blood cells ... It is the final stage of infection with HIV. Not everyone with HIV develops AIDS. HIV most ...

Detection and Phenotypic Characterization of Adult Neurogenesis

PubMed Central

Kuhn, H. Georg; Eisch, Amelia J.; Spalding, Kirsty; Peterson, Daniel A.

2016-01-01

Studies of adult neurogenesis have greatly expanded in the last decade, largely as a result of improved tools for detecting and quantifying neurogenesis. In this review, we summarize and critically evaluate detection methods for neurogenesis in mammalian and human brain tissue. Besides thymidine analog labeling, cell-cycle markers are discussed, as well as cell stage and lineage commitment markers. Use of these histological tools is critically evaluated in terms of their strengths and limitations, as well as possible artifacts. Finally, we discuss the method of radiocarbon dating for determining cell and tissue turnover in humans. PMID:26931327

Deriving excitatory neurons of the neocortex from pluripotent stem cells

PubMed Central

Hansen, David V.; Rubenstein, John L.R.; Kriegstein, Arnold R.

2011-01-01

The human cerebral cortex is an immensely complex structure that subserves critical functions that can be disrupted in developmental and degenerative disorders. Recent innovations in cellular reprogramming and differentiation techniques have provided new ways to study the cellular components of the cerebral cortex. Here we discuss approaches to generate specific subtypes of excitatory cortical neurons from pluripotent stem cells. We review spatial and temporal aspects of cortical neuron specification that can guide efforts to produce excitatory neuron subtypes with increased resolution. Finally, we discuss distinguishing features of human cortical development and their translational ramifications for cortical stem cell technologies. PMID:21609822

hCG, the wonder of today's science

PubMed Central

2012-01-01

Background hCG is a wonder. Firstly, because hCG is such an extreme molecule. hCG is the most acidic glycoprotein containing the highest proportion of sugars. Secondly, hCG exists in 5 common forms. Finally, it has so many functions ranging from control of human pregnancy to human cancer. This review examines these molecules in detail. Content These 5 molecules, hCG, sulfated hCG, hyperglycosylated hCG, hCG free beta and hyperglycosylated free beta are produced by placental syncytiotrophoblast cells and pituitary gonadotrope cells (group 1), and by placental cytotrophoblast cells and human malignancies (group 2). Group 1 molecules are both hormones that act on the hCG/LH receptor. These molecules are central to human menstrual cycle and human pregnancy. Group 2 molecules are autocrines, that act by antagonizing a TGF beta receptor. These molecules are critical to all advanced malignancies. Conclusions The hCG groups are molecules critical to both the molecules of pregnancy or human life, and to the advancement of cancer, or human death. PMID:22455390

Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data.

PubMed

Martins, José Paulo; Santos, Jorge Miguel; de Almeida, Joana Marto; Filipe, Mariana Alves; de Almeida, Mariana Vargas Teixeira; Almeida, Sílvia Cristina Paiva; Água-Doce, Ana; Varela, Alexandre; Gilljam, Mari; Stellan, Birgitta; Pohl, Susanne; Dittmar, Kurt; Lindenmaier, Werner; Alici, Evren; Graça, Luís; Cruz, Pedro Estilita; Cruz, Helder Joaquim; Bárcia, Rita Nogueira

2014-01-17

Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties. We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.

Phloretin induces apoptosis in H-Ras MCF10A human breast tumor cells through the activation of p53 via JNK and p38 mitogen-activated protein kinase signaling.

PubMed

Kim, Mi-Sung; Kwon, Jung Yeon; Kang, Nam Joo; Lee, Ki Won; Lee, Hyong Joo

2009-08-01

Mutations in Ras play a critical role in the development of human cancers, including breast cancer. We investigated the possible antiproliferative effects of the naturally occurring dihydrochalcone phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on H-Ras-transformed MCF10A human breast epithelial (H-Ras MCF10A) cells. Phloretin suppressed H-Ras MCF10A cell proliferation in a dose-dependent manner and induced nuclear condensation in the cells, indicating that phloretin-induced cell death occurs mainly via the induction of apoptosis. Prominent upregulation of p53 and Bax and cleavage of poly (ADP)-ribose polymerase were also detected in the phloretin-treated cells. Finally, phloretin markedly increased caspase-3 activity as well as JNK and p38 mitogen-activated protein kinase signaling. Our findings suggest that the phloretin-induced apoptosis of breast tumor cells contributes to the chemopreventive potential of phloretin against breast cancer.

[Hepatic cell transplantation. Technical and methodological aspects].

PubMed

Pareja, Eugenia; Martínez, Amparo; Cortés, Miriam; Bonora, Ana; Moya, Angel; Sanjuán, Fernando; Gómez-Lechón, M José; Mir, José

2010-03-01

Hepatic cell transplantation consists of grafting already differentiated cells such as hepatocytes. Human hepatocytes are viable and functionally active. Liver cell transplantation is carried out by means of a 3-step method: isolation of hepatocytes from donor liver rejected for orthotopic transplantation, preparing a cell suspension for infusion and, finally, hepatocytes are implanted into the recipient. There are established protocols for the isolation of human hepatocytes from unused segments of donor livers, based on collagenase digestion of cannulated liver tissue at 37 degrees C. The hepatocytes can be used fresh or cryopreserved. Cryopreservation of isolated human hepatocytes would then be available for planned use. In cell transplant, the important aspects are: infusion route, number of cells, number of infusions and viability of the cells. The cells are infused into the patient through a catheter inserted via portal vein or splenic artery. Liver cell transplantation allows liver tissue to be used that would, otherwise, be discarded, enabling multiple patients to be treated with hepatocytes from a single tissue donor. Copyright 2009 AEC. Published by Elsevier Espana. All rights reserved.

Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method.

PubMed

Ohnishi, Hiroe; Skerleva, Desislava; Kitajiri, Shin-ichiro; Sakamoto, Tatsunori; Yamamoto, Norio; Ito, Juichi; Nakagawa, Takayuki

2015-07-10

Disease-specific induced pluripotent stem cells (iPS) cells are expected to contribute to exploring useful tools for studying the pathophysiology of inner ear diseases and to drug discovery for treating inner ear diseases. For this purpose, stable induction methods for the differentiation of human iPS cells into inner ear hair cells are required. In the present study, we examined the efficacy of a simple induction method for inducing the differentiation of human iPS cells into hair cells. The induction of inner ear hair cell-like cells was performed using a stepwise method mimicking inner ear development. Human iPS cells were sequentially transformed into the preplacodal ectoderm, otic placode, and hair cell-like cells. As a first step, preplacodal ectoderm induction, human iPS cells were seeded on a Matrigel-coated plate and cultured in a serum free N2/B27 medium for 8 days according to a previous study that demonstrated spontaneous differentiation of human ES cells into the preplacodal ectoderm. As the second step, the cells after preplacodal ectoderm induction were treated with basic fibroblast growth factor (bFGF) for induction of differentiation into otic-placode-like cells for 15 days. As the final step, cultured cells were incubated in a serum free medium containing Matrigel for 48 days. After preplacodal ectoderm induction, over 90% of cultured cells expressed the genes that express in preplacodal ectoderm. By culture with bFGF, otic placode marker-positive cells were obtained, although their number was limited. Further 48-day culture in serum free media resulted in the induction of hair cell-like cells, which expressed a hair cell marker and had stereocilia bundle-like constructions on their apical surface. Our results indicate that hair cell-like cells are induced from human iPS cells using a simple stepwise method with only bFGF, without the use of xenogeneic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A survey of human brain transcriptome diversity at the single cell level.

PubMed

Darmanis, Spyros; Sloan, Steven A; Zhang, Ye; Enge, Martin; Caneda, Christine; Shuer, Lawrence M; Hayden Gephart, Melanie G; Barres, Ben A; Quake, Stephen R

2015-06-09

The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

HIV/AIDS in Women

MedlinePlus

HIV stands for human immunodeficiency virus. It harms your immune system by destroying the white blood cells ... It is the final stage of infection with HIV. Not everyone with HIV develops AIDS. HIV often ...

Living with HIV/AIDS

MedlinePlus

HIV stands for human immunodeficiency virus. It harms your immune system by destroying the white blood cells ... It is the final stage of infection with HIV. Not everyone with HIV develops AIDS. Infection with ...

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering.

PubMed

Maffioletti, Sara Martina; Sarcar, Shilpita; Henderson, Alexander B H; Mannhardt, Ingra; Pinton, Luca; Moyle, Louise Anne; Steele-Stallard, Heather; Cappellari, Ornella; Wells, Kim E; Ferrari, Giulia; Mitchell, Jamie S; Tyzack, Giulia E; Kotiadis, Vassilios N; Khedr, Moustafa; Ragazzi, Martina; Wang, Weixin; Duchen, Michael R; Patani, Rickie; Zammit, Peter S; Wells, Dominic J; Eschenhagen, Thomas; Tedesco, Francesco Saverio

2018-04-17

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.

PubMed

Su, Lin; Chen, Song-Sen; Yang, Ke-Gong; Liu, Chang-Zheng; Zhang, Yan-Li; Liang, Zhi-Quan

2006-06-01

Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

PubMed

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells.

PubMed Central

Shamah, S M; Stiles, C D; Guha, A

1993-01-01

Malignant astrocytoma is the most common primary human brain tumor. Most astrocytomas express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could close an autocrine loop. It is not known whether these autocrine loops contribute to the transformed phenotype of astrocytoma cells or are incidental to that phenotype. Here we show that dominant-negative mutants of the PDGF ligand break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene. Then, we show that these mutants are selective in that they do not alter the phenotype of 3T3 cells transformed by an activated Ha-ras or v-src gene or by simian virus 40. Finally, we show that these mutants revert the transformed phenotype of two independent human astrocytoma cell lines. They have no effect on the growth of human medulloblastoma, bladder carcinoma, or colon carcinoma cell lines. These observations are consistent with the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas. Images PMID:8246942

Neuroprotective Properties of Endocannabinoids N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine Examined in Neuronal Precursors Derived from Human Pluripotent Stem Cells.

PubMed

Novosadova, E V; Arsenyeva, E L; Manuilova, E S; Khaspekov, L G; Bobrov, M Yu; Bezuglov, V V; Illarioshkin, S N; Grivennikov, I A

2017-11-01

Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine (NADA) and N-docosahexaenoyl dopamine (DHDA) were examined in neuronal precursor cells differentiated from human induced pluripotent stem cells and subjected to oxidative stress. Both compounds exerted neuroprotective activity, which was enhanced by elevating the concentration of the endocannabinoids within the 0.1-10 µM range. However, both agents at 10 µM concentration showed a marked toxic effect resulting in death of ~30% of the cells. Finally, antagonists of cannabinoid receptors as well as the receptor of the TRPV1 endovanilloid system did not hamper the neuroprotective effects of these endocannabinoids.

Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation.

PubMed

Chu, Chung-Ching; Ali, Niwa; Karagiannis, Panagiotis; Di Meglio, Paola; Skowera, Ania; Napolitano, Luca; Barinaga, Guillermo; Grys, Katarzyna; Sharif-Paghaleh, Ehsan; Karagiannis, Sophia N; Peakman, Mark; Lombardi, Giovanna; Nestle, Frank O

2012-05-07

Human skin immune homeostasis, and its regulation by specialized subsets of tissue-residing immune sentinels, is poorly understood. In this study, we identify an immunoregulatory tissue-resident dendritic cell (DC) in the dermis of human skin that is characterized by surface expression of CD141, CD14, and constitutive IL-10 secretion (CD141(+) DDCs). CD141(+) DDCs possess lymph node migratory capacity, induce T cell hyporesponsiveness, cross-present self-antigens to autoreactive T cells, and induce potent regulatory T cells that inhibit skin inflammation. Vitamin D(3) (VitD3) promotes certain phenotypic and functional properties of tissue-resident CD141(+) DDCs from human blood DCs. These CD141(+) DDC-like cells can be generated in vitro and, once transferred in vivo, have the capacity to inhibit xeno-graft versus host disease and tumor alloimmunity. These findings suggest that CD141(+) DDCs play an essential role in the maintenance of skin homeostasis and in the regulation of both systemic and tumor alloimmunity. Finally, VitD3-induced CD141(+) DDC-like cells have potential clinical use for their capacity to induce immune tolerance.

Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation

PubMed Central

Chu, Chung-Ching; Ali, Niwa; Karagiannis, Panagiotis; Di Meglio, Paola; Skowera, Ania; Napolitano, Luca; Barinaga, Guillermo; Grys, Katarzyna; Sharif-Paghaleh, Ehsan; Karagiannis, Sophia N.; Peakman, Mark; Lombardi, Giovanna

2012-01-01

Human skin immune homeostasis, and its regulation by specialized subsets of tissue-residing immune sentinels, is poorly understood. In this study, we identify an immunoregulatory tissue-resident dendritic cell (DC) in the dermis of human skin that is characterized by surface expression of CD141, CD14, and constitutive IL-10 secretion (CD141+ DDCs). CD141+ DDCs possess lymph node migratory capacity, induce T cell hyporesponsiveness, cross-present self-antigens to autoreactive T cells, and induce potent regulatory T cells that inhibit skin inflammation. Vitamin D3 (VitD3) promotes certain phenotypic and functional properties of tissue-resident CD141+ DDCs from human blood DCs. These CD141+ DDC-like cells can be generated in vitro and, once transferred in vivo, have the capacity to inhibit xeno-graft versus host disease and tumor alloimmunity. These findings suggest that CD141+ DDCs play an essential role in the maintenance of skin homeostasis and in the regulation of both systemic and tumor alloimmunity. Finally, VitD3-induced CD141+ DDC-like cells have potential clinical use for their capacity to induce immune tolerance. PMID:22547651

The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells

PubMed Central

Hoffmann, Markus; Krüger, Nadine; Zmora, Pawel; Wrensch, Florian; Herrler, Georg; Pöhlmann, Stefan

2016-01-01

New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin. PMID:27028521

Humanized Mouse Model of Skin Inflammation Is Characterized by Disturbed Keratinocyte Differentiation and Influx of IL-17A Producing T Cells

PubMed Central

de Oliveira, Vivian L.; Keijsers, Romy R. M. C.; van de Kerkhof, Peter C. M.; Seyger, Marieke M. B.; Fasse, Esther; Svensson, Lars; Latta, Markus; Norsgaard, Hanne; Labuda, Tord; Hupkens, Pieter; van Erp, Piet E. J.; Joosten, Irma; Koenen, Hans J. P. M.

2012-01-01

Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response. As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human β-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin. In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our understanding of the dermal immunopathology in humans and benefit the development of novel therapeutics for controlling inflammatory skin diseases. PMID:23094018

Stem cell research in Germany: ethics of healing vs. human dignity.

PubMed

Oduncu, Fuat S

2003-01-01

On 25 April 2002, the German Parliament has passed a strict new law referring to stem cell research. This law took effect on July 1, 2002. The so-called embryonic Stem Cell Act ("Stammzellgesetz-StZG") permits the import of embryonic stem (ES) cells isolated from surplus lvF-embryos for research reasons. The production itself of ES cells from human blastocysts has been prohibited by the German Embryo Protection Act of 1990, with the exception of the use of ES cells which exist already. The debate on the legitimate use of ES cells escalated, after the main German research funding agency, the Deutsche Forschungsgemeinschaft (DFG), unexpectedly published new guidelines recommending a restricted use of human ES cells for research. Meanwhile, the debate has ethically divided society, political parties, government and church members into a group supporting and a group rejecting ES cell research. The arguments in favour of such a research can be summarized as arguments derived from a new "ethics of healing" calling for a therapeutic imperative, whereas the arguments against can be summarized as arguments violating the fundamental principle of human dignity as they imply the destruction of human embryos. This article will try to present and evaluate various ethical arguments founded on the latest biological and medical data on the potential use of stem cell technologies. It will finally come to the conclusion that ES cell research is opposed to human dignity, since the procedures of isolating ES cells require the destruction and instrumentalization of human embryos. Human embryos are human beings at a very early stage of their development, fully possessing the ability of completing their development. At this very early stage, human embryos are extremely dependent and fragile, and thus vulnerable corporealities. Vulnerability and human dignity demand the protection of the embryo's corporeal integrity. Hence, this essay will try to propagate research with adult stem (AS) cells, a procedure which does not require the destruction of human embryos; with regard to the necessary plasticity, it should be emphasized that AS cells very much resemble ES cells.

Cytotoxicity induced by cypermethrin in Human Neuroblastoma Cell Line SH-SY5Y.

PubMed

Raszewski, Grzegorz; Lemieszek, Marta Kinga; Łukawski, Krzysztof

2016-01-01

The purpose of this study was to evaluate the cytotoxic potential of Cypermethrin (CM) on cultured human Neuroblastoma SH-SY5Y cells. SH-SY5Y cells were treated with CM at 0-200µM for 24, 48, and 72 h, in vitro. It was found that CM induced the cell death of Neuroblastoma cells in a dose- and time-dependent manner, as shown by LDH assays. Next, some aspects of the process of cell death triggered by CM in the human SH-SY5Y cell line were investigated. It was revealed that the pan-caspase inhibitor Q-VD-OPh, sensitizes SH-SY5Y cells to necroptosis caused by CM. Furthermore, signal transduction inhibitors PD98059, SL-327, SB202190, SP600125 failed to attenuate the effect of the pesticide. Finally, it was shown that inhibition of TNF-a by Pomalidomide (PLD) caused statistically significant reduction in CM-induced cytotoxicity. Overall, the data obtained suggest that CM induces neurotoxicity in SH-SY5Y cells by necroptosis.

Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin αVβ8.

PubMed

Stockis, Julie; Liénart, Stéphanie; Colau, Didier; Collignon, Amandine; Nishimura, Stephen L; Sheppard, Dean; Coulie, Pierre G; Lucas, Sophie

2017-11-21

Human regulatory T cells (Tregs) suppress other T cells by converting the latent, inactive form of TGF-β1 into active TGF-β1. In Tregs, TGF-β1 activation requires GARP, a transmembrane protein that binds and presents latent TGF-β1 on the surface of Tregs stimulated through their T cell receptor. However, GARP is not sufficient because transduction of GARP in non-Treg T cells does not induce active TGF-β1 production. RGD-binding integrins were shown to activate TGF-β1 in several non-T cell types. Here we show that αVβ8 dimers are present on stimulated human Tregs but not in other T cells, and that antibodies against αV or β8 subunits block TGF-β1 activation in vitro. We also show that αV and β8 interact with GARP/latent TGF-β1 complexes in human Tregs. Finally, a blocking antibody against β8 inhibited immunosuppression by human Tregs in a model of xenogeneic graft-vs.-host disease induced by the transfer of human T cells in immunodeficient mice. These results show that TGF-β1 activation on the surface of human Tregs implies an interaction between the integrin αVβ8 and GARP/latent TGF-β1 complexes. Immunosuppression by human Tregs can be inhibited by antibodies against GARP or against the integrin β8 subunit. Such antibodies may prove beneficial against cancer or chronic infections.

Nanofibers of Human Tropoelastin-inspired peptides: Structural characterization and biological properties.

PubMed

Secchi, Valeria; Franchi, Stefano; Fioramonti, Marco; Polzonetti, Giovanni; Iucci, Giovanna; Bochicchio, Brigida; Battocchio, Chiara

2017-08-01

Regenerative medicine is taking great advantage from the use of biomaterials in the treatments of a wide range of diseases and injuries. Among other biomaterials, self-assembling peptides are appealing systems due to their ability to spontaneously form nanostructured hydrogels that can be directly injected into lesions. Indeed, self-assembling peptide scaffolds are expected to behave as biomimetic matrices able to surround cells, to promote specific interactions, and to control and modify cell behavior by mimicking the native environment as well. We selected three pentadecapeptides inspired by Human Tropoelastin, a natural protein of the extracellular matrix, expected to show high biocompatibility. Moreover, the here proposed self-assembling peptides (SAPs) are able to spontaneously aggregate in nanofibers in biological environment, as revealed by AFM (Atomic Force Microscopy). Peptides were characterized by XPS (X-ray Photoelectron Spectroscopy) and IRRAS (Infrared Reflection Absorption Spectroscopy) both as lyophilized (not aggregated) and as aggregated (nanofibers) samples in order to investigate some potential differences in their chemical composition and intermolecular interactions, and to analyze the surface and interface of nanofibers. Finally, an accurate investigation of the biological properties of the SAPs and of their interaction with cells was performed by culturing for the first time human Mesenchymal Stem Cells (hMSCs) in presence of SAPs. The final aim of this work was to assess if Human Tropoelastin-inspired nanostructured fibers could exert a cytotoxic effect and to evaluate their biocompatibility, cellular adhesion and proliferation. Copyright © 2017 Elsevier B.V. All rights reserved.

Human Embryonic Stem Cell Therapy in Crohn’s Disease: A Case Report

PubMed Central

Shroff, Geeta

2016-01-01

Patient: Male, 21 Final Diagnosis: Crohn’s disease Symptoms: Intolerance to specific foods • abdominal pain and diarrhea Medication: Human embryonic stem cell therapy Clinical Procedure: Human embryonic stem cell transplantation Specialty: Gastroenterology Objective: Unusual or unexpected effect of treatment Background: Crohn’s disease is a chronic inflammatory disease of the intestines, mainly the colon and ileum, related with ulcers and fistulae. It is estimated to affect 565 000 people in the United States. Currently available therapies, such as antibiotics, thiopurines, and anti-tumor necrosis factor-alpha agents, are only observed to reduce the complications associated with Crohn’s disease and to improve quality of life, but cannot cure the disease. Stem cell therapy appears to have certain advantages over conventional therapies. Our study aimed to evaluate the efficacy of human embryonic stem cell therapy in a patient with Crohn’s disease. Case Report: A 21-year-old male with chief complaints of intolerance to specific foods, abdominal pain, and diarrhea underwent human embryonic stem cell therapy for two months. After undergoing human embryonic stem cell therapy, the patient showed symptomatic relief. He had no complaints of back pain, abdominal pain, or diarrhea and had improved digestion. The patient had no signs and symptoms of skin infection, and had improved limb stamina, strength, and endurance. The condition of patient was stable after the therapy. Conclusions: Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn’s disease. PMID:26923312

Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next-generation human artificial chromosomes for Duchenne muscular dystrophy.

PubMed

Benedetti, Sara; Uno, Narumi; Hoshiya, Hidetoshi; Ragazzi, Martina; Ferrari, Giulia; Kazuki, Yasuhiro; Moyle, Louise Anne; Tonlorenzi, Rossana; Lombardo, Angelo; Chaouch, Soraya; Mouly, Vincent; Moore, Marc; Popplewell, Linda; Kazuki, Kanako; Katoh, Motonobu; Naldini, Luigi; Dickson, George; Messina, Graziella; Oshimura, Mitsuo; Cossu, Giulio; Tedesco, Francesco Saverio

2018-02-01

Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS-HAC into DMD satellite cell-derived myoblasts and perivascular cell-derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next-generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Bovine brain ribonuclease is the functional homolog of human ribonuclease 1.

PubMed

Eller, Chelcie H; Lomax, Jo E; Raines, Ronald T

2014-09-19

Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Neocortical glial cell numbers in human brains.

PubMed

Pelvig, D P; Pakkenberg, H; Stark, A K; Pakkenberg, B

2008-11-01

Stereological cell counting was applied to post-mortem neocortices of human brains from 31 normal individuals, age 18-93 years, 18 females (average age 65 years, range 18-93) and 13 males (average age 57 years, range 19-87). The cells were differentiated in astrocytes, oligodendrocytes, microglia and neurons and counting were done in each of the four lobes. The study showed that the different subpopulations of glial cells behave differently as a function of age; the number of oligodendrocytes showed a significant 27% decrease over adult life and a strong correlation to the total number of neurons while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males, a difference of 24% with a high biological variance. These numbers can serve as reference values in quantitative studies of the human neocortex.

Discovery of potent cytotoxic ortho-aryl chalcones as new scaffold targeting tubulin and mitosis with affinity-based fluorescence.

PubMed

Zhu, Cuige; Zuo, Yinglin; Wang, Ruimin; Liang, Baoxia; Yue, Xin; Wen, Gesi; Shang, Nana; Huang, Lei; Chen, Yu; Du, Jun; Bu, Xianzhang

2014-08-14

A series of new ortho-aryl chalcones have been designed and synthesized. Many of these compounds were found to exhibit significant antiproliferation activity toward a panel of cancer cell lines. Selected compounds show potent cytotoxicity against several drug resistant cell lines including paclitaxel (Taxol) resistant human ovarian carcinoma cells, vincristine resistant human ileocecum carcinoma cells, and doxorubicin resistant human breast carcinoma cells. Further investigation revealed that active analogues could inhibit the microtubule polymerization by binding to colchicine site and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of cancer cells. Furthermore, affinity-based fluorescence enhancement was observed during the binding of active compounds with tubulin, which greatly facilitated the determination of tubulin binding site of the compounds. Finally, selected compound 26 was found to exhibit obvious in vivo antitumor activity in A549 tumor xenografts model. Our systematic studies implied a new scaffold targeting tubulin and mitosis for novel antitumor drug discovery.

Francisella philomiragia Infection and Lethality in Mammalian Tissue Culture Cell Models, Galleria mellonella, and BALB/c Mice

PubMed Central

Propst, Crystal N.; Pylypko, Stephanie L.; Blower, Ryan J.; Ahmad, Saira; Mansoor, Mohammad; van Hoek, Monique L.

2016-01-01

Francisella (F.) philomiragia is a Gram-negative bacterium with a preference for brackish environments that has been implicated in causing bacterial infections in near-drowning victims. The purpose of this study was to characterize the ability of F. philomiragia to infect cultured mammalian cells, a commonly used invertebrate model, and, finally, to characterize the ability of F. philomiragia to infect BALB/c mice via the pulmonary (intranasal) route of infection. This study shows that F. philomiragia infects J774A.1 murine macrophage cells, HepG2 cells and A549 human Type II alveolar epithelial cells. However, replication rates vary depending on strain at 24 h. F. philomiragia infection after 24 h was found to be cytotoxic in human U937 macrophage-like cells and J774A.1 cells. This is in contrast to the findings that F. philomiragia was non-cytotoxic to human hepatocellular carcinoma cells, HepG2 cells and A549 cells. Differential cytotoxicity is a point for further study. Here, it was demonstrated that F. philomiragia grown in host-adapted conditions (BHI, pH 6.8) is sensitive to levofloxacin but shows increased resistance to the human cathelicidin LL-37 and murine cathelicidin mCRAMP when compared to related the Francisella species, F. tularensis subsp. novicida and F. tularensis subsp. LVS. Previous findings that LL-37 is strongly upregulated in A549 cells following F. tularensis subsp. novicida infection suggest that the level of antimicrobial peptide expression is not sufficient in cells to eradicate the intracellular bacteria. Finally, this study demonstrates that F. philomiragia is lethal in two in vivo models; Galleria mellonella via hemocoel injection, with a LD50 of 1.8 × 103, and BALB/c mice by intranasal infection, with a LD50 of 3.45 × 103. In conclusion, F. philomiragia may be a useful model organism to study the genus Francisella, particularly for those researchers with interest in studying microbial ecology or environmental strains of Francisella. Additionally, the Biosafety level 2 status of F. philomiragia makes it an attractive model for virulence and pathogenesis studies. PMID:27252681

Ethyl acetate fraction from methanol extraction of Vitis thunbergii var. taiwaniana induced G0 /G1 phase arrest via inhibition of cyclins D and E and induction of apoptosis through caspase-dependent and -independent pathways in human prostate carcinoma DU145 cells.

PubMed

Lin, Chia-Hsin; Chan, Hsiao-Sung; Tsay, Hsin-Sheng; Funayama, Shinji; Kuo, Chao-Lin; Chung, Jing-Gung

2018-01-01

Vitis thunbergii var. taiwaniana (VTT) is a wild grape native to Taiwan, belonging to the Vitaceae family and Vitis genus, and widely used as folk herbal medicine. It is traditionally used for the treatment of diarrhea, hypertension, neuroprotection, jaundice, and arthritis. We used the wild-collected VTT and sterilized them to establish the plant tissue culture, and then took the leaves for DNA sequencing to determine its original base. We use methanol to extract VTT in four different solvents: 1-butanol, n-hexane, ethyl acetate, and water. These four preliminary extracts were used to treat human prostate cancer DU145 cells in vitro. We use the flow cytometry to check the cell survival situation. Finally, we found the ethyl acetate layer roughing product (referred VTEA) in human prostate cancer apoptotic effects of cell line DU-145. In the present studies, we use the crude extract of VTT to examine whether or not it can induce apoptosis of DU145 cells in vitro. Viability assays for extracts of VTT treatment showed that it had dose-dependent effect on human prostate cancer DU145 cells. We also found that the extract of VTT induces time-dependent mitochondrial and intrinsic-dependent apoptosis pathways. The in vitro cytotoxic effects were investigated by cell cycle analysis and the determination of apoptotic DNA fragmentation in DU145 cells. The cell cycle analysis showed that extracts of VTT induced a significant increase in the number of cells in G 0 /G 1 phase. The extract of VTT induced chromatin changes and apoptosis of DU145 cells also were confirmed by DAPI and PI staining that were measured by fluorescence microscopy and flow cytometry, respectively. Finally, the expression of relevant proteins was analyzed by Western blot analysis. These results promoted us to further evaluate apoptosis associated proteins and elucidate the possible signal pathway in DU-145 cells after treated with the extract of VTT. © 2017 Wiley Periodicals, Inc.

Localization, distribution, and connectivity of neuropeptide Y in the human and porcine retinas-A comparative study.

PubMed

Christiansen, Anders Tolstrup; Kiilgaard, Jens Folke; Klemp, Kristian; Woldbye, David Paul Drucker; Hannibal, Jens

2018-04-17

Neuropeptide Y (NPY) is a peptide neurotransmitter abundantly expressed in the mammalian retina. Since its discovery, NPY has been studied in retinas of several species, but detailed characterization of morphology, cell-type, and connectivity has never been conducted in larger mammals including humans and pigs. As the pig due to size and cellular composition is a well-suited animal for retinal research, we chose to compare the endogenous NPY system of the human retina to that of pigs to support future research in this field. In the present study, using immunohistochemistry, confocal microscopy and 3D reconstructions, we found NPY to be expressed in GABAergic and calretinin-immunoreactive (-ir) amacrine cells of both species as well as parvalbumin-ir amacrine cells of humans. Furthermore, we identified at least two different types of medium- to wide-field NPY-ir amacrine cells. Finally, we detected likely synaptic appositions between the NPY-ir amacrine cells and melanopsin- and nonmelanopsin-ir ganglion cells, GABAergic and dopaminergic amacrine cells, rod bipolar cells, and horizontal cells, suggesting that NPY-ir cells play diverse roles in modulation of both image and non-image forming retinal signaling. These findings extend existing knowledge on NPY and NPY-expressing cells in the human and porcine retina showing a high degree of comparability. The extensive distribution and connectivity of NPY-ir cells described in the present study further highlights the potential importance of NPY signaling in retinal function. © 2018 Wiley Periodicals, Inc.

Environmentally Induced Gene Silencing in Breast Cancer

DTIC Science & Technology

2007-07-01

fibrosarcoma cell line (HTD114), and a human breast cancer cell line (MCF7). The MLH1 promoter was only tested in the MCG7 cells. The control TRE-Luc...TRE- Luc MLH1 - Luc step in silencing is quite unstable. Nonetheless, cells that exhibit stable silencing of the HPRT construct can arise in...mechanism (i.e., gene repression). Finally, during the last year we have isolated or acquired functional promoters for the BRCA-1, MLH1 , and E

3D culture models of Alzheimer's disease: a road map to a "cure-in-a-dish".

PubMed

Choi, Se Hoon; Kim, Young Hye; Quinti, Luisa; Tanzi, Rudolph E; Kim, Doo Yeon

2016-12-09

Alzheimer's disease (AD) transgenic mice have been used as a standard AD model for basic mechanistic studies and drug discovery. These mouse models showed symbolic AD pathologies including β-amyloid (Aβ) plaques, gliosis and memory deficits but failed to fully recapitulate AD pathogenic cascades including robust phospho tau (p-tau) accumulation, clear neurofibrillary tangles (NFTs) and neurodegeneration, solely driven by familial AD (FAD) mutation(s). Recent advances in human stem cell and three-dimensional (3D) culture technologies made it possible to generate novel 3D neural cell culture models that recapitulate AD pathologies including robust Aβ deposition and Aβ-driven NFT-like tau pathology. These new 3D human cell culture models of AD hold a promise for a novel platform that can be used for mechanism studies in human brain-like environment and high-throughput drug screening (HTS). In this review, we will summarize the current progress in recapitulating AD pathogenic cascades in human neural cell culture models using AD patient-derived induced pluripotent stem cells (iPSCs) or genetically modified human stem cell lines. We will also explain how new 3D culture technologies were applied to accelerate Aβ and p-tau pathologies in human neural cell cultures, as compared the standard two-dimensional (2D) culture conditions. Finally, we will discuss a potential impact of the human 3D human neural cell culture models on the AD drug-development process. These revolutionary 3D culture models of AD will contribute to accelerate the discovery of novel AD drugs.

Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system.

PubMed

Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

2016-03-09

Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production.

The boundary vector cell model of place cell firing and spatial memory

PubMed Central

Barry, Caswell; Lever, Colin; Hayman, Robin; Hartley, Tom; Burton, Stephen; O'Keefe, John; Jeffery, Kate; Burgess, Neil

2009-01-01

We review evidence for the boundary vector cell model of the environmental determinants of the firing of hippocampal place cells. Preliminary experimental results are presented concerning the effects of addition or removal of environmental boundaries on place cell firing and evidence that boundary vector cells may exist in the subiculum. We review and update computational simulations predicting the location of human search within a virtual environment of variable geometry, assuming that boundary vector cells provide one of the input representations of location used in mammalian spatial memory. Finally, we extend the model to include experience-dependent modification of connection strengths through a BCM-like learning rule, and compare the effects to experimental data on the firing of place cells under geometrical manipulations to their environment. The relationship between neurophysiological results in rats and spatial behaviour in humans is discussed. PMID:16703944

Unsolved mysteries: How does lipid peroxidation cause ferroptosis?

PubMed Central

Feng, Huizhong

2018-01-01

Ferroptosis is a cell death process driven by damage to cell membranes and linked to numerous human diseases. Ferroptosis is caused by loss of activity of the key enzyme that is tasked with repairing oxidative damage to cell membranes—glutathione peroxidase 4 (GPX4). GPX4 normally removes the dangerous products of iron-dependent lipid peroxidation, protecting cell membranes from this type of damage; when GPX4 fails, ferroptosis ensues. Ferroptosis is distinct from apoptosis, necroptosis, necrosis, and other modes of cell death. Several key mysteries regarding how cells die during ferroptosis remain unsolved. First, the drivers of lipid peroxidation are not yet clear. Second, the subcellular location of lethal lipid peroxides remains an outstanding question. Finally, how exactly lipid peroxidation leads to cell death is an unsolved mystery. Answers to these questions will provide insights into the mechanisms of ferroptotic cell death and associated human diseases, as well as new therapeutic strategies for such diseases. PMID:29795546

Deregulated activation of oncoprotein kinase Tpl2/Cot in HTLV-I-transformed T cells.

PubMed

Babu, Geetha; Waterfield, Michael; Chang, Mikyoung; Wu, Xuefeng; Sun, Shao-Cong

2006-05-19

Protein kinase Tpl2/Cot is encoded by a protooncogene that is cis-activated by retroviral insertion in murine T cell lymphomas. It has remained unclear whether this oncoprotein kinase is mutated or post-translationally activated in human cancer cells. We have shown here that Tpl2/Cot is constitutively activated in human leukemia cell lines transformed by the human T cell leukemia virus type I (HTLV-I). The kinase activity of Tpl2/Cot is normally suppressed through its physical interaction with an inhibitor, the NF-kappaB1 precursor protein p105. Interestingly, a large pool of Tpl2/Cot is liberated from p105 and exhibits constitutive kinase activity in HTLV-I-transformed T cells. In contrast to its labile property in normal cells, the pathologically activated Tpl2/Cot is remarkably stable. Further, whereas the physiological activation of Tpl2/Cot involves its long isoform, the HTLV-activated Tpl2/Cot is predominantly the short isoform. We have also shown that the HTLV-I-encoded Tax protein is able to activate Tpl2/Cot in transfected cells. Finally, Tpl2/Cot participates in the activation of NF-kappaB by Tax. These findings indicate that deregulated activation of Tpl2/Cot may occur in human cancer cells.

Splicing-factor alterations in cancers

PubMed Central

Anczuków, Olga; Krainer, Adrian R.

2016-01-01

Tumor-associated alterations in RNA splicing result either from mutations in splicing-regulatory elements or changes in components of the splicing machinery. This review summarizes our current understanding of the role of splicing-factor alterations in human cancers. We describe splicing-factor alterations detected in human tumors and the resulting changes in splicing, highlighting cell-type-specific similarities and differences. We review the mechanisms of splicing-factor regulation in normal and cancer cells. Finally, we summarize recent efforts to develop novel cancer therapies, based on targeting either the oncogenic splicing events or their upstream splicing regulators. PMID:27530828

A promising sword of tomorrow: Human γδ T cell strategies reconcile allo-HSCT complications.

PubMed

Hu, Yongxian; Cui, Qu; Luo, Chao; Luo, Yi; Shi, Jimin; Huang, He

2016-05-01

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially a curative therapeutic option for hematological malignancies. In clinical practice, transplantation associated complications greatly affected the final therapeutical outcomes. Currently, primary disease relapse, graft-versus-host disease (GVHD) and infections remain the three leading causes of a high morbidity and mortality in allo-HSCT patients. Various strategies have been investigated in the past several decades including human γδ T cell-based therapeutical regimens. In different microenvironments, human γδ T cells assume features reminiscent of classical Th1, Th2, Th17, NKT and regulatory T cells, showing diverse biological functions. The cytotoxic γδ T cells could be utilized to target relapsed malignancies, and recently regulatory γδ T cells are defined as a novel implement for GVHD management. In addition, human γδ Τ cells facilitate control of post-transplantation infections and participate in tissue regeneration and wound healing processes. These features potentiate γδ T cells a versatile therapeutical agent to target transplantation associated complications. This review focuses on insights of applicable potentials of human γδ T cells reconciling complications associated with allo-HSCT. We believe an improved understanding of pertinent γδ T cell functions would be further exploited in the design of innovative immunotherapeutic approaches in allo-HSCT, to reduce mortality and morbidity, as well as improve quality of life for patients after transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human Induced Pluripotent Stem Cell-Derived Macrophages for Unraveling Human Macrophage Biology.

PubMed

Zhang, Hanrui; Reilly, Muredach P

2017-11-01

Despite a substantial appreciation for the critical role of macrophages in cardiometabolic diseases, understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM), as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of the role of macrophages in human diseases. In this review, we summarize current literature in the differentiation and characterization of IPSDM at phenotypic, functional, and transcriptomic levels. We emphasize the progress in differentiating iPSC to tissue resident macrophages, and in understanding the ontogeny of in vitro differentiated IPSDM that resembles primitive hematopoiesis, rather than adult definitive hematopoiesis. We review the application of IPSDM in modeling both Mendelian genetic disorders and host-pathogen interactions. Finally, we highlighted the potential areas of research using IPSDM in functional validation of coronary artery disease loci in genome-wide association studies, functional genomic analyses, drug testing, and cell therapeutics in cardiovascular diseases. © 2017 American Heart Association, Inc.

Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening.

PubMed

Cheng, Chialin; Fass, Daniel M; Folz-Donahue, Kat; MacDonald, Marcy E; Haggarty, Stephen J

2017-01-11

Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical-like neurons from these NPC through inducible expression of the pro-neural transcription factor Neurogenin 2 (iNgn2-NPC). Finally, we describe methodology for the use of iNgn2-NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high-content, single-cell-level automated microscopy assays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia

PubMed Central

Karjalainen, Katja; Pasqualini, Renata; Cortes, Jorge E.; Kornblau, Steven M.; Lichtiger, Benjamin; O'Brien, Susan; Kantarjian, Hagop M.; Sidman, Richard L.; Arap, Wadih; Koivunen, Erkki

2015-01-01

Background We introduce an ex vivo methodology to perform drug library screening against human leukemia. Method Our strategy relies on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. We demonstrate several advantages: (I) partial recapitulation of the leukemia microenvironment, (ii) use of native hemoglobin oxygenation as real-time sensor/reporter, (iii) cost-effectiveness, (iv) species-specificity, and (v) format that enables high-throughput screening. Results As a proof-of-concept, we screened a chemical library (size ∼20,000) against human leukemia cells. We identified 70 compounds (“hit” rate=0.35%; Z-factor=0.71) with activity; we examined 20 to find 18 true-positives (90%). Finally, we show that carbonohydraxonic diamide group-containing compounds are potent anti-leukemia agents that induce cell death in leukemia cells and patient-derived samples. Conclusions This unique functional assay can identify novel drug candidates as well as find future applications in personalized drug selection for leukemia patients. PMID:24496871

Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors

PubMed Central

Patsialou, Antonia; Bravo-Cordero, Jose Javier; Wang, Yarong; Entenberg, David; Liu, Huiping; Clarke, Michael; Condeelis, John S.

2014-01-01

Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. In this report, we have used intravital multiphoton microscopy to visualize the different migration patterns of human breast tumor cells in live primary tumors. We used xenograft tumors of MDA-MB-231 cells as well as a low passage xenograft tumor from orthotopically injected patient-derived breast tumor cells. Direct visualization of human tumor cells in vivo shows two patterns of high-speed migration inside primary tumors: a. single cells and b. multicellular streams (i.e., cells following each other in a single file but without cohesive cell junctions). Critically, we found that only streaming and not random migration of single cells was significantly correlated with proximity to vessels, with intravasation and with numbers of elevated circulating tumor cells in the bloodstream. Finally, although the two human tumors were derived from diverse genetic backgrounds, we found that their migratory tumor cells exhibited coordinated gene expression changes that led to the same end-phenotype of enhanced migration involving activating actin polymerization and myosin contraction. Our data are the first direct visualization and assessment of in vivo migration within a live patient-derived breast xenograft tumor. PMID:25013744

Using the cost-effectiveness of allogeneic islet transplantation to inform induced pluripotent stem cell-derived β-cell therapy reimbursement.

PubMed

Archibald, Peter R T; Williams, David J

2015-11-01

In the present study a cost-effectiveness analysis of allogeneic islet transplantation was performed and the financial feasibility of a human induced pluripotent stem cell-derived β-cell therapy was explored. Previously published cost and health benefit data for islet transplantation were utilized to perform the cost-effectiveness and sensitivity analyses. It was determined that, over a 9-year time horizon, islet transplantation would become cost saving and 'dominate' the comparator. Over a 20-year time horizon, islet transplantation would incur significant cost savings over the comparator (GB£59,000). Finally, assuming a similar cost of goods to islet transplantation and a lack of requirement for immunosuppression, a human induced pluripotent stem cell-derived β-cell therapy would dominate the comparator over an 8-year time horizon.

Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

PubMed Central

Pezzolla, Daniela; López-Beas, Javier; Lachaud, Christian C.; Domínguez-Rodríguez, Alejandro; Smani, Tarik; Hmadcha, Abdelkrim; Soria, Bernat

2015-01-01

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process. PMID:25774684

Analysis of Large Seeds from Three Different Medicago truncatula Ecotypes Reveals a Potential Role of Hormonal Balance in Final Size Determination of Legume Grains

PubMed Central

Bandyopadhyay, Kaustav; Uluçay, Orhan; Şakiroğlu, Muhammet; Udvardi, Michael K.; Verdier, Jerome

2016-01-01

Legume seeds are important as protein and oil source for human diet. Understanding how their final seed size is determined is crucial to improve crop yield. In this study, we analyzed seed development of three accessions of the model legume, Medicago truncatula, displaying contrasted seed size. By comparing two large seed accessions to the reference accession A17, we described mechanisms associated with large seed size determination and potential factors modulating the final seed size. We observed that early events during embryogenesis had a major impact on final seed size and a delayed heart stage embryo development resulted to large seeds. We also observed that the difference in seed growth rate was mainly due to a difference in embryo cell number, implicating a role of cell division rate. Large seed accessions could be explained by an extended period of cell division due to a longer embryogenesis phase. According to our observations and recent reports, we observed that auxin (IAA) and abscisic acid (ABA) ratio could be a key determinant of cell division regulation at the end of embryogenesis. Overall, our study highlights that timing of events occurring during early seed development play decisive role for final seed size determination. PMID:27618017

Genoprotective properties of astaxanthin revealed by ionizing radiation exposure in vitro on human peripheral blood lymphocytes.

PubMed

Pilinska, M A; Кurinnyi, D A; Rushkovsky, S R; Dybska, O B

2016-12-01

to identify possible radioprotective properties of astaxanthin by means of cytogenetic criteria. Cultivation of peripheral blood lymphocytes from five apparently healthy volunteers; treatment of lym phocytes' cultures by astaxanthin in final concentrations 20 μg/ml in Go phase of mitotic cycle, prior to ? irradia tion in vitro in a dose 1 Gy; cytogenetic analysis the uniformly stained slides of metaphase chromosomes. The elec trophoresis of individual cells (Comet assay); visualization of results under fluorescent microscope; accounting the number of nucleoid the fourth grade that correspond to apoptosis of the cells. Established that astaxanthin in final concentration 20.0 μg/ml exposed to the culture of human peripher al blood lymphocytes in the early G0 phase of mitotic cycle leads to significant reduction of cytogenetic effects induced by gamma irradiation in vitro in dose 1.0 Gy (from 26.05 ± 1.81 to 9.08 ± 0.78 per 100 cells, respectively) and to significant increase the frequency of apoptotic cells at the 48 hour of cultivation (from (3.78 ± 0.24) to (8.26 ± 0.91) %, respectively). The results obtained show the ability of astaxanthin to considerable weakening of radioinduced muta genic effect in human peripheral blood lymphocytes, which testify its powerful radioprotective potential. M. А. Pilinska, D. А. Кurinnyi, S. R. Rushkovsky, О. B. Dybska.

Modeling Alzheimer’s disease with human induced pluripotent stem (iPS) cells

PubMed Central

Mungenast, Alison E.; Siegert, Sandra; Tsai, Li-Huei

2018-01-01

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer’s disease (AD) have been often failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD. PMID:26657644

Modeling Alzheimer's disease with human induced pluripotent stem (iPS) cells.

PubMed

Mungenast, Alison E; Siegert, Sandra; Tsai, Li-Huei

2016-06-01

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer's disease (AD) have been failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas9 will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD. Copyright © 2015 Elsevier Inc. All rights reserved.

A Simple and Efficient Method of Slow Freezing for Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

PubMed

Imaizumi, Keitaro; Iha, Momoe; Nishishita, Naoki; Kawamata, Shin; Nishikawa, Shinichi; Akuta, Teruo

2016-01-01

Protocols available for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells are very inefficient and laborious compared to those for the cryopreservation of murine ES/iPS cells or other general cell lines. While the vitrification method may be adequate when working with small numbers of human ES/iPS cells, it requires special skills and is unsuitable when working with large cell numbers. Here, we describe a simple and efficient method for the cryopreservation of hES/hiPS cells that is based on a conventional slow freezing method that uses a combination of Pronase/EDTA for Stem™ and CP-5E™ [final concentrations: 6 % hydroxyethyl starch, 5 % DMSO, and 5 % ethylene glycol in saline]. CP-5E™ is highly effective for the cryopreservation of small cell clumps produced by hES/hiPS colony detachment in the presence of Pronase and EDTA (Pronase/EDTA for Stem™, a formulation containing multiple digestive enzymes from Streptomyces griseus). This novel method would be quite useful for large-scale hES/iPS cell banking for use in clinical applications.

PM2.5 induces autophagy-mediated cell death via NOS2 signaling in human bronchial epithelium cells

PubMed Central

Zhu, Xiao-Ming; Wang, Qin; Xing, Wei-Wei; Long, Min-Hui; Fu, Wen-Liang; Xia, Wen-Rong; Jin, Chen; Guo, Ning; Xu, Dong-Qun; Xu, Dong-Gang

2018-01-01

The biggest victim of ambient air pollution is the respiratory system. Mainly because of the harmful components, especially the particulate matters with an aerodynamic diameter of ≤ 2.5µm (PM2.5), can be directly inhaled and deeply penetrate into the lung alveoli, thus causing severe lung dysfunction, including chronic cough, bronchitis and asthma, even lung cancer. Unfortunately, the toxicological mechanisms of PM2.5 associations with these adverse respiratory outcomes have still not been clearly unveiled. Here, we found that PM2.5 rapidly induced inflammatory responses, oxidative injure and cell death in human bronchial epithelium cells through upregulation of IL-6 expression, ROS production and apoptosis. Furthermore, PM2.5 specifically induced nitric oxide synthase 2 (NOS2) expression and NO generation to elevate excessive autophagy. Finally, disruption of NOS2 signaling effectively blocked autophayosome formation and the subsequent cell death. Our novel findings systemically reveled the role of autophagy-mediated cell death in PM2.5-treated human bronchial epithelium cells and provided potential strategy for future clinic intervention.

It’s alive: microbes and cells in human milk and their potential benefits to mother and infant.

PubMed

Bode, Lars; McGuire, Mark; Rodriguez, Juan M; Geddes, Donna T; Hassiotou, Foteini; Hartmann, Peter E; McGuire, Michelle K

2014-09-01

Human milk is the optimal source of nutrition for the nursing infant. Classically, the nutrients (water, protein, lipid, carbohydrate, vitamins, and minerals) were studied as the critical components of milk serving the growth needs of the infant for optimum growth. However, human milk contains factors other than the classically defined nutrients for which researchers are investigating potential roles in infant and maternal health, development, and well-being. The symposium addressed some of the exciting factors being studied, including microbes and maternal cells found within milk. Drs. Michelle McGuire and Juan M. Rodríguez addressed the presence of a bacterial community in human milk produced by healthy and mastitic mothers, potential sources of those bacteria, and the impact of milk-derived bacteria on the nursing infant. Drs. Donna Geddes, Peter Hartmann, and Foteini Hassiotou discussed the potential importance of maternal cells. For years, immune cells were known to be present in human milk, but recent evidence suggests that their impact is as much on the infant as on the health of the lactating mammary gland. Finally, the existence of highly plastic stem cells in human milk opens doors for previously unforeseen developmental “training” of the nursing infant.

Replication and Oncolytic Activity of an Avian Orthoreovirus in Human Hepatocellular Carcinoma Cells

PubMed Central

Kozak, Robert A.; Hattin, Larissa; Biondi, Mia J.; Corredor, Juan C.; Walsh, Scott; Xue-Zhong, Max; Manuel, Justin; McGilvray, Ian D.; Morgenstern, Jason; Lusty, Evan; Cherepanov, Vera; McBey, Betty-Anne; Leishman, David; Feld, Jordan J.; Bridle, Byram; Nagy, Éva

2017-01-01

Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies. PMID:28441762

Inhibition of Fatty Acid Metabolism Reduces Human Myeloma Cells Proliferation

PubMed Central

Tirado-Vélez, José Manuel; Joumady, Insaf; Sáez-Benito, Ana; Cózar-Castellano, Irene; Perdomo, Germán

2012-01-01

Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40–70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma. PMID:23029529

The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

PubMed

Singh, D; Paduch, D A; Schlegel, P N; Orwig, K E; Mielnik, A; Bolyakov, A; Wright, W W

2017-05-01

Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype. We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches. N/A. There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature. We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production. This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy

PubMed Central

da Silva, Rafaela J.; Gomes, Angelica O.; Franco, Priscila S.; Pereira, Ariane S.; Milian, Iliana C. B.; Ribeiro, Mayara; Fiorenzani, Paolo; dos Santos, Maria C.; Mineo, José R.; da Silva, Neide M.; Ferro, Eloisa A. V.; de Freitas Barbosa, Bellisa

2017-01-01

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure. PMID:28798905

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

PubMed

da Silva, Rafaela J; Gomes, Angelica O; Franco, Priscila S; Pereira, Ariane S; Milian, Iliana C B; Ribeiro, Mayara; Fiorenzani, Paolo; Dos Santos, Maria C; Mineo, José R; da Silva, Neide M; Ferro, Eloisa A V; de Freitas Barbosa, Bellisa

2017-01-01

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

PlGF gene knockdown in human retinal pigment epithelial cells.

PubMed

Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Ahmadieh, Hamid; Rezaeikanavi, Mozhgan; Samiei, Shahram; Khalooghi, Keynoush

2011-04-01

To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells. Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs). At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs. Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

Gen-27, a newly synthesized flavonoid, inhibits glycolysis and induces cell apoptosis via suppression of hexokinase II in human breast cancer cells.

PubMed

Tao, Lei; Wei, Libing; Liu, Yishi; Ding, Yang; Liu, Xiuting; Zhang, Xin; Wang, Xiaoping; Yao, Yuyuan; Lu, Jinrong; Wang, Qing; Hu, Rong

2017-02-01

We have previously reported that Gen-27, a newly synthesized flavonoid, exhibits anticancer effects against human colorectal cancer cells. In this study, we investigated the anticancer effects in human breast cancer cell lines and its underlying mechanisms. We demonstrated that Gen-27 inhibited the growth and proliferation of human breast cancer cells in concentration and time-dependent manners. It was found that Gen-27 induced mitochondrial-mediated apoptosis, characterized by the dissipation of mitochondrial membrane potential (ΔΨm), cytochrome c (Cyt c) release from mitochondria to cytosol, activation of caspases and induction of poly (ADP-ribose) polymerase (PARP). In addition, Gen-27 inhibited the glycolysis in human breast cancer cells. After treatment with Gen-27, the expression of HKII was down-regulated, accompanied by weakened interaction of HKII and VDAC. Further research revealed that the induction of mitochondrial apoptosis was associated with the decrease of HKII expression by Gen-27. Finally, in vivo studies demonstrated that Gen-27 significantly suppressed the growth and promoted apoptosis of MDA-MB-231 breast cancer orthotopic tumors with low systemic toxicity. In conclusion, the results showed that Gen-27 had significant anticancer effects against human breast cancer and it may potentially be used as a novel anticancer agent for the treatment of breast cancer. Copyright © 2016. Published by Elsevier Inc.

Human Cytomegalovirus UL99-Encoded pp28 Is Required for the Cytoplasmic Envelopment of Tegument-Associated Capsids

PubMed Central

Silva, Maria C.; Yu, Qian-Chun; Enquist, Lynn; Shenk, Thomas

2003-01-01

The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm. PMID:12970444

Genetics of the connective tissue proteins: Assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids*

PubMed Central

Raj, Cholappadi V. Sundar; Church, Robert L.; Klobutcher, Lawrence A.; Ruddle, Frank H.

1977-01-01

Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [3H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17. Images PMID:412188

Drosophila TIEG Is a Modulator of Different Signalling Pathways Involved in Wing Patterning and Cell Proliferation

PubMed Central

Rodriguez, Isabel

2011-01-01

Acquisition of a final shape and size during organ development requires a regulated program of growth and patterning controlled by a complex genetic network of signalling molecules that must be coordinated to provide positional information to each cell within the corresponding organ or tissue. The mechanism by which all these signals are coordinated to yield a final response is not well understood. Here, I have characterized the Drosophila ortholog of the human TGF-β Inducible Early Gene 1 (dTIEG). TIEG are zinc-finger proteins that belong to the Krüppel-like factor (KLF) family and were initially identified in human osteoblasts and pancreatic tumor cells for the ability to enhance TGF-β response. Using the developing wing of Drosophila as “in vivo” model, the dTIEG function has been studied in the control of cell proliferation and patterning. These results show that dTIEG can modulate Dpp signalling. Furthermore, dTIEG also regulates the activity of JAK/STAT pathway suggesting a conserved role of TIEG proteins as positive regulators of TGF-β signalling and as mediators of the crosstalk between signalling pathways acting in a same cellular context. PMID:21494610

Pathogen inactivation of human serum facilitates its clinical use for islet cell culture and subsequent transplantation.

PubMed

Ståhle, Magnus U; Brandhorst, Daniel; Korsgren, Olle; Knutson, Folke

2011-01-01

Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept® technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3-4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

PubMed

Boso, Guney; Somia, Nikunj V

2015-01-01

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.

Transdifferentiation of human periodontal ligament stem cells into pancreatic cell lineage.

PubMed

Lee, Jeong Seok; An, Seong Yeong; Kwon, Il Keun; Heo, Jung Sun

2014-10-01

Human periodontal ligament-derived stem cells (PDLSCs) demonstrate self-renewal capacity and multilineage differentiation potential. In this study, we investigated the transdifferentiation potential of human PDLSCs into pancreatic islet cells. To form three-dimensional (3D) clusters, PDLSCs were cultured in Matrigel with media containing differentiation-inducing agents. We found that after 6 days in culture, PDLSCs underwent morphological changes resembling pancreatic islet-like cell clusters (ICCs). The morphological characteristics of PDLSC-derived ICCs were further assessed using scanning electron microscopy analysis. Using reverse transcription-polymerase chain reaction analysis, we found that pluripotency genes were downregulated, whereas early endoderm and pancreatic differentiation genes were upregulated, in PDLSC-derived ICCs compared with undifferentiated PDLSCs. Furthermore, we found that PDLSC-derived ICCs were capable of secreting insulin in response to high concentrations of glucose, validating their functional differentiation into islet cells. Finally, we also performed dithizone staining, as well as immunofluorescence assays and fluorescence-activated cell sorting analysis for pancreatic differentiation markers, to confirm the differentiation status of PDLSC-derived ICCs. These results demonstrate that PDLSCs can transdifferentiate into functional pancreatic islet-like cells and provide a novel, alternative cell population for pancreatic repair. Copyright © 2014 John Wiley & Sons, Ltd.

Generation of high-yield insulin producing cells from human bone marrow mesenchymal stem cells.

PubMed

Jafarian, Arefeh; Taghikhani, Mohammad; Abroun, Saeid; Pourpak, Zahra; Allahverdi, Amir; Soleimani, Masoud

2014-07-01

Allogenic islet transplantation is a most efficient approach for treatment of diabetes mellitus. However, the scarcity of islets and long term need for an immunosuppressant limits its application. Recently, cell replacement therapies that generate of unlimited sources of β cells have been developed to overcome these limitations. In this study we have described a stage specific differentiation protocol for the generation of insulin producing islet-like clusters from human bone marrow mesenchymal stem cells (hBM-MSCs). This specific stepwise protocol induced differentiation of hMSCs into definitive endoderm, pancreatic endoderm and pancreatic endocrine cells that expressed of sox17, foxa2, pdx1, ngn3, nkx2.2, insulin, glucagon, somatostatin, pancreatic polypeptide, and glut2 transcripts respectively. In addition, immunocytochemical analysis confirmed protein expression of the above mentioned genes. Western blot analysis discriminated insulin from proinsulin in the final differentiated cells. In derived insulin producing cells (IPCs), secreted insulin and C-peptide was in a glucose dependent manner. We have developed a protocol that generates effective high-yield human IPCs from hBM-MSCs in vitro. These finding suggest that functional IPCs generated by this procedure can be used as a cell-based approach for insulin dependent diabetes mellitus.

Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.

PubMed

Vera-Rodriguez, M; Diez-Juan, A; Jimenez-Almazan, J; Martinez, S; Navarro, R; Peinado, V; Mercader, A; Meseguer, M; Blesa, D; Moreno, I; Valbuena, D; Rubio, C; Simon, C

2018-04-01

What is the origin and composition of cell-free DNA in human embryo spent culture media? Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. Trophectoderm biopsies and culture media samples (20 μl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. This study was limited by the sample size and the number of cells analyzed by FISH. This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. This work was funded by Igenomix S.L. There are no competing interests.

Host cell proteins in biotechnology-derived products: A risk assessment framework.

PubMed

de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

2015-11-01

To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

Of mice and men: how animal models advance our understanding of T-cell function in RA.

PubMed

Kobezda, Tamás; Ghassemi-Nejad, Sheida; Mikecz, Katalin; Glant, Tibor T; Szekanecz, Zoltán

2014-03-01

The involvement of autoreactive T cells in the pathogenesis of rheumatoid arthritis (RA) as well as in autoimmune animal models of arthritis has been well established; however, unanswered questions, such as the role of joint-homing T cells, remain. Animal models of arthritis are superb experimental tools in demonstrating how T cells trigger joint inflammation, and thus can help to further our knowledge of disease mechanisms and potential therapies. In this Review, we discuss the similarities and differences in T-cell subsets and functions between RA and mouse arthritis models. For example, various T-cell subsets are involved in both human and mouse arthritis, but differences might exist in the cytokine regulation and plasticity of these cells. With regard to joint-homing T cells, an abundance of synovial T cells is present in humans compared with mice. On the other hand, local expansion of type 17 T-helper (TH17) cells is observed in some animal models, but not in RA. Finally, whereas T-cell depletion therapy essentially failed in RA, antibody targeting of T cells can work, at least preventatively, in most arthritis models. Clearly, additional human and animal studies are needed to fill the gap in our understanding of the specific contribution of T-cell subsets to arthritis in mice and men.

The pyrimidin analogue cyclopentenyl cytosine induces alloantigen-specific non-responsiveness of human T lymphocytes

PubMed Central

Nikolaeva, N; Bemelman, F J; Yong, S-L; Verschuur, A; van Lier, R A W; ten Berge, I J M

2008-01-01

Cyclopentenyl cytosine (CPEC) has been shown to induce apoptosis in human T lymphoblastic cell lines and T cells from leukaemia patients. In this study we have addressed the question of whether CPEC is able to decrease proliferation and effector functions of human alloresponsive T lymphocytes and induce T cell anergy. The proliferative capacity of human peripheral blood mononuclear cells in response to allogeneic stimulation was measured by 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester staining. Flow cytometric analysis was performed using surface CD4, CD8, CD25, CD103 and intracellular perforin, granzyme A, granzyme B, caspase-3 and forkhead box P3 (FoxP3) markers. The in vivo immunosuppressive capacity was tested in a murine skin graft model. Addition of CPEC at a concentration of 20 nM strongly decreased the expansion and cytotoxicity of alloreactive T cells. Specific restimulation in the absence of CPEC showed that the cells became anergic. The drug induced caspase-dependent apoptosis of alloreactive T lymphocytes. Finally, CPEC increased the percentage of CD25high FoxP3+ CD4+ and CD103+ CD8+ T cells, and potentiated the effect of rapamycin in increasing the numbers of alloreactive regulatory T cells. Treatment with CPEC of CBA/CA mice transplanted with B10/Br skin grafts significantly prolonged graft survival. We conclude that CPEC inhibits proliferation and cytotoxicity of human alloreactive T cells and induces alloantigen non-responsiveness in vitro. PMID:18062797

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

PubMed

Cigan, Alexander D; Roach, Brendan L; Nims, Robert J; Tan, Andrea R; Albro, Michael B; Stoker, Aaron M; Cook, James L; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

2016-06-14

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

PubMed

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules

PubMed Central

Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Singh, A. J. A. Ranjith; Peng, I-Chia; Priya, Sivan Padma; Hamat, Rukman Awang; Higuchi, Akon

2014-01-01

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation. PMID:25526563

Human and feline adipose-derived mesenchymal stem cells have comparable phenotype, immunomodulatory functions, and transcriptome.

PubMed

Clark, Kaitlin C; Fierro, Fernando A; Ko, Emily Mills; Walker, Naomi J; Arzi, Boaz; Tepper, Clifford G; Dahlenburg, Heather; Cicchetto, Andrew; Kol, Amir; Marsh, Lyndsey; Murphy, William J; Fazel, Nasim; Borjesson, Dori L

2017-03-20

Adipose-derived mesenchymal stem cells (ASCs) are a promising cell therapy to treat inflammatory and immune-mediated diseases. Development of appropriate pre-clinical animal models is critical to determine safety and attain early efficacy data for the most promising therapeutic candidates. Naturally occurring diseases in cats already serve as valuable models to inform human clinical trials in oncologic, cardiovascular, and genetic diseases. The objective of this study was to complete a comprehensive side-by-side comparison of human and feline ASCs, with an emphasis on their immunomodulatory capacity and transcriptome. Human and feline ASCs were evaluated for phenotype, immunomodulatory profile, and transcriptome. Additionally, transwells were used to determine the role of cell-cell contact in ASC-mediated inhibition of lymphocyte proliferation in both humans and cats. Similar to human ASCs, feline ASCs were highly proliferative at low passages and fit the minimal criteria of multipotent stem cells including a compatible surface protein phenotype, osteogenic capacity, and normal karyotype. Like ASCs from all species, feline ASCs inhibited mitogen-activated lymphocyte proliferation in vitro, with or without direct ASC-lymphocyte contact. Feline ASCs mimic human ASCs in their mediator secretion pattern, including prostaglandin E2, indoleamine 2,3 dioxygenase, transforming growth factor beta, and interleukin-6, all augmented by interferon gamma secretion by lymphocytes. The transcriptome of three unactivated feline ASC lines were highly similar. Functional analysis of the most highly expressed genes highlighted processes including: 1) the regulation of apoptosis; 2) cell adhesion; 3) response to oxidative stress; and 4) regulation of cell differentiation. Finally, feline ASCs had a similar gene expression profile to noninduced human ASCs. Findings suggest that feline ASCs modulate lymphocyte proliferation using soluble mediators that mirror the human ASC secretion pattern. Uninduced feline ASCs have similar gene expression profiles to uninduced human ASCs, as revealed by transcriptome analysis. These data will help inform clinical trials using cats with naturally occurring diseases as surrogate models for human clinical trials in the regenerative medicine arena.

Molecular cloning of metaphase chromosome protein 1 (MCP1), a novel human autoantigen that associates with condensed chromosomes during mitosis.

PubMed

Bronze-da-Rocha, E; Catita, J A; Sunkel, C E

1998-02-01

Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human chromosome-associated proteins. Previous immunolocalization studies in human and murine tissue culture cells showed that some of these monoclonal antibodies recognize nuclear antigens that associate with condensed chromosomes during mitosis. One antibody was selected for screening a human HeLa S3 cDNA expression library, and cDNAs that code for an antigen of 31-33 kDa were isolated. Immunological, biochemical and cell fractionation data indicate that the 31- to 33-kDa antigen corresponds to the chromosome-associated protein recognized by the original monoclonal antibody. Sequence analysis shows that we isolated a novel human gene. Immunolocalization to human tissue culture cells shows that during interphase the antigen is dispersed in the nucleus and that during mitosis it associates exclusively with condensed chromosomes. A similar pattern of localization was also observed in mouse fibroblasts, suggesting that the antigen is conserved among different species. Finally, we show that part of the antigen remains bound to the scaffold/matrix component, even after high salt extraction.

The efficacy of 9-cis retinoic acid in experimental models of cancer.

PubMed

Gottardis, M M; Lamph, W W; Shalinsky, D R; Wellstein, A; Heyman, R A

1996-01-01

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

PubMed Central

Chaikind, Brian; Bessen, Jeffrey L.; Thompson, David B.; Hu, Johnny H.; Liu, David R.

2016-01-01

We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases. PMID:27515511

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

PubMed

Lan, Xiaoyang; Jörg, David J; Cavalli, Florence M G; Richards, Laura M; Nguyen, Long V; Vanner, Robert J; Guilhamon, Paul; Lee, Lilian; Kushida, Michelle M; Pellacani, Davide; Park, Nicole I; Coutinho, Fiona J; Whetstone, Heather; Selvadurai, Hayden J; Che, Clare; Luu, Betty; Carles, Annaick; Moksa, Michelle; Rastegar, Naghmeh; Head, Renee; Dolma, Sonam; Prinos, Panagiotis; Cusimano, Michael D; Das, Sunit; Bernstein, Mark; Arrowsmith, Cheryl H; Mungall, Andrew J; Moore, Richard A; Ma, Yussanne; Gallo, Marco; Lupien, Mathieu; Pugh, Trevor J; Taylor, Michael D; Hirst, Martin; Eaves, Connie J; Simons, Benjamin D; Dirks, Peter B

2017-09-14

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Stem cell senescence drives age-attenuated induction of pituitary tumours in mouse models of paediatric craniopharyngioma.

PubMed

Mario Gonzalez-Meljem, Jose; Haston, Scott; Carreno, Gabriela; Apps, John R; Pozzi, Sara; Stache, Christina; Kaushal, Grace; Virasami, Alex; Panousopoulos, Leonidas; Neda Mousavy-Gharavy, Seyedeh; Guerrero, Ana; Rashid, Mamunur; Jani, Nital; Goding, Colin R; Jacques, Thomas S; Adams, David J; Gil, Jesus; Andoniadou, Cynthia L; Martinez-Barbera, Juan Pedro

2017-11-28

Senescent cells may promote tumour progression through the activation of a senescence-associated secretory phenotype (SASP), whether these cells are capable of initiating tumourigenesis in vivo is not known. Expression of oncogenic β-catenin in Sox2+ young adult pituitary stem cells leads to formation of clusters of stem cells and induction of tumours resembling human adamantinomatous craniopharyngioma (ACP), derived from Sox2- cells in a paracrine manner. Here, we uncover the mechanisms underlying this paracrine tumourigenesis. We show that expression of oncogenic β-catenin in Hesx1+ embryonic precursors also results in stem cell clusters and paracrine tumours. We reveal that human and mouse clusters are analogous and share a common signature of senescence and SASP. Finally, we show that mice with reduced senescence and SASP responses exhibit decreased tumour-inducing potential. Together, we provide evidence that senescence and a stem cell-associated SASP drive cell transformation and tumour initiation in vivo in an age-dependent fashion.

Synthetic PAMAM-RGD conjugates target and bind to odontoblast-like MDPC 23 cells and the predentin in tooth organ cultures.

PubMed

Hill, Elliott; Shukla, Rameshwer; Park, Steve S; Baker, James R

2007-01-01

Screening techniques now allow for the identification of small peptides that bind specifically to molecules like cells. However, despite the enthusiasm for this approach, single peptides often lack the binding affinity to target in vivo and regulate cell function. We took peptides containing the Arg-Gly Asp(RGD) motif that bind to the alpha Vbeta 3 integrin and have shown potential as therapeutics. To improve their binding affinity, we synthesized polyamidoamine (PAMAM) dendrimer-RGD conjugates that that contain 12-13 copies of the peptide. When cultured with human dermal microvessel endothelial cells (HDMEC), human vascular endothelial cells (HUVEC), or odontoblast-like MDPC-23 cells, the PAMAM dendrimer conjugate targets this receptor in a manner that is both time- and dose-dependent. Finally, this conjugate selectively targets RGD binding sites in the predentin of human tooth organ cultures. Taken together, these studies provide proof of principle that synthetic PAMAM-RGD conjugates could prove useful as carriers for the tissue-specific delivery of integrin-targeted therapeutics or imaging agents and could be used to engineer tissue regeneration.

Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

PubMed

Lee, Patrick C; Truong, Brian; Vega-Crespo, Agustin; Gilmore, W Blake; Hermann, Kip; Angarita, Stephanie Ak; Tang, Jonathan K; Chang, Katherine M; Wininger, Austin E; Lam, Alex K; Schoenberg, Benjamen E; Cederbaum, Stephen D; Pyle, April D; Byrne, James A; Lipshutz, Gerald S

2016-11-29

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

Energy Metabolism in Human Pluripotent Stem Cells and Their Differentiated Counterparts

PubMed Central

Moura, Michelle B.; Momcilovic, Olga; Easley, Charles A.; Ramalho-Santos, João; Van Houten, Bennett; Schatten, Gerald

2011-01-01

Background Human pluripotent stem cells have the ability to generate all cell types present in the adult organism, therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly, many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines, namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells. Methodology/Principal Findings We compared the energy metabolism of hESCs, IPSCs, and their somatic counterparts. Focusing on mitochondria, we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism, including glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer, as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism. Conclusions/Findings Our results demonstrate that, although the metabolic signature of IPSCs is not identical to that of hESCs, nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels, lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore, our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates, such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH). PMID:21698063

Unique and Common Features of Innate-Like Human Vδ2+ γδT Cells and Mucosal-Associated Invariant T Cells.

PubMed

Provine, Nicholas M; Binder, Benedikt; FitzPatrick, Michael E B; Schuch, Anita; Garner, Lucy C; Williamson, Kate D; van Wilgenburg, Bonnie; Thimme, Robert; Klenerman, Paul; Hofmann, Maike

2018-01-01

Mucosal-associated invariant T (MAIT) cells are innate-like T cells abundant in humans that can be activated in a TCR-independent manner by inflammatory and antiviral cytokines. In humans, the capacity for TCR-independent activation is functionally linked to a transcriptional program that can be identified by the expression of the C-type lectin receptor, CD161. In addition to MAIT cells, it has been demonstrated that a subset of γδT cells expresses CD161 and can be activated by TCR-independent cytokine stimulation. In this study, we sought to clarify the nature of cytokine-responsive human γδT cells. We could link CD161 expression on Vδ2 + versus Vδ1 + γδT cells to the observation that Vδ2 + γδT cells, but not Vδ1 + γδT cells, robustly produced IFN-γ upon stimulation with a variety of cytokine combinations. Interestingly, both CD161 + and CD161 - Vδ2 + γδT cells responded to these stimuli, with increased functionality within the CD161 + subset. This innate-like responsiveness corresponded to high expression of PLZF and IL-18Rα, analogous to MAIT cells. Vδ2 + γδT cells in human duodenum and liver maintained a CD161 + IL-18Rα + phenotype and produced IFN-γ in response to IL-12 and IL-18 stimulation. In contrast to MAIT cells, we could not detect IL-17A production but observed higher steady-state expression of Granzyme B by Vδ2 + γδT cells. Finally, we investigated the frequency and functionality of γδT cells in the context of chronic hepatitis C virus infection, as MAIT cells are reduced in frequency in this disease. By contrast, Vδ2 + γδT cells were maintained in frequency and displayed unimpaired IFN-γ production in response to cytokine stimulation. In sum, human Vδ2 + γδT cells are a functionally distinct population of cytokine-responsive innate-like T cells that is abundant in blood and tissues with similarities to human MAIT cells.

Expression pattern of RAGE and IGF-1 in the human fetal ovary and ovarian serous carcinoma.

PubMed

Poljicanin, Ana; Filipovic, Natalija; Vukusic Pusic, Tanja; Soljic, Violeta; Caric, Ana; Saraga-Babic, Mirna; Vukojevic, Katarina

2015-01-01

The expression pattern of RAGE and IGF-1 proteins in different ovarian cell lineages was histologically analyzed in six fetal, nine adult human ovaries, and nine serous ovarian carcinomas (OSC) using immunohistochemical methods. Mild expression of IGF-1 in ovarian surface epithelium (Ose) and oocytes in the 15-week human ovaries increased to moderate or strong in the stromal cells, oocytes and follicular cells in week 22. Occasional mild RAGE expression was observed in Ose during week 15, while strong expression characterized primordial follicles in week 22. In the reproductive human ovary, IGF-1 was mildly to moderately expressed in all ovarian cell lineages except in theca cells of the tertiary follicle where IGF-1 was negative. RAGE was strongly positive in the granulosa cells and some theca cells of the tertiary follicle, while negative to mildly positive in all cells of the secondary follicle. In the postmenopausal human ovary IGF-1 and RAGE were mildly expressed in Ose and stroma. In OSC, cells were strongly positive to IGF-1 and RAGE, except for some negative stromal cells. Different levels of IGF-1 and RAGE co-expression characterized fetal ovarian cells during development. In reproductive ovaries, IGF-1 and RAGE were co-localized in the granulosa and theca interna cells of tertiary follicles, while in postmenopausal ovaries and OSC, IGF-1 and RAGE were co-localized in Ose and OSC cells respectively. Our results indicate that intracellular levels of IGF-1 and RAGE protein might regulate the final destiny of the ovarian cell populations prior and during folliculogenesis, possibly controlling the metastatic potential of OSC as well. Copyright © 2015. Published by Elsevier GmbH.

Three dimensional living neural networks

NASA Astrophysics Data System (ADS)

Linnenberger, Anna; McLeod, Robert R.; Basta, Tamara; Stowell, Michael H. B.

2015-08-01

We investigate holographic optical tweezing combined with step-and-repeat maskless projection micro-stereolithography for fine control of 3D positioning of living cells within a 3D microstructured hydrogel grid. Samples were fabricated using three different cell lines; PC12, NT2/D1 and iPSC. PC12 cells are a rat cell line capable of differentiation into neuron-like cells NT2/D1 cells are a human cell line that exhibit biochemical and developmental properties similar to that of an early embryo and when exposed to retinoic acid the cells differentiate into human neurons useful for studies of human neurological disease. Finally induced pluripotent stem cells (iPSC) were utilized with the goal of future studies of neural networks fabricated from human iPSC derived neurons. Cells are positioned in the monomer solution with holographic optical tweezers at 1064 nm and then are encapsulated by photopolymerization of polyethylene glycol (PEG) hydrogels formed by thiol-ene photo-click chemistry via projection of a 512x512 spatial light modulator (SLM) illuminated at 405 nm. Fabricated samples are incubated in differentiation media such that cells cease to divide and begin to form axons or axon-like structures. By controlling the position of the cells within the encapsulating hydrogel structure the formation of the neural circuits is controlled. The samples fabricated with this system are a useful model for future studies of neural circuit formation, neurological disease, cellular communication, plasticity, and repair mechanisms.

BMP signaling in the human fetal ovary is developmentally regulated and promotes primordial germ cell apoptosis.

PubMed

Childs, Andrew J; Kinnell, Hazel L; Collins, Craig S; Hogg, Kirsten; Bayne, Rosemary A L; Green, Samira J; McNeilly, Alan S; Anderson, Richard A

2010-08-01

Primordial germ cells (PGCs) are the embryonic precursors of gametes in the adult organism, and their development, differentiation, and survival are regulated by a combination of growth factors collectively known as the germ cell niche. Although many candidate niche components have been identified through studies on mouse PGCs, the growth factor composition of the human PGC niche has not been studied extensively. Here we report a detailed analysis of the expression of components of the bone morphogenetic protein (BMP) signaling apparatus in the human fetal ovary, from postmigratory PGC proliferation to the onset of primordial follicle formation. We find developmentally regulated and reciprocal patterns of expression of BMP2 and BMP4 and identify germ cells to be the exclusive targets of ovarian BMP signaling. By establishing long-term cultures of human fetal ovaries in which PGCs are retained within their physiological niche, we find that BMP4 negatively regulates postmigratory PGC numbers in the human fetal ovary by promoting PGC apoptosis. Finally, we report expression of both muscle segment homeobox (MSX)1 and MSX2 in the human fetal ovary and reveal a selective upregulation of MSX2 expression in human fetal ovary in response to BMP4, suggesting this gene may act as a downstream effector of BMP-induced apoptosis in the ovary, as in other systems. These data reveal for the first time growth factor regulation of human PGC development in a physiologically relevant context and have significant implications for the development of cultures systems for the in vitro maturation of germ cells, and their derivation from pluripotent stem cells.

mAb C19 targets a novel surface marker for the isolation of human cardiac progenitor cells from human heart tissue and differentiated hESCs.

PubMed

Leung, Hau Wan; Moerkamp, Asja T; Padmanabhan, Jayanthi; Ng, Sze-Wai; Goumans, Marie-José; Choo, Andre

2015-05-01

Cardiac progenitor cells (CPCs) have been isolated from adult and developing hearts using an anti-mouse Sca-1 antibody. However, the absence of a human Sca-1 homologue has hampered the clinical application of the CPCs. Therefore, we generated novel monoclonal antibodies (mAbs) specifically raised against surface markers expressed by resident human CPCs. Here, we explored the suitability of one of these mAbs, mAb C19, for the identification, isolation and characterization of CPCs from fetal heart tissue and differentiating cultures of human embryonic stem cells (hESCs). Using whole-cell immunization, mAbs were raised against Sca-1+ CPCs and screened for reactivity to various CPC lines by flow cytometry. mAb C19 was found to be specific for Sca-1+ CPCs, with high cell surface binding capabilities. mAb C19 stained small stem-like cells in cardiac tissue sections. Moreover, during differentiation of hESCs towards cardiomyocytes, a transient population of cells with mAb C19 reactivity was identified and isolated using magnetic-activated cell sorting. Their cell fate was tracked and found to improve cardiomyocyte purity from hESC-derived cultures. mAb C19+ CPCs, from both hESC differentiation and fetal heart tissues, were maintained and expanded in culture, while retaining their CPC-like characteristics and their ability to further differentiate into cardiomyocytes by stimulation with TGFβ1. Finally, gene expression profiling of these mAb C19+ CPCs suggested a highly angiogenic nature, which was further validated by cell-based angiogenesis assays. mAb C19 is a new surface marker for the isolation of multipotent CPCs from both human heart tissues and differentiating hESCs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Neuronal connections, cell formation and cell migration in the perinatal human hippocampal dentate gyrus.

PubMed

Seress, L

1998-06-01

Jean Piaget's "stage theory" suggests that cognitive development proceeds in discrete steps, among which the first is the sensorimotor period that occupies the first two years. In recent years it became clear that an intact and mature hippocampus is necessary for memory formation both in experimental animals and in human. In the present experiments the perinatal morphological development of the human hippocampus was studied to describe structural changes that may correlate with the developmental changes of intellectual growth. Our results suggest that cell formation in the human hippocampus terminates several weeks before birth, but immature cells migrate to their final positions through the first six postnatal months. The newborn hippocampus contains all cell types and cell layers that are characteristic for the adult hippocampus. However, changes of the light microscopic features of the postsynaptic target neurons of hippocampal granule cells indicate that connections between granule cells and their target neurons are immature at birth and develop through an extended period of time that may last for three years. Since this neuronal connection is the first link in the chain of the main hippocampal synaptic circuitry, it may be suggested that human hippocampus is functionally impaired at birth. This period of light microscopic morphological maturation correlates well with the time period of Piaget's first stage of cognitive development. It can also be suggested that the prolonged postnatal development of some neuronal circuitries in the human hippocampus may be responsible for the psychological phenomenon of "infantile amnesia", that is the lack of memory traces from the early postnatal period.

Clinical significance of the correlation between PLCE 1 and PRKCA in esophageal inflammation and esophageal carcinoma

PubMed Central

Ma, Ming; Zhang, Shanshan; Zhang, Yongmeng; Yuan, Ming; Liu, Bing; Yang, Yiqiong; Cui, Wen; Ansong, Emmanuel; Dong, Huali; Macias, Virgilia; Yang, Wancai

2017-01-01

Esophagitis and Barrett's esophagus are linked to esophageal squamous cell carcinoma and adenocarcinoma, respectively. However, the underlying mechanisms are still unclear. This study analyzed the expression levels of and correlation between PLCE1 and PRKCA in human esophagitis, carcinogen NMBA-induced rat esophagus, PLCE1 genetic deficient mouse esophageal epithelial tissues and human esophageal cancer cell line, integrated with Online oncology data sets. We found that the expression levels of both PLCE1 and PRKCA were significantly elevated in human esophagitis, esophageal squamous cell carcinoma, Barrett's esophagus, esophageal adenocarcinoma and in NMBA-treated rat esophageal epithelia. However, PRKCA and cytokines were significantly downregulated in PLCE1-deficient mouse esophageal epithelia, and knockdown of PLCE1 in human esophageal cancer cells led to reduction of PRKCA and cytokines. Finally, high expression of both PLCE1 and PRKCA is significantly associated with poor outcomes of the patients with esophageal cancers. In conclusion, this study defined the initiation and progression of esophageal inflammation and malignant transformation, in which the positive correlation of PLCE1 and PRKCA exhibits critical clinical significance. PMID:28402280

Engineering of a Potent Recombinant Lectin-Toxin Fusion Protein to Eliminate Human Pluripotent Stem Cells.

PubMed

Tateno, Hiroaki; Saito, Sayoko

2017-07-10

The use of human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in regenerative medicine is hindered by their tumorigenic potential. Previously, we developed a recombinant lectin-toxin fusion protein of the hPSC-specific lectin rBC2LCN, which has a 23 kDa catalytic domain (domain III) of Pseudomonas aeruginosa exotoxin A (rBC2LCN-PE23). This fusion protein could selectively eliminate hPSCs following its addition to the cell culture medium. Here we conjugated rBC2LCN lectin with a 38 kDa domain of exotoxin A containing domains Ib and II in addition to domain III (PE38). The developed rBC2LCN-PE38 fusion protein could eliminate 50% of 201B7 hPSCs at a concentration of 0.003 μg/mL (24 h incubation), representing an approximately 556-fold higher activity than rBC2LCN-PE23. Little or no effect on human fibroblasts, human mesenchymal stem cells, and hiPSC-derived hepatocytes was observed at concentrations lower than 1 μg/mL. Finally, we demonstrate that rBC2LCN-PE38 selectively eliminates hiPSCs from a mixed culture of hiPSCs and hiPSC-derived hepatocytes. Since rBC2LCN-PE38 can be prepared from soluble fractions of E. coli culture at a yield of 9 mg/L, rBC2LCN-PE38 represents a practical reagent to remove human pluripotent stem cells residing in cultured cells destined for transplantation.

Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2

PubMed Central

Ma, Tianzhong; Su, Zhongjing; Chen, Ling; Liu, Shuyan; Zhu, Ningxia; Wen, Lifeng; Yuan, Yan; Lv, Leili; Chen, Xiancai; Huang, Jianmin; Chen, Haibin

2012-01-01

Background and Objectives Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study. PMID:22655088

Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

PubMed

Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

2008-06-01

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

Human GIP(3-30)NH2 inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors.

PubMed

Gabe, Maria Buur Nordskov; Sparre-Ulrich, Alexander Hovard; Pedersen, Mie Fabricius; Gasbjerg, Lærke Smidt; Inoue, Asuka; Bräuner-Osborne, Hans; Hartmann, Bolette; Rosenkilde, Mette Marie

2018-04-01

GIP(3-30)NH 2 is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3-30)NH 2 in human adipocytes. Finally, we determine the receptor selectivity of GIP(3-30)NH 2 among other human and animal GPCRs. cAMP accumulation and β-arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real-time internalization in transiently transfected HEK293A and in HEK293A β-arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS-7 cells using human 125 I-GIP(3-30)NH 2 . The selectivity of human GIP(3-30)NH 2 was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH 2 inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA 2 and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in β-arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in β-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either β-arrestin 1 or 2. Moreover, GIP(3-30)NH 2 inhibited GIP(1-42)-induced internalization in a concentration-dependent manner and notably also inhibited GIP-mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species-specific with high affinity binding to the human and non-human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (K d values of 2.0, 2.5, 31.6 and 100 nM, respectively). In conclusion, human GIP(3-30)NH 2 is a selective and species-specific GIPR antagonist with broad inhibition of signaling and internalization in transfected cells as well as in human adipocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

Optimization of human tendon tissue engineering: peracetic acid oxidation for enhanced reseeding of acellularized intrasynovial tendon.

PubMed

Woon, Colin Y L; Pridgen, Brian C; Kraus, Armin; Bari, Sina; Pham, Hung; Chang, James

2011-03-01

Tissue engineering of human flexor tendons combines tendon scaffolds with recipient cells to create complete cell-tendon constructs. Allogenic acellularized human flexor tendon has been shown to be a useful natural scaffold. However, there is difficulty repopulating acellularized tendon with recipient cells, as cell penetration is restricted by a tightly woven tendon matrix. The authors evaluated peracetic acid treatment in optimizing intratendinous cell penetration. Cadaveric human flexor tendons were harvested, acellularized, and divided into experimental groups. These groups were treated with peracetic acid in varying concentrations (2%, 5%, and 10%) and for varying time periods (4 and 20 hours) to determine the optimal treatment protocol. Experimental tendons were analyzed for differences in tendon microarchitecture. Additional specimens were reseeded by incubation in a fibroblast cell suspension at 1 × 10(6) cells/ml. This group was then analyzed for reseeding efficacy. A final group underwent biomechanical studies for strength. The optimal treatment protocol comprising peracetic acid at 5% concentration for 4 hours produced increased scaffold porosity, improving cell penetration and migration. Treated scaffolds did not show reduced collagen or glycosaminoglycan content compared with controls (p = 0.37 and p = 0.65, respectively). Treated scaffolds were cytotoxic to neither attached cells nor the surrounding cell suspension. Treated scaffolds also did not show inferior ultimate tensile stress or elastic modulus compared with controls (p = 0.26 and p = 0.28, respectively). Peracetic acid treatment of acellularized tendon scaffolds increases matrix porosity, leading to greater reseeding. It may prove to be an important step in tissue engineering of human flexor tendon using natural scaffolds.

The Mechanism of Gene Targeting in Human Somatic Cells

PubMed Central

Kan, Yinan; Ruis, Brian; Lin, Sherry; Hendrickson, Eric A.

2014-01-01

Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells. PMID:24699519

Short-term application of dexamethasone on stem cells derived from human gingiva reduces the expression of RUNX2 and β-catenin.

PubMed

Kim, Bo-Bae; Kim, Minji; Park, Yun-Hee; Ko, Youngkyung; Park, Jun-Beom

2017-06-01

Objective Next-generation sequencing was performed to evaluate the effects of short-term application of dexamethasone on human gingiva-derived mesenchymal stem cells. Methods Human gingiva-derived stem cells were treated with a final concentration of 10 -7  M dexamethasone and the same concentration of vehicle control. This was followed by mRNA sequencing and data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis of RUNX2 and β-catenin. Results In total, 26,364 mRNAs were differentially expressed. Comparison of the results of dexamethasone versus control at 2 hours revealed that 7 mRNAs were upregulated and 25 mRNAs were downregulated. The application of dexamethasone reduced the expression of RUNX2 and β-catenin in human gingiva-derived mesenchymal stem cells. Conclusion The effects of dexamethasone on stem cells were evaluated with mRNA sequencing, and validation of the expression was performed with qualitative real-time polymerase chain reaction and western blot analysis. The results of this study can provide new insights into the role of mRNA sequencing in maxillofacial areas.

Heterologous mitochondrial DNA recombination in human cells.

PubMed

D'Aurelio, Marilena; Gajewski, Carl D; Lin, Michael T; Mauck, William M; Shao, Leon Z; Lenaz, Giorgio; Moraes, Carlos T; Manfredi, Giovanni

2004-12-15

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs.

PubMed

Bertero, Alessandro; Pawlowski, Matthias; Ortmann, Daniel; Snijders, Kirsten; Yiangou, Loukia; Cardoso de Brito, Miguel; Brown, Stephanie; Bernard, William G; Cooper, James D; Giacomelli, Elisa; Gambardella, Laure; Hannan, Nicholas R F; Iyer, Dharini; Sampaziotis, Fotios; Serrano, Felipe; Zonneveld, Mariëlle C F; Sinha, Sanjay; Kotter, Mark; Vallier, Ludovic

2016-12-01

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development. © 2016. Published by The Company of Biologists Ltd.

Production, characterisation, and cytocompatibility of porous titanium-based particulate scaffolds.

PubMed

Luthringer, B J C; Ali, F; Akaichi, H; Feyerabend, F; Ebel, T; Willumeit, R

2013-10-01

Despite its non-matching mechanical properties titanium remains the preferred metal implant material in orthopaedics. As a consequence in some cases stress shielding effect occurs, leading to implant loosening, osteopenia, and finally revision surgery. Porous metal scaffolds to allow easier specialised cells ingrowth with mechanical properties closer to the ones of bone can overcome this problem. This should improve healing processes, implant integration, and dynamic strength of implants retaining. Three Ti-6Al-4V materials were metal injection moulded and tailored porosities were effectively achieved. After microstructural and mechanical characterisation, two different primary cells of mesenchymal origin (human umbilical cord perivascular cells and human bone derived cells which revealed to be two pertinent models) as well as one cell line originated from primary osteogenic sarcoma, Saos-2, were bestowed to investigate cell-material interaction on genomic and proteome levels. Biological examinations disclosed that no material has negative impact on early adhesion, proliferation or cell viability. An efficient cell ingrowth into material with an average porosity of 25-50 μm was proved.

Sickle red cell-endothelium interactions.

PubMed

Kaul, Dhananjay K; Finnegan, Eileen; Barabino, Gilda A

2009-01-01

Periodic recurrence of painful vaso-occlusive crisis is the defining feature of sickle cell disease. Among multiple pathologies associated with this disease, sickle red cell-endothelium interaction has been implicated as a potential initiating mechanism in vaso-occlusive events. This review focuses on various interrelated mechanisms involved in human sickle red cell adhesion. We discuss in vitro and microcirculatory findings on sickle red cell adhesion, its potential role in vaso-occlusion, and the current understanding of receptor-ligand interactions involved in this pathological phenomenon. In addition, we discuss the contribution of other cellular interactions (leukocytes recruitment and leukocyte-red cell interaction) to vaso-occlusion, as observed in transgenic sickle mouse models. Emphasis is given to recently discovered adhesion molecules that play a predominant role in mediating human sickle red cell adhesion. Finally, we analyze various therapeutic approaches for inhibiting sickle red cell adhesion by targeting adhesion molecules and also consider therapeutic strategies that target stimuli involved in endothelial activation and initiation of adhesion.

The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ji-meng; State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101; Zhao, Hong-xi

2013-05-10

Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expressionmore » of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.« less

Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas.

PubMed

Würth, Roberto; Barbieri, Federica; Pattarozzi, Alessandra; Gaudenzi, Germano; Gatto, Federico; Fiaschi, Pietro; Ravetti, Jean-Louis; Zona, Gianluigi; Daga, Antonio; Persani, Luca; Ferone, Diego; Vitale, Giovanni; Florio, Tullio

2017-09-01

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

PubMed Central

Chou, Bin-Kuan; Gu, Haihui; Gao, Yongxing; Dowey, Sarah N.; Wang, Ying; Shi, Jun; Li, Yanxin; Ye, Zhaohui; Cheng, Tao

2015-01-01

Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10–50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. PMID:25742692

Cell specific radiation dosimetry in skeleton from life-span carcinogenesis studies. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Webster, S.S.J.

1993-04-05

The osteogenic sarcoma is the dominant life-threatening pathology in lifespan studies of beagles exposed to alpha-emitting bone-seeking radionuclides. It was deduced from these studies that certain skeletal sites are more prone to develop tumors. This project sought to determine the bone cells at risk and their cell-specific radiation dose. The cell-specific radiation dose values are related to loss and high Ra-226 and Pu-239 induced osteogenic sarcoma sites, to test different dose response hypothesis and predict the extent of effects in humans.

Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells

PubMed Central

Coufal, Nicole G.; Garcia-Perez, Josè Luis; Peng, Grace E.; Marchetto, Maria C. N.; Muotri, Alysson R.; Mu, Yangling; Carson, Christian T.; Macia, Angela; Moran, John V.; Gage, Fred H.

2011-01-01

Long interspersed element-1 (L1) retrotransposons compose ∼20% of the mammalian genome, and ongoing L1 retrotransposition events can impact genetic diversity by various mechanisms. Previous studies have demonstrated that endogenous L1 retrotransposition can occur in the germ line and during early embryonic development. In addition, recent data indicate that engineered human L1s can undergo somatic retrotransposition in human neural progenitor cells and that an increase in human-specific L1 DNA content can be detected in the brains of normal controls, as well as in Rett syndrome patients. Here, we demonstrate an increase in the retrotransposition efficiency of engineered human L1s in cells that lack or contain severely reduced levels of ataxia telangiectasia mutated, a serine/threonine kinase involved in DNA damage signaling and neurodegenerative disease. We demonstrate that the increase in L1 retrotransposition in ataxia telangiectasia mutated-deficient cells most likely occurs by conventional target-site primed reverse transcription and generate either longer, or perhaps more, L1 retrotransposition events per cell. Finally, we provide evidence suggesting an increase in human-specific L1 DNA copy number in postmortem brain tissue derived from ataxia telangiectasia patients compared with healthy controls. Together, these data suggest that cellular proteins involved in the DNA damage response may modulate L1 retrotransposition. PMID:22159035

Presence of MUC4 in human milk and at the luminal surfaces of blood vessels.

PubMed

Zhang, Jin; Perez, Aymee; Yasin, Mohammad; Soto, Pedro; Rong, Min; Theodoropoulos, George; Carothers Carraway, Coralie A; Carraway, Kermit L

2005-07-01

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP-1 (MUC4alpha) and a transmembrane subunit ASGP-2 (MUC4beta), which has been implicated in the protection of epithelial cell surfaces. Surprisingly, development and characterization of a new monoclonal antibody (mAb), called 1G8, against ASGP-2 demonstrated by immunohistochemistry the presence of MUC4 at the luminal surfaces of blood vessels of both normal tissues and tumors. Muc4 was detected with 1G8 and other Muc4 antibodies in blood vessels from humans, rats and mice. This expression of MUC4 in endothelial cells was confirmed by immunoblotting with 1G8 in human umbilical vein endothelial cells (HUVECs), human iliac artery endothelial cells (HIAECs), and human microvascular endothelial cells (HMVECs). MUC4 could be observed on HUVECs grown on either plastic or Matrigel. Finally, MUC4 expression in the three types of endothelial cell lines was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). These results provide, to our knowledge, the first demonstration of a member of the MUC gene family and membrane mucin in blood vessels. As a luminal surface component, the MUC4 is situated to contribute to the non-adhesive luminal surface and to act as an intrinsic protection and survival factor. (c) 2004 Wiley-Liss, Inc.

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Espinoza, B.; Wharton, W.

1986-08-01

Cholera toxin produced a dose-dependent decrease in the restimulation of G0/G1 traverse in density-arrested human fibroblasts but did not inhibit the stimulation of cells arrested in G0 after serum starvation at low density. In addition, cholera toxin did not inhibit the proliferation of sparse logarithmically growing human fibroblasts, even when low concentrations of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were also present. However, the final density to which sparse cells grew was limited by cholera toxin, when added either alone or together with low concentrations of IBMX. In contrast, high concentrations of the phosphodiesterase inhibitor alone produced a profound inhibition inmore » the growth of sparse human fibrobasts. IBMX produced an inhibition both in the G1 and in the G2 phases of the cell cycle by a mechanism(s) that was not related to the magnitude of the increases in adenosine 3,5-cyclic monophosphate concentrations.« less

Mutant KRAS promotes malignant pleural effusion formation

PubMed Central

Αgalioti, Theodora; Giannou, Anastasios D.; Krontira, Anthi C.; Kanellakis, Nikolaos I.; Kati, Danai; Vreka, Malamati; Pepe, Mario; Spella, Μagda; Lilis, Ioannis; Zazara, Dimitra E.; Nikolouli, Eirini; Spiropoulou, Nikolitsa; Papadakis, Andreas; Papadia, Konstantina; Voulgaridis, Apostolos; Harokopos, Vaggelis; Stamou, Panagiota; Meiners, Silke; Eickelberg, Oliver; Snyder, Linda A.; Antimisiaris, Sophia G.; Kardamakis, Dimitrios; Psallidas, Ioannis; Μarazioti, Antonia; Stathopoulos, Georgios T.

2017-01-01

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition. PMID:28508873

May I Cut in? Gene Editing Approaches in Human Induced Pluripotent Stem Cells.

PubMed

Brookhouser, Nicholas; Raman, Sreedevi; Potts, Christopher; Brafman, David A

2017-02-06

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.

High-throughput identification of antigen-specific TCRs by TCR gene capture.

PubMed

Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia; Kluin, Roelof J C; Bolotin, Dmitriy A; Chen, Xiaojing; Bresser, Kaspar; Nieuwland, Marja; Schotte, Remko; Michels, Samira; Gomez-Eerland, Raquel; Jahn, Lorenz; Hombrink, Pleun; Legrand, Nicolas; Shu, Chengyi Jenny; Mamedov, Ilgar Z; Velds, Arno; Blank, Christian U; Haanen, John B A G; Turchaninova, Maria A; Kerkhoven, Ron M; Spits, Hergen; Hadrup, Sine Reker; Heemskerk, Mirjam H M; Blankenstein, Thomas; Chudakov, Dmitriy M; Bendle, Gavin M; Schumacher, Ton N M

2013-11-01

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

May I Cut in? Gene Editing Approaches in Human Induced Pluripotent Stem Cells

PubMed Central

Brookhouser, Nicholas; Raman, Sreedevi; Potts, Christopher; Brafman, David. A.

2017-01-01

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods. PMID:28178187

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

PubMed

Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

2016-06-01

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Human immune system mouse models of Ebola virus infection.

PubMed

Spengler, Jessica R; Prescott, Joseph; Feldmann, Heinz; Spiropoulou, Christina F

2017-08-01

Human immune system (HIS) mice, immunodeficient mice engrafted with human cells (with or without donor-matched tissue), offer a unique opportunity to study pathogens that cause disease predominantly or exclusively in humans. Several HIS mouse models have recently been used to study Ebola virus (EBOV) infection and disease. The results of these studies are encouraging and support further development and use of these models in Ebola research. HIS mice provide a small animal model to study EBOV isolates, investigate early viral interactions with human immune cells, screen vaccines and therapeutics that modulate the immune system, and investigate sequelae in survivors. Here we review existing models, discuss their use in pathogenesis studies and therapeutic screening, and highlight considerations for study design and analysis. Finally, we point out caveats to current models, and recommend future efforts for modeling EBOV infection in HIS mice. Published by Elsevier B.V.

Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

PubMed

Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

2016-11-01

To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Identification of a nuclear-localized nuclease from wheat cells undergoing programmed cell death that is able to trigger DNA fragmentation and apoptotic morphology on nuclei from human cells

PubMed Central

Domínguez, Fernando; Cejudo, Francisco J.

2006-01-01

PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed. PMID:16613587

The expression patterns of pro-apoptotic and anti-apoptotic factors in human fetal and adult ovary.

PubMed

Poljicanin, Ana; Vukusic Pusic, Tanja; Vukojevic, Katarina; Caric, Ana; Vilovic, Katarina; Tomic, Snjezana; Soljic, Violeta; Saraga-Babic, Mirna

2013-07-01

The influence of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins on the cell death (caspase-3, TUNEL) of different ovarian cell lineages was immunohistochemically analyzed in six fetal and five adult human ovaries in order to disclose possible mechanisms of cell number control. Mild to moderate expression of Bcl-2 characterized ovarian surface epithelium, follicular cells and oocytes of 15 and 22 week human ovaries, while expression of Bax and caspase-3 gradually increased in all ovarian cell populations, except caspase-3 in the ovarian surface epithelium. Different levels of Bax and Bcl-2 proteins co-expression characterized fetal ovarian cells, while TUNEL and caspase-3 co-expression was found only in some of them. In adult ovaries, Bcl-2 was moderately and Bax strongly expressed in the surface ovarian epithelium and stroma. Bcl-2 and Bax expression in granulosa and theca interna cells varied depending on the stage of follicular atresia. Caspase-3 apoptotic cells characterized granulosa cells of adult atretic follicles. Our results indicate that intracellular levels of Bcl-2 and Bax protein might regulate the final destiny of developing germ cells. Caspase-3 dependent apoptosis seems to be the most important, but not the only cell death pathway in ovaries. In adult ovaries, caspase-dependent cell death characterized granulosa cells, but not the germ cells. Copyright © 2012 Elsevier GmbH. All rights reserved.

Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel.

PubMed

Trojani, Christophe; Weiss, Pierre; Michiels, Jean-François; Vinatier, Claire; Guicheux, Jérôme; Daculsi, Guy; Gaudray, Patrick; Carle, Georges F; Rochet, Nathalie

2005-09-01

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material.

A natural product-like JAK2/STAT3 inhibitor induces apoptosis of malignant melanoma cells

PubMed Central

Zhong, Hai-Jing; Dong, Zhen-Zhen; Vellaisamy, Kasipandi; Lu, Jin-Jian; Chen, Xiu-Ping; Chiu, Pauline; Kwong, Daniel W. J.; Han, Quan-Bin; Ma, Dik-Lung

2017-01-01

The JAK2/STAT3 signaling pathway plays a critical role in tumorigenesis, and has been suggested as a potential molecular target for anti-melanoma therapeutics. However, few JAK2 inhibitors were being tested for melanoma therapy. In this study, eight amentoflavone analogues were evaluated for their activity against human malignant melanoma cells. The most potent analogue, compound 1, inhibited the phosphorylation of JAK2 and STAT3 in human melanoma cells, but had no discernible effect on total JAK2 and STAT3 levels. A cellular thermal shift assay was performed to identify that JAK2 is engaged by 1 in cell lysates. Moreover, compound 1 showed higher antiproliferative activity against human melanoma A375 cells compared to a panel of cancer and normal cell lines. Compound 1 also activated caspase-3 and cleaved PARP, which are markers of apoptosis, and suppressed the anti-apoptotic Bcl-2 level. Finally, compound 1 induced apoptosis in 80% of treated melanoma cells. To our knowledge, compound 1 is the first amentoflavone-based JAK2 inhibitor to be investigated for use as an anti-melanoma agent. PMID:28570563

Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

PubMed Central

Quast, Robert B.; Ballion, Biljana; Stech, Marlitt; Sonnabend, Andrei; Varga, Balázs R.; Wüstenhagen, Doreen A.; Kele, Péter; Schiller, Stefan M.; Kubick, Stefan

2016-01-01

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system. PMID:27670253

Surface Enhanced Raman Spectroscopy for the Rapid Detection and Identification of Microbial Pathogens in Human Serum

DTIC Science & Technology

2014-12-11

and 1 mm depth. Bacterial culture and cell count determination Bacterial species of Acinetobacter baumannii (A. baumannii, ST-3), Escherichia coli...remove all broth components followed by a final resuspension of the pellet in ddH2O back to 1 OD. Cell count was determined by plating the 10 4 , 10 3...10 2 and 10 1 cell dilutions on TSB Nutrient Agar media. Colony forming units (CFU) were counted the following day to confirm bacterial species

The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44.

PubMed

Allcock, Richard J N; Barrow, Alexander D; Forbes, Simon; Beck, Stephan; Trowsdale, John

2003-02-01

We have characterized a cluster of single immunoglobulin variable (IgV) domain receptors centromeric of the major histocompatibility complex (MHC) on human chromosome 6. In addition to triggering receptor expressed on myeloid cells (TREM)-1 and TREM2, the cluster contains NKp44, a triggering receptor whose expression is limited to NK cells. We identified three new related genes and two gene fragments within a cluster of approximately 200 kb. Two of the three new genes lack charged residues in their transmembrane domain tails. Further, one of the genes contains two potential immunotyrosine Inhibitory motifs in its cytoplasmic tail, suggesting that it delivers inhibitory signals. The human and mouse TREM clusters appear to have diverged such that there are unique sequences in each species. Finally, each gene in the TREM cluster was expressed in a different range of cell types.

Induction of autophagy by spermidine promotes longevity.

PubMed

Eisenberg, Tobias; Knauer, Heide; Schauer, Alexandra; Büttner, Sabrina; Ruckenstuhl, Christoph; Carmona-Gutierrez, Didac; Ring, Julia; Schroeder, Sabrina; Magnes, Christoph; Antonacci, Lucia; Fussi, Heike; Deszcz, Luiza; Hartl, Regina; Schraml, Elisabeth; Criollo, Alfredo; Megalou, Evgenia; Weiskopf, Daniela; Laun, Peter; Heeren, Gino; Breitenbach, Michael; Grubeck-Loebenstein, Beatrix; Herker, Eva; Fahrenkrog, Birthe; Fröhlich, Kai-Uwe; Sinner, Frank; Tavernarakis, Nektarios; Minois, Nadege; Kroemer, Guido; Madeo, Frank

2009-11-01

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.

Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer.

PubMed

Staquicini, Fernanda I; Qian, Ming D; Salameh, Ahmad; Dobroff, Andrey S; Edwards, Julianna K; Cimino, Daniel F; Moeller, Benjamin J; Kelly, Patrick; Nunez, Maria I; Tang, Ximing; Liu, Diane D; Lee, J Jack; Hong, Waun Ki; Ferrara, Fortunato; Bradbury, Andrew R M; Lobb, Roy R; Edelman, Martin J; Sidman, Richard L; Wistuba, Ignacio I; Arap, Wadih; Pasqualini, Renata

2015-03-20

Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Production of lnterleukin-1 Receptor Antagonist by Human Bronchogenic Carcinoma

PubMed Central

Smith, Daniel R.; Kunkel, Steven L.; Standiford, Theodore J.; Chensue, Stephen W.; Rolfe, Mark W.; Orringer, Mark B.; Whyte, Richard I.; Burdick, Marie D.; Danforth, Jean M.; Gilbert, Andrew R.; Strieter, Robert M.

1993-01-01

Bronchogenic carcinoma displays an aggressive clinical course that may reflect a capacity to evade host defenses. We postulated that tumors may elaborate interleukin-1 receptor antagonist protein (IRAP) to escape host interleukin-1-dependent responses. Homogenates of human bronchogenic lung tumors demonstrated significant increases of IRAP compared with normal lung tissue controls (n = 48). There was no significant difference in interleukin-1 β levels between tumor and normal lung tissue. Immunohistochemical staining localized IRAP to tumor cells. Semiquantitative pathological analysis demonstrated a modest inflammatory cell infiltrate with qualitative differences between tumors of different histology. Western blot analysis of tumor homogenates demonstrated several molecular weight forms of IRAP. Finally, antigenic IRAP was detected in supernatants of the human bronchogenic carcinoma cell line (A549) maintained in vitro. These findings illustrate the capacity of bronchogenic tumors to produce and secrete IRAP that may be important in tumor evasion of host defenses. ImagesFigure 3Figure 4 PMID:8362978

Male germ cells support long-term propagation of Zika virus.

PubMed

Robinson, Christopher L; Chong, Angie C N; Ashbrook, Alison W; Jeng, Ginnie; Jin, Julia; Chen, Haiqi; Tang, Elizabeth I; Martin, Laura A; Kim, Rosa S; Kenyon, Reyn M; Do, Eileen; Luna, Joseph M; Saeed, Mohsan; Zeltser, Lori; Ralph, Harold; Dudley, Vanessa L; Goldstein, Marc; Rice, Charles M; Cheng, C Yan; Seandel, Marco; Chen, Shuibing

2018-05-29

Evidence of male-to-female sexual transmission of Zika virus (ZIKV) and viral RNA in semen and sperm months after infection supports a potential role for testicular cells in ZIKV propagation. Here, we demonstrate that germ cells (GCs) are most susceptible to ZIKV. We found that only GCs infected by ZIKV, but not those infected by dengue virus and yellow fever virus, produce high levels of infectious virus. This observation coincides with decreased expression of interferon-stimulated gene Ifi44l in ZIKV-infected GCs, and overexpression of Ifi44l results in reduced ZIKV production. Using primary human testicular tissue, we demonstrate that human GCs are also permissive for ZIKV infection and production. Finally, we identified berberine chloride as a potent inhibitor of ZIKV infection in both murine and human testes. Together, these studies identify a potential cellular source for propagation of ZIKV in testes and a candidate drug for preventing sexual transmission of ZIKV.

[Apoptosis of human leukemic cells induced by topoisomerase I and II inhibitors].

PubMed

Solary, E; Dubrez, L; Eymin, B; Bertrand, R; Pommier, Y

1996-03-01

Comparison between five human leukemic lines (BV173, HL60, U937, K562, KCL22) suggest that the main determinant of their sensitivity to topoisomerase I (camptothecin) and II (VP-16) inhibitors is their ability to regulate cell cycle progression in response to specific DNA damage, then to die through apoptosis: the more the cells inhibit cell cycle progression, the less sensitive they are. The final pathway of apoptosis induction involves a cytoplasmic signal, active at neutral pH, needing magnesium, sensitive to various protease inhibitors and activated directly by staurosporine. Modulators of intracellular signaling (calcium chelators, calmodulin inhibitors, PKC modulators, kinase and phosphatase inhibitors) have no significant influence upon apoptosis induction. Conversely, apoptosis induction pathway is modified during monocytic differentiation of HL60 cells induced by phorbol esters. Lastly, poly(ADP-ribosyl)ation and chromatine structure should regulate apoptotic DNA fragmentation that is prevented by 3-aminobenzamide and spermine, respectively.

ATM kinase is required for telomere elongation in mouse and human cells

PubMed Central

Lee, Stella Suyong; Bohrson, Craig; Pike, Alexandra Mims; Wheelan, Sarah Jo; Greider, Carol Widney

2015-01-01

Summary Short telomeres induce a DNA damage response, senescence and apoptosis; thus, maintaining telomere length equilibrium is essential for cell viability. Telomerase addition of telomere repeats is tightly regulated in cells. To probe pathways that regulate telomere addition, we developed the ADDIT assay to measure new telomere addition at a single telomere in vivo. Sequence analysis showed telomerase specific addition of repeats onto a new telomere occurred in just 48 hr. Using the ADDIT assay, we found that ATM is required for addition of new repeats onto telomeres in mouse cells. Evaluation of bulk telomeres, in both human and mouse cells, showed that blocking ATM inhibited telomere elongation. Finally, the activation of ATM through the inhibition of PARP1 resulted in increased telomere elongation, supporting the central role of the ATM pathway in regulating telomere addition. Understanding this role of ATM may yield new areas for possible therapeutic intervention in telomere-mediated disease. PMID:26586427

Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function.

PubMed

Estrada, Leonardo D; Ağaç, Didem; Farrar, J David

2016-08-01

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis.

PubMed

Bras, Stéphanie; Martin-Lannerée, Séverine; Gobert, Vanessa; Augé, Benoît; Breig, Osman; Sanial, Matthieu; Yamaguchi, Masamitsu; Haenlin, Marc; Plessis, Anne; Waltzer, Lucas

2012-03-27

Defining the function of the genes that, like RUNX1, are deregulated in blood cell malignancies represents an important challenge. Myeloid leukemia factors (MLFs) constitute a poorly characterized family of conserved proteins whose founding member, MLF1, has been associated with acute myeloid leukemia in humans. To gain insight into the functions of this family, we investigated the role of the Drosophila MLF homolog during blood cell development. Here we report that mlf controls the homeostasis of the Drosophila hematopoietic system. Notably, mlf participates in a positive feedback loop to fine tune the activity of the RUNX transcription factor Lozenge (LZ) during development of the crystal cells, one of the two main blood cell lineages in Drosophila. At the molecular level, our data in cell cultures and in vivo strongly suggest that MLF controls the number of crystal cells by protecting LZ from degradation. Remarkably, it appears that the human MLF1 protein can substitute for MLF in the crystal cell lineage. In addition, MLF stabilizes the human oncogenic fusion protein RUNX1-ETO and is required for RUNX1-ETO-induced blood cell disorders in a Drosophila model of leukemia. Finally, using the human leukemic blood cell line Kasumi-1, we show that MLF1 depletion impairs RUNX1-ETO accumulation and reduces RUNX1-ETO-dependent proliferation. Thus, we propose that the regulation of RUNX protein levels is a conserved feature of MLF family members that could be critical for normal and pathological blood cell development.

Upregulation of Glucose Uptake and Hexokinase Activity of Primary Human CD4+ T Cells in Response to Infection with HIV-1

PubMed Central

Kavanagh Williamson, Maia; Coombes, Naomi; Juszczak, Florian; Athanasopoulos, Marios; Khan, Mariam B.; Eykyn, Thomas R.; Srenathan, Ushani; Dias Zeidler, Julianna; Huthoff, Hendrik

2018-01-01

Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT) and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC) protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out. PMID:29518929

Human Lung Fibroblasts Present Bacterial Antigens to Autologous Lung Th Cells.

PubMed

Hutton, Andrew J; Polak, Marta E; Spalluto, C Mirella; Wallington, Joshua C; Pickard, Chris; Staples, Karl J; Warner, Jane A; Wilkinson, Tom M A

2017-01-01

Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4 + Th cells. During severe viral infection and chronic inflammation, the submucosa is susceptible to bacterial invasion by lung microbiota such as nontypeable Haemophilus influenzae (NTHi). Given their proximity in tissue, we hypothesized that human lung fibroblasts play an important role in modulating Th cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4 + T cell Ag-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to IFN-γ. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. Although unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the costimulatory molecules 4-1BBL, OX-40L, and CD70, all of which are related to memory T cell activation and maintenance. CD4 + T cells isolated from the lung were predominantly (mean 97.5%) CD45RO + memory cells. Finally, cultured fibroblasts activated IFN-γ and IL-17A cytokine production by autologous, NTHi-specific lung CD4 + T cells, and cytokine production was inhibited by a HLA-DR blocking Ab. These results indicate a novel role for human lung fibroblasts in contributing to responses against bacterial infection through activation of bacteria-specific CD4 + T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

Potential transfer of neurotoxic amino acid β-N-methylamino-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines.

PubMed

Andersson, Marie; Ersson, Lisa; Brandt, Ingvar; Bergström, Ulrika

2017-04-01

β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [ 14 C]l-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [ 14 C]l-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here, we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [ 14 C]l- and [ 14 C]d-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [ 14 C]l- and [ 14 C]d-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [ 14 C]l-and [ 14 C]d-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [ 14 C]l-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant. Copyright © 2017. Published by Elsevier Inc.

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway

PubMed Central

Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

2016-01-01

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients. PMID:27367026

Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway.

PubMed

Tao, Na-Na; Zhou, Hong-Zhong; Tang, Hua; Cai, Xue-Fei; Zhang, Wen-Lu; Ren, Ji-Hua; Zhou, Li; Chen, Xiang; Chen, Ke; Li, Wan-Yu; Liu, Bo; Yang, Qiu-Xia; Cheng, Sheng-Tao; Huang, Li-Xia; Huang, Ai-Long; Chen, Juan

2016-08-02

SIRT3, a class III histone deacetylase, has been implicated in various cancers as a novel therapeutic target. In hepatocellular carcinoma (HCC), we previously reported that SIRT3 induced cell apoptosis by regulating GSK-3β/Bax signaling pathway. Downregulation of SIRT3 in HCC cells facilitates tumor cell survival. In this study, we found that chemotherapeutic agents (doxorubicin, cisplatin and epirubicin) and sorafenib treatment downregulated SIRT3 mRNA and protein levels in three HCC cell lines. MTS assay found that SIRT3 overexpression sensitized liver cancer cells to chemotherapeutic agents and sorafenib in SMMC-7721, Huh-7 and PLC/PRF/5 cell lines. Moreover, SIRT3 overexpression promoted chemotherapeutic agents-induced or sorafenib-induced apoptosis as evidenced by flow cytometry, enhanced PARP cleavage and enhanced Caspase-9 cleavage in three HCC cells. In contrast, SIRT3 silencing increased drug resistance of HCC cells to chemotherapeutic agents. Mechanistic study found that SIRT3 downregulated the mRNA and protein levels of glutathione S-transferase pi 1 (GSTP1), which is a member of phase II detoxification enzymes families involved in metabolizing for chemotherapeutic agents. Moreover, SIRT3 decreased the amount of GSTP1 that was associated with JNK, which finally contributed the activation of JNK activity and activation of downstream target c-Jun and Bim. Importantly, GSTP1 overexpression or JNK inhibitor abolished SIRT3-induced apoptosis in HCC cells exposed to chemotherapeutic agents. Finally, there was a negative correlation between SIRT3 expression and GSTP1 expression in human HCC tissues. Together, our findings revealed SIRT3 could enhance the drug sensitivity of HCC cells to an array of chemotherapeutic agents. SIRT3 may serve as a potential target for improving the chemosensitivity of HCC patients.

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

PubMed

Canfield, Scott G; Stebbins, Matthew J; Morales, Bethsymarie Soto; Asai, Shusaku W; Vatine, Gad D; Svendsen, Clive N; Palecek, Sean P; Shusta, Eric V

2017-03-01

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843. © 2016 International Society for Neurochemistry.

Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility

PubMed Central

Wissing, Silke; Muñoz-Lopez, Martin; Macia, Angela; Yang, Zhiyuan; Montano, Mauricio; Collins, William; Garcia-Perez, Jose Luis; Moran, John V.; Greene, Warner C.

2012-01-01

Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ∼10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs. PMID:21989055

Overview of measles and mumps vaccine: origin, present, and future of vaccine production.

PubMed

Betáková, T; Svetlíková, D; Gocník, M

2013-01-01

Measles and mumps are common viral childhood diseases that can cause serious complications. Vaccination remains the most efficient way to control the spread of these viruses. The manufacturing capability for viral vaccines produced in embryonated hen eggs and conventional/classical cell substrates, such as chicken embryo fibroblast or primary dog kidney cell substrates, is no longer sufficient. This limitation can be overcome by utilizing other recognized cell substrates such as Madin Darby Canine Kidney (MDCK), Chinese Hamster Ovary (CHO), Vero (monkey origin) cells, MRC-5 (human diploid) or as an alternative, introducing new cell substrates of human or avian origin. A very important factor in vaccine production is the safety and immunogenicity of the final vaccine, where the proper choice of cell substrate used for virus propagation is made. All substrates used in vaccine production must be fully characterized to avoid the contamination of hidden unknown pathogens which is difficult to achieve in primary cell substrates.

The promise of human embryonic stem cells in aging-associated diseases

PubMed Central

Yabut, Odessa; Bernstein, Harold S.

2011-01-01

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. PMID:21566262

Cell Systems to Investigate the Impact of Polyphenols on Cardiovascular Health

PubMed Central

Grootaert, Charlotte; Kamiloglu, Senem; Capanoglu, Esra; Van Camp, John

2015-01-01

Polyphenols are a diverse group of micronutrients from plant origin that may serve as antioxidants and that contribute to human health in general. More specifically, many research groups have investigated their protective effect against cardiovascular diseases in several animal studies and human trials. Yet, because of the excessive processing of the polyphenol structure by human cells and the residing intestinal microbial community, which results in a large variability between the test subjects, the exact mechanisms of their protective effects are still under investigation. To this end, simplified cell culture systems have been used to decrease the inter-individual variability in mechanistic studies. In this review, we will discuss the different cell culture models that have been used so far for polyphenol research in the context of cardiovascular diseases. We will also review the current trends in cell culture research, including co-culture methodologies. Finally, we will discuss the potential of these advanced models to screen for cardiovascular effects of the large pool of bioactive polyphenols present in foods and their metabolites. PMID:26569293

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

PubMed

Pérès, Eléonore; Blin, Juliana; Ricci, Emiliano P; Artesi, Maria; Hahaut, Vincent; Van den Broeke, Anne; Corbin, Antoine; Gazzolo, Louis; Ratner, Lee; Jalinot, Pierre; Duc Dodon, Madeleine

2018-03-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice

PubMed Central

Artesi, Maria; Jalinot, Pierre

2018-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization. PMID:29566098

Dimethyl Sulfoxide (DMSO) Increases Percentage of CXCR4(+) Hematopoietic Stem/Progenitor Cells, Their Responsiveness to an SDF-1 Gradient, Homing Capacities, and Survival.

PubMed

Jarocha, Danuta; Zuba-Surma, Ewa; Majka, Marcin

2016-01-01

Cryopreservation of bone marrow (BM), mobilized peripheral blood (mPB), and cord blood (CB) hematopoietic stem/progenitor cells (HSPCs) is a routine procedure before transplantation. The most commonly used cryoprotectant for HSPCs is dimethyl sulfoxide (DMSO). The objective of this study was to evaluate the influence of DMSO on surface receptor expression and chemotactic activities of HSPCs. We found that 10 min of incubation of human mononuclear cells (MNCs) with 10% DMSO significantly increases the percentage of CXCR4(+), CD38(+), and CD34(+) cells, resulting in an increase of CD34(+), CD34(+)CXCR4(+), and CD34(+)CXCR4(+)CD38(-) subpopulations. Furthermore, DMSO significantly increased chemotactic responsiveness of MNCs and CXCR4(+) human hematopoietic Jurkat cell line to a stromal cell-derived factor-1 (SDF-1) gradient. Furthermore, we demonstrated enhanced chemotaxis of human clonogenic progenitor cells to an SDF-1 gradient, which suggests that DMSO directly enhances the chemotactic responsiveness of early human progenitors. DMSO preincubation also caused lower internalization of the CXCR4 receptor. In parallel experiments, we found that approximately 30% more of DMSO-preincubated human CD45(+) and CD45(+)CD34(+) cells homed to the mouse BM 24 h after transplantation in comparison to control cells. Finally, we demonstrated considerably higher (25 days) survival of mice transplanted with DMSO-exposed MNCs than those transplanted with the control cells. We show in this study an unexpected beneficial influence of DMSO on HSPC homing and suggest that a short priming with DMSO before transplantation could be considered a new strategy to enhance cell homing and engraftment.

A Semi-Supervised Approach for Refining Transcriptional Signatures of Drug Response and Repositioning Predictions

PubMed Central

Iorio, Francesco; Shrestha, Roshan L.; Levin, Nicolas; Boilot, Viviane; Garnett, Mathew J.; Saez-Rodriguez, Julio; Draviam, Viji M.

2015-01-01

We present a novel strategy to identify drug-repositioning opportunities. The starting point of our method is the generation of a signature summarising the consensual transcriptional response of multiple human cell lines to a compound of interest (namely the seed compound). This signature can be derived from data in existing databases, such as the connectivity-map, and it is used at first instance to query a network interlinking all the connectivity-map compounds, based on the similarity of their transcriptional responses. This provides a drug neighbourhood, composed of compounds predicted to share some effects with the seed one. The original signature is then refined by systematically reducing its overlap with the transcriptional responses induced by drugs in this neighbourhood that are known to share a secondary effect with the seed compound. Finally, the drug network is queried again with the resulting refined signatures and the whole process is carried on for a number of iterations. Drugs in the final refined neighbourhood are then predicted to exert the principal mode of action of the seed compound. We illustrate our approach using paclitaxel (a microtubule stabilising agent) as seed compound. Our method predicts that glipizide and splitomicin perturb microtubule function in human cells: a result that could not be obtained through standard signature matching methods. In agreement, we find that glipizide and splitomicin reduce interphase microtubule growth rates and transiently increase the percentage of mitotic cells–consistent with our prediction. Finally, we validated the refined signatures of paclitaxel response by mining a large drug screening dataset, showing that human cancer cell lines whose basal transcriptional profile is anti-correlated to them are significantly more sensitive to paclitaxel and docetaxel. PMID:26452147

Adult human pancreas-derived cells expressing stage-specific embryonic antigen 4 differentiate into Sox9-expressing and Ngn3-expressing pancreatic ducts in vivo.

PubMed

Lee, Song; Lee, Chan Mi; Kim, Song Cheol

2016-11-11

Tissue-specific stem/progenitor cells are found in various adult tissues and may have the capacity for lineage-specific differentiation, facilitating applications in autologous transplantation. Stage-specific embryonic antigen 4 (SSEA-4), an early embryonic glycolipid antigen, is expressed in cells derived from adult human pancreas exocrine tissue. Here, we examined the characteristics and lineage-specific differentiation capacity of SSEA-4 + cells. Human adult partial pancreas tissues were obtained from different donors and cultured in vitro. SSEA-4 + and CA19-9 + cells were isolated from adult human pancreas exocrine cells using magnetic-activated cell sorting, and gene expression was validated by quantitative polymerase chain reaction. To confirm in-vivo differentiation, SSEA-4 + and CA19-9 + cells were transplanted into the dorsal subcutaneous region of mice. Finally, morphological features of differentiated areas were confirmed by immunostaining and morphometric analysis. SSEA-4-expressing cells were detected in isolated pancreas exocrine cells from adult humans. These SSEA-4 + cells exhibited coexpression of CA19-9, a marker of pancreatic duct cells, but not amylase expression, as shown by immunostaining and flow cytometry. SSEA-4 + cells exhibited higher relative expression of Oct4, Nanog, Klf4, Sox2, and c-Myc mRNAs than CA19-9 + cells. Pancreatic intralobular ducts (PIDs) were generated from SSEA-4 + or CA19-9 + cells in vivo at 5 weeks after transplantation. However, newly formed PIDs from CA19-9 + cells were less abundant and showed an incomplete PID morphology. In contrast, newly formed PIDs from SSEA-4 + cells were abundant in the transplanted area and showed a crowded morphology, typical of PIDs. Sox9 and Ngn3, key transcription factors associated with pancreatic development and regeneration, were expressed in PIDs from SSEA-4 + cells. SSEA-4-expressing cells in the adult human pancreas may have the potential for regeneration of the pancreas and may be used as a source of stem/progenitor cells for pancreatic cell lineage-specific differentiation.

Human T cells engineered to express a programmed death 1/28 costimulatory retargeting molecule display enhanced antitumor activity.

PubMed

Ankri, Chen; Shamalov, Katerina; Horovitz-Fried, Miryam; Mauer, Shmuel; Cohen, Cyrille J

2013-10-15

Adoptive transfer of T cells genetically modified to express cancer-specific receptors can mediate impressive tumor regression in terminally ill patients. However, T cell function and persistence over time could be hampered by the activation of inhibitory costimulatory pathways, such as programmed death 1 (PD1)/programmed death ligand 1, leading to T cell exhaustion and providing tumor cells with an escape mechanism from immunosurveillance. In addition, the lack of positive costimulation at the tumor site can further dampen T cell response. Thus, as T cell genetic engineering has become clinically relevant, we aimed at enhancing T cell antitumor activity by genetically diverting T cell-negative costimulatory signals into positive ones using chimeric costimulatory retargeting molecules and which are composed of the PD1 extracellular domain fused to the signaling domains of positive costimulatory molecules such as CD28 and 4-1BB. After characterizing the optimal PD1 chimera, we designed and optimized a tripartite retroviral vector that enables the simultaneous expression of this chimeric molecule in conjunction with a cancer-specific TCR. Human T cells, transduced to express a PD1/28 chimeric molecule, exhibited enhanced cytokine secretion and upregulation of activation markers upon coculture with tumor cells. These engineered cells also proliferated better compared with control cells. Finally, we tested the function of these cells in two xenograft models of human melanoma tumors and show that PD1/28-engineered human T cells demonstrated superior antitumor function. Overall, we propose that engineering T cells with a costimulatory retargeting molecule can enhance their function, which bears important implications for the improvement of T cell immunotherapy.

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

PubMed Central

Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

2013-01-01

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

DUF1220 protein domains drive proliferation in human neural stem cells and are associated with increased cortical volume in anthropoid primates.

PubMed

Keeney, J G; Davis, J M; Siegenthaler, J; Post, M D; Nielsen, B S; Hopkins, W D; Sikela, J M

2015-09-01

Genome sequences encoding DUF1220 protein domains show a burst in copy number among anthropoid species and especially humans, where they have undergone the greatest human lineage-specific copy number expansion of any protein coding sequence in the genome. While DUF1220 copy number shows a dosage-related association with brain size in both normal populations and in 1q21.1-associated microcephaly and macrocephaly, a function for these domains has not yet been described. Here we provide multiple lines of evidence supporting the view that DUF1220 domains function as drivers of neural stem cell proliferation among anthropoid species including humans. First, we show that brain MRI data from 131 individuals across 7 anthropoid species shows a strong correlation between DUF1220 copy number and multiple brain size-related measures. Using in situ hybridization analyses of human fetal brain, we also show that DUF1220 domains are expressed in the ventricular zone and primarily during human cortical neurogenesis, and are therefore expressed at the right time and place to be affecting cortical brain development. Finally, we demonstrate that in vitro expression of DUF1220 sequences in neural stem cells strongly promotes proliferation. Taken together, these data provide the strongest evidence so far reported implicating DUF1220 dosage in anthropoid and human brain expansion through mechanisms involving increasing neural stem cell proliferation.

Microglial Dynamics During Human Brain Development

PubMed Central

Menassa, David A.; Gomez-Nicola, Diego

2018-01-01

Microglial cells are thought to colonize the human cerebrum between the 4th and 24th gestational weeks. Rodent studies have demonstrated that these cells originate from yolk sac progenitors though it is not clear whether this directly pertains to human development. Our understanding of microglial cell dynamics in the developing human brain comes mostly from postmortem studies demonstrating that the beginning of microglial colonization precedes the appearance of the vasculature, the blood–brain barrier, astrogliogenesis, oligodendrogenesis, neurogenesis, migration, and myelination of the various brain areas. Furthermore, migrating microglial populations cluster by morphology and express differential markers within the developing brain and according to developmental age. With the advent of novel technologies such as RNA-sequencing in fresh human tissue, we are beginning to identify the molecular features of the adult microglial signature. However, this is may not extend to the much more dynamic and rapidly changing antenatal microglial population and this is further complicated by the scarcity of tissue resources. In this brief review, we first describe the various historic schools of thought that had debated the origin of microglial cells while examining the evidence supporting the various theories. We then proceed to examine the evidence we have accumulated on microglial dynamics in the developing human brain, present evidence from rodent studies on the functional role of microglia during development and finally identify limitations for the used approaches in human studies and highlight under investigated questions. PMID:29881376

Integrating human stem cell expansion and neuronal differentiation in bioreactors

PubMed Central

Serra, Margarida; Brito, Catarina; Costa, Eunice M; Sousa, Marcos FQ; Alves, Paula M

2009-01-01

Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. PMID:19772662

Adenovirus small interfering RNA targeting ezrin induces apoptosis and inhibits metastasis of human osteosarcoma MG-63 cells.

PubMed

Tao, Zhi-Wei; Zou, Ping-An

2018-06-13

Osteosarcoma is a disease prone to recurrence and metastasis, and adenovirus expression vector is frequently studied as a therapeutic target of osteosarcoma in recent year. This study attempts to explore the effect of adenovirus-mediated small interfering RNA (siRNA) targeting ezrin on the proliferation, migration, invasion and apoptosis of human osteosarcoma MG-63 cells. Human osteosarcoma MG-63 cell line was selected for construction of recombinant adenovirus vector. The mRNA and protein levels of ezrin, Bcl2-associated X protein (Bax), B cell lymphoma-2 (Bcl-2), p21, p53, Caspase-3, matrix metalloproteinase 2 (MMP-2) and MMP-9, Cyclin D1, and cyclin-dependent kinase 4a (CDK4a) were determined. Through ELISA, the levels of Caspase-3, MMP-2 and MMP-9 were examined. Finally, human osteosarcoma MG-63 cell viability, growth, invasion, migration, and apoptosis were detected. Initially, adenovirus expression vector of ezrin was constructed by ezrin 2 siRNA sequence. Adenovirus-mediated siRNA targeting ezrin reduced expression of ezrin in MG-63 cells. The results revealed that adenovirus-mediated siRNA targeting ezrin elevated expression levels of Bax, P21, P53, and Caspase-3, Cyclin D1, and CDK4a and reduced expression levels of Bcl-2, MMP-2, and MMP-9. Furthermore, adenovirus-mediated siRNA targeting ezrin inhibited human osteosarcoma MG-63 cell viability, growth, invasion, and migration, and promoted apoptosis. Our study demonstrates that adenovirus-mediated siRNA targeting ezrin can induce apoptosis and inhibit the proliferation, migration and invasion of human osteosarcoma MG-63 cells. ©2018 The Author(s).

Modulation of human endothelial cell proliferation and migration by fucoidan and heparin.

PubMed

Giraux, J L; Matou, S; Bros, A; Tapon-Bretaudière, J; Letourneur, D; Fischer, A M

1998-12-01

Fucoidan is a sulfated polysaccharide extracted from brown seaweeds. It has anticoagulant and antithrombotic properties and inhibits, as well as heparin, vascular smooth muscle cell growth. In this study, we investigated, in the presence of serum and human recombinant growth factors, the effects of fucoidan and heparin on the growth and migration of human umbilical vein endothelial cells (HUVEC) in culture. We found that fucoidan stimulated fetal bovine serum-induced HUVEC proliferation, whereas heparin inhibited it. In the presence of fibroblast growth factor-1 (FGF-1), both fucoidan and heparin potentiated HUVEC growth. In contrast, fucoidan and heparin inhibited HUVEC proliferation induced by FGF-2, but did not influence the mitogenic activity of vascular endothelial growth factor (VEGF). In the in vitro migration assay from a denuded area of confluent cells, the two sulfated polysaccharides markedly enhanced the migration of endothelial cells in the presence of FGF-1. Finally, a weak inhibitory effect on cell migration was found only with the two polysaccharides at high concentrations (> or = 100 micro/ml) in presence of serum or combined with FGF-2. All together, the results indicated that heparin and fucoidan can be used as tools to further investigate the cellular mechanisms regulating the proliferation and migration of human vascular cells. Moreover, the data already suggest a potential role of fucoidan as a new therapeutic agent of vegetal origin in the vascular endothelium wound repair.

Involvement of matrix metalloproteinase-13 in stromal-cell-derived factor 1 alpha-directed invasion of human basal cell carcinoma cells.

PubMed

Chu, C-Y; Cha, S-T; Chang, C-C; Hsiao, C-H; Tan, C-T; Lu, Y-C; Jee, S-H; Kuo, M-L

2007-04-12

Basal cell carcinoma (BCC) is one of the most common skin neoplasms in humans and is usually characterized by local aggressiveness with little metastatic potential, although deep invasion, recurrence, and regional and distant metastases may occur. Here, we studied the mechanism of BCC invasion. We found that human BCC tissues and a BCC cell line had significant expression of CXCR4, which was higher in invasive than non-invasive BCC types. Further, of 19 recurrent tumors among 390 BCCs diagnosed during the past 12 years, 17/19 (89.5%) had high CXCR4 expression. We found that the CXCR4 ligand, stromal-cell-derived factor 1alpha (SDF-1alpha), directed BCC invasion and that this was mediated by time-dependent upregulation of mRNA expression and gelatinase activity of matrix metalloproteinase-13 (MMP-13). The transcriptional regulation of MMP-13 by SDF-1alpha was mediated by phosphorylation of extracellular signal-related kinase 1/2 and activation of the AP-1 component c-Jun. Finally, CXCR4-transfected BCC cells injected into nude mice induced aggressive BCCs that co-expressed CXCR4 and MMP-13. The identification of SDF-1alpha/CXCR4 as an important factor in BCC invasiveness may contribute insight into mechanisms involved in the aggressive potential of human BCC and may improve therapy for invasive BCCs.

Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL.

PubMed

Plaisance, Valérie; Brajkovic, Saška; Tenenbaum, Mathie; Favre, Dimitri; Ezanno, Hélène; Bonnefond, Amélie; Bonner, Caroline; Gmyr, Valéry; Kerr-Conte, Julie; Gauthier, Benoit R; Widmann, Christian; Waeber, Gérard; Pattou, François; Froguel, Philippe; Abderrahmani, Amar

2016-01-01

Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment.

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

PubMed

Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-05-01

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line

PubMed Central

Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P.; Parton, Robert G.; Keller, Charles; Fanzani, Alessandro

2015-01-01

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS. PMID:26086601

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

PubMed

Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P; Parton, Robert G; Keller, Charles; Fanzani, Alessandro

2015-01-01

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

PubMed

Nemkov, Travis; Sun, Kaiqi; Reisz, Julie A; Song, Anren; Yoshida, Tatsuro; Dunham, Andrew; Wither, Matthew J; Francis, Richard O; Roach, Robert C; Dzieciatkowska, Monika; Rogers, Stephen C; Doctor, Allan; Kriebardis, Anastasios; Antonelou, Marianna; Papassideri, Issidora; Young, Carolyn T; Thomas, Tiffany A; Hansen, Kirk C; Spitalnik, Steven L; Xia, Yang; Zimring, James C; Hod, Eldad A; D'Alessandro, Angelo

2018-02-01

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13 C 1 -aspartate or 13 C 5 -adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and - preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates. Copyright© 2018 Ferrata Storti Foundation.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage

PubMed Central

Nemkov, Travis; Sun, Kaiqi; Reisz, Julie A.; Song, Anren; Yoshida, Tatsuro; Dunham, Andrew; Wither, Matthew J.; Francis, Richard O.; Roach, Robert C.; Dzieciatkowska, Monika; Rogers, Stephen C.; Doctor, Allan; Kriebardis, Anastasios; Antonelou, Marianna; Papassideri, Issidora; Young, Carolyn T.; Thomas, Tiffany A.; Hansen, Kirk C.; Spitalnik, Steven L.; Xia, Yang; Zimring, James C.; Hod, Eldad A.; D’Alessandro, Angelo

2018-01-01

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates. PMID:29079593

APOPTIC POTANCY AND CYTOTOXIC EFFECTS OF DIBROMOMOACETONITRILE IN HUMAN LEUKEMIA CELLS. (R825955)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Immunogenic apoptosis in human acute myeloid leukemia (AML): primary human AML cells expose calreticulin and release heat shock protein (HSP) 70 and HSP90 during apoptosis.

PubMed

Fredly, Hanne; Ersvær, Elisabeth; Gjertsen, Bjørn-Tore; Bruserud, Oystein

2011-06-01

Several previous studies have demonstrated that both conventional cytotoxic drugs as well as targeted therapeutics can induce apoptosis in primary human acute myelogenous leukemia (AML) cells. However, the apoptotic phenotype of dying AML cells has been less extensively characterized. Even though specific antileukemic immune reactivity is important in AML, especially for allotransplanted patients, it has not been investigated whether dying primary human AML cells show phenotypic characteristics consistent with immunogenic apoptosis [calreticulin exposure, heat shock protein (HSP) release]. We therefore investigated whether in vitro cultured primary human acute myeloid leukemia (AML) cells show calreticulin exposure and HSP70/HSP90 release during spontaneous (stress-induced) apoptosis when cultured in medium alone and when cultured in the presence of antileukemic drugs. Both surface exposure of calreticulin and release of HSP70 and HSP90 was detected but showed a wide variation between patients. This variation was also maintained when the AML cells were cultured in the presence of cytotoxic drugs (cytarabine, daunorubicin, mitomycin), all-trans retinoic acid (ATRA) and valproic acid. Finally, AML cells collected during in vivo ATRA therapy showed increased calreticulin exposure during spontaneous in vitro apoptosis, suggesting that in vivo pharmacotherapy can modulate the apoptotic phenotype. To conclude, apoptotic AML cells can show phenotypic characteristics consistent with immunogenic apoptosis, but there is a wide variation between patients and the level of calreticulin exposure/HSP release seems to depend on individual patient characteristics rather than the apoptosis-inducing agent.

Reversal of Human Papillomavirus-Specific T Cell Immune Suppression through TLR Agonist Treatment of Langerhans Cells Exposed to Human Papillomavirus Type 161

PubMed Central

Fahey, Laura M.; Raff, Adam B.; Da Silva, Diane M.; Kast, W. Martin

2009-01-01

Human papillomavirus (HPV) type 16 infects the epithelial layer of cervical mucosa and is causally associated with the generation of cervical cancer. Langerhans cells (LC) are the resident antigen-presenting cells at the site of infection and therefore are responsible for initiating an immune response against HPV16. On the contrary, LC exposed to HPV16 do not induce a specific T cell immune response, which leads to the immune evasion of HPV16. Demonstrating that Toll-like receptor 7 (TLR7) and TLR8 are expressed on human LC, we hypothesized that imidazoquinolines would activate LC exposed to HPV16, leading to the induction of an HPV16-specific cell-mediated immune response. Surprisingly both phenotypic and functional hallmarks of activation are not observed when LC are exposed to HPV16 virus-like particles (VLP) and treated with imiquimod (TLR7 agonist). However, we found that LC are activated by 3M-002 (TLR8 agonist) and resiquimod (TLR8/7 agonist). LC exposed to HPV16 VLP and subsequently treated with 3M-002 or resiquimod highly up-regulate surface activation markers, secrete pro-inflammatory cytokines and chemokines, induce CCL21-directed migration, and initiate an HPV16-specific CD8+ T cell response. These data strongly indicate that 3M-002 and resiquimod are promising therapeutics for treatment of HPV-infections and HPV-induced cervical lesions. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.” PMID:19234187

Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate.

PubMed

Ebhardt, H Alexander; Sabidó, Eduard; Hüttenhain, Ruth; Collins, Ben; Aebersold, Ruedi

2012-04-01

Selected or multiple reaction monitoring is a targeted mass spectrometry method (S/MRM-MS), in which many peptides are simultaneously and consistently analyzed during a single liquid chromatography-mass spectrometry (LC-S/MRM-MS) measurement. These capabilities make S/MRM-MS an attractive method to monitor a consistent set of proteins over various experimental conditions. To increase throughput for S/MRM-MS it is advantageous to use scheduled methods and unfractionated protein extracts. Here, we established the practically measurable dynamic range of proteins reliably detectable and quantifiable in an unfractionated protein extract from a human cell line using LC-S/MRM-MS. Initially, we analyzed S/MRM transition peak groups in terms of interfering signals and compared S/MRM transition peak groups to MS1-triggered MS2 spectra using dot-product analysis. Finally, using unfractionated protein extract from human cell lysate, we quantified the upper boundary of copies per cell to be 35 million copies per cell, while 7500 copies per cell represents a lower boundary using a single 35 min linear gradient LC-S/MRM-MS measurement on a current, standard commercial instrument. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation: Progress and Clinical Applications.

PubMed

Di Baldassarre, Angela; Cimetta, Elisa; Bollini, Sveva; Gaggi, Giulia; Ghinassi, Barbara

2018-05-25

Human-induced pluripotent stem cells (hiPSCs) are reprogrammed cells that have hallmarks similar to embryonic stem cells including the capacity of self-renewal and differentiation into cardiac myocytes. The improvements in reprogramming and differentiating methods achieved in the past 10 years widened the use of hiPSCs, especially in cardiac research. hiPSC-derived cardiac myocytes (CMs) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models and useful tools for drug discovery and toxicology testing. In addition, hiPSCs can be used as sources of cells for cardiac regeneration in animal models. Here, we review the advances in the genetic and epigenetic control of cardiomyogenesis that underlies the significant improvement of the induced reprogramming of somatic cells to CMs; the methods used to improve scalability of throughput assays for functional screening and drug testing in vitro; the phenotypic characteristics of hiPSCs-derived CMs and their ability to rescue injured CMs through paracrine effects; we also cover the novel approaches in tissue engineering for hiPSC-derived cardiac tissue generation, and finally, their immunological features and the potential use in biomedical applications.

Auto-catalysed progression of aneuploidy explains the Hayflick limit of cultured cells, carcinogen-induced tumours in mice, and the age distribution of human cancer.

PubMed Central

Rasnick, D

2000-01-01

Evidence continues to accumulate that aneuploidy, an imbalance in the number of chromosomes, is responsible for the characteristic phenotypes of cancer, including the abnormal cellular size and morphology of cancer cells, the appearance of tumour-associated antigens, as well as the high levels of membrane-bound and secreted proteins responsible for invasiveness and loss of contact inhibition. Aneuploidy has also been demonstrated to be the self-perpetuating source of the karyotypic instability of cancer cells. Here it is shown that the auto-catalysed progression of aneuploidy explains the kinetics of the finite lifetime of diploid cells in culture, the time course of the appearance of papillomas and carcinomas in benzo[a]pyrene-treated mice, and the age-dependence of human cancers. Modelling studies indicate that the ease of spontaneous transformation of mouse cells in culture may be due to a chaotic progression of aneuploidy. Conversely, the strong preference towards senescence and resistance to transformation of human cells in culture may be the result of a non-chaotic progression of aneuploidy. Finally, a method is proposed for quantifying the aneuploidogenic potencies of carcinogens. PMID:10839979

Auto-catalysed progression of aneuploidy explains the Hayflick limit of cultured cells, carcinogen-induced tumours in mice, and the age distribution of human cancer.

PubMed

Rasnick, D

2000-06-15

Evidence continues to accumulate that aneuploidy, an imbalance in the number of chromosomes, is responsible for the characteristic phenotypes of cancer, including the abnormal cellular size and morphology of cancer cells, the appearance of tumour-associated antigens, as well as the high levels of membrane-bound and secreted proteins responsible for invasiveness and loss of contact inhibition. Aneuploidy has also been demonstrated to be the self-perpetuating source of the karyotypic instability of cancer cells. Here it is shown that the auto-catalysed progression of aneuploidy explains the kinetics of the finite lifetime of diploid cells in culture, the time course of the appearance of papillomas and carcinomas in benzo[a]pyrene-treated mice, and the age-dependence of human cancers. Modelling studies indicate that the ease of spontaneous transformation of mouse cells in culture may be due to a chaotic progression of aneuploidy. Conversely, the strong preference towards senescence and resistance to transformation of human cells in culture may be the result of a non-chaotic progression of aneuploidy. Finally, a method is proposed for quantifying the aneuploidogenic potencies of carcinogens.

Biomaterials-based 3D cell printing for next-generation therapeutics and diagnostics.

PubMed

Jang, Jinah; Park, Ju Young; Gao, Ge; Cho, Dong-Woo

2018-02-01

Building human tissues via 3D cell printing technology has received particular attention due to its process flexibility and versatility. This technology enables the recapitulation of unique features of human tissues and the all-in-one manufacturing process through the design of smart and advanced biomaterials and proper polymerization techniques. For the optimal engineering of tissues, a higher-order assembly of physiological components, including cells, biomaterials, and biomolecules, should meet the critical requirements for tissue morphogenesis and vascularization. The convergence of 3D cell printing with a microfluidic approach has led to a significant leap in the vascularization of engineering tissues. In addition, recent cutting-edge technology in stem cells and genetic engineering can potentially be adapted to the 3D tissue fabrication technique, and it has great potential to shift the paradigm of disease modeling and the study of unknown disease mechanisms required for precision medicine. This review gives an overview of recent developments in 3D cell printing and bioinks and provides technical requirements for engineering human tissues. Finally, we propose suggestions on the development of next-generation therapeutics and diagnostics. Copyright © 2017 Elsevier Ltd. All rights reserved.

The use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering.

PubMed

Wang, Zhong-Shan; Feng, Zhi-Hong; Wu, Guo-Feng; Bai, Shi-Zhu; Dong, Yan; Chen, Fa-Ming; Zhao, Yi-Min

2016-06-21

Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament tissue (PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells (PDLSCs) and jaw bone mesenchymal stem cells (JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Platelet-rich fibrin (PRF) derived from human venous blood was then fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, the in vivo potential of a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets to form periodontal tissue was assessed in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space comprising human treated dentin matrix (TDM) and hydroxyapatite (HA)/tricalcium phosphate (TCP) frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL-like tissues, while the JBMSC sheets tended to produce predominantly bone-like tissues. In addition, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal tissue-like structures containing PDL- and bone-like tissues. Further improvements in this cell transplantation design may have the potential to provide an effective approach for future periodontal tissue regeneration.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Costantini, Lindsey M.; Irvin, Susan C.; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFPmore » enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.« less

Inter-Individual Variability in Human Response to Low-Dose Ionizing Radiation, Final Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rocke, David

2016-08-01

In order to investigate inter-individual variability in response to low-dose ionizing radiation, we are working with three models, 1) in-vivo irradiated human skin, for which we have a realistic model, but with few subjects, all from a previous project, 2) ex-vivo irradiated human skin, for which we also have a realistic model, though with the limitations involved in keeping skin pieces alive in media, and 3) MatTek EpiDermFT skin plugs, which provides a more realistic model than cell lines, which is more controllable than human samples.

Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1

PubMed Central

Ebrahimi, Mina; Kazemi, Tohid; Ganjalikhani-hakemi, Mazdak; Majidi, Jafar; khanahmad, Hossein; Rahimmanesh, Ilnaz; Homayouni, Vida; Kohpayeh, Shirin

2015-01-01

Background Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases. Objectives In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. Materials and Methods The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F’. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. Results The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Conclusions Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1. PMID:28959306

Ionophore-A23187-induced cellular cytotoxicity: a cell fragment mediated process.

PubMed Central

Nash, G S; Niedt, G W; MacDermott, R P

1980-01-01

Calcium ionophore A23187 was found to induce human white blood cells to kill human red blood cells. Optimal conditions for ionophore-induced cellular cytotoxicity (IICC) included an 18 h time period, an incubation temperature of 25 degrees, a 25:1 or 50:1 killer:target cell ratio,and a final ionophore concentration of 2 . 5 microgram/ml. WBC or granulocytes which were either frozen and thawed three times or sonicated were capable of mediating IICC. As intact cells, granulocytes (67 . 2% cytotoxicity), monocytes (34 . 8%), B cells (22 . 0%) and Null cells (19 . 3%) were effector cells but T cells (7 . 4%) were not. After fragmenting these cells, all cell types including T cells were able to mediate IICC. When cell lines (K562, Chang, and NCTC) were used as effectors, none would mediate IICC when intact. After freezing and thawing, Chang and NCTC would not mediate IICC, whereas K562 cells did. These studies may be indicative of a calcium-dependent, membrane-localized mechanism in cellular cytotoxic processes, and may provide a useful indicator system for isolation of the enzyme systems involved in cellular cytotoxicity. PMID:6773881

Oncolytic Activity of Avian Influenza Virus in Human Pancreatic Ductal Adenocarcinoma Cell Lines

PubMed Central

Pizzuto, Matteo S.; Silic-Benussi, Micol; Pavone, Silvia; Ciminale, Vincenzo; Capua, Ilaria

2014-01-01

ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas in vivo, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3- and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas. IMPORTANCE Despite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis in vitro and antitumor effects in vivo. These results suggest that slightly pathogenic IAVs may prove to be effective for oncolytic virotherapy of PDA and provide grounds for further studies to develop specific and targeted viruses, with the aim of testing their efficacy in clinical contexts. PMID:24899201

Post-thymic maturation: young T cells assert their individuality.

PubMed

Fink, Pamela J; Hendricks, Deborah W

2011-07-22

T cell maturation was once thought to occur entirely within the thymus. Now, evidence is mounting that the youngest peripheral T cells in both mice and humans comprise a distinct population from their more mature, yet still naive, counterparts. These cells, termed recent thymic emigrants (RTEs), undergo a process of post-thymic maturation that can be monitored at the levels of cell phenotype and immune function. Understanding this final maturation step in the process of generating useful and safe T cells is of clinical relevance, given that RTEs are over-represented in neonates and in adults recovering from lymphopenia. Post-thymic maturation may function to ensure T cell fitness and self tolerance.

Experience of microbiological screening of human hepatocytes for clinical transplantation.

PubMed

Lehec, Sharon C; Hughes, Robin D; Mitry, Ragai R; Graver, Michelle A; Verma, Anita; Wade, Jim J; Dhawan, Anil

2009-01-01

Hepatocyte transplantation is being used in patients with liver-based metabolic disorders and acute liver failure. Hepatocytes are isolated from unused donor liver tissue under GMP conditions. Cells must be free of microbiological contamination to be safe for human use. The experience of microbiological screening during 72 hepatocyte isolation procedures at one center is reported. Samples were taken at different stages of the process and tested using a blood culture bottle system and Gram stain. Bacterial contamination was detected in 37.5% of the UW organ preservative solutions used to transport the liver tissue to the Cell Isolation Unit. After tissue processing the contamination was reduced to 7% overall in the final hepatocyte product, irrespective of the presence of initial contamination of the transport solution. The most common organisms recovered were coagulase-negative staphylococci, a skin commensal. A total of 41 preparations of fresh or cryopreserved hepatocytes were used for cell transplantation in children with liver-based metabolic disorders without any evidence of sepsis due to infusion of hepatocytes. In conclusion, the incidence of bacterial contamination of the final product was low, confirming the suitability of the organs used, hepatocyte isolation procedure, and the environmental conditions of the clean room.

CD8 T-cell-mediated protection against liver-stage malaria: lessons from a mouse model

PubMed Central

Van Braeckel-Budimir, Natalija; Harty, John T.

2014-01-01

Malaria is a major global health problem, with severe mortality in children living in sub-Saharan Africa, and there is currently no licensed, effective vaccine. However, vaccine-induced protection from Plasmodium infection, the causative agent of malaria, was established for humans in small clinical trials and for rodents in the 1960s. Soon after, a critical role for memory CD8 T cells in vaccine-induced protection against Plasmodium liver-stage infection was established in rodent models and is assumed to apply to humans. However, these seminal early studies have led to only modest advances over the ensuing years in our understanding the basic features of memory CD8 T cells required for protection against liver-stage Plasmodium infection, an issue which has likely impeded the development of effective vaccines for humans. Given the ethical and practical limitations in gaining mechanistic insight from human vaccine and challenge studies, animal models still have an important role in dissecting the basic parameters underlying memory CD8 T-cell immunity to Plasmodium. Here, we will highlight recent data from our own work in the mouse model of Plasmodium infection that identify quantitative and qualitative features of protective memory CD8 T-cell responses. Finally, these lessons will be discussed in the context of recent findings from clinical trials of vaccine-induced protection in controlled human challenge models. PMID:24936199

Characterization of mesenchymal stem cells from human dental pulp, preapical follicle and periodontal ligament.

PubMed

Navabazam, Ali Reza; Sadeghian Nodoshan, Fatemeh; Sheikhha, Mohammad Hasan; Miresmaeili, Sayyed Mohsen; Soleimani, Mehrdad; Fesahat, Farzaneh

2013-03-01

Human dental stem cells have high proliferative potential for self-renewal that is important to the regenerative capacity of the tissue. Objective : The aim was to isolate human dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC) and periapical follicle stem cells (PAFSC) for their potential role in tissue regeneration. In this experimental study, the postnatal stem cells were isolated from dental pulp, preapical follicle and periodontal ligament .The cells were stained for different stem cell markers by immunocytochemistry. To investigate the mesenchymal nature of cells, differentiation potential along osteoblastic and adipogenic lineages and gene expression profile were performed. For proliferation potential assay, Brdu staining and growth curve tests were performed. Finally, all three cell types were compared together regarding their proliferation, differentiation and displaying phenotype. The isolated cell populations have similar fibroblastic like morphology and expressed all examined cell surface molecule markers. These cells were capable of differentiating into osteocyte with different capability and adipocyte with the same rate. PAFSCs showed more significant proliferation rate than others. Reverse transcriptase PCR (RT-PCR) for nanog, oct4, Alkaline phosphatase (ALP) and glyceraldehydes-3-phosphate dehydrogenease (GADPH) as control gene showed strong positive expression of these genes in all three isolated cell types. PDLSCs, DPSCs and PAFSCs exist in various tissues of the teeth and can use as a source of mesenchymal stem cells for developing bioengineered organs and also in craniomaxillofacial reconstruction with varying efficiency in differentiation and proliferation.

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, K.; Chubb, C.; Huberman, E.

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors.

PubMed

Patin, Etienne; Hasan, Milena; Bergstedt, Jacob; Rouilly, Vincent; Libri, Valentina; Urrutia, Alejandra; Alanio, Cécile; Scepanovic, Petar; Hammer, Christian; Jönsson, Friederike; Beitz, Benoît; Quach, Hélène; Lim, Yoong Wearn; Hunkapiller, Julie; Zepeda, Magge; Green, Cherie; Piasecka, Barbara; Leloup, Claire; Rogge, Lars; Huetz, François; Peguillet, Isabelle; Lantz, Olivier; Fontes, Magnus; Di Santo, James P; Thomas, Stéphanie; Fellay, Jacques; Duffy, Darragh; Quintana-Murci, Lluís; Albert, Matthew L

2018-03-01

The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

PubMed Central

Lan, Xiaoyang; Jörg, David J.; Cavalli, Florence M. G.; Richards, Laura M.; Nguyen, Long V.; Vanner, Robert J.; Guilhamon, Paul; Lee, Lilian; Kushida, Michelle; Pellacani, Davide; Park, Nicole I.; Coutinho, Fiona J.; Whetstone, Heather; Selvadurai, Hayden J.; Che, Clare; Luu, Betty; Carles, Annaick; Moksa, Michelle; Rastegar, Naghmeh; Head, Renee; Dolma, Sonam; Prinos, Panagiotis; Cusimano, Michael D.; Das, Sunit; Bernstein, Mark; Arrowsmith, Cheryl H.; Mungall, Andrew J.; Moore, Richard A.; Ma, Yussanne; Gallo, Marco; Lupien, Mathieu; Pugh, Trevor J.; Taylor, Michael D.; Hirst, Martin; Eaves, Connie J.; Simons, Benjamin D.; Dirks, Peter B.

2017-01-01

Summary Human glioblastomas (GBMs) harbour a subpopulation of glioblastoma stem cells (GSCs) that drive tumourigenesis. However, the origin of intra-tumoural functional heterogeneity between GBM cells remains poorly understood. Here we study the clonal evolution of barcoded GBM cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of GBM clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in GSCs. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, that in turn generates non-proliferative cells. We also identify rare “outlier” clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant GSCs. Finally, we show that functionally distinct GSCs can be separately targeted using epigenetic compounds, suggesting new avenues for GBM targeted therapy. PMID:28854171

Activity of botulinum neurotoxin type D (strain 1873) in human neurons

PubMed Central

Pellett, Sabine; Tepp, William H.; Scherf, Jacob M.; Pier, Christina L.; Johnson, Eric A.

2015-01-01

Botulinum Neurotoxin type D (BoNT/D) causes periodic outbreaks of botulism in cattle and horses, but is rarely associated with human botulism. Previous studies have shown that humans responded poorly to peripheral injection of up to 10 U of BoNT/D. Isolated human pyramidalis muscle preparations were resistant to BoNT/D, whereas isolated human intercostal muscle preparations responded to BoNT/D similarly as to other BoNT serotypes. In vitro data indicate that BoNT/D does not cleave human VAMP1 efficiently, and differential expression of the VAMP 1 and 2 isoforms may be responsible for the above observations. Here we examined sensitivity of cultured human neurons derived from human induced pluripotent stem cells to BoNT/D. Our data indicate that BoNT/D can enter and cleave VAMP 2 in human neurons, but at significantly lower efficiency than other BoNT serotypes. In addition, BoNT/D had a short duration of action in the cultured neurons, similar to that of BoNT/E. In vivo analyses indicated a slower time to death in mice, as well as a later onset and shorter duration of action than BoNT/A1. Finally, examination of BoNT/D activity in various rodent and human cell models resulted in dramatic differences in sensitivity, indicating a unique cell entry mechanism of BoNT/D. PMID:25937339

Baicalein inhibits the migration and invasive properties of human hepatoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiu, Yung-Wei; Institute of Medicine, Chung Shan Medical University, Taiwan; Lin, Tseng-Hsi

Flavonoids have been demonstrated to exert health benefits in humans. We investigated whether the flavonoid baicalein would inhibit the adhesion, migration, invasion, and growth of human hepatoma cell lines, and we also investigated its mechanism of action. The separate effects of baicalein and baicalin on the viability of HA22T/VGH and SK-Hep1 cells were investigated for 24 h. To evaluate their invasive properties, cells were incubated on matrigel-coated transwell membranes in the presence or absence of baicalein. We examined the effect of baicalein on the adhesion of cells, on the activation of matrix metalloproteinases (MMPs), protein kinase C (PKC), and p38more » mitogen-activated protein kinase (MAPK), and on tumor growth in vivo. We observed that baicalein suppresses hepatoma cell growth by 55%, baicalein-treated cells showed lower levels of migration than untreated cells, and cell invasion was significantly reduced to 28%. Incubation of hepatoma cells with baicalein also significantly inhibited cell adhesion to matrigel, collagen I, and gelatin-coated substrate. Baicalein also decreased the gelatinolytic activities of the matrix metalloproteinases MMP-2, MMP-9, and uPA, decreased p50 and p65 nuclear translocation, and decreased phosphorylated I-kappa-B (IKB)-{beta}. In addition, baicalein reduced the phosphorylation levels of PKC{alpha} and p38 proteins, which regulate invasion in poorly differentiated hepatoma cells. Finally, when SK-Hep1 cells were grown as xenografts in nude mice, intraperitoneal (i.p.) injection of baicalein induced a significant dose-dependent decrease in tumor growth. These results demonstrate the anticancer properties of baicalein, which include the inhibition of adhesion, invasion, migration, and proliferation of human hepatoma cells in vivo. - Highlight: > Baicalein inhibits several essential steps in the onset of metastasis.« less

Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, A.D.; Bender, M.A.; Harris, E.A.S.

1988-11-01

Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitatesmore » an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.« less

Sustained synchronized neuronal network activity in a human astrocyte co-culture system

PubMed Central

Kuijlaars, Jacobine; Oyelami, Tutu; Diels, Annick; Rohrbacher, Jutta; Versweyveld, Sofie; Meneghello, Giulia; Tuefferd, Marianne; Verstraelen, Peter; Detrez, Jan R.; Verschuuren, Marlies; De Vos, Winnok H.; Meert, Theo; Peeters, Pieter J.; Cik, Miroslav; Nuydens, Rony; Brône, Bert; Verheyen, An

2016-01-01

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer’s disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal)function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases. PMID:27819315

Red Blood Cell Susceptibility to Pneumolysin

PubMed Central

Bokori-Brown, Monika; Petrov, Peter G.; Khafaji, Mawya A.; Mughal, Muhammad K.; Naylor, Claire E.; Shore, Angela C.; Gooding, Kim M.; Casanova, Francesco; Mitchell, Tim J.; Titball, Richard W.; Winlove, C. Peter

2016-01-01

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell. PMID:26984406

Physiologically relevant organs on chips

PubMed Central

Yum, Kyungsuk; Hong, Soon Gweon; Lee, Luke P.

2015-01-01

Recent advances in integrating microengineering and tissue engineering have generated promising microengineered physiological models for experimental medicine and pharmaceutical research. Here we review the recent development of microengineered physiological systems, or organs on chips, that reconstitute the physiologically critical features of specific human tissues and organs and their interactions. This technology uses microengineering approaches to construct organ-specific microenvironments, reconstituting tissue structures, tissue–tissue interactions and interfaces, and dynamic mechanical and biochemical stimuli found in specific organs, to direct cells to assemble into functional tissues. We first discuss microengineering approaches to reproduce the key elements of physiologically important, dynamic mechanical microenvironments, biochemical microenvironments, and microarchitectures of specific tissues and organs in microfluidic cell culture systems. This is followed by examples of microengineered individual organ models that incorporate the key elements of physiological microenvironments into single microfluidic cell culture systems to reproduce organ-level functions. Finally, microengineered multiple organ systems that simulate multiple organ interactions to better represent human physiology, including human responses to drugs, is covered in this review. This emerging organs-on-chips technology has the potential to become an alternative to 2D and 3D cell culture and animal models for experimental medicine, human disease modeling, drug development, and toxicology. PMID:24357624

Efficient Transduction of Human and Rhesus Macaque Primary T Cells by a Modified Human Immunodeficiency Virus Type 1-Based Lentiviral Vector.

PubMed

He, Huan; Xue, Jing; Wang, Weiming; Liu, Lihong; Ye, Chaobaihui; Cong, Zhe; Kimata, Jason T; Qin, Chuan; Zhou, Paul

2017-03-01

Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors efficiently transduce genes to human, but not rhesus, primary T cells and hematopoietic stem cells (HSCs). The poor transduction of HIV-1 vectors to rhesus cells is mainly due to species-specific restriction factors such as rhesus TRIM5α. Previously, several strategies to modify HIV-1 vectors were developed to overcome rhesus TRIM5α restriction. While the modified HIV-1 vectors efficiently transduce rhesus HSCs, they remain suboptimal for rhesus primary T cells. Recently, HIV-1 variants that encode combinations of LNEIE mutations in capsid (CA) protein and SIVmac239 Vif were found to replicate efficiently in rhesus primary T cells. Thus, the present study tested whether HIV-1 vectors packaged by a packaging construct containing these CA substitutions could efficiently transduce both human and rhesus primary CD4 T cells. To accomplish this, LNEIE mutations were made in the packaging construct CEMΔ8.9, and recombinant HIV-1 vectors packaged by Δ8.9 WT or Δ8.9 LNEIE were generated. Transduction rates, CA stability, and vector integration in CEMss-CCR5 and CEMss-CCR5-rhTRIM5α/green fluorescent protein cells, as well as transduction rates in human and rhesus primary CD4 T cells by Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors, were compared. Finally, the influence of rhesus TRIM5α variations in transduction rates to primary CD4 T cells from a cohort of 37 Chinese rhesus macaques was studied. While it maintains efficient transduction for human T-cell line and primary CD4 T cells, Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α-mediated CA degradation, resulting in significantly higher transduction efficiency of rhesus primary CD4 T cells than Δ8.9 WT-packaged HIV-1 vector. Rhesus TRIM5α variations strongly influence transduction efficiency of rhesus primary CD4 T cells by both Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors. Thus, it is concluded that Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α restriction and efficiently transduces both human and rhesus primary T cells.

Efficient Transduction of Human and Rhesus Macaque Primary T Cells by a Modified Human Immunodeficiency Virus Type 1–Based Lentiviral Vector

PubMed Central

He, Huan; Xue, Jing; Wang, Weiming; Liu, Lihong; Ye, Chaobaihui; Cong, Zhe; Kimata, Jason T.; Qin, Chuan; Zhou, Paul

2017-01-01

Human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors efficiently transduce genes to human, but not rhesus, primary T cells and hematopoietic stem cells (HSCs). The poor transduction of HIV-1 vectors to rhesus cells is mainly due to species-specific restriction factors such as rhesus TRIM5α. Previously, several strategies to modify HIV-1 vectors were developed to overcome rhesus TRIM5α restriction. While the modified HIV-1 vectors efficiently transduce rhesus HSCs, they remain suboptimal for rhesus primary T cells. Recently, HIV-1 variants that encode combinations of LNEIE mutations in capsid (CA) protein and SIVmac239 Vif were found to replicate efficiently in rhesus primary T cells. Thus, the present study tested whether HIV-1 vectors packaged by a packaging construct containing these CA substitutions could efficiently transduce both human and rhesus primary CD4 T cells. To accomplish this, LNEIE mutations were made in the packaging construct CEMΔ8.9, and recombinant HIV-1 vectors packaged by Δ8.9 WT or Δ8.9 LNEIE were generated. Transduction rates, CA stability, and vector integration in CEMss-CCR5 and CEMss-CCR5-rhTRIM5α/green fluorescent protein cells, as well as transduction rates in human and rhesus primary CD4 T cells by Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors, were compared. Finally, the influence of rhesus TRIM5α variations in transduction rates to primary CD4 T cells from a cohort of 37 Chinese rhesus macaques was studied. While it maintains efficient transduction for human T-cell line and primary CD4 T cells, Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α-mediated CA degradation, resulting in significantly higher transduction efficiency of rhesus primary CD4 T cells than Δ8.9 WT-packaged HIV-1 vector. Rhesus TRIM5α variations strongly influence transduction efficiency of rhesus primary CD4 T cells by both Δ8.9 WT or Δ8.9 LNEIE-packaged HIV-1 vectors. Thus, it is concluded that Δ8.9 LNEIE-packaged HIV-1 vector overcomes rhesus TRIM5α restriction and efficiently transduces both human and rhesus primary T cells. PMID:28042947

SILICA AND PM1648 MODIFY HUMAN ALVEOLAR MACROPHAGE ANTIGEN PRESENTING CELL ACTIVITY IN VITRO. (R826782)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Targeting neuropilin-1 in human leukemia and lymphoma.

PubMed

Karjalainen, Katja; Jaalouk, Diana E; Bueso-Ramos, Carlos E; Zurita, Amado J; Kuniyasu, Akihiko; Eckhardt, Bedrich L; Marini, Frank C; Lichtiger, Benjamin; O'Brien, Susan; Kantarjian, Hagop M; Cortes, Jorge E; Koivunen, Erkki; Arap, Wadih; Pasqualini, Renata

2011-01-20

Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short, cell-internalizing peptide ligands that could serve as directive agents for specific drug delivery in hematologic malignancies. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence Phe-Phe/Tyr-Any-Leu-Arg-Ser (F(F)/(Y)XLRS), which bound to different leukemia cell lines and to patient-derived bone marrow samples. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, we observed a potent anti-leukemia cell effect when the targeting motif was synthesized in tandem to the pro-apoptotic sequence (D)(KLAKLAK)₂. Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with acute lymphoblastic leukemia or acute myelogenous leukemia compared with normal bone marrow. These results indicate that NRP-1 could potentially be used as a target for ligand-directed therapy in human leukemias and lymphomas and that the prototype CGFYWLRSC-GG-(D)(KLAKLAK)₂ is a promising drug candidate in this setting.

Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

PubMed

Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz

2009-03-01

Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

Recapitulating Human Gastric Cancer Pathogenesis: Experimental Models of Gastric Cancer

PubMed Central

Ding, Lin; El Zaatari, Mohamad

2017-01-01

Overview Gastric cancer has been traditionally defined by the Correa paradigm as a progression of sequential pathological events that begins with chronic inflammation [1]. Infection with Helicobacter pylori (H. pylori) is the typical explanation for why the stomach becomes chronically inflamed. Acute gastric inflammation then leads to chronic gastritis, atrophy particularly of acid-secreting parietal cells, metaplasia due to mucous neck cell expansion from trans-differentiation of zymogenic cells to dysplasia and eventually carcinoma [2]. The chapter contains an overview of gastric anatomy and physiology to set the stage for signaling pathways that play a role in gastric tumorigenesis. Finally, the major known mouse models of gastric transformation are critiqued in terms of the rationale behind their generation and contribution to our understanding of human cancer subtypes. PMID:27573785

Bis-pyridinium quadrupolar derivatives. High Stokes shift selective probes for bio-imaging

NASA Astrophysics Data System (ADS)

Salice, Patrizio; Versari, Silvia; Bradamante, Silvia; Meinardi, Francesco; Macchi, Giorgio; Pagani, Giorgio A.; Beverina, Luca

2013-11-01

We describe the design, synthesis and characterization of five high Stokes shift quadrupolar heteroaryl compounds suitable as fluorescent probes in bio-imaging. In particular, we characterize the photophysical properties and the intracellular localization in Human Umbilical Vein Endothelial Cells (HUVEC) and Human Mesenchymal Stem Cells (HMSCs) for each dye. We show that, amongst all of the investigated derivatives, the 2,5-bis[1-(4-N-methylpyridinium)ethen-2-yl)]- N-methylpyrrole salt is the best candidates as selective mitochondrial tracker. Finally, we recorded the full emission spectrum of the most performing - exclusively mitochondrial selective - fluorescent probe directly from HUVEC stained cells. The emission spectrum collected from the stained mitochondria shows a remarkably more pronounced vibronic structure with respect to the emission of the free fluorophore in solution.

Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress

PubMed Central

Li, Xiaoming; Bai, Jing; Li, Jianchun; Li, Shenghao; Wang, Zeming; Zhou, Mingrui

2017-01-01

Dihydroartemisinin is an effective antimalarial agent with multiple biological activities. In the present investigation, we elucidated its therapeutic potential and working mechanism on human tongue squamous cell carcinoma (TSCC). It was demonstrated that dihydroartemisinin could significantly inhibit cell growth in a dose- and time-dependent manner by the Cell Counting Kit-8 and colony formation assay in vitro. Meanwhile, autophagy was promoted in the Cal-27 cells treated by dihydroartemisinin, evidenced by increased LC3B-II level, increased autophagosome formation, and increased Beclin-1 level compared to dihydroartemisinin-untreated cells. Importantly, dihydroartemisinin caused DNA double-strand break with simultaneously increased γH2AX foci and oxidative stress; this inhibited the nuclear localization of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), finally leading to autophagic cell death. Furthermore, the antitumor effect of dihydroartemisinin-monotherapy was confirmed with a mouse xenograft model, and no kidney injury associated with toxic effect was observed after intraperitoneal injection with dihydroartemisinin for 3 weeks in vivo. In the present study, it was revealed that dihydroartemisinin-induced DNA double-strand break promoted oxidative stress, which decreased p-STAT3 (Tyr705) nuclear localization, and successively increased autophagic cell death in the Cal-27 cells. Thus, dihydroartemisinin alone may represent an effective and safe therapeutic agent for human TSCC. PMID:28526807

Synthetic Substrata to Instruct Human Pluripotent Stem Cell Fate: From Novel Ligands to Functional Biomaterials

NASA Astrophysics Data System (ADS)

Musah, Samira

Human pluripotent stem (hPS) cells have the remarkable capacity to self-renew indefinitely and differentiate into desired cell types. They can serve as a virtually unlimited supply of cells for applications ranging from drug screening to cell therapies to understanding human development. Reaping the promise of hPS cells hinges on effective defined culture and differentiation conditions. Efforts to generate chemically-defined environments for hPS cell propagation and directed differentiation have been hindered by access to only a handful of ligands to target hPS cells. Additionally, progress has been limited also by lack of knowledge regarding the relevant functional properties of the cell culture substratum. To address these problems, I first employed forward-chemical-genetics coupled with self-assembled monolayer technology to identify novel peptides that bind to hPS cell-surface receptors. I then developed a controlled synthesis of hydrogels with tailored peptide display and mechanical properties. This approach yielded synthetic hydrogels with specific mechanical properties that function in a defined medium to robustly support hPS cell self-renewal. Finally, by starting from molecular level understanding that matrix elasticity regulates developmental pathways, I generated a highly efficient hydrogel platform that restricts hPS cell differentiation to neurons, even without soluble inductive factors. These results indicate that insoluble cues can be important information conduits to guide hPS cell fate decisions. I envision that the blueprint provided by this work can be utilized to devise new materials to guide hPS cell fate.

Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.

PubMed

Santini, Roberta; Vinci, Maria C; Pandolfi, Silvia; Penachioni, Junia Y; Montagnani, Valentina; Olivito, Biagio; Gattai, Riccardo; Pimpinelli, Nicola; Gerlini, Gianni; Borgognoni, Lorenzo; Stecca, Barbara

2012-09-01

The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment. Copyright © 2012 AlphaMed Press.

Freeze-Dried Human Red Blood Cells.

DTIC Science & Technology

1992-07-15

conducted with the approval of the appropriate Institutional Review Board. The design and dosage of these studies parallels what would be used in any...components by standard blood bank procedures. The red blood cells were mixed with standard lyophilization buffers at a standard blood to buffer ratio. Samples...3% -1% - Yeas I Year 2 i Final poduct sterility (at Inluslon stage). Not done O "emonstrated Year I Year 2 Shell Life: Refrlgerated storage. vt10

Potential transfer of neurotoxic amino acid β-N-methylamino-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersson, Marie

β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [{sup 14}C]L-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [{sup 14}C]L-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here,more » we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [{sup 14}C]L- and [{sup 14}C]D-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [{sup 14}C]L- and [{sup 14}C]D-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [{sup 14}C]L-and [{sup 14}C]D-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [{sup 14}C]L-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant. - Highlights: • Transport of BMAA in human intestinal, mammary and CNS cell lines was examined. • The transport of L-BMAA over intestinal cell monolayers was unidirectional. • Enantiomer-selective uptake of L-BMAA in breast, neuron and glia cells was evident. • Competition experiments indicate that L-BMAA uptake involved several transporters. • A potential for mother to infant transfer of BMAA is proposed.« less

A Novel Human Adipocyte-derived Basement Membrane for Tissue Engineering Applications

NASA Astrophysics Data System (ADS)

Damm, Aaron

Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis

PubMed Central

Bras, Stéphanie; Martin-Lannerée, Séverine; Gobert, Vanessa; Augé, Benoît; Breig, Osman; Sanial, Matthieu; Yamaguchi, Masamitsu; Haenlin, Marc; Plessis, Anne; Waltzer, Lucas

2012-01-01

Defining the function of the genes that, like RUNX1, are deregulated in blood cell malignancies represents an important challenge. Myeloid leukemia factors (MLFs) constitute a poorly characterized family of conserved proteins whose founding member, MLF1, has been associated with acute myeloid leukemia in humans. To gain insight into the functions of this family, we investigated the role of the Drosophila MLF homolog during blood cell development. Here we report that mlf controls the homeostasis of the Drosophila hematopoietic system. Notably, mlf participates in a positive feedback loop to fine tune the activity of the RUNX transcription factor Lozenge (LZ) during development of the crystal cells, one of the two main blood cell lineages in Drosophila. At the molecular level, our data in cell cultures and in vivo strongly suggest that MLF controls the number of crystal cells by protecting LZ from degradation. Remarkably, it appears that the human MLF1 protein can substitute for MLF in the crystal cell lineage. In addition, MLF stabilizes the human oncogenic fusion protein RUNX1-ETO and is required for RUNX1-ETO–induced blood cell disorders in a Drosophila model of leukemia. Finally, using the human leukemic blood cell line Kasumi-1, we show that MLF1 depletion impairs RUNX1-ETO accumulation and reduces RUNX1-ETO–dependent proliferation. Thus, we propose that the regulation of RUNX protein levels is a conserved feature of MLF family members that could be critical for normal and pathological blood cell development. PMID:22411814

Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression.

PubMed

Lanuza, Pilar M; Vigueras, Alan; Olivan, Sara; Prats, Anne C; Costas, Santiago; Llamazares, Guillermo; Sanchez-Martinez, Diego; Ayuso, José María; Fernandez, Luis; Ochoa, Ignacio; Pardo, Julián

2018-01-01

Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.

Effects of the Particulate Matter₂.₅ (PM₂.₅) on Lipoprotein Metabolism, Uptake and Degradation, and Embryo Toxicity.

PubMed

Kim, Jae-Yong; Lee, Eun-Young; Choi, Inho; Kim, Jihoe; Cho, Kyung-Hyun

2015-12-01

Particulate matter2.5 (PM2.5) is notorious for its strong toxic effects on the cardiovascular, skin, nervous, and reproduction systems. However, the molecular mechanism by which PM2.5 aggravates disease progression is poorly understood, especially in a water-soluble state. In the current study, we investigated the putative physiological effects of aqueous PM2.5 solution on lipoprotein metabolism. Collected PM2.5 from Seoul, Korea was dissolved in water, and the water extract (final 3 and 30 ppm) was treated to human serum lipoproteins, macrophages, and dermal cells. PM2.5 extract resulted in degradation and aggregation of high-density lipoprotein (HDL) as well as low-density lipoprotein (LDL); apoA-I in HDL aggregated and apo-B in LDL disappeared. PM2.5 treatment (final 30 ppm) also induced cellular uptake of oxidized LDL (oxLDL) into macrophages, especially in the presence of fructose (final 50 mM). Uptake of oxLDL along with production of reactive oxygen species was accelerated by PM2.5 solution in a dose-dependent manner. Further, PM2.5 solution caused cellular senescence in human dermal fibroblast cells. Microinjection of PM2.5 solution into zebrafish embryos induced severe mortality accompanied by impairment of skeletal development. In conclusion, water extract of PM2.5 induced oxidative stress as a precursor to cardiovascular toxicity, skin cell senescence, and embryonic toxicity via aggregation and proteolytic degradation of serum lipoproteins.

Effects of the Particulate Matter2.5 (PM2.5) on Lipoprotein Metabolism, Uptake and Degradation, and Embryo Toxicity

PubMed Central

Kim, Jae-Yong; Lee, Eun-Young; Choi, Inho; Kim, Jihoe; Cho, Kyung-Hyun

2015-01-01

Particulate matter2.5 (PM2.5) is notorious for its strong toxic effects on the cardiovascular, skin, nervous, and reproduction systems. However, the molecular mechanism by which PM2.5 aggravates disease progression is poorly understood, especially in a water-soluble state. In the current study, we investigated the putative physiological effects of aqueous PM2.5 solution on lipoprotein metabolism. Collected PM2.5 from Seoul, Korea was dissolved in water, and the water extract (final 3 and 30 ppm) was treated to human serum lipoproteins, macrophages, and dermal cells. PM2.5 extract resulted in degradation and aggregation of high-density lipoprotein (HDL) as well as low-density lipoprotein (LDL); apoA-I in HDL aggregated and apo-B in LDL disappeared. PM2.5 treatment (final 30 ppm) also induced cellular uptake of oxidized LDL (oxLDL) into macrophages, especially in the presence of fructose (final 50 mM). Uptake of oxLDL along with production of reactive oxygen species was accelerated by PM2.5 solution in a dose-dependent manner. Further, PM2.5 solution caused cellular senescence in human dermal fibroblast cells. Microinjection of PM2.5 solution into zebrafish embryos induced severe mortality accompanied by impairment of skeletal development. In conclusion, water extract of PM2.5 induced oxidative stress as a precursor to cardiovascular toxicity, skin cell senescence, and embryonic toxicity via aggregation and proteolytic degradation of serum lipoproteins. PMID:26615830

Genome-wide maps of nuclear lamina interactions in single human cells.

PubMed

Kind, Jop; Pagie, Ludo; de Vries, Sandra S; Nahidiazar, Leila; Dey, Siddharth S; Bienko, Magda; Zhan, Ye; Lajoie, Bryan; de Graaf, Carolyn A; Amendola, Mario; Fudenberg, Geoffrey; Imakaev, Maxim; Mirny, Leonid A; Jalink, Kees; Dekker, Job; van Oudenaarden, Alexander; van Steensel, Bas

2015-09-24

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

HTLV-1-induced leukotriene B4 secretion by T cells promotes T cell recruitment and virus propagation

PubMed Central

Percher, Florent; Curis, Céline; Pérès, Eléonore; Artesi, Maria; Rosewick, Nicolas; Jeannin, Patricia; Gessain, Antoine; Gout, Olivier; Mahieux, Renaud; Ceccaldi, Pierre-Emmanuel; Van den Broeke, Anne; Duc Dodon, Madeleine; Afonso, Philippe V.

2017-01-01

The human T-lymphotropic virus type 1 (HTLV-1) is efficiently transmitted through cellular contacts. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied in vitro, those facilitating the encounter between infected and target cells remain unknown. In this study, we demonstrate that HTLV-1-infected CD4 T cells secrete a potent chemoattractant, leukotriene B4 (LTB4). LTB4 secretion is dependent on Tax-induced transactivation of the pla2g4c gene, which encodes the cytosolic phospholipase A2 gamma. Inhibition of LTB4 secretion or LTB4 receptor knockdown on target cells reduces T-cell recruitment, cellular contact formation and virus propagation in vitro. Finally, blocking the synthesis of LTB4 in a humanized mouse model of HTLV-1 infection significantly reduces proviral load. This results from a decrease in the number of infected clones while their expansion is not impaired. This study shows the critical role of LTB4 secretion in HTLV-1 transmission both in vitro and in vivo. PMID:28639618

Genotoxicity in primary human peripheral lymphocytes after exposure to lithium titanate nanoparticles in vitro.

PubMed

Akbaba, Giray B; Turkez, Hasan; Sönmez, Erdal; Tatar, Abdulgani; Yilmaz, Mehmet

2016-08-01

Lithium titanate (Li 2 TiO 3 ) nanoparticles (LTT NPs; <100 nm) are widely used in battery technology, porcelain enamels, and ceramic insulating bodies. With the increased applications of LTT NPs, the concerns about their potential human toxicity effects and their environmental impact were also increased. However, toxicity data for LTT NPs relating to human health are very limited. Therefore, the purpose of this study was to evaluate whether LTT NPs are able to induce genetic damage in human peripheral lymphocytes in vitro when taking into consideration that DNA damage plays an important role in carcinogenesis. With this aim, the chromosome aberrations (CA), sister chromatid exchanges (SCE), and micronucleus (MN) assays were used as genotoxicity end points. Human peripheral lymphocytes obtained from five healthy male volunteers were exposed to LTT NPs at final dispersed concentrations ranging from 0 to 1000 μg/mL for 72 h at 37°C. The obtained results indicated that LTT NPs compound did not induce DNA damage in human peripheral lymphocytes as depicted by CA/cell, SCE/cell, and MN/1000 cell values in all concentrations tested. In summary, our results revealed that exposure to LTT NPs is not capable of inducing DNA lesions in human peripheral lymphocytes for the first time. © The Author(s) 2014.

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors.

PubMed

Barbieri, Federica; Thellung, Stefano; Ratto, Alessandra; Carra, Elisa; Marini, Valeria; Fucile, Carmen; Bajetto, Adriana; Pattarozzi, Alessandra; Würth, Roberto; Gatti, Monica; Campanella, Chiara; Vito, Guendalina; Mattioli, Francesca; Pagano, Aldo; Daga, Antonio; Ferrari, Angelo; Florio, Tullio

2015-04-07

Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.

[Using of cell biocomposite material in tissue engineering of the urinary bladder].

PubMed

Glybochko, P V; Olefir, Yu V; Alyaev, Yu G; Butnaru, D V; Bezrukov, E A; Chaplenko, A A; Zharikova, T M

2017-06-01

In a systematic review, to present an overview of the current situation in the field of tissue engineering of urinary bladder related to the use of cell lines pre-cultured on matrices. The selection of eligible publications was conducted according to the method described in the article Glybochko P.V. et al. "Tissue engineering of urinary bladder using acellular matrix." At the final stage, studies investigating the application of matrices with human and animal cell lines were analyzed. Contemporary approaches to using cell-based tissue engineering of the bladder were analyzed, including the formation of 3D structures from several types of cells, cell layers and genetic modification of injected cells. The most commonly used cell lines are urothelial cells, mesenchymal stem cells and fibroblasts. The safety and efficacy of any types of composite cell structures used in the cell-based bladder tissue engineering has not been proven sufficiently to warrant clinical studies of their usefulness. The results of cystoplasty of rat bladder are almost impossible to extrapolate to humans; besides, it is difficult to predict possible side effects. For the transition to clinical trials, additional studies on relevant animal models are needed.

P21 and p27: roles in carcinogenesis and drug resistance.

PubMed

Abukhdeir, Abde M; Park, Ben Ho

2008-07-01

Human cancers arise from an imbalance of cell growth and cell death. Key proteins that govern this balance are those that mediate the cell cycle. Several different molecular effectors have been identified that tightly regulate specific phases of the cell cycle, including cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors. Notably, loss of expression or function of two G1-checkpoint CDK inhibitors - p21 (CDKN1A) and p27 (CDKN1B) - has been implicated in the genesis or progression of many human malignancies. Additionally, there is a growing body of evidence suggesting that functional loss of p21 or p27 can mediate a drug-resistance phenotype. However, reports in the literature have also suggested p21 and p27 can promote tumours, indicating a paradoxical effect. Here, we review historic and recent studies of these two CDK inhibitors, including their identification, function, importance to carcinogenesis and finally their roles in drug resistance.

The Notch/Hes1 pathway sustains NF-κB activation through CYLD repression in T cell leukemia

PubMed Central

Espinosa, Lluis; Cathelin, Severine; D’Altri, Teresa; Trimarchi, Thomas; Statnikov, Alexander; Guiu, Jordi; Rodilla, Veronica; Inglés-Esteve, Julia; Nomdedeu, Josep; Bellosillo, Beatriz; Besses, Carles; Abdel-Wahab, Omar; Kucine, Nicole; Sun, Shao-Cong; Song, Guangchan; Mullighan, Charles C.; Levine, Ross L.; Rajewsky, Klaus; Aifantis, Iannis; Bigas, Anna

2010-01-01

SUMMARY It was previously shown that the NF-κB pathway is downstream of oncogenic Notch1 in T cell acute lymphoblastic leukemia (T-ALL). Here we visualize Notch-induced NF-κB activation using both human T-ALL cell lines and animal models. We demonstrate that Hes1, a canonical Notch target and transcriptional repressor, is responsible for sustaining IKK activation in T-ALL. Hes1 exerts its effects by repressing the deubiquitinase CYLD, a negative IKK complex regulator. CYLD expression was found to be significantly suppressed in primary T-ALL. Finally, we demonstrate that IKK inhibition is a promising option for the targeted therapy of T-ALL as specific suppression of IKK expression and function affected both the survival of human T-ALL cells and the maintenance of the disease in vivo. PMID:20832754

The Notch/Hes1 pathway sustains NF-κB activation through CYLD repression in T cell leukemia.

PubMed

Espinosa, Lluis; Cathelin, Severine; D'Altri, Teresa; Trimarchi, Thomas; Statnikov, Alexander; Guiu, Jordi; Rodilla, Veronica; Inglés-Esteve, Julia; Nomdedeu, Josep; Bellosillo, Beatriz; Besses, Carles; Abdel-Wahab, Omar; Kucine, Nicole; Sun, Shao-Cong; Song, Guangchan; Mullighan, Charles C; Levine, Ross L; Rajewsky, Klaus; Aifantis, Iannis; Bigas, Anna

2010-09-14

It was previously shown that the NF-κB pathway is downstream of oncogenic Notch1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we visualize Notch-induced NF-κB activation using both human T-ALL cell lines and animal models. We demonstrate that Hes1, a canonical Notch target and transcriptional repressor, is responsible for sustaining IKK activation in T-ALL. Hes1 exerts its effects by repressing the deubiquitinase CYLD, a negative IKK complex regulator. CYLD expression was found to be significantly suppressed in primary T-ALL. Finally, we demonstrate that IKK inhibition is a promising option for the targeted therapy of T-ALL as specific suppression of IKK expression and function affected both the survival of human T-ALL cells and the maintenance of the disease in vivo. Copyright © 2010 Elsevier Inc. All rights reserved.

Differentiation of human pluripotent stem cells into highly functional classical brown adipocytes.

PubMed

Nishio, Miwako; Saeki, Kumiko

2014-01-01

We describe a detailed method for directed differentiation of human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), into functional classical brown adipocytes (BAs) under serum-free and feeder-free conditions. It is a two-tiered culture system, based on very simple techniques, a floating culture and a subsequent adherent culture. It does not require gene transfer. The entire process can be carried out in about 10 days. The key point is the usage of our special hematopoietic cytokine cocktail. Almost all the differentiated cells express uncoupling protein 1, a BA-selective marker, as determined by immunostaining. The differentiated cells show characteristics of classical BA as assessed by morphology and gene/protein expression. Moreover, the expression of myoblast marker genes is transiently induced during the floating culture step. hESC/hiPSC-derived BAs show significantly higher oxygen consumption rates (OCRs) than white adipocytes generated from human mesenchymal stem cell. They also show responsiveness to adrenergic stimuli, with about twofold upregulation in OCR by β-adrenergic receptor (β-AR) agonist treatments. hESC/hiPSC-derived BAs exert in vivo calorigenic activities in response to β-AR agonist treatments as assessed by thermography. Finally, lipid and glucose metabolisms are significantly improved in hESC/hiPSC-derived BA-transplanted mice. Our system provides a highly feasible way to produce functional classical BA bearing metabolism-improving capacities from hESC/hiPSC under a feeder-free and serum-free condition without gene transfer. © 2014 Elsevier Inc. All rights reserved.

Dehydroandrographolide, an iNOS inhibitor, extracted from Andrographis paniculata (Burm.f.) Nees, induces autophagy in human oral cancer cells.

PubMed

Hsieh, Ming-Ju; Lin, Chiao-Wen; Chiou, Hui-Ling; Yang, Shun-Fa; Chen, Mu-Kuan

2015-10-13

Autophagy, which is constitutively executed at the basal level in all cells, promotes cellular homeostasis by regulating the turnover of organelles and proteins. Andrographolide and dehydroandrographolide (DA) are the two principle components of Andrographis paniculata (Burm.f.) Nees. and are the main contributors to its therapeutic properties. However, the pharmacological activities of dehydroandrographolide (DA) remain unclear. In this study, DA induces oral cancer cell death by activating autophagy. Treatment with autophagy inhibitors inhibited DA-induced human oral cancer cell death. In addition, DA increased LC3-II expression and reduced p53 expression in a time- and concentration-dependent manner. Furthermore, DA induced autophagy and decreased cell viability through modulation of p53 expression. DA-induced autophagy was triggered by an activation of JNK1/2 and an inhibition of Akt and p38. In conclusion, this study demonstrated that DA induced autophagy in human oral cancer cells by modulating p53 expression, activating JNK1/2, and inhibiting Akt and p38. Finally, an administration of DA effectively suppressed the tumor formation in the oral carcinoma xenograft model in vivo. This is the first study to reveal the novel function of DA in activating autophagy, suggesting that DA could serve as a new and potential chemopreventive agent for treating human oral cancer.

Dehydroandrographolide, an iNOS inhibitor, extracted from from Andrographis paniculata (Burm.f.) Nees, induces autophagy in human oral cancer cells

PubMed Central

Hsieh, Ming-Ju; Lin, Chiao-Wen; Chiou, Hui-Ling; Yang, Shun-Fa; Chen, Mu-Kuan

2015-01-01

Autophagy, which is constitutively executed at the basal level in all cells, promotes cellular homeostasis by regulating the turnover of organelles and proteins. Andrographolide and dehydroandrographolide (DA) are the two principle components of Andrographis paniculata (Burm.f.) Nees. and are the main contributors to its therapeutic properties. However, the pharmacological activities of dehydroandrographolide (DA) remain unclear. In this study, DA induces oral cancer cell death by activating autophagy. Treatment with autophagy inhibitors inhibited DA-induced human oral cancer cell death. In addition, DA increased LC3-II expression and reduced p53 expression in a time- and concentration-dependent manner. Furthermore, DA induced autophagy and decreased cell viability through modulation of p53 expression. DA-induced autophagy was triggered by an activation of JNK1/2 and an inhibition of Akt and p38. In conclusion, this study demonstrated that DA induced autophagy in human oral cancer cells by modulating p53 expression, activating JNK1/2, and inhibiting Akt and p38. Finally, an administration of DA effectively suppressed the tumor formation in the oral carcinoma xenograft model in vivo. This is the first study to reveal the novel function of DA in activating autophagy, suggesting that DA could serve as a new and potential chemopreventive agent for treating human oral cancer. PMID:26356821

Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

PubMed

Mullins, Christina S; Hühns, Maja; Krohn, Mathias; Peters, Sven; Cheynet, Valérie; Oriol, Guy; Guillotte, Michèle; Ducrot, Sandrine; Mallet, François; Linnebacher, Michael

2016-01-01

A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wei; Man, Xiao-Yong; Li, Chun-Ming

Vascular endothelial growth factor (VEGF) is one of the strongest regulators of physiological and pathological angiogenesis. VEGF receptor 2 (VEGFR-2), the primary receptor for VEGF, is thought to mediate major functional effects of VEGF. Previously, we have localized both VEGF and VEGFR-2 in human hair follicles. In this study, we further defined the expression and roles of VEGFR-2 on human hair follicle dermal papilla (DP) cells. The expression of VEGFR-2 on DP cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis separately, and localization of VEGFR-2 was defined by immunofluorescence. The effect of VEGF on DPmore » cells was analyzed by MTT assays and specific inhibitors. Finally, the role of VEGF involved in the signaling pathways was investigated by Western blot. RT-PCR and Western blot analysis demonstrated the expression of VEGFR-2 on DP cells. Immunostaining for VEGFR-2 showed strong signal on cultured human DP cells in vitro. Exogenous VEGF{sub 165} stimulated proliferation of DP cells in a dose-dependent manner. Furthermore, this stimulation was blocked by a VEGFR-2 neutralizing antibody (MAB3571) and an ERK inhibitor (PD98059). VEGF{sub 165}-induced phosphorylation of ERK1/2 was abolished by MAB3571 and PD98059, while the phosphorylation of p38, JNK and AKT were not changed by VEGF{sub 165}. Taken together, VEGFR-2 is expressed on primary human hair follicle DP cells and VEGF induces proliferation of DP cells through VEGFR-2/ERK pathway, but not p38, JNK or AKT signaling. -- Highlights: Black-Right-Pointing-Pointer We examine the expression of VEGFR-2 on cultured human dermal papilla (DP) cells. Black-Right-Pointing-Pointer VEGF{sub 165} stimulated proliferation of human DP cells in a dose-dependent manner. Black-Right-Pointing-Pointer This stimulation was through VEGFR-2-mediated activation of ERK.« less

Regulatory role of periodontal ligament fibroblasts for innate immune cell function and differentiation.

PubMed

Konermann, Anna; Stabenow, Dirk; Knolle, Percy A; Held, Stefanie A E; Deschner, James; Jäger, Andreas

2012-10-01

Innate immunity is crucial for an effective host defense against pathogenic microorganisms in periodontal tissues. As periodontal ligament (PDL) cells synthesize immunomodulatory cytokines, the aim of this in vitro study was to investigate whether these cells can interact with innate immune cells. Resting and inflammatory primed (IL-1β, TNF-α, HMGB1) human PDL cells were co-cultured with human monocyte-derived dendritic cells or macrophages. Migration, phenotypic maturation and modulation of phagocytosis of Porphyromonas gingivalis by immune cells were investigated upon co-culture with PDL cells and/or their released soluble factors. PDL cells interacted with immune cells under both non-inflammatory and inflammatory conditions. Immune cell migration was significantly enhanced by co-culture with PDL cells, which also affected their phenotypic maturation both through cell-cell contact and through released soluble mediators. The dendritic cell maturation markers CD83 and CD86 were upregulated as much as both 'alternatively activated' M2 macrophage maturation markers CD23 and CD163. In contrast, the 'classically activated' M1 macrophage maturation marker CD64 was downregulated. Finally, PDL cells significantly enhanced the phagocytosis of Porphyromonas gingivalis by immune cells. Our experiments revealed that PDL cells are not only structural elements of the periodontium, but actively influence immune responses by interaction with innate immune cells.

Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*

PubMed Central

Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc

2009-01-01

High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226

High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain.

PubMed

Zhou, Ting; Tan, Lei; Cederquist, Gustav Y; Fan, Yujie; Hartley, Brigham J; Mukherjee, Suranjit; Tomishima, Mark; Brennand, Kristen J; Zhang, Qisheng; Schwartz, Robert E; Evans, Todd; Studer, Lorenz; Chen, Shuibing

2017-08-03

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

PubMed

Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A; Scharenberg, Andrew M; Rawlings, David J

2015-09-30

Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties. Copyright © 2015, American Association for the Advancement of Science.

METABOLITES OF BENZO[A]FLUORANTHENE ARE POTENT CYP1 INDUCERS IN T-47D HUMAN BREAST CANCER CELLS. (R827180)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

21 CFR 610.15 - Constituent materials.

Code of Federal Regulations, 2012 CFR

2012-04-01

... evidence that it does not affect adversely the safety or potency of the product. The amount of aluminum in... culture produced vaccines. Extraneous protein known to be capable of producing allergenic effects in human subjects shall not be added to a final virus medium of cell culture produced vaccines intended for...

21 CFR 610.15 - Constituent materials.

Code of Federal Regulations, 2013 CFR

2013-04-01

... evidence that it does not affect adversely the safety or potency of the product. The amount of aluminum in... culture produced vaccines. Extraneous protein known to be capable of producing allergenic effects in human subjects shall not be added to a final virus medium of cell culture produced vaccines intended for...

21 CFR 610.15 - Constituent materials.

Code of Federal Regulations, 2014 CFR

2014-04-01

... evidence that it does not affect adversely the safety or potency of the product. The amount of aluminum in... culture produced vaccines. Extraneous protein known to be capable of producing allergenic effects in human subjects shall not be added to a final virus medium of cell culture produced vaccines intended for...

EFFECT OF PHENYTOIN ON GENE EXPRESSION, OXIDATIVE DAMAGE AND CELL VIABILITY OF CULTURED HUMAN FETAL LIVER SLICES. (R827441)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

SIZE-FRACTIONATED AMBIENT PARTICULATE MATTER INDUCES GM-CSF IN HUMAN BRONCHIAL EPITHELIAL CELLS BY MAPK PATHWAYS. (R827351C003)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Nitric Oxide: Perspectives and Emerging Studies of a Well Known Cytotoxin

PubMed Central

Paradise, William A.; Vesper, Benjamin J.; Goel, Ajay; Waltonen, Joshua D.; Altman, Kenneth W.; Haines, G. Kenneth; Radosevich, James A.

2010-01-01

The free radical nitric oxide (NO•) is known to play a dual role in human physiology and pathophysiology. At low levels, NO• can protect cells; however, at higher levels, NO• is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO• in cancer has to date been tissue-specific, we herein review underlying commonalities of NO• which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO• in human physiology and pathophysiology, including the very important relationship between NO• and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO• in three main areas of epigenetics—DNA methylation, microRNAs, and histone modifications—is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO• (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO• in cancer. PMID:20717533

[Use of recombinant Human Growth Hormone (rHGH)].

PubMed

Calzada-León, Raúl

2017-01-01

Recombinant human growth hormone, synthesized in E.coli or mammalian cells cultures, is since 1985, a useful therapeutic resource to increase growth velocity and final height. In this paper are discussed the four phases (aims, security and efficacy, utility and efficiency) indispensables to define the start of treatment, as well as the absolute, relative and metabolic indications and the transitory and permanent conditions that contraindicate its use. It is commented the way to optimize the results (simple but indispensables indications for the physician, the patients and their family). Finally it is analyzed the results of treatment in patients with growth hormone deficiency, Turner syndrome, chronic renal failure, Prader-Willi syndrome, Noonan syndrome, SHOX deficiency, intrauterine growth retardation and idiopathic short stature.

Antagonism between apoptotic (Bax/Bcl-2) and anti-apoptotic (IAP) signals in human osteoblastic cells under vector-averaged gravity condition.

PubMed

Nakamura, Hiroshi; Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Keiichi; Shinomiya, Kenichi

2003-12-01

A functional disorder associated with weightlessness is well documented in osteoblasts. The apototic features of this disorder are poorly understood. Harmful stress induces apoptosis in cells via mitochondria and/or Fas. The Bax triggers cytochrome c release from mitochondria, which can be blocked by the Bcl-2. Released cytochrome c then activates the initiator caspase, caspase-9, which can be blocked by the anti-apototic (IAP) family of molecules. The effector caspase, caspase-3, finally exerts DNA fragmentation. We conducted this study to examine the apoptotic effects of vector-averaged gravity on normal human osteoblastic cells. Cell culture flasks were incubated on the clinostat, which generated vector-averaged gravity condition (simulated microgravity) for 12, 24, 48, and 96 hours. Upon termination of clinostat cultures, the cell number and cell viability were assessed. DNA fragmentation was analyzed on the agarose-gel electrophoresis. The mRNA levels for Bax, Bcl-2, XIAP, and caspase-3 genes were analyzed by semi-quantitative RT-PCR. Twenty-four hours after starting clinostat rotation, the ratios of Bax/Bcl-2 mRNA levels (indicator of apoptosis) were significantly increased to 136% of the 1G static controls. However, the XIAP mRNA levels (anti-apoptotic molecule) were increased concomitantly to 138% of the 1G static controls. Thus, cell proliferation or cell viability was not affected by vector-averaged gravity. DNA fragmentation was not observed in clinostat group as well as in control group. Finally, the caspase-3 mRNA levels were not affected by vector-averaged gravity. Simulated microgravity might modulate some apoptotic signals upstream the mitochondrial pathway.

Attenuated PfSPZ Vaccine induces strain-transcending T cells and durable protection against heterologous controlled human malaria infection.

PubMed

Lyke, Kirsten E; Ishizuka, Andrew S; Berry, Andrea A; Chakravarty, Sumana; DeZure, Adam; Enama, Mary E; James, Eric R; Billingsley, Peter F; Gunasekera, Anusha; Manoj, Anita; Li, Minglin; Ruben, Adam J; Li, Tao; Eappen, Abraham G; Stafford, Richard E; Kc, Natasha; Murshedkar, Tooba; Mendoza, Floreliz H; Gordon, Ingelise J; Zephir, Kathryn L; Holman, LaSonji A; Plummer, Sarah H; Hendel, Cynthia S; Novik, Laura; Costner, Pamela J M; Saunders, Jamie G; Berkowitz, Nina M; Flynn, Barbara J; Nason, Martha C; Garver, Lindsay S; Laurens, Matthew B; Plowe, Christopher V; Richie, Thomas L; Graham, Barney S; Roederer, Mario; Sim, B Kim Lee; Ledgerwood, Julie E; Hoffman, Stephen L; Seder, Robert A

2017-03-07

A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), confers sterile protection against controlled human malaria infection (CHMI) with Plasmodium falciparum (Pf) parasites homologous to the vaccine strain up to 14 mo after final vaccination. No injectable malaria vaccine has demonstrated long-term protection against CHMI using Pf parasites heterologous to the vaccine strain. Here, we conducted an open-label trial with PfSPZ Vaccine at a dose of 9.0 × 10 5 PfSPZ administered i.v. three times at 8-wk intervals to 15 malaria-naive adults. After CHMI with homologous Pf parasites 19 wk after final immunization, nine (64%) of 14 (95% CI, 35-87%) vaccinated volunteers remained without parasitemia compared with none of six nonvaccinated controls ( P = 0.012). Of the nine nonparasitemic subjects, six underwent repeat CHMI with heterologous Pf7G8 parasites 33 wk after final immunization. Five (83%) of six (95% CI, 36-99%) remained without parasitemia compared with none of six nonvaccinated controls. PfSPZ-specific T-cell and antibody responses were detected in all vaccine recipients. Cytokine production by T cells from vaccinated subjects after in vitro stimulation with homologous (NF54) or heterologous (7G8) PfSPZ were highly correlated. Interestingly, PfSPZ-specific T-cell responses in the blood peaked after the first immunization and were not enhanced by subsequent immunizations. Collectively, these data suggest durable protection against homologous and heterologous Pf parasites can be achieved with PfSPZ Vaccine. Ongoing studies will determine whether protective efficacy can be enhanced by additional alterations in the vaccine dose and number of immunizations.

The proapoptotic effect of formononetin in human osteosarcoma cells: involvement of inactivation of ERK and Akt pathways.

PubMed

Liu, Yun; He, JinJie; Chen, XiaoMing; Li, Jian; Shen, MaoRong; Yu, WenJun; Yang, Yuan; Xiao, ZengMing

2014-01-01

Previous studies have shown that some phytoestrogens inhibits proliferation and induces apoptosis in estrogen-dependent cancers via estrogen receptor (ER)-mediated signaling pathway. In view of the expression of ER in human osteosarcoma cells, the purpose of this study is to investigate whether formononetin and calycosin, two of the major isoflavones in Radix astragali, could also elicit anti-tumor activity against osteosarcoma, along with the underlying mechanism. Human osteosarcoma cells U2OS were respectively treated with various concentrations of formononetin or calycosin. Cell proliferation was determined by MTT assay, while apoptosis by flow cytometry. Next, the expression levels of apoptosis-related genes ERK, Akt, Bcl-2, Bax and caspase-3 were quantified by real-time PCR and Western blotting. Formononetin exhibited higher anti-proliferative activities toward human osteosarcoma cells U2OS, when compared with calycosin. Therefore, U2OS cells were then respectively treated with various concentrations of formononetin, in order to elucidate the isoflavones-related signaling pathway. It was found that formononetin dose-dependently triggered apoptosis of U2OS cells in vitro. Furthermore, treatment of formononetin led to significant inactivation of ERK and Akt, followed by downregulation of Bcl-2, upregulation of Bax and finally increased expression of caspase-3. Formononetin is more effective than calycosin at promoting cell death of U2OS cells by induction of apoptosis, which is mediated by inactivation of ERK and Akt signaling pathways. Thus isoflavones, especially formononetin, may be useful as anti-cancer drugs for osteosarcoma through their apoptosis-inducing effects. © 2014 S. Karger AG, Basel.

The humanized anti-human AMHRII mAb 3C23K exerts an anti-tumor activity against human ovarian cancer through tumor-associated macrophages.

PubMed

Bougherara, Houcine; Némati, Fariba; Nicolas, André; Massonnet, Gérald; Pugnière, Martine; Ngô, Charlotte; Le Frère-Belda, Marie-Aude; Leary, Alexandra; Alexandre, Jérôme; Meseure, Didier; Barret, Jean-Marc; Navarro-Teulon, Isabelle; Pèlegrin, André; Roman-Roman, Sergio; Prost, Jean-François; Donnadieu, Emmanuel; Decaudin, Didier

2017-11-21

Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient's tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials.

The humanized anti-human AMHRII mAb 3C23K exerts an anti-tumor activity against human ovarian cancer through tumor-associated macrophages

PubMed Central

Bougherara, Houcine; Némati, Fariba; Nicolas, André; Massonnet, Gérald; Pugnière, Martine; Ngô, Charlotte; Le Frère-Belda, Marie-Aude; Leary, Alexandra; Alexandre, Jérôme; Meseure, Didier; Barret, Jean-Marc; Navarro-Teulon, Isabelle; Pèlegrin, André; Roman-Roman, Sergio; Prost, Jean-François; Donnadieu, Emmanuel; Decaudin, Didier

2017-01-01

Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient’s tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials. PMID:29245952

Glycoconjugates reveal diversity of human neural stem cells (hNSCs) derived from human induced pluripotent stem cells (hiPSCs).

PubMed

Kandasamy, Majury; Roll, Lars; Langenstroth, Daniel; Brüstle, Oliver; Faissner, Andreas

2017-06-01

Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487 LeX , 5750 LeX and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage, 487 LeX -, 5750 LeX - and 473HD-related glycans were differently expressed. Later, cells of the three germ layers in embryoid bodies (hEBs) and, even after neuralization of hEBs, subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC), LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCs FGF-2/EGF derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCs FGF-2/EGF . Finally, we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487 LeX , 5750 LeX and 473HD are promising tools for identifying distinct stages during neural differentiation.

The importance of the Non Obese Diabetic (NOD) mouse model in autoimmune diabetes

PubMed Central

Pearson, James A; Wong, F. Susan; Wen, Li

2016-01-01

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the pancreatic infiltration of immune cells resulting in T cell-mediated destruction of the insulin-producing beta cells. The successes of the Non Obese Diabetic (NOD) mouse model have come in multiple forms including identifying key genetic and environmental risk factors e.g. Idd loci and effects of microorganisms including the gut microbiota, respectively, and how they may contribute to disease susceptibility and pathogenesis. Furthermore, the NOD model also provides insights into the roles of the innate immune cells as well as the B cells in contributing to the T cell-mediated disease. Unlike many autoimmune disease models, the NOD mouse develops spontaneous disease and has many similarities to human T1D. Through exploiting these similarities many targets have been identified for immune-intervention strategies. Although many of these immunotherapies did not have a significant impact on human T1D, they have been shown to be effective in the NOD mouse in early stage disease, which is not equivalent to trials in newly-diagnosed patients with diabetes. However, the continued development of humanized NOD mice would enable further clinical developments, bringing T1D research to a new translational level. Therefore, it is the aim of this review to discuss the importance of the NOD model in identifying the roles of the innate immune system and the interaction with the gut microbiota in modifying diabetes susceptibility. In addition, the role of the B cells will also be discussed with new insights gained through B cell depletion experiments and the impact on translational developments. Finally, this review will also discuss the future of the NOD mice and the development of humanized NOD mice, providing novel insights into human T1D. PMID:26403950

Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

PubMed

Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

2017-02-28

Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells

PubMed Central

Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

2011-01-01

The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their ‘fresh’ host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its ‘fresh’ host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA. PMID:21729006

Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology.

PubMed

Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

2016-03-01

Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system. Published by Elsevier B.V.

Spatiotemporal dynamics of neocortical excitation and inhibition during human sleep.

PubMed

Peyrache, Adrien; Dehghani, Nima; Eskandar, Emad N; Madsen, Joseph R; Anderson, William S; Donoghue, Jacob A; Hochberg, Leigh R; Halgren, Eric; Cash, Sydney S; Destexhe, Alain

2012-01-31

Intracranial recording is an important diagnostic method routinely used in a number of neurological monitoring scenarios. In recent years, advancements in such recordings have been extended to include unit activity of an ensemble of neurons. However, a detailed functional characterization of excitatory and inhibitory cells has not been attempted in human neocortex, particularly during the sleep state. Here, we report that such feature discrimination is possible from high-density recordings in the neocortex by using 2D multielectrode arrays. Successful separation of regular-spiking neurons (or bursting cells) from fast-spiking cells resulted in well-defined clusters that each showed unique intrinsic firing properties. The high density of the array, which allowed recording from a large number of cells (up to 90), helped us to identify apparent monosynaptic connections, confirming the excitatory and inhibitory nature of regular-spiking and fast-spiking cells, thus categorized as putative pyramidal cells and interneurons, respectively. Finally, we investigated the dynamics of correlations within each class. A marked exponential decay with distance was observed in the case of excitatory but not for inhibitory cells. Although the amplitude of that decline depended on the timescale at which the correlations were computed, the spatial constant did not. Furthermore, this spatial constant is compatible with the typical size of human columnar organization. These findings provide a detailed characterization of neuronal activity, functional connectivity at the microcircuit level, and the interplay of excitation and inhibition in the human neocortex.

LTR12 promoter activation in a broad range of human tumor cells by HDAC inhibition

PubMed Central

Krönung, Sonja K.; Beyer, Ulrike; Chiaramonte, Maria Luisa; Dolfini, Diletta; Mantovani, Roberto; Dobbelstein, Matthias

2016-01-01

A considerable proportion of the human genome consists of transposable elements, including the long terminal repeats (LTRs) of endogenous retroviruses. During evolution, such LTRs were occasionally inserted upstream of protein-coding genes, contributing to their regulation. We previously identified the LTR12 from endogenous retrovirus 9 (ERV9) as a regulator of proapoptotic genes such as TP63 or TNFRSF10B. The promoter activity of LTR12 is largely confined to the testes, silenced in testicular carcinoma, but reactivated in testicular cancer cells by broad-range histone deacetylase (HDAC) inhibitors. Here we show that inhibition of HDAC1-3 is sufficient for LTR12 activation. Importantly, HDAC inhibitors induce LTR12 activity not only in testicular cancer cells, but also in cells derived from many additional tumor species. Finally, we characterize the transcription factor NF-Y as a mediator of LTR12 promoter activity and HDAC inhibitor-induced apoptosis, in the context of widespread genomic binding of NF-Y to specific LTR12 sequences. Thus, HDAC inhibitor-driven LTR12 activation represents a generally applicable means to induce proapoptotic genes in human cancer cells. PMID:27172897

TLR10 suppresses the activation and differentiation of monocytes with effects on DC-mediated adaptive immune responses

PubMed Central

Hess, Nicholas J.; Felicelli, Christopher; Grage, Jennifer; Tapping, Richard I.

2017-01-01

TLRs are important pattern-recognition receptors involved in the activation of innate immune responses against foreign pathogens. TLR10 is the only TLR family member without a known ligand, signaling pathway, or clear cellular function. Previous work has shown that TLR10 suppresses proinflammatory cytokine production in response to TLR agonists in a mixed human mononuclear cell population. We report that TLR10 is preferentially expressed on monocytes and suppresses proinflammatory cytokine production resulting from either TLR or CD40 stimulation. TLR10 engagement affects both the MAPK and Akt signaling pathways, leading to changes in the transcriptome of isolated human monocytes. Differentiation of monocytes into dendritic cells in the presence of an αTLR10 mAb reduced the expression of maturation markers and the induction of proinflammatory cytokines, again in response to either TLR or CD40 stimulation. Finally, in coculture experiments, TLR10 differentiated dendritic cells exhibited a decreased capacity to activate T cells as measured by IL-2 and IFN-γ production. These data demonstrate that TLR10 is a novel regulator of innate immune responses and of the differentiation of primary human monocytes into effective dendritic cells. PMID:28235773

The U24 Protein from Human Herpesvirus 6 and 7 Affects Endocytic Recycling▿

PubMed Central

Sullivan, Brian M.; Coscoy, Laurent

2010-01-01

Modulation of T-cell receptor expression and signaling is essential to the survival of many viruses. The U24 protein expressed by human herpesvirus 6A, a ubiquitous human pathogen, has been previously shown to downregulate the T-cell receptor. Here, we show that U24 also mediates cell surface downregulation of a canonical early endosomal recycling receptor, the transferrin receptor, indicating that this viral protein acts by blocking early endosomal recycling. We present evidence that U24 is a C-tail-anchored protein that is dependent for its function on TRC40/Asna-1, a component of a posttranslational membrane insertion pathway. Finally, we find that U24 proteins from other roseoloviruses have a similar genetic organization and a conserved function that is dependent on a proline-rich motif. Inhibition of a basic cellular process by U24 has interesting implications not only for the pathogenicity of roseoloviruses but also for our understanding of the biology of endosomal transport. PMID:19923186

3,3′-Diindolylmethane Induces G1 Arrest and Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia Cells

PubMed Central

Shorey, Lyndsey E.; Hagman, Amanda M.; Williams, David E.; Ho, Emily; Dashwood, Roderick H.; Benninghoff, Abby D.

2012-01-01

Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL. PMID:22514694

Overexpression of SAMD9 suppresses tumorigenesis and progression during non small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Qing; Yu, Tao; Ren, Yao-Yao

2014-11-07

Highlights: • SAMD9 is down-regulated in human non-small cell lung cancer (NSCLC). • Knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro. • Overexpression of SAMD9 suppressed proliferation and invasion in A549 cells in vitro. • Depletion of SAMD9 increases tumor formation in vivo. - Abstract: The Sterile Alpha Motif Domain-containing 9 (SAMD9) gene has been recently emphasized during the discovery that it is expressed at a lower level in aggressive fibromatosis and some cases of breast and colon cancer, however, the underlying mechanisms are poorly understood. Here, we found that SAMD9 ismore » down-regulated in human non-small cell lung cancer (NSCLC). Furthermore, knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro and overexpression of SAMD9 suppressed proliferation and invasion in A549 cells. Finally, depletion of SAMD9 increases tumor formation in vivo. Our results may provide a strategy for blocking NSCLC tumorigenesis and progression.« less

Interaction of rotavirus with human peripheral blood mononuclear cells: plasmacytoid dendritic cells play a role in stimulating memory rotavirus specific T cells in vitro.

PubMed

Mesa, Martha C; Rodríguez, Luz-Stella; Franco, Manuel A; Angel, Juana

2007-09-15

We studied the interaction of RV with human peripheral blood mononuclear cells (PBMC) from adult volunteers. After exposure of PBMC to rhesus RV (RRV), T and B lymphocytes, NK cells, monocytes, and myeloid and plasmacytoid dendritic cells expressed RV non-structural proteins, at variable levels. Expression of these RV proteins was abolished if infection was done in the presence of anti-VP7 neutralizing antibodies or 10% autologous serum. Supernatants of RRV exposed PBMC contained TNF-alpha, IL-6, IFN-alpha, IFN-gamma, IL-2 and IL-10. Plasmacytoid DC were found to be the main source of IFN-alpha production, and in their absence the production of IFN-gamma and the frequency of RV specific T cells that secrete IFN-gamma diminished. Finally, we could not detect RV-antigen associated with the PBMC or expression of RV non-structural proteins in PBMC of acutely RV-infected children. Thus, although PBMC are susceptible to the initial steps of RV infection, most PBMC of children with RV-gastroenteritis are not infected.

PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination.

PubMed

Garinot, Marie; Fiévez, Virginie; Pourcelle, Vincent; Stoffelbach, François; des Rieux, Anne; Plapied, Laurence; Theate, Ivan; Freichels, Hélène; Jérôme, Christine; Marchand-Brynaert, Jacqueline; Schneider, Yves-Jacques; Préat, Véronique

2007-07-31

To improve the efficiency of orally delivered vaccines, PEGylated PLGA-based nanoparticles displaying RGD molecules at their surface were designed to target human M cells. RGD grafting was performed by an original method called "photografting" which covalently linked RGD peptides mainly on the PEG moiety of the PCL-PEG, included in the formulation. First, three non-targeted formulations with size and zeta potential adapted to M cell uptake and stable in gastro-intestinal fluids, were developed. Their transport by an in vitro model of the human Follicle associated epithelium (co-cultures) was largely increased as compared to mono-cultures (Caco-2 cells). RGD-labelling of nanoparticles significantly increased their transport by co-cultures, due to interactions between the RGD ligand and the beta(1) intregrins detected at the apical surface of co-cultures. In vivo studies demonstrated that RGD-labelled nanoparticles particularly concentrated in M cells. Finally, ovalbumin-loaded nanoparticles were orally administrated to mice and induced an IgG response, attesting antigen ability to elicit an immune response after oral delivery.

Translating Research into Clinical Scale Manufacturing of Mesenchymal Stromal Cells

PubMed Central

Bieback, Karen; Kinzebach, Sven; Karagianni, Marianna

2010-01-01

It sounds simple to obtain sufficient numbers of cells derived from fetal or adult human tissues, isolate and/or expand the stem cells, and then transplant an appropriate number of these cells into the patient at the correct location. However, translating basic research into routine therapies is a complex multistep process which necessitates product regulation. The challenge relates to managing the expected therapeutic benefits with the potential risks and to balance the fast move to clinical trials with time-consuming cautious risk assessment. This paper will focus on the definition of mesenchymal stromal cells (MSCs), and challenges and achievements in the manufacturing process enabling their use in clinical studies. It will allude to different cellular sources, special capacities of MSCs, but also to current regulations, with a special focus on accessory material of human or animal origin, like media supplements. As cellular integrity and purity, formulation and lot release testing of the final product, validation of all procedures, and quality assurance are of utmost necessity, these topics will be addressed. PMID:21318154

Evaluation of Accessory Lacrimal Gland in Muller's Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease.

PubMed

Ali, Marwan; Shah, Dhara; Pasha, Zeeshan; Jassim, Sarmad H; Jassim Jaboori, Assraa; Setabutr, Pete; Aakalu, Vinay K

2017-04-01

The accessory lacrimal glands (ALGs) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential, and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. ALG tissues obtained from Muller's muscle conjunctival resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2, and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Thus, we report for the first time that human ALG tissues contain precursor marker-positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES, and isolate candidate precursor cells from ALG tissue.

Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

PubMed

Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

2013-03-01

Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification.

PubMed

Nims, Raymond W; Sykes, Greg; Cottrill, Karin; Ikonomi, Pranvera; Elmore, Eugene

2010-12-01

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line's species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification.

Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

PubMed

Antonucci, Ivana; Di Pietro, Roberta; Alfonsi, Melissa; Centurione, Maria Antonietta; Centurione, Lucia; Sancilio, Silvia; Pelagatti, Francesca; D'Amico, Maria Angela; Di Baldassarre, Angela; Piattelli, Adriano; Tetè, Stefano; Palka, Giandomenico; Borlongan, Cesar V; Stuppia, Liborio

2014-01-01

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oligodendrocytes.

PubMed

De Filippis, Lidia; Lamorte, Giuseppe; Snyder, Evan Y; Malgaroli, Antonio; Vescovi, Angelo L

2007-09-01

The discovery and study of neural stem cells have revolutionized our understanding of the neurogenetic process, and their inherent ability to adopt expansive growth behavior in vitro is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high-throughput assays for drug development and gene discovery dictate the need for rapid, reproducible, long-term expansion of human neural stem cells (hNSCs). In this view, the complement of wild-type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (immortalized human NSCs [IhNSCs]) by v-myc-mediated immortalization of previously derived wild-type hNSCs. These cells demonstrate three- to fourfold faster proliferation than wild-type cells in response to growth factors but retain rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy, and electrophysiology, we show that upon growth factor removal, IhNSCs completely downregulate v-myc expression, cease proliferation, and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes, and neurons. The latter are functional, mature cells displaying clear-cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the GABAergic, glutamatergic, and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to 20% of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high-throughput gene and drug screening that are essential in both diagnostics and cell therapy studies.

Role for early-differentiated natural killer cells in infectious mononucleosis

PubMed Central

Azzi, Tarik; Lünemann, Anna; Murer, Anita; Ueda, Seigo; Béziat, Vivien; Malmberg, Karl-Johan; Staubli, Georg; Gysin, Claudine; Berger, Christoph; Münz, Christian

2014-01-01

A growing body of evidence suggests that the human natural killer (NK)-cell compartment is phenotypically and functionally heterogeneous and is composed of several differentiation stages. Moreover, NK-cell subsets have been shown to exhibit adaptive immune features during herpes virus infection in experimental mice and to expand preferentially during viral infections in humans. However, both phenotype and role of NK cells during acute symptomatic Epstein-Barr virus (EBV) infection, termed infectious mononucleosis (IM), remain unclear. Here, we longitudinally assessed the kinetics, the differentiation, and the proliferation of subsets of NK cells in pediatric IM patients. Our results indicate that acute IM is characterized by the preferential proliferation of early-differentiated CD56dim NKG2A+ immunoglobulin-like receptor- NK cells. Moreover, this NK-cell subset exhibits features of terminal differentiation and persists at higher frequency during at least the first 6 months after acute IM. Finally, we demonstrate that this NK-cell subset preferentially degranulates and proliferates on exposure to EBV-infected B cells expressing lytic antigens. Thus, early-differentiated NK cells might play a key role in the immune control of primary infection with this persistent tumor-associated virus. PMID:25205117

Role for early-differentiated natural killer cells in infectious mononucleosis.

PubMed

Azzi, Tarik; Lünemann, Anna; Murer, Anita; Ueda, Seigo; Béziat, Vivien; Malmberg, Karl-Johan; Staubli, Georg; Gysin, Claudine; Berger, Christoph; Münz, Christian; Chijioke, Obinna; Nadal, David

2014-10-16

A growing body of evidence suggests that the human natural killer (NK)-cell compartment is phenotypically and functionally heterogeneous and is composed of several differentiation stages. Moreover, NK-cell subsets have been shown to exhibit adaptive immune features during herpes virus infection in experimental mice and to expand preferentially during viral infections in humans. However, both phenotype and role of NK cells during acute symptomatic Epstein-Barr virus (EBV) infection, termed infectious mononucleosis (IM), remain unclear. Here, we longitudinally assessed the kinetics, the differentiation, and the proliferation of subsets of NK cells in pediatric IM patients. Our results indicate that acute IM is characterized by the preferential proliferation of early-differentiated CD56(dim) NKG2A(+) immunoglobulin-like receptor(-) NK cells. Moreover, this NK-cell subset exhibits features of terminal differentiation and persists at higher frequency during at least the first 6 months after acute IM. Finally, we demonstrate that this NK-cell subset preferentially degranulates and proliferates on exposure to EBV-infected B cells expressing lytic antigens. Thus, early-differentiated NK cells might play a key role in the immune control of primary infection with this persistent tumor-associated virus. © 2014 by The American Society of Hematology.

CIP-36, a novel topoisomerase II-targeting agent, induces the apoptosis of multidrug-resistant cancer cells in vitro.

PubMed

Cao, Bo; Chen, Hong; Gao, Ying; Niu, Cong; Zhang, Yuan; Li, Ling

2015-03-01

The need to overcome cancer multidrug resistance (MDR) has fueled considerable interest in the development of novel synthetic antitumor agents with cytotoxicity against cancer cell lines with MDR. In this study, we aimed to investigate CIP-36, a novel podophyllotoxin derivative, for its inhibitory effects on human cancer cells from multiple sources, particularly cells with MDR in vitro. The human leukemia cell line, K562, and the adriamycin-resistant subline, K562/A02, were exposed to CIP-36 or anticancer agents, and various morphological and biochemical properties were assessed by Hoechst 33342 staining under a fluorescence microscope. Subsequently, cytotoxicity, cell growth curves and the cell cycle were analyzed. Finally, the effects of CIP-36 on topoisomerase IIα (Topo IIα) activity were determined. Treatment with CIP-36 significantly inhibited the growth of the K562 and MDR K562/A02 cells. Our data demonstrated that CIP-36 induced apoptosis, inhibited cell cycle progression and inhibited Topo IIα activity. These findings suggest that CIP-36 has the potential to overcome the multidrug resistance of K562/A02 cells by mediating Topo IIα activity.

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Shan; Wang, Ming-Wei; Yao, Xiaoqiang

2009-05-15

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newlymore » developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.« less

Flavonoids and Tannins from Smilax china L. Rhizome Induce Apoptosis Via Mitochondrial Pathway and MDM2-p53 Signaling in Human Lung Adenocarcinoma Cells.

PubMed

Fu, San; Yang, Yanfang; Liu, Dan; Luo, Yan; Ye, Xiaochuan; Liu, Yanwen; Chen, Xin; Wang, Song; Wu, Hezhen; Wang, Yuhang; Hu, Qiwei; You, Pengtao

2017-01-01

In vitro evidence indicates that Smilax china L. rhizome (SCR) can inhibit cell proliferation. Therefore, in the present study, we analyzed the effects in vitro of SCR extracts on human lung adenocarcinoma A549 cells. Our results showed that A549 cell growth was inhibited in a dose- and time-dependent manner after treatment with SCR extracts. Total flavonoids and total tannins from SCR induced A549 apoptosis in a dose-dependent manner, as shown by our flow cytometry analysis, which was consistent with the alterations in nuclear morphology we observed. In addition, the total apoptotic rate induced by total tannins was higher than the rate induced by total flavonoids at the same dose. Cleaved-caspase-3 protein levels in A549 cells after treatment with total flavonoids or total tannins were increased in a dose-dependent manner, followed by the activation of caspase-8 and caspase-9, finally triggering to PARP cleavage. Furthermore, total flavonoids and total tannins increased the expression of Bax, decreased the expression of Bcl-2, and promoted cytochrome [Formula: see text] release. Moreover, MDM2 and p-MDM2 proteins were decreased, while p53 and p-p53 proteins were increased, both in a dose-dependent manner, after A549 treatment with total flavonoids and total tannins. Finally, cleaved-caspase-3 protein levels in the total flavonoids or total tannins-treated H1299 (p53 null) and p53-knockdown A549 cells were increased. Our results indicated that total flavonoids and total tannins from SCR exerted a remarkable effect in reducing A549 growth through their action on mitochondrial pathway and disruption of MDM2-p53 balance. Hence, our findings demonstrated a potential application of total flavonoids and total tannins from SCR in the treatment of human lung adenocarcinoma.

Norm- and hypo-fractionated radiotherapy is capable of activating human dendritic cells.

PubMed

Kulzer, Lorenz; Rubner, Yvonne; Deloch, Lisa; Allgäuer, Andrea; Frey, Benjamin; Fietkau, Rainer; Dörrie, Jan; Schaft, Niels; Gaipl, Udo S

2014-10-01

Despite the transient immunosuppressive properties of local radiotherapy (RT), this classical treatment modality of solid tumors is capable of inducing immunostimulatory forms of tumor-cell death. The resulting 'immunotoxicity' in the tumor, but not in healthy tissues, may finally lead to immune-mediated destruction of the tumor. However, little is known about the best irradiation scheme in this setting. This study examines the immunological effects of differently irradiated human colorectal tumor cells on human monocyte-derived dendritic cells (DC). Human SW480 tumor cells were irradiated with a norm-fractionation scheme (5 × 2 Gy), a hypo-fractionated protocol (3 × 5 Gy), and with a high single irradiation dose (radiosurgery; 1 × 15 Gy). Subsequently, human immature DC (iDC) were co-incubated with supernatants (SN) of these differently treated tumor cells. Afterwards, DC were analyzed regarding the expression of maturation markers, the release of cytokines, and the potential to stimulate CD4(+) T-cells. The co-incubation of iDC with SN of tumor cells exposed to norm- or hypo-fractionated RT resulted in a significantly increased secretion of the immune activating cytokines IL-12p70, IL-8, IL-6, and TNFα, compared to iDC co-incubated with SN of tumor cells that received a high single irradiation dose or were not irradiated. In addition, DC-maturation markers CD80, CD83, and CD25 were also exclusively elevated after co-incubation with the SN of fractionated irradiated tumor cells. Furthermore, the SN of tumor cells that were irradiated with norm- or hypo-fractionated RT triggered iDC to stimulate CD4(+) T-cells not only in an allogenic, but also in an antigen-specific manner like mature DC. Collectively, these results demonstrate that norm- and hypo-fractionated RT induces a fast human colorectal tumor-cell death with immunogenic potential that can trigger DC maturation and activation in vitro. Such findings may contribute to the improvement of irradiation protocols for the most beneficial induction of anti-tumor immunity.

Hepatitis B virus PreS1 facilitates hepatocellular carcinoma development by promoting appearance and self-renewal of liver cancer stem cells.

PubMed

Liu, Zhixin; Dai, Xuechen; Wang, Tianci; Zhang, Chengcheng; Zhang, Wenjun; Zhang, Wei; Zhang, Qi; Wu, Kailang; Liu, Fang; Liu, Yingle; Wu, Jianguo

2017-08-01

Hepatitis B virus (HBV) is a major etiologic agent of hepatocellular carcinoma (HCC). However, the molecular mechanism by which HBV infection contributes to HCC development is not fully understood. Here, we initially showed that HBV stimulates the production of cancer stem cells (CSCs)-related markers (CD133, CD117 and CD90) and CSCs-related genes (Klf4, Sox2, Nanog, c-Myc and Oct4) and facilitates the self-renewal of CSCs in human hepatoma cells. Cellular and clinical studies revealed that HBV facilitates hepatoma cell growth and migration, enhances white blood cell (WBC) production in the sera of patients, stimulates CD133 and CD117 expression in HCC tissues, and promotes the CSCs generation of human hepatoma cells and clinical cancer tissues. Detailed studies revealed that PreS1 protein of HBV is required for HBV-mediated CSCs generation. PreS1 activates CD133, CD117 and CD90 expression in normal hepatocyte derived cell line (L02) and human hepatoma cell line (HepG2 and Huh-7); facilitates L02 cells migration, growth and sphere formation; and finally enhances the abilities of L02 cells and HepG2 cells to induce tumorigeneses in nude mice. Thus, PreS1 acts as a new oncoprotein to play a key role in the appearance and self-renewal of CSCs during HCC development. Copyright © 2017 Elsevier B.V. All rights reserved.

The use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering

PubMed Central

Wang, Zhong-Shan; Feng, Zhi-Hong; Wu, Guo-Feng; Bai, Shi-Zhu; Dong, Yan; Chen, Fa-Ming; Zhao, Yi-Min

2016-01-01

Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament tissue (PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells (PDLSCs) and jaw bone mesenchymal stem cells (JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Platelet-rich fibrin (PRF) derived from human venous blood was then fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, the in vivo potential of a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets to form periodontal tissue was assessed in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space comprising human treated dentin matrix (TDM) and hydroxyapatite (HA)/tricalcium phosphate (TCP) frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL-like tissues, while the JBMSC sheets tended to produce predominantly bone-like tissues. In addition, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal tissue-like structures containing PDL- and bone-like tissues. Further improvements in this cell transplantation design may have the potential to provide an effective approach for future periodontal tissue regeneration. PMID:27324079

Androgen receptor-negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms.

PubMed

Li, Zhi Gang; Mathew, Paul; Yang, Jun; Starbuck, Michael W; Zurita, Amado J; Liu, Jie; Sikes, Charles; Multani, Asha S; Efstathiou, Eleni; Lopez, Adriana; Wang, Jing; Fanning, Tina V; Prieto, Victor G; Kundra, Vikas; Vazquez, Elba S; Troncoso, Patricia; Raymond, Austin K; Logothetis, Christopher J; Lin, Sue-Hwa; Maity, Sankar; Navone, Nora M

2008-08-01

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor-negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer-induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor-null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells.

Androgen receptor–negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms

PubMed Central

Li, Zhi Gang; Mathew, Paul; Yang, Jun; Starbuck, Michael W.; Zurita, Amado J.; Liu, Jie; Sikes, Charles; Multani, Asha S.; Efstathiou, Eleni; Lopez, Adriana; Wang, Jing; Fanning, Tina V.; Prieto, Victor G.; Kundra, Vikas; Vazquez, Elba S.; Troncoso, Patricia; Raymond, Austin K.; Logothetis, Christopher J.; Lin, Sue-Hwa; Maity, Sankar; Navone, Nora M.

2008-01-01

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor–negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer–induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor–null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells. PMID:18618013

Human bone perivascular niche-on-a-chip for studying metastatic colonization.

PubMed

Marturano-Kruik, Alessandro; Nava, Michele Maria; Yeager, Keith; Chramiec, Alan; Hao, Luke; Robinson, Samuel; Guo, Edward; Raimondi, Manuela Teresa; Vunjak-Novakovic, Gordana

2018-02-06

Eight out of 10 breast cancer patients die within 5 years after the primary tumor has spread to the bones. Tumor cells disseminated from the breast roam the vasculature, colonizing perivascular niches around blood capillaries. Slow flows support the niche maintenance by driving the oxygen, nutrients, and signaling factors from the blood into the interstitial tissue, while extracellular matrix, endothelial cells, and mesenchymal stem cells regulate metastatic homing. Here, we show the feasibility of developing a perfused bone perivascular niche-on-a-chip to investigate the progression and drug resistance of breast cancer cells colonizing the bone. The model is a functional human triculture with stable vascular networks within a 3D native bone matrix cultured on a microfluidic chip. Providing the niche-on-a-chip with controlled flow velocities, shear stresses, and oxygen gradients, we established a long-lasting, self-assembled vascular network without supplementation of angiogenic factors. We further show that human bone marrow-derived mesenchymal stem cells, which have undergone phenotypical transition toward perivascular cell lineages, support the formation of capillary-like structures lining the vascular lumen. Finally, breast cancer cells exposed to interstitial flow within the bone perivascular niche-on-a-chip persist in a slow-proliferative state associated with increased drug resistance. We propose that the bone perivascular niche-on-a-chip with interstitial flow promotes the formation of stable vasculature and mediates cancer cell colonization.

Lipidomics of human brain aging and Alzheimer's disease pathology.

PubMed

Naudí, Alba; Cabré, Rosanna; Jové, Mariona; Ayala, Victoria; Gonzalo, Hugo; Portero-Otín, Manuel; Ferrer, Isidre; Pamplona, Reinald

2015-01-01

Lipids stimulated and favored the evolution of the brain. Adult human brain contains a large amount of lipids, and the largest diversity of lipid classes and lipid molecular species. Lipidomics is defined as "the full characterization of lipid molecular species and of their biological roles with respect to expression of proteins involved in lipid metabolism and function, including gene regulation." Therefore, the study of brain lipidomics can help to unravel the diversity and to disclose the specificity of these lipid traits and its alterations in neural (neurons and glial) cells, groups of neural cells, brain, and fluids such as cerebrospinal fluid and plasma, thus helping to uncover potential biomarkers of human brain aging and Alzheimer disease. This review will discuss the lipid composition of the adult human brain. We first consider a brief approach to lipid definition, classification, and tools for analysis from the new point of view that has emerged with lipidomics, and then turn to the lipid profiles in human brain and how lipids affect brain function. Finally, we focus on the current status of lipidomics findings in human brain aging and Alzheimer's disease pathology. Neurolipidomics will increase knowledge about physiological and pathological functions of brain cells and will place the concept of selective neuronal vulnerability in a lipid context. © 2015 Elsevier Inc. All rights reserved.

A scalable strategy for high-throughput GFP tagging of endogenous human proteins.

PubMed

Leonetti, Manuel D; Sekine, Sayaka; Kamiyama, Daichi; Weissman, Jonathan S; Huang, Bo

2016-06-21

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

A novel functional site of extracellular matrix metalloproteinase inducer (EMMPRIN) that limits the migration of human uterine cervical carcinoma cells.

PubMed

Sato, Takashi; Watanabe, Mami; Hashimoto, Kei; Ota, Tomoko; Akimoto, Noriko; Imada, Keisuke; Nomizu, Motoyoshi; Ito, Akira

2012-01-01

EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147, a membrane-bound glycoprotein with two extracellular loop domains (termed loops I and II), progresses tumor invasion and metastasis by increasing the production of matrix metalloproteinase (MMP) in peritumoral stoma cells. EMMPRIN has also been associated with the control of migration activity in some tumor cells, but little is known about how EMMPRIN regulates tumor cell migration. In the present study, EMMPRIN siRNA suppressed the gene expression and production of EMMPRIN in human uterine cervical carcinoma SKG-II cells. An in vitro scratch wound assay showed enhancement of migration of EMMPRIN-knockdown SKG-II cells. In addition, the SKG-II cell migration was augmented by adding an E. coli-expressed human EMMPRIN mutant with two extracellular loop domains (eEMP-I/II), which bound to the cell surface of SKG-II cells. However, eEMP-I/II suppressed the native EMMPRIN-mediated augmentation of proMMP-1/procollagenase-1 production in a co-culture of the SKG-II cells and human uterine cervical fibroblasts, indicating that the augmentation of SKG-II cell migration resulted from the interference of native EMMPRIN functions by eEMP-I/II on the cell surface. Furthermore, a systematic peptide screening method using nine synthetic EMMPRIN peptides coding the loop I and II domains (termed EM1-9) revealed that EM9 (170HIENLNMEADPGQYR184) facilitated SKG-II cell migration. Moreover, SKG-II cell migration was enhanced by administration of an antibody against EM9, but not EM1 which is a crucial site for the MMP inducible activity of EMMPRIN. Therefore, these results provide novel evidence that EMMPRIN on the cell surface limits the cell migration of human uterine cervical carcinoma cells through 170HIENLNMEADPGQYR184 in the loop II domain. Finally, these results should provide an increased understanding of the functions of EMMPRIN in malignant cervical carcinoma cells, and could contribute to the development of clinical strategies for cervical cancer therapy.

Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

PubMed

Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

2015-02-01

Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Regulatory Eosinophils Suppress T Cells Partly through Galectin-10.

PubMed

Lingblom, Christine; Andersson, Jennie; Andersson, Kerstin; Wennerås, Christine

2017-06-15

Eosinophils have the capacity to regulate the function of T cell subsets. Our aim was to test the hypothesis of the existence of a regulatory subset of eosinophils. Human eosinophils were incubated with T cells that were stimulated with allogeneic leukocytes or CD3/CD28 cross-linking. After 2 d of coculture, 11% of the eosinophils gained CD16 expression. A CD16 hi subset of eosinophils, encompassing 1-5% of all eosinophils, was also identified in the blood of healthy subjects. FACS sorting showed that these CD16 hi eosinophils were significantly stronger suppressors of T cell proliferation than were conventional CD16 neg eosinophils. Human eosinophils contain stores of the immunoregulatory protein galectin-10. We found that Ab-mediated neutralization of galectin-10 partially abrogated the suppressive function of the eosinophils. Moreover, recombinant galectin-10 by itself was able to suppress T cell proliferation. Finally, we detected galectin-10-containing immune synapses between eosinophils and lymphocytes. To conclude, we describe a subset of suppressive eosinophils expressing CD16 that may escape detection because CD16-based negative selection is the standard procedure for the isolation of human eosinophils. Moreover, we show that galectin-10 functions as a T cell-suppressive molecule in eosinophils. Copyright © 2017 by The American Association of Immunologists, Inc.

Nitric oxide signaling depends on biotin in Jurkat human lymphoma cells.

PubMed

Rodriguez-Melendez, Rocio; Zempleni, Janos

2009-03-01

Biotin affects gene expression through a diverse array of cell signaling pathways. Previous studies provided evidence that cGMP-dependent signaling also depends on biotin, but the mechanistic sequence of cGMP regulation by biotin is unknown. Here we tested the hypothesis that the effects of biotin in cGMP-dependent cell signaling are mediated by nitric oxide (NO). Human lymphoid (Jurkat) cells were cultured in media containing deficient (0.025 nmol/L), physiological (0.25 nmol/L), and pharmacological (10 nmol/L) concentrations of biotin for 5 wk. Both levels of intracellular biotin and NO exhibited a dose-dependent relationship in regard to biotin concentrations in culture media. Effects of biotin on NO levels were disrupted by the NO synthase (NOS) inhibitor N-monomethyl-arginine. Biotin-dependent production of NO was linked with biotin-dependent expression of endothelial and neuronal NOS, but not inducible NOS. Previous studies revealed that NO is an activator of guanylate cyclase. Consistent with these previous observations, biotin-dependent generation of NO increased the abundance of cGMP in Jurkat cells. Finally, the biotin-dependent generation of cGMP increased protein kinase G activity. Collectively, the results of this study are consistent with the hypothesis that biotin-dependent cGMP signaling in human lymphoid cells is mediated by NO.

A p53-dependent response limits the viability of mammalian haploid cells

PubMed Central

Olbrich, Teresa; Mayor-Ruiz, Cristina; Vega-Sendino, Maria; Gomez, Carmen; Ortega, Sagrario; Ruiz, Sergio; Fernandez-Capetillo, Oscar

2017-01-01

The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell–sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells. PMID:28808015

Melatonin: an Inhibitor of Breast Cancer

PubMed Central

Hill, Steven M.; Belancio, Victoria P.; Dauchy, Robert T.; Xiang, Shulin; Brimer, Samantha; Mao, Lulu; Hauch, Adam; Lundberg, Peter W.; Summers, Whitney; Yuan, Lin; Frasch, Tripp; Blask, David E.

2015-01-01

This review discusses recent work on melatonin-mediated circadian regulation and metabolic and molecular signaling mechanisms involved in human breast cancer growth and associated consequences of circadian disruption by exposure to light at night (LEN). The anti-cancer actions of the circadian melatonin signal in human breast cancer cell lines and xenografts heavily involve MT1 receptor-mediated mechanisms. In estrogen receptor alpha (ERα)-positive human breast cancer, melatonin, via the MT1 receptor, suppresses ERα mRNA expression and ERα transcriptional activity. As well, melatonin regulates the transactivation of other members of the nuclear receptor super-family, estrogen metabolizing enzymes, and the expression of core clock and clock-related genes. Furthermore, melatonin also suppresses tumor aerobic metabolism (Warburg effect), and, subsequently, cell-signaling pathways critical to cell proliferation, cell survival, metastasis, and drug resistance. Melatonin demonstrates both cytostatic and cytotoxic activity in breast cancer cells that appears to be cell type specific. Melatonin also possesses anti-invasive/anti-metastatic actions that involve multiple pathways including inhibition of p38 MAPK and repression of epithelial-to-mesenchymal transition. Studies demonstrate that melatonin promotes genomic stability by inhibiting the expression of LINE-1 retrotransposons. Finally, research in animal and human models indicate that LEN induced disruption of the circadian nocturnal melatonin signal promotes the growth, metabolism, and signaling of human breast cancer to drive breast tumors to endocrine and chemotherapeutic resistance. These data provide the strongest understanding and support of the mechanisms underpinning the epidemiologic demonstration of elevated breast cancer risk in night shift workers and other individuals increasingly exposed to LEN. PMID:25876649

Identification and Quantification of the Main Active Anticancer Alkaloids from the Root of Glaucium flavum

PubMed Central

Bournine, Lamine; Bensalem, Sihem; Wauters, Jean-Noël; Iguer-Ouada, Mokrane; Maiza-Benabdesselam, Fadila; Bedjou, Fatiha; Castronovo, Vincent; Bellahcène, Akeila; Tits, Monique; Frédérich, Michel

2013-01-01

Glaucium flavum is used in Algerian folk medicine to remove warts (benign tumors). Its local appellations are Cheqiq el-asfar and Qarn el-djedyane. We have recently reported the anti-tumoral activity of Glaucium flavum root alkaloid extract against human cancer cells, in vitro and in vivo. The principal identified alkaloid in the extract was protopine. This study aims to determine which component(s) of Glaucium flavum root extract might possess potent antitumor activity on human cancer cells. Quantitative estimation of Glaucium flavum alkaloids was realized by HPLC-DAD. Glaucium flavum effect on human normal and cancer cell viability was determined using WST-1 assay. Quantification of alkaloids in Glaucium flavum revealed that the dried root part contained 0.84% of protopine and 0.07% of bocconoline (w/w), while the dried aerial part contained only 0.08% of protopine, glaucine as the main alkaloid, and no bocconoline. In vitro evaluation of the growth inhibitory activity on breast cancer and normal cells demonstrated that purified protopine did not reproduce the full cytotoxic activity of the alkaloid root extract on cancer cell lines. On the other hand, bocconoline inhibited strongly the viability of cancer cells with an IC50 of 7.8 μM and only a low cytotoxic effect was observed against normal human cells. Our results showed for the first time that protopine is the major root alkaloid of Glaucium flavum. Finally, we are the first to demonstrate a specific anticancer effect of Glaucium flavum root extract against breast cancer cells, which can be attributed, at least in part, to bocconoline. PMID:24317429

Transplantation of Human Embryonic Stem Cells in Patients with Multiple Sclerosis and Lyme Disease

PubMed Central

Shroff, Geeta

2016-01-01

Case series Patient: Male, 42 • Female, 30 Final Diagnosis: Human embryonic stem cells showed good therapeutic potential for treatment of multiple sclerosis with lyme disease Symptoms: Fatigue • weakness in limbs Medication: — Clinical Procedure: Human embryonic stem cells transplantation Specialty: Transplantology Objective: Rare disease Background: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease in which the myelin sheath of nerve cells is damaged. It can cause delayed neurologic symptoms similar to those seen in Lyme disease (LD) patients. Thymus derived T-cells (myelin reactive) migrate to the blood brain barrier and stimulate an inflammatory cascade in the central nervous system. Cell based therapies play an important role in treating neurological diseases such as MS and LD. Case Report: Human embryonic stem cell (hESC) therapy was used to treat two patients with both MS and LD. The hESCs were administered via different routes including intramuscular, intravenous, and supplemental routes (e.g., deep spinal, caudal, intercostal through eye drops) to regenerate the injured cells. Both the patients showed remarkable improvement in their functional skills, overall stamina, cognitive abilities, and muscle strength. Furthermore, the improvement in the patients’ conditions were assessed by magnetic resonance tractography and single photon emission computed tomography (SPECT). Conclusions: Therapy with hESCs might emerge as an effective and safe treatment for patients with both MS and LD. Well-designed clinical trials and follow-up studies are needed to prove the long-term efficacy and safety of hESC therapy in the treatment of patients with MS and LD. PMID:27956736

Identification and quantification of the main active anticancer alkaloids from the root of Glaucium flavum.

PubMed

Bournine, Lamine; Bensalem, Sihem; Wauters, Jean-Noël; Iguer-Ouada, Mokrane; Maiza-Benabdesselam, Fadila; Bedjou, Fatiha; Castronovo, Vincent; Bellahcène, Akeila; Tits, Monique; Frédérich, Michel

2013-12-02

Glaucium flavum is used in Algerian folk medicine to remove warts (benign tumors). Its local appellations are Cheqiq el-asfar and Qarn el-djedyane. We have recently reported the anti-tumoral activity of Glaucium flavum root alkaloid extract against human cancer cells, in vitro and in vivo. The principal identified alkaloid in the extract was protopine. This study aims to determine which component(s) of Glaucium flavum root extract might possess potent antitumor activity on human cancer cells. Quantitative estimation of Glaucium flavum alkaloids was realized by HPLC-DAD. Glaucium flavum effect on human normal and cancer cell viability was determined using WST-1 assay. Quantification of alkaloids in Glaucium flavum revealed that the dried root part contained 0.84% of protopine and 0.07% of bocconoline (w/w), while the dried aerial part contained only 0.08% of protopine, glaucine as the main alkaloid, and no bocconoline. In vitro evaluation of the growth inhibitory activity on breast cancer and normal cells demonstrated that purified protopine did not reproduce the full cytotoxic activity of the alkaloid root extract on cancer cell lines. On the other hand, bocconoline inhibited strongly the viability of cancer cells with an IC50 of 7.8 µM and only a low cytotoxic effect was observed against normal human cells. Our results showed for the first time that protopine is the major root alkaloid of Glaucium flavum. Finally, we are the first to demonstrate a specific anticancer effect of Glaucium flavum root extract against breast cancer cells, which can be attributed, at least in part, to bocconoline.

Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study

PubMed Central

Palmela, Inês; Correia, Leonor; Silva, Rui F. M.; Sasaki, Hiroyuki; Kim, Kwang S.; Brites, Dora; Brito, Maria A.

2015-01-01

Ursodeoxycholic acid and its main conjugate glycoursodeoxycholic acid are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 activation and cell death, as well as interleukin-6 release and a loss of blood-brain barrier integrity. Here, we tested the preventive and restorative effects of these bile acids regarding the disruption of blood-brain barrier properties by UCB in in vitro conditions mimicking severe neonatal hyperbilirubinemia and using the same experimental blood-brain barrier model. Both bile acids reduced the apoptotic cell death induced by UCB, but only glycoursodeoxycholic acid significantly counteracted caspase-3 activation. Bile acids also prevented the upregulation of interleukin-6 mRNA, whereas only ursodeoxycholic acid abrogated cytokine release. Regarding barrier integrity, only ursodeoxycholic acid abrogated UCB-induced barrier permeability. Better protective effects were obtained by bile acid pre-treatment, but a strong efficacy was still observed by their addition after UCB treatment. Finally, both bile acids showed ability to cross confluent monolayers of human brain microvascular endothelial cells in a time-dependent manner. Collectively, data disclose a therapeutic time-window for preventive and restorative effects of ursodeoxycholic acid and glycoursodeoxycholic acid against UCB-induced blood-brain barrier disruption and damage to human brain microvascular endothelial cells. PMID:25821432

Autonomous molecular cascades for evaluation of cell surfaces

NASA Astrophysics Data System (ADS)

Rudchenko, Maria; Taylor, Steven; Pallavi, Payal; Dechkovskaia, Alesia; Khan, Safana; Butler, Vincent P., Jr.; Rudchenko, Sergei; Stojanovic, Milan N.

2013-08-01

Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs. Previously studied nucleic acid-based automata include game-playing molecular devices (MAYA automata) and finite-state automata for the analysis of nucleic acids, with the latter inspiring circuits for the analysis of RNA species inside cells. Here, we describe automata based on strand-displacement cascades directed by antibodies that can analyse cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells.

A novel strategy to adapt SHIV-E1 carrying env from an RV144 volunteer to rhesus macaques: coreceptor switch and final recovery of a pathogenic virus with exclusive R5 tropism.

PubMed

Scinto, Hanna B; Gupta, Sandeep; Thorat, Swati; Mukhtar, Muhammad M; Griffiths, Anthony; Delgado, Jennifer; Plake, Elizabeth; Vyas, Hemant K; Strickland, Amanda; Byrareddy, Siddappa N; Montefiori, David C; LaBranche, Celia; Pal, Ranajit; Treece, Jim; Orndorff, Sharon; Ferrari, Maria Grazia; Weiss, Deborah; Chenine, Agnes-Laurence; McLinden, Robert; Michael, Nelson; Kim, Jerome H; Robb, Merlin; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Ruprecht, Ruth M

2018-05-09

The Phase III RV144 HIV vaccine trial conducted in Thailand remains the only study to show efficacy in decreasing HIV acquisition risk. In Thailand, circulating recombinant forms of HIV clades A/E (CRF01_AE) predominate; in such viruses, env originates from clade E (HIV-E). We constructed a simian-human immunodeficiency virus (SHIV) chimera carrying env isolated from an RV144 placebo recipient in the SHIV-1157ipd3N4 backbone. The latter contains long terminal repeats (LTRs) with duplicated NF-κB sites, thus resembling HIV LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV-E1, to rhesus macaques: simultaneous depletion of B and CD8 + cells followed by intramuscular inoculation of proviral DNA and repeated administrations of cell-free virus. High viremia and CD4 + T-cell depletion ensued. Passage 3 virus unexpectedly caused acute, irreversible CD4 + T-cell loss; the partially adapted SHIV had become dual-tropic. Virus and IMCs with exclusive R5 tropism were reisolated from earlier passages, combined, and used to complete the adaptation through additional macaques. The final isolate, SHIV-E1p5, remained solely R5-tropic. It had a tier 2 neutralization phenotype, was mucosally transmissible, and pathogenic. Deep sequencing revealed 99% Env amino acid sequence conservation; X4-only and dual-tropic strains had evolved independently from an early branch of parental SHIV-E1. To conclude, our primate model data reveal that SHIV-E1p5 recapitulates important aspects of HIV transmission and pathobiology in humans. IMPORTANCE Understanding the protective principles that lead to a safe, effective vaccine against HIV in non-human primate (NHP) models requires test viruses that allow evaluation of anti-HIV envelope responses. Reduced HIV acquisition risk in RV144 has been linked to non-neutralizing IgG antibodies with a range of effector activities. Definitive experiments to decipher the mechanisms of the partial protection observed in RV144 require passive immunization studies in NHPs with a relevant test virus. We have generated such a virus by inserting env from an RV144 placebo recipient into a SHIV backbone with HIV-like LTRs. The final SHIV-E1p5 isolate, grown in rhesus monkey peripheral blood mononuclear cells, was mucosally transmissible and pathogenic. Earlier SHIV-E passages showed coreceptor switch, again mimicking HIV biology in humans. Thus, our series of SHIV-E strains mirrors HIV transmission and disease progression in humans. SHIV-E1p5 represents a biologically relevant tool to assess prevention strategies. Copyright © 2018 American Society for Microbiology.

Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

PubMed

Terrasso, Ana Paula; Silva, Ana Carina; Filipe, Augusto; Pedroso, Pedro; Ferreira, Ana Lúcia; Alves, Paula Marques; Brito, Catarina

Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates. Copyright © 2016 Elsevier Inc. All rights reserved.

Human a-L-fucosidase-1 attenuates the invasive properties of thyroid cancer.

PubMed

Vecchio, Giancarlo; Parascandolo, Alessia; Allocca, Chiara; Ugolini, Clara; Basolo, Fulvio; Moracci, Marco; Strazzulli, Andrea; Cobucci-Ponzano, Beatrice; Laukkanen, Mikko O; Castellone, Maria Domenica; Tsuchida, Nobuo

2017-04-18

Glycans containing α-L-fucose participate in diverse interactions between cells and extracellular matrix. High glycan expression on cell surface is often associated with neoplastic progression. The lysosomal exoenzyme, α-L-fucosidase-1 (FUCA-1) removes fucose residues from glycans. The FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors. However, the role of FUCA-1 in tumor progression remains unclear. It is speculated that its inactivation perturbs glycosylation of proteins involved in cell adhesion and promotes cancer. FUCA-1 expression of various thyroid normal and cancer tissues assayed by immunohistochemical (IHC) staining was high in normal thyroids and papillary thyroid carcinomas (PTC), whereas it progressively decreased in poorly differentiated, metastatic and anaplastic thyroid carcinomas (ATC). FUCA-1 mRNA expression from tissue samples and cell lines and protein expression levels and enzyme activity in thyroid cancer cell lines paralleled those of IHC staining. Furthermore, ATC-derived 8505C cells adhesion to human E-selectin and HUVEC cells was inhibited by bovine α-L-fucosidase or Lewis antigens, thus pointing to an essential role of fucose residues in the adhesive phenotype of this cancer cell line. Finally, 8505C cells transfected with a FUCA-1 containing plasmid displayed a less invasive phenotype versus the parental 8505C. These results demonstrate that FUCA-1 is down-regulated in ATC compared to PTC and normal thyroid tissues and cell lines. As shown for other human cancers, the down-regulation of FUCA-1 correlates with increased aggressiveness of the cancer type. This is the first report indicating that the down-regulation of FUCA-1 is related to the increased aggressiveness of thyroid cancer.

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells.

PubMed

Zambelli, Filippo; Mertens, Joke; Dziedzicka, Dominika; Sterckx, Johan; Markouli, Christina; Keller, Alexander; Tropel, Philippe; Jung, Laura; Viville, Stephane; Van de Velde, Hilde; Geens, Mieke; Seneca, Sara; Sermon, Karen; Spits, Claudia

2018-06-07

In this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system.

PubMed

Cheng, Liang; Zhang, Zheng; Li, Guangming; Li, Feng; Wang, Li; Zhang, Liguo; Zurawski, Sandra M; Zurawski, Gerard; Levy, Yves; Su, Lishan

2017-10-27

TLR ligands (TLR-Ls) represent a class of novel vaccine adjuvants. However, their immunologic effects in humans remain poorly defined in vivo. Using a humanized mouse model with a functional human immune system, we investigated how different TLR-Ls stimulated human innate immune response in vivo and their applications as vaccine adjuvants for enhancing human cellular immune response. We found that splenocytes from humanized mice showed identical responses to various TLR-Ls as human PBMCs in vitro. To our surprise, various TLR-Ls stimulated human cytokines and chemokines differently in vivo compared to that in vitro. For example, CpG-A was most efficient to induce IFN-α production in vitro. In contrast, CpG-B, R848 and Poly I:C stimulated much more IFN-α than CpG-A in vivo. Importantly, the human innate immune response to specific TLR-Ls in humanized mice was different from that reported in C57BL/6 mice, but similar to that reported in nonhuman primates. Furthermore, we found that different TLR-Ls distinctively activated and mobilized human plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs) and monocytes in different organs. Finally, we showed that, as adjuvants, CpG-B, R848 and Poly I:C can all enhance antigen specific CD4 + T cell response, while only R848 and Poly I:C induced CD8 + cytotoxic T cells response to a CD40-targeting HIV vaccine in humanized mice, correlated with their ability to activate human mDCs but not pDCs. We conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sterigmatocystin induces G1 arrest in primary human esophageal epithelial cells but induces G2 arrest in immortalized cells: key mechanistic differences in these two models.

PubMed

Wang, Juan; Huang, Shujuan; Xing, Lingxiao; Cui, Jinfeng; Tian, Ziqiang; Shen, Haitao; Jiang, Xiujuan; Yan, Xia; Wang, Junling; Zhang, Xianghong

2015-11-01

Sterigmatocystin (ST), a mycotoxin commonly found in food and feed commodities, has been classified as a "possible human carcinogen." Our previous studies suggested that ST exposure might be a risk factor for esophageal cancer and that ST may induce DNA damage and G2 phase arrest in immortalized human esophageal epithelial cells (Het-1A). To further confirm and explore the cellular responses of ST in human esophageal epithelia, we comparatively evaluated DNA damage, cell cycle distribution and the relative mechanisms in primary cultured human esophageal epithelial cells (EPC), which represent a more representative model of the in vivo state, and Het-1A cells. In this study, we found that ST could induce DNA damage in both EPC and Het-1A cells but led to G1 phase arrest in EPC cells and G2 phase arrest in Het-1A cells. Furthermore, our results indicated that the activation of the ATM-Chk2 pathway was involved in ST-induced G1 phase arrest in EPC cells, whereas the p53-p21 pathway activation in ST-induced G2 phase arrest in Het-1A cells. Studies have demonstrated that SV40 large T-antigen (SV40LT) may disturb cell cycle progression by inactivating some of the proteins involved in the G1/S checkpoint. Het-1A is a non-cancerous epithelial cell line immortalized by SV40LT. To evaluate the possible perturbation effect of SV40LT on ST-induced cell cycle disturbance in Het-1A cells, we knocked down SV40LT of Het-1A cells with siRNA and found that under this condition, ST-induced G2 arrest was significantly attenuated, whereas the proportion of cells in the G1 phase was significantly increased. Furthermore, SV40LT-siRNA also inhibited the activation of the p53-p21 signaling pathway induced by ST. In conclusion, our data indicated that ST could induce DNA damage in both primary cultured and immortalized esophageal epithelial cells. In primary human esophageal epithelial cells, ST induced DNA damage and then triggered the ATM-Chk2 pathway, resulting in G1 phase arrest, whereas in SV40LT-immortalized human esophageal epithelial cells, SV40LT-mediated G1 checkpoint inactivation occurred, and ST-DNA damage activated p53-p21 signaling pathway, up-regulating G2/M phase regulatory proteins and finally leading to a G2 phase arrest. Thus, the SV40LT-mediated G1 checkpoint inactivation is responsible for the difference in the cell cycle arrest by ST between immortalized and primary cultured human esophageal epithelial cells.

Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins.

PubMed

Barrera, G J; Tortolero, G Sanchez

2016-01-01

Trefoil factors are effector molecules in gastrointestinal tract physiology. Each one improves healing of the gastrointestinal tract. Trefoil factors may be grouped into three classes: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Significant amounts of TFF3 are present in human breast milk. Previously, we have reported that trefoil factor 3 isolated from human breast milk produces down regulation of cytokines and promotes human beta defensins expression in intestinal epithelial cells. This study aimed to determine the molecular mechanism involved. Here we showed that the presence of TFF3 strongly correlated with protease activated receptors 2 (PAR-2) activation in human intestinal cells. Intracellular calcium ((Ca2+)i)mobilization was induced by the treatment with: 1) TFF3, 2) synthetic PAR-2 agonist peptide. The co-treatment with a synthetic PAR-2 antagonist peptide and TFF3 eliminates the latter's effect. Additionally, we demonstrated the existence of interactions among TFF3 and PAR-2 receptors through far Western blot and co-precipitation. Finally, down regulation of PAR-2 by siRNA resulted in a decrease of TFF3 induced intracellular (Ca2+)i mobilization, cytokine regulation and defensins expression. These findings suggest that TFF3 activates intestinal cells through PAR-2 (Fig. 4, Ref. 19).

Ketone bodies and two-compartment tumor metabolism

PubMed Central

Martinez-Outschoorn, Ubaldo E.; Lin, Zhao; Whitaker-Menezes, Diana; Howell, Anthony; Lisanti, Michael P.; Sotgia, Federica

2012-01-01

We have previously suggested that ketone body metabolism is critical for tumor progression and metastasis. Here, using a co-culture system employing human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts, we provide new evidence to directly support this hypothesis. More specifically, we show that the enzymes required for ketone body production are highly upregulated within cancer-associated fibroblasts. This appears to be mechanistically controlled by the stromal expression of caveolin-1 (Cav-1) and/or serum starvation. In addition, treatment with ketone bodies (such as 3-hydroxy-butyrate, and/or butanediol) is sufficient to drive mitochondrial biogenesis in human breast cancer cells. This observation was also validated by unbiased proteomic analysis. Interestingly, an MCT1 inhibitor was sufficient to block the onset of mitochondrial biogenesis in human breast cancer cells, suggesting a possible avenue for anticancer therapy. Finally, using human breast cancer tumor samples, we directly confirmed that the enzymes associated with ketone body production (HMGCS2, HMGCL and BDH1) were preferentially expressed in the tumor stroma. Conversely, enzymes associated with ketone re-utilization (ACAT1) and mitochondrial biogenesis (HSP60) were selectively associated with the epithelial tumor cell compartment. Our current findings are consistent with the “two-compartment tumor metabolism” model. Furthermore, they suggest that we should target ketone body metabolism as a new area for drug discovery, for the prevention and treatment of human cancers. PMID:23082721

The heat shock protein 60 promotes progesterone synthesis in mitochondria of JEG-3 cells.

PubMed

Monreal-Flores, Jessica; Espinosa-García, María Teresa; García-Regalado, Alejandro; Arechavaleta-Velasco, Fabian; Martínez, Federico

2017-06-01

Progesterone synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental progesterone synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in progesterone synthesis. Here, we examined the role of HSP60 in progesterone synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes progesterone synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes progesterone synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial progesterone synthesis. These findings provide novel insights into progesterone synthesis in the human placenta and its role in maintaining pregnancy. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes.

PubMed

Xue, Ruidan; Lynes, Matthew D; Dreyfuss, Jonathan M; Shamsi, Farnaz; Schulz, Tim J; Zhang, Hongbin; Huang, Tian Lian; Townsend, Kristy L; Li, Yiming; Takahashi, Hirokazu; Weiner, Lauren S; White, Andrew P; Lynes, Maureen S; Rubin, Lee L; Goodyear, Laurie J; Cypess, Aaron M; Tseng, Yu-Hua

2015-07-01

Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

ONTOGENIC DIFFERENCES IN HUMAN LIVER 4-HYDROXYNONENAL DETOXIFICATION ARE ASSOCIATED WITH IN VITRO INJURY TO FETAL HEMATOPOIETIC STEM CELLS. (R827441)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

CONFORMATIONAL AND MOLECULAR BASIS FOR INDUCTION OF APOPTOSIS BY A P53 C-TERMINAL PEPTIDE IN HUMAN CANCER CELLS. (R826685)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

POLYCYCLIC AROMATIC HYDROCARBON/METAL MIXTURES: EFFECT ON PAH INDUCTION OF CYP1A1 IN HUMAN HEPG2 CELLS. (R827180)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

[Microsurgical transplantation of bone tumors of uncertain prognosis in athymic nude mice].

PubMed

Duprez, A; Féry, A; Sommelet, J

1986-01-01

Eight cases of human bone or soft tissue tumours were transplanted to nude mice. After such transplants to nude mice which are immunologically deficient, the malignant tumors developed like benign tumours, but maintaining malignant cytological characteristics. The transplants of normal human tissues or of benign tumours decreased in size or remained stable. The technique allowed a change of an original diagnosis of osteosarcoma to a final diagnosis of chondrosarcoma. It made it possible to diagnose a benign osteoblastoma, the diagnosis of which was doubtful before the transplant between osteosarcoma and chondroblastoma. It was possible to diagnose the malignancy of a haemangiopericytoma of muscle. Two aggressive tumours--a non-ossifying fibroma and a giant-cell tumour--were rated as benign after transplantation. This technique also allowed a more precise diagnosis of the grade of one chondrosarcoma and one osteosarcoma. Finally, transplantation also made it possible to test the efficacy of chemotherapy. In a patient so treated, the extreme cell proliferation after transplantation to the nude mouse led to a change in the drugs administered.

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

PubMed Central

Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

2015-01-01

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field. PMID:26317353

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation.

PubMed

Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

2015-01-01

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.

Degradation of the human mitotic checkpoint kinase Mps1 is cell cycle-regulated by APC-cCdc20 and APC-cCdh1 ubiquitin ligases.

PubMed

Cui, Yongping; Cheng, Xiaolong; Zhang, Ce; Zhang, Yanyan; Li, Shujing; Wang, Chuangui; Guadagno, Thomas M

2010-10-22

Mps1 is a dual specificity protein kinase with key roles in regulating the spindle assembly checkpoint and chromosome-microtubule attachments. Consistent with these mitotic functions, Mps1 protein levels fluctuate during the cell cycle, peaking at early mitosis and abruptly declining during mitotic exit and progression into the G(1) phase. Although evidence in budding yeast indicates that Mps1 is targeted for degradation at anaphase by the anaphase-promoting complex (APC)-c(Cdc20) complex, little is known about the regulatory mechanisms that govern Mps1 protein levels in human cells. Here, we provide evidence for the ubiquitin ligase/proteosome pathway in regulating human Mps1 levels during late mitosis through G(1) phase. First, we showed that treatment of HEK 293T cells with the proteosome inhibitor MG132 resulted in an increase in both the polyubiquitination and the accumulation of Mps1 protein levels. Next, Mps1 was shown to co-precipitate with APC and its activators Cdc20 and Cdh1 in a cell cycle-dependent manner. Consistent with this, overexpression of Cdc20 or Cdh1 led to a marked reduction of endogenous Mps1 levels during anaphase or G(1) phase, respectively. In contrast, depletion of Cdc20 or Cdh1 by RNAi treatment both led to the stabilization of Mps1 protein during mitosis or G(1) phase, respectively. Finally, we identified a single D-box motif in human Mps1 that is required for its ubiquitination and degradation. Failure to appropriately degrade Mps1 is sufficient to trigger centrosome amplification and mitotic abnormalities in human cells. Thus, our results suggest that the sequential actions of the APC-c(Cdc20) and APC-c(Cdh1) ubiquitin ligases regulate the clearance of Mps1 levels and are critical for Mps1 functions during the cell cycle in human cells.

Physiologically relevant organs on chips.

PubMed

Yum, Kyungsuk; Hong, Soon Gweon; Healy, Kevin E; Lee, Luke P

2014-01-01

Recent advances in integrating microengineering and tissue engineering have generated promising microengineered physiological models for experimental medicine and pharmaceutical research. Here we review the recent development of microengineered physiological systems, or also known as "ogans-on-chips", that reconstitute the physiologically critical features of specific human tissues and organs and their interactions. This technology uses microengineering approaches to construct organ-specific microenvironments, reconstituting tissue structures, tissue-tissue interactions and interfaces, and dynamic mechanical and biochemical stimuli found in specific organs, to direct cells to assemble into functional tissues. We first discuss microengineering approaches to reproduce the key elements of physiologically important, dynamic mechanical microenvironments, biochemical microenvironments, and microarchitectures of specific tissues and organs in microfluidic cell culture systems. This is followed by examples of microengineered individual organ models that incorporate the key elements of physiological microenvironments into single microfluidic cell culture systems to reproduce organ-level functions. Finally, microengineered multiple organ systems that simulate multiple organ interactions to better represent human physiology, including human responses to drugs, is covered in this review. This emerging organs-on-chips technology has the potential to become an alternative to 2D and 3D cell culture and animal models for experimental medicine, human disease modeling, drug development, and toxicology. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pre-existing immunity against Ad vectors: humoral, cellular, and innate response, what's important?.

PubMed

Fausther-Bovendo, Hugues; Kobinger, Gary P

2014-01-01

Pre-existing immunity against human adenovirus (HAd) serotype 5 derived vector in the human population is widespread, thus hampering its clinical use. Various components of the immune system, including neutralizing antibodies (nAbs), Ad specific T cells and type I IFN activated NK cells, contribute to dampening the efficacy of Ad vectors in individuals with pre-existing Ad immunity. In order to circumvent pre-existing immunity to adenovirus, numerous strategies, such as developing alternative Ad serotypes, varying immunization routes and utilizing prime-boost regimens, are under pre-clinical or clinical phases of development. However, these strategies mainly focus on one arm of pre-existing immunity. Selection of alternative serotypes has been largely driven by the absence in the human population of nAbs against them with little attention paid to cross-reactive Ad specific T cells. Conversely, varying the route of immunization appears to mainly rely on avoiding Ad specific tissue-resident T cells. Finally, prime-boost regimens do not actually circumvent pre-existing immunity but instead generate immune responses of sufficient magnitude to confer protection despite pre-existing immunity. Combining the above strategies and thus taking into account all components regulating pre-existing Ad immunity will help further improve the development of Ad vectors for animal and human use.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells.

PubMed

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa E; Cuervo, Patricia; Jesus, José Batista de

2017-10-01

Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

PubMed Central

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa e; Cuervo, Patricia; de Jesus, José Batista

2017-01-01

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis. PMID:28953994

Single-cell gene expression analysis reveals diversity among human spermatogonia.

PubMed

Neuhaus, N; Yoon, J; Terwort, N; Kliesch, S; Seggewiss, J; Huge, A; Voss, R; Schlatt, S; Grindberg, R V; Schöler, H R

2017-02-10

Is the molecular profile of human spermatogonia homogeneous or heterogeneous when analysed at the single-cell level? Heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the existence of a heterogeneous stem cell population. Despite the fact that many studies have sought to identify specific markers for human spermatogonia, the molecular fingerprint of these cells remains hitherto unknown. Testicular tissues from patients with spermatogonial arrest (arrest, n = 1) and with qualitatively normal spermatogenesis (normal, n = 7) were selected from a pool of 179 consecutively obtained biopsies. Gene expression analyses of cell populations and single-cells (n = 105) were performed. Two OCT4-positive individual cells were selected for global transcriptional capture using shallow RNA-seq. Finally, expression of four candidate markers was assessed by immunohistochemistry. Histological analysis and blood hormone measurements for LH, FSH and testosterone were performed prior to testicular sample selection. Following enzymatic digestion of testicular tissues, differential plating and subsequent micromanipulation of individual cells was employed to enrich and isolate human spermatogonia, respectively. Endpoint analyses were qPCR analysis of cell populations and individual cells, shallow RNA-seq and immunohistochemical analyses. Unexpectedly, single-cell expression data from the arrest patient (20 cells) showed heterogeneous expression profiles. Also, from patients with normal spermatogenesis, heterogeneous expression patterns of undifferentiated (OCT4, UTF1 and MAGE A4) and differentiated marker genes (BOLL and PRM2) were obtained within each spermatogonia cluster (13 clusters with 85 cells). Shallow RNA-seq analysis of individual human spermatogonia was validated, and a spermatogonia-specific heterogeneous protein expression of selected candidate markers (DDX5, TSPY1, EEF1A1 and NGN3) was demonstrated. The heterogeneity of human spermatogonia at the RNA and protein levels is a snapshot. To further assess the functional meaning of this heterogeneity and the dynamics of stem cell populations, approaches need to be developed to facilitate the repeated analysis of individual cells. Our data suggest that heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the model of a heterogeneous stem cell population. Future studies will assess the dynamics of spermatogonial populations in fertile and infertile patients. RNA-seq data is published in the GEO database: GSE91063. This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft DFG-Research Unit FOR 1041 Germ Cell Potential (grant numbers SCHO 340/7-1, SCHL394/11-2). The authors declare that there is no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Toward a CRISPR Picture: Use of CRISPR/Cas9 to Model Diseases in Human Stem Cells In Vitro.

PubMed

Freiermuth, Jamie L; Powell-Castilla, Ian J; Gallicano, G Ian

2018-01-01

Human induced pluripotent stem cells (iPSCs) can be differentiated into any cell in the body unlocking enormous research potential. Combined with the recent discovery of CRISPR/Cas9 endonucleases in bacteria and their modification for use in biomedical research, these methods have the potential to revolutionize the field of genetic engineering and open the door to generating in vitro models that more closely resemble the in vivo system than ever before. Use of CRISPR/Cas9 has created a whirlwind within the scientific community in the last few years, as the race to move beyond just disease analysis and toward the goal of gene and cell therapy moves further. This review will detail the CRISPR/Cas9 method and its use in stem cells as well as highlight recent studies that demonstrate its use in creating robust disease models. Finally, recent results and current controversies in the field are reviewed and lingering challenges to further development are explored. J. Cell. Biochem. 119: 62-68, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Antibodies and tuberculosis: finally coming of age?

PubMed

Li, Hao; Javid, Babak

2018-06-05

Are antibodies important for protection against tuberculosis? The jury has been out for more than 100 years. B cell depletion in experimental Mycobacterium tuberculosis infection failed to identify a major role for these cells in immunity to tuberculosis. However, recent identification of naturally occurring antibodies in humans that are protective during M. tuberculosis infection has reignited the debate. Here, we discuss the evidence for a protective role for antibodies in tuberculosis and consider the feasibility of designing novel tuberculosis vaccines targeting humoral immunity.

Nicotine Promotes Cholangiocarcinoma Growth in Xenograft Mice.

PubMed

Martínez, Allyson K; Jensen, Kendal; Hall, Chad; O'Brien, April; Ehrlich, Laurent; White, Tori; Meng, Fanyin; Zhou, Tianhao; Greene, John; Bernuzzi, Francesca; Invernizzi, Pietro; Dostal, David E; Lairmore, Terry; Alpini, Gianfranco; Glaser, Shannon S

2017-05-01

Nicotine, the main addictive substance in tobacco, is known to play a role in the development and/or progression of a number of malignant tumors. However, nicotine's involvement in the pathogenesis of cholangiocarcinoma is controversial. Therefore, we studied the effects of nicotine on the growth of cholangiocarcinoma cells in vitro and the progression of cholangiocarcinoma in a mouse xenograft model. The predominant subunit responsible for nicotine-mediated proliferation in normal and cancer cells, the α7 nicotinic acetylcholine receptor (α7-nAChR), was more highly expressed in human cholangiocarcinoma cell lines compared with normal human cholangiocytes. Nicotine also stimulated the proliferation of cholangiocarcinoma cell lines and promoted α7-nAChR-dependent activation of proliferation and phosphorylation of extracellular-regulated kinase in Mz-ChA-1 cells. In addition, nicotine and PNU282987 (α7-nAChR agonist) accelerated the growth of the cholangiocarcinoma tumors in our xenograft mouse model and increased fibrosis, proliferation of the tumor cells, and phosphorylation of extracellular-regulated kinase activation. Finally, α7-nAChR was expressed at significantly higher levels in human cholangiocarcinoma compared with normal human control liver samples. Taken together, results of this study suggest that nicotine acts through α7-nAChR and plays a novel role in the pathogenesis of cholangiocarcinoma. Furthermore, nicotine may act as a mitogen in cholestatic liver disease processes, thereby facilitating malignant transformation. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples

PubMed Central

Mullins, Christina S.; Hühns, Maja; Krohn, Mathias; Peters, Sven; Cheynet, Valérie; Oriol, Guy; Guillotte, Michèle; Ducrot, Sandrine; Mallet, François; Linnebacher, Michael

2016-01-01

Introduction A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). Results The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. Conclusion Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut. PMID:27119520

Epigenetic inactivation of CHFR in human tumors

PubMed Central

Toyota, Minoru; Sasaki, Yasushi; Satoh, Ayumi; Ogi, Kazuhiro; Kikuchi, Takefumi; Suzuki, Hiromu; Mita, Hiroaki; Tanaka, Nobuyuki; Itoh, Fumio; Issa, Jean-Pierre J.; Jair, Kam-Wing; Schuebel, Kornel E.; Imai, Kohzoh; Tokino, Takashi

2003-01-01

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin–ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected. PMID:12810945

A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

DTIC Science & Technology

2006-11-01

fibers that served as the junction for the replacement of the OAdV7 tail domain, as well as other common sequences, is highlighted. The final Ad5Luc1-OvF...Fold increase in luciferase activity vs. Ad5 b Reference CHO Hamster ovary L/N 22 Soudais et al., 2000 RD Rhabdomyosarcoma L/N 1.5 Dmitriev et al., 1998...of OV-3 cells (human ovarian cancer, 23-fold) and CAR-deficient CHO cells (Chinese hamster ovary, 22-fold), suggesting that RD cells do not express

CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

PubMed

Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

2015-03-01

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells. © 2015 The Authors.

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line.

PubMed

Niu, Chao; Chauhan, Uday; Gargus, Matthew; Shaker, Anisa

2016-01-01

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.

Advances in reprogramming somatic cells to induced pluripotent stem cells.

PubMed

Patel, Minal; Yang, Shuying

2010-09-01

Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

Tissue Regeneration in the Chronically Inflamed Tumor Environment: Implications for Cell Fusion Driven Tumor Progression and Therapy Resistant Tumor Hybrid Cells

PubMed Central

Dittmar, Thomas; Zänker, Kurt S.

2015-01-01

The biological phenomenon of cell fusion in a cancer context is still a matter of controversial debates. Even though a plethora of in vitro and in vivo data have been published in the past decades the ultimate proof that tumor hybrid cells could originate in (human) cancers and could contribute to the progression of the disease is still missing, suggesting that the cell fusion hypothesis is rather fiction than fact. However, is the lack of this ultimate proof a valid argument against this hypothesis, particularly if one has to consider that appropriate markers do not (yet) exist, thus making it virtually impossible to identify a human tumor cell clearly as a tumor hybrid cell. In the present review, we will summarize the evidence supporting the cell fusion in cancer concept. Moreover, we will refine the cell fusion hypothesis by providing evidence that cell fusion is a potent inducer of aneuploidy, genomic instability and, most likely, even chromothripsis, suggesting that cell fusion, like mutations and aneuploidy, might be an inducer of a mutator phenotype. Finally, we will show that “accidental” tissue repair processes during cancer therapy could lead to the origin of therapy resistant cancer hybrid stem cells. PMID:26703575

Tissue Regeneration in the Chronically Inflamed Tumor Environment: Implications for Cell Fusion Driven Tumor Progression and Therapy Resistant Tumor Hybrid Cells.

PubMed

Dittmar, Thomas; Zänker, Kurt S

2015-12-19

The biological phenomenon of cell fusion in a cancer context is still a matter of controversial debates. Even though a plethora of in vitro and in vivo data have been published in the past decades the ultimate proof that tumor hybrid cells could originate in (human) cancers and could contribute to the progression of the disease is still missing, suggesting that the cell fusion hypothesis is rather fiction than fact. However, is the lack of this ultimate proof a valid argument against this hypothesis, particularly if one has to consider that appropriate markers do not (yet) exist, thus making it virtually impossible to identify a human tumor cell clearly as a tumor hybrid cell. In the present review, we will summarize the evidence supporting the cell fusion in cancer concept. Moreover, we will refine the cell fusion hypothesis by providing evidence that cell fusion is a potent inducer of aneuploidy, genomic instability and, most likely, even chromothripsis, suggesting that cell fusion, like mutations and aneuploidy, might be an inducer of a mutator phenotype. Finally, we will show that "accidental" tissue repair processes during cancer therapy could lead to the origin of therapy resistant cancer hybrid stem cells.

Mechanical Properties of Human Cells Change during Neoplastic Processes

NASA Astrophysics Data System (ADS)

Guthold, Martin; Guo, Xinyi; Bonin, Keith; Scarpinato, Karin

2014-03-01

Using an AFM with a spherical probe of 5.3 μm, we determined mechanical properties of individual human mammary epithelial cells that have progressed through four stages of neoplastic transformation: normal, immortal, tumorigenic, and metastatic. Measurements on cells in all four stages were taken over both the nucleus and the cytoplasm. Moreover, the measurements were made for cells outside of a colony (isolated), on the periphery of a colony, and inside a colony. By fitting the AFM force vs. indentation curves to a Hertz model, we determined the Young's modulus, E. We found a distinct contrast in the influence a cell's colony environment has on its stiffness depending on whether the cells are normal or cancer cells. We also found that cells become softer as they advance to the tumorigenic stage and then stiffen somewhat in the final step to metastatic cells. For cells averaged over all locations the stiffness values of the nuclear region for normal, immortal, tumorigenic, and metastatic cells were (mean +/- sem) 880 +/- 50, 940+/-50, 400 +/- 20, and 600 +/-20 Pa respectively. Cytoplasmic regions followed a similar trend. These results point to a complex picture of the mechanical changes that occur as cells undergo neoplastic transformation. This work is supported by NSF Materials and Surface Engineering grant CMMI-1152781.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wyrobek, Andrew J.; Mulvihill, John J.; Wassom, John S.

Although numerous germ-cell mutagens have been identified inanimal model systems, to date, no human germ-cell mutagens have beenconfirmed. Because the genomic integrity of our germ cells is essentialfor the continuation of the human species, a resolution of this enduringconundrum is needed. To facilitate such a resolution, we organized aworkshop at The Jackson Laboratory in Bar Harbor, Maine on September28-30, 2004. This interactive workshop brought together scientists from awide range of disciplines to assess the applicability of emergingmolecular methods for genomic analysis to the field of human germ-cellmutagenesis. Participants recommended that focused, coordinated humangerm-cell mutation studies be conducted in relation tomore » important societalexposures. Because cancer survivors represent a unique cohort withwell-defined exposures, there was a consensus that studies should bedesigned to assess the mutational impact on children born to parents whohad received certain types of mutagenic cancer chemotherapy prior toconceiving their children. Within this high-risk cohort, parents andchildren could be evaluated for inherited changes in (a) gene sequencesand chromosomal structure, (b) repeat sequences and minisatelliteregions, and (c) global gene expression and chromatin. Participants alsorecommended studies to examine trans-generational effects in humansinvolving mechanisms such as changes in imprinting and methylationpatterns, expansion of nucleotide repeats, or induction of mitochondrialDNA mutations. Workshop participants advocated establishment of abio-bank of human tissue samples that could be used to conduct amultiple-endpoint, comprehensive, and collaborative effort to detectexposure-induced heritable alterations in the human genome. Appropriateanimal models of human germ-cell mutagenes is should be used in parallelwith human studies to provide insights into the mechanisms of mammaliangerm-cell mutagenesis. Finally, participants recommended that scientificspecialty groups be convened to address specific questions regarding thepotential germ-cell mutagenicity of environmental, occupational, andlifestyle exposures. Strong support from relevant funding agencies andengagement of scientists outside the fields of genomics and germ-cellmutagenesis will be required to launch a full-scale assault on some ofthe most pressing and enduring questions in environmental mutagenesis: Dohuman germ-cell mutagens exist, what risk do they pose to futuregenerations, and are some parents at higher risk than others foracquiring and transmitting germ-cell mutations?« less

Capsaicin Induces Autophagy and Apoptosis in Human Nasopharyngeal Carcinoma Cells by Downregulating the PI3K/AKT/mTOR Pathway.

PubMed

Lin, Yu-Tsai; Wang, Hung-Chen; Hsu, Yi-Chiang; Cho, Chung-Lung; Yang, Ming-Yu; Chien, Chih-Yen

2017-06-23

Capsaicin is a potential chemotherapeutic agent for different human cancers. In Southeast China, nasopharyngeal carcinoma (NPC) has the highest incidence of all cancers, but final treatment outcomes are unsatisfactory. However, there is a lack of information regarding the anticancer activity of capsaicin in NPC cells, and its effects on the signaling transduction pathways related to apoptosis and autophagy remain unclear. In the present study, the precise mechanisms by which capsaicin exerts anti-proliferative effects, cell cycle arrest, autophagy and apoptosis were investigated in NPC-TW01 cells. Exposure to capsaicin inhibited cancer cell growth and increased G1 phase cell cycle arrest. Western blotting and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) were used to measure capsaicin-induced autophagy via involvement of the class III PI3K/Beclin-1/Bcl-2 signaling pathway. Capsaicin induced autophagy by increasing levels of the autophagy markers LC3-II and Atg5, enhancing p62 and Fap-1 degradation and increasing caspase-3 activity to induce apoptosis, suggesting a correlation of blocking the PI3K/Akt/mTOR pathway with the above-mentioned anticancer activities. Taken together, these data confirm that capsaicin inhibited the growth of human NPC cells and induced autophagy, supporting its potential as a therapeutic agent for cancer.

The mucin MUC4 and its membrane partner ErbB2 regulate biological properties of human CAPAN-2 pancreatic cancer cells via different signalling pathways.

PubMed

Jonckheere, Nicolas; Skrypek, Nicolas; Merlin, Johann; Dessein, Anne Frédérique; Dumont, Patrick; Leteurtre, Emmanuelle; Harris, Ann; Desseyn, Jean-Luc; Susini, Christiane; Frénois, Frédéric; Van Seuningen, Isabelle

2012-01-01

The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells.

Synaptotagmin 4 Regulates Pancreatic β Cell Maturation by Modulating the Ca2+ Sensitivity of Insulin Secretion Vesicles.

PubMed

Huang, Chen; Walker, Emily M; Dadi, Prasanna K; Hu, Ruiying; Xu, Yanwen; Zhang, Wenjian; Sanavia, Tiziana; Mun, Jisoo; Liu, Jennifer; Nair, Gopika G; Tan, Hwee Yim Angeline; Wang, Sui; Magnuson, Mark A; Stoeckert, Christian J; Hebrok, Matthias; Gannon, Maureen; Han, Weiping; Stein, Roland; Jacobson, David A; Gu, Guoqiang

2018-05-07

Islet β cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca 2+ concentrations, suggesting differences in the Ca 2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca 2+ binding paralog of the β cell Ca 2+ sensor Syt7, increased by ∼8-fold during β cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-βH1 cells, a human β cell line. These findings reveal the role that altered Ca 2+ sensing plays in regulating β cell maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

PubMed

Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

2016-05-15

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

Inhibition of Excessive Monoamine Oxidase A/B Activity Protects Against Stress-induced Neuronal Death in Huntington Disease.

PubMed

Ooi, Jolene; Hayden, Michael R; Pouladi, Mahmoud A

2015-12-01

Monoamine oxidases (MAO) are important components of the homeostatic machinery that maintains the levels of monoamine neurotransmitters, including dopamine, in balance. Given the imbalance in dopamine levels observed in Huntington disease (HD), the aim of this study was to examine MAO activity in a mouse striatal cell model of HD and in human neural cells differentiated from control and HD patient-derived induced pluripotent stem cell (hiPSC) lines. We show that mouse striatal neural cells expressing mutant huntingtin (HTT) exhibit increased MAO expression and activity. We demonstrate using luciferase promoter assays that the increased MAO expression reflects enhanced epigenetic activation in striatal neural cells expressing mutant HTT. Using cellular stress paradigms, we further demonstrate that the increase in MAO activity in mutant striatal neural cells is accompanied by enhanced susceptibility to oxidative stress and impaired viability. Treatment of mutant striatal neural cells with MAO inhibitors ameliorated oxidative stress and improved cellular viability. Finally, we demonstrate that human HD neural cells exhibit increased MAO-A and MAO-B expression and activity. Altogether, this study demonstrates abnormal MAO expression and activity and suggests a potential use for MAO inhibitors in HD.

THE EFFECT OF POLIOMYELITIS VIRUS ON HUMAN BRAIN CELLS IN TISSUE CULTURE

PubMed Central

Hogue, M. J.; McAllister, R.; Greene, A. E.; Coriell, L. L.

1955-01-01

Poliomyelitis virus I, Mahoney strain, affected human brain cells grown in tissue cultures usually causing death of the cells in 3 days. The neurons reacted in different ways to the virus, some died with their neurites extended, others contracted one or more of their neurites. Terminal bulbs were frequently formed at the tips of the neurites when they were being drawn into the cell body. The final contraction of the cell body and the change into a mass of granules were often very sudden. Vacuoles often developed in the neuron. There was no recovery. Astrocytes, oligodendroglia, and macrophages were affected by the virus but not as quickly as the neurons. The age of the tissue culture was not a factor when the cells were in good condition. The age of the individual donor of the brain tissue was a factor; the fetal brain cells appeared to be more sensitive to the virus than the adult brain cells. The fetal neurons often reacted ½ hour after inoculation while the adult neurons reacted more slowly, 2 to 24 hours after inoculation. All these changes seemed to be caused by virus infection because they were prevented by specific antiserum or by preheating the virus. PMID:14392238

[Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells].

PubMed

Xiaojun, Yang; Yongmei, Tan; Zhihui, Tian; Ting, Zhou; Wanghong, Zhao; Jin, Hou

2017-04-01

This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues. The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry. P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues. Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
.

Retinoic Acid Signalling and the Control of Meiotic Entry in the Human Fetal Gonad

PubMed Central

Kinnell, Hazel L.; Anderson, Richard A.; Saunders, Philippa T. K.

2011-01-01

The development of mammalian fetal germ cells along oogenic or spermatogenic fate trajectories is dictated by signals from the surrounding gonadal environment. Germ cells in the fetal testis enter mitotic arrest, whilst those in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by selective exposure of fetal ovarian germ cells to mesonephros-derived retinoic acid (RA). Aspects of this model are hard to reconcile with the spatiotemporal pattern of germ cell differentiation in the human fetal ovary, however. We have therefore examined the expression of components of the RA synthesis, metabolism and signalling pathways, and their downstream effectors and inhibitors in germ cells around the time of the initiation of meiosis in the human fetal gonad. Expression of the three RA-synthesising enzymes, ALDH1A1, 2 and 3 in the fetal ovary and testis was equal to or greater than that in the mesonephros at 8–9 weeks gestation, indicating an intrinsic capacity within the gonad to synthesise RA. Using immunohistochemistry to detect RA receptors RARα, β and RXRα, we find germ cells to be the predominant target of RA signalling in the fetal human ovary, but also reveal widespread receptor nuclear localization indicative of signalling in the testis, suggesting that human fetal testicular germ cells are not efficiently shielded from RA by the action of the RA-metabolising enzyme CYP26B1. Consistent with this, expression of CYP26B1 was greater in the human fetal ovary than testis, although the sexually-dimorphic expression patterns of the germ cell-intrinsic regulators of meiotic initiation, STRA8 and NANOS2, appear conserved. Finally, we demonstrate that RA induces a two-fold increase in STRA8 expression in cultures of human fetal testis, but is not sufficient to cause widespread meiosis-associated gene expression. Together, these data indicate that while local production of RA within the fetal ovary may be important in regulating the onset of meiosis in the human fetal ovary, mechanisms other than CYP26B1-mediated metabolism of RA may exist to inhibit the entry of germ cells into meiosis in the human fetal testis. PMID:21674038

Protection by polyphenol extract from olive stones against apoptosis produced by oxidative stress in human neuroblastoma cells

PubMed

Cortés-Castell, Ernesto; Veciana-Galindo, Carmen; Torró-Montell, Luis; Palazón-Bru, Antonio; Sirvent-Segura, Elia; Gil-Guillén, Vicente; Rizo-Baeza, Mercedes

2016-02-16

We evaluated the protective activity of an extract from a by-product such as olive stones, through its ability to inhibit H202 induced apoptosis in the SH-SY5Y human neuroblastoma cell line. To such end, 20,000 cells/well were cultivated and differentiation with retinoic acid was initiated. Once the cells were differentiated, apoptosis was induced with and without H2O2 extract. Finally, cDNA extraction was performed, and pro-apoptotic genes Bax and anti-apoptotic genes Bcl-2 were analyzed. Quantification of the gene expression was performed using the GAPDH gene marker. Cell viability with the extract is 97.6% (SD 5.7) with 10 mg/l and 62.8% (SD 1.2) to 50 mg/l, using 10 mg/l for the biomarker assay. The retinoic acid differentiated SH-S cell line (10 μM) shows a clear apoptosis when treated with H2O2 150 μM, with a Bax/Bcl-2 ratio of 3.75 (SD 0.80) in contrast to the differentiated control cells subjected to H2O2 and with extract, which have the same ratio of 1.02 (SD 0.01-0.03). The olive stone extract shows anti-apoptotic activity in the provoked cell death of SH-SY5Y human neuroblastoma cells in their normal state, defending them from oxidative stress which produces a significant increase in the apoptotic gene ratio in contrast to anti-apoptotic genes (Bax/Bcl-2).

Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montoudis, Alain; Delvin, Edgard; Canadian Institute of Health Research, Group of the Functional Development and Physiopathology of the Digestive Tract, and Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, Que., Canada J1H 5N4

2006-01-06

Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferationmore » and viability. Studies using these transfected cells cultured with [{sup 14}C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [{sup 35}S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.« less

Kidins220/ARMS depletion is associated with the neural-to Schwann-like transition in a human neuroblastoma cell line model.

PubMed

Rogers, Danny A; Schor, Nina F

2013-03-10

Peripheral neuroblastic tumors exist as a heterogeneous mixture of neuroblastic (N-type) cells and Schwannian stromal (S-type) cells. These stromal cells not only represent a differentiated and less aggressive fraction of the tumor, but also have properties that can influence the further differentiation of nearby malignant cells. In vitro neuroblastoma cultures exhibit similar heterogeneity with N-type and S-type cells representing the neuroblastic and stromal portions of the tumor, respectively, in behavior, morphology, and molecular expression patterns. In this study, we deplete kinase D-interacting substrate of 220kD (Kidins220) with an shRNA construct and thereby cause morphologic transition of the human SH-SY5Y neuroblastoma cell line from N-type to S-type. The resulting cells have similar morphology and expression profile to SH-EP1 cells, a native S-type cell line from the same parent cell line, and to SH-SY5Y cells treated with BrdU, a treatment that induces S-type morphology. Specifically, both Kidins220-deficient SH-SY5Y cells and native SH-EP1 cells demonstrate down-regulation of the genes DCX and STMN2, markers for the neuronal lineage. We further show that Kidins220, DCX and STMN2 are co-down-regulated in cells of S-type morphology generated by methods other than Kidins220 depletion. Finally, we report that the association of low Kidins220 expression with S-type morphology and low DCX and STMN2 expression is demonstrated in spontaneously occurring human peripheral neuroblastic tumors. We propose that Kidins220 is critical in N- to S-type transition of neural crest tumor cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Three-Step Method for Proliferation and Differentiation of Human Embryonic Stem Cell (hESC)-Derived Male Germ Cells

PubMed Central

Lim, Jung Jin; Shim, Myung Sun; Lee, Jeoung Eun; Lee, Dong Ryul

2014-01-01

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells. PMID:24690677

Autonomous parvoviruses neither stimulate nor are inhibited by the type I interferon response in human normal or cancer cells.

PubMed

Paglino, Justin C; Andres, Wells; van den Pol, Anthony N

2014-05-01

Members of the genus Parvovirus are small, nonenveloped single-stranded DNA viruses that are nonpathogenic in humans but have potential utility as cancer therapeutics. Because the innate immune response to parvoviruses has received relatively little attention, we compared the response to parvoviruses to that of several other types of viruses in human cells. In normal human glia, fibroblasts, or melanocytes, vesicular stomatitis virus evoked robust beta interferon (IFN-β) responses. Cytomegalovirus, pseudorabies virus, and Sindbis virus all evoked a 2-log-unit or greater upregulation of IFN-β in glia; in contrast, LuIII and MVMp parvoviruses did not evoke a detectable IFN-β or interferon-stimulated gene (ISG; MX1, oligoadenylate synthetase [OAS], IFIT-1) response in the same cell types. The lack of response raised the question of whether parvoviral infection can be attenuated by IFN; interestingly, we found that IFN did not decrease parvovirus (MVMp, LuIII, and H-1) infectivity in normal human glia, fibroblasts, or melanocytes. The same was true in human cancers, including glioma, sarcoma, and melanoma. Similarly, IFN failed to attenuate transduction by the dependovirus vector adeno-associated virus type 2. Progeny production of parvoviruses was also unimpaired by IFN in both glioma and melanoma, whereas vesicular stomatitis virus replication was blocked. Sarcoma cells with upregulated IFN signaling that show high levels of resistance to other viruses showed strong infection by LuIII. Unlike many other oncolytic viruses, we found no evidence that impairment of innate immunity in cancer cells plays a role in the oncoselectivity of parvoviruses in human cells. Parvoviral resistance to the effects of IFN in cancer cells may constitute an advantage in the virotherapy of some tumors. Understanding the interactions between oncolytic viruses and the innate immune system will facilitate employing these viruses as therapeutic agents in cancer patients. The cancer-selective nature of some oncolytic viruses is based on the impaired innate immunity of many cancer cells. The parvoviruses H-1, LuIII, and MVM target cancer cells; however, their relationship with the innate immune system is relatively uncharacterized. Surprisingly, we found that these parvoviruses do not evoke an interferon response in normal human fibroblasts, glia, or melanocytes. Furthermore, unlike most other types of virus, we found that parvovirus infectivity is unaffected by interferon treatment of human normal or tumor cells. Finally, parvoviral replication was unimpaired by interferon in four human tumor types, including those with residual interferon functionality. We conclude that deficits in the interferon antiviral response of cancer cells do not contribute to parvoviral oncoselectivity in human cells. The interferon-resistant phenotype of parvoviruses may give them an advantage over interferon-sensitive oncolytic viruses in tumors showing residual interferon functionality.

Autonomous Parvoviruses neither Stimulate nor Are Inhibited by the Type I Interferon Response in Human Normal or Cancer Cells

PubMed Central

Paglino, Justin C.; Andres, Wells

2014-01-01

ABSTRACT Members of the genus Parvovirus are small, nonenveloped single-stranded DNA viruses that are nonpathogenic in humans but have potential utility as cancer therapeutics. Because the innate immune response to parvoviruses has received relatively little attention, we compared the response to parvoviruses to that of several other types of viruses in human cells. In normal human glia, fibroblasts, or melanocytes, vesicular stomatitis virus evoked robust beta interferon (IFN-β) responses. Cytomegalovirus, pseudorabies virus, and Sindbis virus all evoked a 2-log-unit or greater upregulation of IFN-β in glia; in contrast, LuIII and MVMp parvoviruses did not evoke a detectable IFN-β or interferon-stimulated gene (ISG; MX1, oligoadenylate synthetase [OAS], IFIT-1) response in the same cell types. The lack of response raised the question of whether parvoviral infection can be attenuated by IFN; interestingly, we found that IFN did not decrease parvovirus (MVMp, LuIII, and H-1) infectivity in normal human glia, fibroblasts, or melanocytes. The same was true in human cancers, including glioma, sarcoma, and melanoma. Similarly, IFN failed to attenuate transduction by the dependovirus vector adeno-associated virus type 2. Progeny production of parvoviruses was also unimpaired by IFN in both glioma and melanoma, whereas vesicular stomatitis virus replication was blocked. Sarcoma cells with upregulated IFN signaling that show high levels of resistance to other viruses showed strong infection by LuIII. Unlike many other oncolytic viruses, we found no evidence that impairment of innate immunity in cancer cells plays a role in the oncoselectivity of parvoviruses in human cells. Parvoviral resistance to the effects of IFN in cancer cells may constitute an advantage in the virotherapy of some tumors. IMPORTANCE Understanding the interactions between oncolytic viruses and the innate immune system will facilitate employing these viruses as therapeutic agents in cancer patients. The cancer-selective nature of some oncolytic viruses is based on the impaired innate immunity of many cancer cells. The parvoviruses H-1, LuIII, and MVM target cancer cells; however, their relationship with the innate immune system is relatively uncharacterized. Surprisingly, we found that these parvoviruses do not evoke an interferon response in normal human fibroblasts, glia, or melanocytes. Furthermore, unlike most other types of virus, we found that parvovirus infectivity is unaffected by interferon treatment of human normal or tumor cells. Finally, parvoviral replication was unimpaired by interferon in four human tumor types, including those with residual interferon functionality. We conclude that deficits in the interferon antiviral response of cancer cells do not contribute to parvoviral oncoselectivity in human cells. The interferon-resistant phenotype of parvoviruses may give them an advantage over interferon-sensitive oncolytic viruses in tumors showing residual interferon functionality. PMID:24554651

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

PubMed Central

Wang, Zhi-Bo; Zhang, Xiaoqing; Li, Xue-Jun

2013-01-01

Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease. Here, we developed a closely representative cell model of SMA by knocking down the disease-determining gene, survival motor neuron (SMN), in human embryonic stem cells (hESCs). Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons. Notably, the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated. Furthermore, these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-Δ7 (lacking exon 7) knockdown, and were specific to spinal motor neurons. Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes, including specific axonal defects and motor neuron loss. Finally, knockdown of SMN-FL led to excessive mitochondrial oxidative stress in human motor neuron progenitors. The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine, a potent antioxidant, which prevented disease-related apoptosis and subsequent motor neuron death. Thus, we report here the successful establishment of an hESC-based SMA model, which exhibits disease gene isoform specificity, cell type specificity, and phenotype reversibility. Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA. PMID:23208423

The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells

PubMed Central

Crozat, Karine; Guiton, Rachel; Contreras, Vanessa; Feuillet, Vincent; Dutertre, Charles-Antoine; Ventre, Erwan; Vu Manh, Thien-Phong; Baranek, Thomas; Storset, Anne K.; Marvel, Jacqueline; Boudinot, Pierre; Hosmalin, Anne; Schwartz-Cornil, Isabelle

2010-01-01

Human BDCA3+ dendritic cells (DCs) were suggested to be homologous to mouse CD8α+ DCs. We demonstrate that human BDCA3+ DCs are more efficient than their BDCA1+ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8+ T cells, which is similar to mouse CD8α+ DCs as compared with CD11b+ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α+, human BDCA3+, and sheep CD26+ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8+ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8+ T lymphocytes. Finally, XCR1−/− mice have decreased early CD8+ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α+ DCs in higher vertebrates and promotes their ability to activate early CD8+ T cell defenses against an intracellular pathogenic bacteria. PMID:20479118

Identification of the G protein-coupled estrogen receptor (GPER) in human prostate: expression site of the estrogen receptor in the benign and neoplastic gland.

PubMed

Rago, V; Romeo, F; Giordano, F; Ferraro, A; Carpino, A

2016-01-01

Estrogens are involved in growth, differentiation and pathogenesis of human prostate through the mediation of the classical estrogen receptors ERα and ERβ. The G protein-coupled estrogen receptor (GPER) is a 'novel' mediator of estrogen signaling which has been recently recognized in some human reproductive tissues, but its expression in the prostate gland is still unknown. Here, we investigated GPER in benign (from 5 patients) and neoplastic prostatic tissues (from 50 patients) by immunohistochemical analysis and Western blotting. Normal areas of benign prostates revealed a strong GPER immunoreactivity in the basal epithelial cells while luminal epithelial cells were unreactive and stromal cells were weakly immunostained. GPER was also immunolocalized in adenocarcinoma samples but the immunoreactivity of tumoral areas decreased from Gleason pattern 2 to Gleason pattern 4. Furthermore, a strong GPER immunostaining was also revealed in cells of pre-neoplastic lesions (high-grade prostatic intra-epithelial neoplasia). Western blot analysis of benign and tumor protein extracts showed the presence of a ~42 kDa band, consistent with the GPER molecular weight. An increase in both pAkt and p cAMP-response-binding protein (pCREB) levels was also observed in poorly differentiated PCa samples. Finally, this work identified GPER in the epithelial basal cells of benign human prostate, with a different localization with respect to the classical estrogen receptors. Furthermore, the expression of GPER in prostatic adenocarcinoma cells was also observed but with a modulation of the immunoreactivity according to tumor cell arrangements. © 2015 American Society of Andrology and European Academy of Andrology.

Basal cell adhesion molecule/lutheran protein. The receptor critical for sickle cell adhesion to laminin.

PubMed Central

Udani, M; Zen, Q; Cottman, M; Leonard, N; Jefferson, S; Daymont, C; Truskey, G; Telen, M J

1998-01-01

Sickle red cells bind significant amounts of soluble laminin, whereas normal red cells do not. Solid phase assays demonstrate that B-CAM/LU binds laminin on intact sickle red cells and that red cell B-CAM/LU binds immobilized laminin, whereas another putative laminin binding protein, CD44, does not. Ligand blots also identify B-CAM/LU as the only erythrocyte membrane protein(s) that binds laminin. Finally, transfection of murine erythroleukemia cells with human B-CAM cDNA induces binding of both soluble and immobilized laminin. Thus, B-CAM/LU appears to be the major laminin-binding protein of sickle red cells. Previously reported overexpression of B-CAM/LU by epithelial cancer cells suggests that this protein may also serve as a laminin receptor in malignant tumors. PMID:9616226

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

PubMed Central

Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

2015-01-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

Expression of hydroxyindole-O-methyltransferase enzyme in the human central nervous system and in pineal parenchymal cell tumors.

PubMed

Fukuda, Takahiro; Akiyama, Nobutake; Ikegami, Masahiro; Takahashi, Hitoshi; Sasaki, Atsushi; Oka, Hidehiro; Komori, Takashi; Tanaka, Yuko; Nakazato, Youichi; Akimoto, Jiro; Tanaka, Masahiko; Okada, Yoshikazu; Saito, Saburo

2010-05-01

Pineal parenchymal tumor (PPT) cells usually show immunoreactivity for synaptophysin, neuron-specific enolase, neurofilament protein, class III beta-tubulin, tau protein, PGP9.5, chromogranin, serotonin, retinal S-antigen, and rhodopsin, but these markers are not specific for PPTs. Melatonin is produced and secreted mainly bypineal parenchymal cells; hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final reaction in melatonin biosynthesis. We hypothesized that HIOMT could serve as a tumor marker of PPTs, and we investigated HIOMT localization and HIOMT expression in samples of normal human tissue and in PPTs, primitive neuroectodermal tumors, and medulloblastomas. In normal tissue, HIOMT was expressed in retinal cells, pineal parenchymal cells, neurons of the Edinger-Westphal nucleus, microglia, macrophages, thyroid follicular epithelium, principal and oxyphil cells of parathyroid gland, adrenal cortical cells, hepatic parenchymal cells, renal tubule epithelium, and enteroendocrine cells of stomach and duodenum. The HIOMT was also expressed in all 46 PPTs studied. The proportions of HIOMT-immunoreactive cells successively decreased in the following tumors: pineocytoma, pineal parenchymal tumor of intermediate differentiation, and pineoblastoma. A few HIOMT-immunoreactive cells were observed in one of 6 primitive neuroectodermal tumors and 23 of 42 medulloblastomas. These results indicate that HIOMT immunohistochemistry may be useful for the diagnosis of PPTs and be a prognostic factor in PPTs.

Human haemodynamic frequency harmonics regulate the inflammatory phenotype of vascular endothelial cells.

PubMed

Feaver, Ryan E; Gelfand, Bradley D; Blackman, Brett R

2013-01-01

Haemodynamic variations are inherent to blood vessel geometries (such as bifurcations) and correlate with regional development of inflammation and atherosclerosis. However, the complex frequency spectrum characteristics from these haemodynamics have never been exploited to test whether frequency variations are critical determinants of endothelial inflammatory phenotype. Here we utilize an experimental Fourier transform analysis to systematically manipulate individual frequency harmonics from human carotid shear stress waveforms applied in vitro to human endothelial cells. The frequency spectrum, specifically the 0 th and 1st harmonics, is a significant regulator of inflammation, including NF-κB activity and downstream inflammatory phenotype. Further, a harmonic-based regression-model predicts eccentric NF-κB activity observed in the human internal carotid artery. Finally, short interfering RNA-knockdown of the mechanosensor PECAM-1 reverses frequency-dependent regulation of NF-κB activity. Thus, PECAM-1 may have a critical role in the endothelium's exquisite sensitivity to complex shear stress frequency harmonics and provide a mechanism for the focal development of vascular inflammation.

Genome-wide DNA methylation drives human embryonic stem cell erythropoiesis by remodeling gene expression dynamics.

PubMed

Liu, Zhijing; Feng, Qiang; Sun, Pengpeng; Lu, Yan; Yang, Minlan; Zhang, Xiaowei; Jin, Xiangshu; Li, Yulin; Lu, Shi-Jiang; Quan, Chengshi

2017-12-01

To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells.

PubMed

Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2015-01-09

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

PubMed Central

Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2015-01-01

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

Assessment of stem cell differentiation based on genome-wide expression profiles.

PubMed

Godoy, Patricio; Schmidt-Heck, Wolfgang; Hellwig, Birte; Nell, Patrick; Feuerborn, David; Rahnenführer, Jörg; Kattler, Kathrin; Walter, Jörn; Blüthgen, Nils; Hengstler, Jan G

2018-07-05

In recent years, protocols have been established to differentiate stem and precursor cells into more mature cell types. However, progress in this field has been hampered by difficulties to assess the differentiation status of stem cell-derived cells in an unbiased manner. Here, we present an analysis pipeline based on published data and methods to quantify the degree of differentiation and to identify transcriptional control factors explaining differences from the intended target cells or tissues. The pipeline requires RNA-Seq or gene array data of the stem cell starting population, derived 'mature' cells and primary target cells or tissue. It consists of a principal component analysis to represent global expression changes and to identify possible problems of the dataset that require special attention, such as: batch effects; clustering techniques to identify gene groups with similar features; over-representation analysis to characterize biological motifs and transcriptional control factors of the identified gene clusters; and metagenes as well as gene regulatory networks for quantitative cell-type assessment and identification of influential transcription factors. Possibilities and limitations of the analysis pipeline are illustrated using the example of human embryonic stem cell and human induced pluripotent cells to generate 'hepatocyte-like cells'. The pipeline quantifies the degree of incomplete differentiation as well as remaining stemness and identifies unwanted features, such as colon- and fibroblast-associated gene clusters that are absent in real hepatocytes but typically induced by currently available differentiation protocols. Finally, transcription factors responsible for incomplete and unwanted differentiation are identified. The proposed method is widely applicable and allows an unbiased and quantitative assessment of stem cell-derived cells.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Author(s).

Myeloproliferative Neoplasm Animal Models

PubMed Central

Mullally, Ann; Lane, Steven W.; Brumme, Kristina; Ebert, Benjamin L.

2012-01-01

Synopsis Myeloproliferative neoplasm (MPN) animal models accurately re-capitulate human disease in mice and have been an important tool for the study of MPN biology and therapy. Transplantation of BCR-ABL transduced bone marrow cells into irradiated syngeneic mice established the field of MPN animal modeling and the retroviral bone marrow transplantation (BMT) assay has been used extensively since. Genetically engineered MPN animal models have enabled detailed characterization of the effects of specific MPN associated genetic abnormalities on the hematopoietic stem and progenitor cell (HSPC) compartment and xenograft models have allowed the study of primary human MPN-propagating cells in vivo. All models have facilitated the pre-clinical development of MPN therapies. JAK2V617F, the most common molecular abnormality in BCR-ABL negative MPN, has been extensively studied using retroviral, transgenic, knock-in and xenograft models. MPN animal models have also been used to investigate additional genetic lesions found in human MPN and to evaluate the bone marrow microenvironment in these diseases. Finally, several genetic lesions, although not common, somatically mutated drivers of MPN in humans induce a MPN phenotype in mice. Future uses for MPN animal models will include modeling compound genetic lesions in MPN and studying myelofibrotic transformation. PMID:23009938

The Complexity of Targeting PI3K-Akt-mTOR Signalling in Human Acute Myeloid Leukaemia: The Importance of Leukemic Cell Heterogeneity, Neighbouring Mesenchymal Stem Cells and Immunocompetent Cells.

PubMed

Brenner, Annette K; Andersson Tvedt, Tor Henrik; Bruserud, Øystein

2016-11-11

Therapeutic targeting of PI3K-Akt-mTOR is considered a possible strategy in human acute myeloid leukaemia (AML); the most important rationale being the proapoptotic and antiproliferative effects of direct PI3K/mTOR inhibition observed in experimental studies of human AML cells. However, AML is a heterogeneous disease and these effects caused by direct pathway inhibition in the leukemic cells are observed only for a subset of patients. Furthermore, the final effect of PI3K-Akt-mTOR inhibition is modulated by indirect effects, i.e., treatment effects on AML-supporting non-leukemic bone marrow cells. In this article we focus on the effects of this treatment on mesenchymal stem cells (MSCs) and monocytes/macrophages; both these cell types are parts of the haematopoietic stem cell niches in the bone marrow. MSCs have unique membrane molecule and constitutive cytokine release profiles, and mediate their support through bidirectional crosstalk involving both cell-cell contact and the local cytokine network. It is not known how various forms of PI3K-Akt-mTOR targeting alter the molecular mechanisms of this crosstalk. The effect on monocytes/macrophages is also difficult to predict and depends on the targeted molecule. Thus, further development of PI3K-Akt-mTOR targeting into a clinical strategy requires detailed molecular studies in well-characterized experimental models combined with careful clinical studies, to identify patient subsets that are likely to respond to this treatment.

BID is a critical factor controlling cell viability regulated by IFN-α.

PubMed

Tsuno, Takaya; Mejido, Josef; Zhao, Tongmao; Phillips, Terry; Myers, Timothy G; Bekisz, Joseph; Zoon, Kathryn C

2012-01-01

Clinical applications of human interferon (IFN)-α have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-α have not been clearly identified. Our previous study indicated that IFN (α, β, and ω) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-α treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-α. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-α treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-α notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-α and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-α and TRAIL from collapsing the mitochondrial membrane potential (ΔΨm). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-α or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-α. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-α resistant tumors.

The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy

PubMed Central

Honigberg, Lee A.; Smith, Ashley M.; Sirisawad, Mint; Verner, Erik; Loury, David; Chang, Betty; Li, Shyr; Pan, Zhengying; Thamm, Douglas H.; Miller, Richard A.; Buggy, Joseph J.

2010-01-01

Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell non-Hodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway. PMID:20615965

IL-27 induces the production of IgG1 by human B cells.

PubMed

Boumendjel, Amel; Tawk, Lina; Malefijt, René de Waal; Boulay, Vera; Yssel, Hans; Pène, Jérôme

2006-12-01

It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human naïve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to naïve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by naïve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.

Acetylshikonin Inhibits Human Pancreatic PANC-1 Cancer Cell Proliferation by Suppressing the NF-κB Activity.

PubMed

Cho, Seok-Cheol; Choi, Bu Young

2015-09-01

Acetylshikonin, a natural naphthoquinone derivative compound, has been used for treatment of inflammation and cancer. In the present study, we have investigated whether acetylshikonin could regulate the NF-κB signaling pathway, thereby leading to suppression of tumorigenesis. We observed that acetylshikonin significantly reduced proliferation of several cancer cell lines, including human pancreatic PANC-1 cancer cells. In addition, acetylshikonin inhibited phorbol 12-myristate 13-acetate (PMA) or tumor necrosis-α (TNF-α)-induced NF-κB reporter activity. Proteome cytokine array and real-time RT-PCR results illustrated that acetylshikonin inhibition of PMA-induced production of cytokines was mediated at the transcriptional level and it was associated with suppression of NF-κB activity and matrix metalloprotenases. Finally, we observed that an exposure of acetylshikonin significantly inhibited the anchorage-independent growth of PANC-1 cells. Together, our results indicate that acetylshikonin could serve as a promising therapeutic agent for future treatment of pancreatic cancer.

Pathological classification of human iPSC-derived neural stem/progenitor cells towards safety assessment of transplantation therapy for CNS diseases.

PubMed

Sugai, Keiko; Fukuzawa, Ryuji; Shofuda, Tomoko; Fukusumi, Hayato; Kawabata, Soya; Nishiyama, Yuichiro; Higuchi, Yuichiro; Kawai, Kenji; Isoda, Miho; Kanematsu, Daisuke; Hashimoto-Tamaoki, Tomoko; Kohyama, Jun; Iwanami, Akio; Suemizu, Hiroshi; Ikeda, Eiji; Matsumoto, Morio; Kanemura, Yonehiro; Nakamura, Masaya; Okano, Hideyuki

2016-09-19

The risk of tumorigenicity is a hurdle for regenerative medicine using induced pluripotent stem cells (iPSCs). Although teratoma formation is readily distinguishable, the malignant transformation of iPSC derivatives has not been clearly defined due to insufficient analysis of histology and phenotype. In the present study, we evaluated the histology of neural stem/progenitor cells (NSPCs) generated from integration-free human peripheral blood mononuclear cell (PBMC)-derived iPSCs (iPSC-NSPCs) following transplantation into central nervous system (CNS) of immunodeficient mice. We found that transplanted iPSC-NSPCs produced differentiation patterns resembling those in embryonic CNS development, and that the microenvironment of the final site of migration affected their maturational stage. Genomic instability of iPSCs correlated with increased proliferation of transplants, although no carcinogenesis was evident. The histological classifications presented here may provide cues for addressing potential safety issues confronting regenerative medicine involving iPSCs.

Control of glioblastoma tumorigenesis by feed-forward cytokine signaling

PubMed Central

Jahani-Asl, Arezu; Yin, Hang; Soleimani, Vahab D; Haque, Takrima; Luchman, H Artee; Chang, Natasha C; Sincennes, Marie-Claude; Puram, Sidharth V; Scott, Andrew M; Lorimer, Ian A J; Perkins, Theodore J; Ligon, Keith L; Weiss, Samuel; Rudnicki, Michael A; Bonni, Azad

2016-01-01

EGFRvIII-STAT3 signaling is important in glioblastoma pathogenesis. Here, we identified the cytokine receptor OSMR as a direct target gene of the transcription factor STAT3 in mouse astrocytes and human brain tumor stem cells (BTSCs). We found that OSMR functioned as an essential co-receptor for EGFRvIII. OSMR formed a physical complex with EGFRvIII, and depletion of OSMR impaired EGFRvIII-STAT3 signaling. Conversely, pharmacological inhibition of EGFRvIII phosphorylation inhibited the EGFRvIII-OSMR interaction and activation of STAT3. EGFRvIII-OSMR signaling in tumors operated constitutively, whereas EGFR-OSMR signaling in nontumor cells was synergistically activated by the ligands EGF and OSM. Finally, knockdown of OSMR strongly suppressed cell proliferation and tumor growth of mouse glioblastoma cells and human BTSC xenografts in mice, and prolonged the lifespan of those mice. Our findings identify OSMR as a critical regulator of glioblastoma tumor growth that orchestrates a feed-forward signaling mechanism with EGFRvIII and STAT3 to drive tumorigenesis. PMID:27110918

Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis.

PubMed

Jafari, Abbas; Qanie, Diyako; Andersen, Thomas L; Zhang, Yuxi; Chen, Li; Postert, Benno; Parsons, Stuart; Ditzel, Nicholas; Khosla, Sundeep; Johansen, Harald Thidemann; Kjærsgaard-Andersen, Per; Delaisse, Jean-Marie; Abdallah, Basem M; Hesselson, Daniel; Solberg, Rigmor; Kassem, Moustapha

2017-02-14

Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

PubMed Central

Liu, Shuzhen; Liu, Hua; Johnston, Andrea; Hanna-Addams, Sarah; Reynoso, Eduardo; Xiang, Yougui

2017-01-01

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α–induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis. PMID:28827318

Titin Mutations in iPS cells Define Sarcomere Insufficiency as a Cause of Dilated Cardiomyopathy

PubMed Central

Hinson, John T.; Chopra, Anant; Nafissi, Navid; Polacheck, William J.; Benson, Craig C.; Swist, Sandra; Gorham, Joshua; Yang, Luhan; Schafer, Sebastian; Sheng, Calvin C.; Haghighi, Alireza; Homsy, Jason; Hubner, Norbert; Church, George; Cook, Stuart A.; Linke, Wolfgang A.; Chen, Christopher S.; Seidman, J. G.; Seidman, Christine E.

2015-01-01

Human mutations that truncate the massive sarcomere protein titin (TTNtv) are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtv, diminish contractile performance and are pathogenic. By combining functional analyses with RNAseq, we explain why truncations in the A-band domain of TTN cause DCM while truncations in the I-band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS-cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodelling. PMID:26315439

Hepatic progenitor cells in canine and feline medicine: potential for regenerative strategies

PubMed Central

2014-01-01

New curative therapies for severe liver disease are urgently needed in both the human and veterinary clinic. It is important to find new treatment modalities which aim to compensate for the loss of parenchymal tissue and to repopulate the liver with healthy hepatocytes. A prime focus in regenerative medicine of the liver is the use of adult liver stem cells, or hepatic progenitor cells (HPCs), for functional recovery of liver disease. This review describes recent developments in HPC research in dog and cat and compares these findings to experimental rodent studies and human pathology. Specifically, the role of HPCs in liver regeneration, key components of the HPC niche, and HPC activation in specific types of canine and feline liver disease will be reviewed. Finally, the potential applications of HPCs in regenerative medicine of the liver are discussed and a potential role is suggested for dogs as first target species for HPC-based trials. PMID:24946932

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.

PubMed

Liu, Shuzhen; Liu, Hua; Johnston, Andrea; Hanna-Addams, Sarah; Reynoso, Eduardo; Xiang, Yougui; Wang, Zhigao

2017-09-05

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α-induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

Genome Editing: A New Approach to Human Therapeutics.

PubMed

Porteus, Matthew

2016-01-01

The ability to manipulate the genome with precise spatial and nucleotide resolution (genome editing) has been a powerful research tool. In the past decade, the tools and expertise for using genome editing in human somatic cells and pluripotent cells have increased to such an extent that the approach is now being developed widely as a strategy to treat human disease. The fundamental process depends on creating a site-specific DNA double-strand break (DSB) in the genome and then allowing the cell's endogenous DSB repair machinery to fix the break such that precise nucleotide changes are made to the DNA sequence. With the development and discovery of several different nuclease platforms and increasing knowledge of the parameters affecting different genome editing outcomes, genome editing frequencies now reach therapeutic relevance for a wide variety of diseases. Moreover, there is a series of complementary approaches to assessing the safety and toxicity of any genome editing process, irrespective of the underlying nuclease used. Finally, the development of genome editing has raised the issue of whether it should be used to engineer the human germline. Although such an approach could clearly prevent the birth of people with devastating and destructive genetic diseases, questions remain about whether human society is morally responsible enough to use this tool.

Genome-wide identification and characterisation of HOT regions in the human genome.

PubMed

Li, Hao; Liu, Feng; Ren, Chao; Bo, Xiaochen; Shu, Wenjie

2016-09-15

HOT (high-occupancy target) regions, which are bound by a surprisingly large number of transcription factors, are considered to be among the most intriguing findings of recent years. An improved understanding of the roles that HOT regions play in biology would be afforded by knowing the constellation of factors that constitute these domains and by identifying HOT regions across the spectrum of human cell types. We characterised and validated HOT regions in embryonic stem cells (ESCs) and produced a catalogue of HOT regions in a broad range of human cell types. We found that HOT regions are associated with genes that control and define the developmental processes of the respective cell and tissue types. We also showed evidence of the developmental persistence of HOT regions at primitive enhancers and demonstrate unique signatures of HOT regions that distinguish them from typical enhancers and super-enhancers. Finally, we performed a dynamic analysis to reveal the dynamical regulation of HOT regions upon H1 differentiation. Taken together, our results provide a resource for the functional exploration of HOT regions and extend our understanding of the key roles of HOT regions in development and differentiation.

A synergetic use of hydrogen and fuel cells in human spaceflight power systems

NASA Astrophysics Data System (ADS)

Belz, S.

2016-04-01

Hydrogen is very flexible in different fields of application of energy conversion. It can be generated by water electrolysis. Stored in tanks it is available for re-electrification by fuel cells. But it is not only the power system, which benefits from use of hydrogen, but also the life support system, which can contain hydrogen consuming technologies for recycling management (e.g. carbon dioxide removal and waste combustion processes). This paper points out various fields of hydrogen use in a human spaceflight system. Depending on mission scenarios, shadow phases, and the need of energy storage, regenerative fuel cell systems can be more efficient than secondary batteries. Here, different power storage concepts are compared by equivalent system mass calculation, thus including impact in the peripheral structure (volume, thermal management, etc.) on the space system. It is also focused on the technical integration aspect, e.g. which peripheral components have to be adapted when hydrogen is also used for life support technologies and what system mass benefit can be expected. Finally, a recommendation is given for the following development steps for a synergetic use of hydrogen and fuel cells in human spaceflight power systems.

CRISPR Genome Engineering for Human Pluripotent Stem Cell Research

PubMed Central

Chaterji, Somali; Ahn, Eun Hyun; Kim, Deok-Ho

2017-01-01

The emergence of targeted and efficient genome editing technologies, such as repurposed bacterial programmable nucleases (e.g., CRISPR-Cas systems), has abetted the development of cell engineering approaches. Lessons learned from the development of RNA-interference (RNA-i) therapies can spur the translation of genome editing, such as those enabling the translation of human pluripotent stem cell engineering. In this review, we discuss the opportunities and the challenges of repurposing bacterial nucleases for genome editing, while appreciating their roles, primarily at the epigenomic granularity. First, we discuss the evolution of high-precision, genome editing technologies, highlighting CRISPR-Cas9. They exist in the form of programmable nucleases, engineered with sequence-specific localizing domains, and with the ability to revolutionize human stem cell technologies through precision targeting with greater on-target activities. Next, we highlight the major challenges that need to be met prior to bench-to-bedside translation, often learning from the path-to-clinic of complementary technologies, such as RNA-i. Finally, we suggest potential bioinformatics developments and CRISPR delivery vehicles that can be deployed to circumvent some of the challenges confronting genome editing technologies en route to the clinic. PMID:29158838

BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

PubMed

Maas Enriquez, Monika; Thrift, John; Garger, Stephen; Katterle, Yvonne

2016-11-01

BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues.

PubMed

Bachmayr-Heyda, Anna; Reiner, Agnes T; Auer, Katharina; Sukhbaatar, Nyamdelger; Aust, Stefanie; Bachleitner-Hofmann, Thomas; Mesteri, Ildiko; Grunt, Thomas W; Zeillinger, Robert; Pils, Dietmar

2015-01-27

Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

PubMed

Toscano, Miguel G; Navarro-Montero, Oscar; Ayllon, Veronica; Ramos-Mejia, Veronica; Guerrero-Carreno, Xiomara; Bueno, Clara; Romero, Tamara; Lamolda, Mar; Cobo, Marien; Martin, Francisco; Menendez, Pablo; Real, Pedro J

2015-01-01

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

Elabela-apelin receptor signaling pathway is functional in mammalian systems.

PubMed

Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

2015-02-02

Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer.

PubMed

Vecchione, A; Fassan, M; Anesti, V; Morrione, A; Goldoni, S; Baldassarre, G; Byrne, D; D'Arca, D; Palazzo, J P; Lloyd, J; Scorrano, L; Gomella, L G; Iozzo, R V; Baffa, R

2009-01-15

Allelic deletions on human chromosome 12q24 are frequently reported in a variety of malignant neoplasms, indicating the presence of a tumor suppressor gene(s) in this chromosomal region. However, no reasonable candidate has been identified so far. In this study, we report the cloning and functional characterization of a novel mitochondrial protein with tumor suppressor activity, henceforth designated MITOSTATIN. Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a approximately 62 kDa, ubiquitously expressed protein with little homology to any known protein. We found homozygous deletions and mutations of MITOSTATIN gene in approximately 5 and approximately 11% of various cancer-derived cells and solid tumors, respectively. When transiently overexpressed, MITOSTATIN inhibited colony formation, tumor cell growth and was proapoptotic, all features shared by established tumor suppressor genes. We discovered a specific link between MITOSTATIN overexpression and downregulation of Hsp27. Conversely, MITOSTATIN knockdown cells showed an increase in cell growth and cell survival rates. Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages. Our findings support the hypothesis that MITOSTATIN has many hallmarks of a classical tumor suppressor in solid tumors and may play an important role in cancer development and progression.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts.

PubMed

Bernstein, Diana L; Le Lay, John E; Ruano, Elena G; Kaestner, Klaus H

2015-05-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

PubMed Central

Bernstein, Diana L.; Le Lay, John E.; Ruano, Elena G.; Kaestner, Klaus H.

2015-01-01

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator–like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics. PMID:25866970

Interleukin-2-dependent long-term cultures of low-density lymphocytes allow the proliferation of lymphokine-activated killer cells with natural killer, Ti gamma/delta or TNK phenotype.

PubMed

Testa, U; Care, A; Montesoro, E; Fossati, C; Giannella, G; Masciulli, R; Fagioli, M; Bulgarini, D; Habetswallner, D; Isacchi, G

1990-01-01

We have developed a culture system for "long-term" growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum antitumor cytotoxicity. The system allows the exponential growth of monocyte-depleted low-density lymphocytes in the presence of human serum and recombinant human interleukin-2 (10(3) U/ml), alone or in combination with interleukin-1 alpha or beta (both at 10 U/ml). Eighteen cultures were established from 18 normal adult donors. The membrane phenotypes of the final LAK cell population, assessed by a panel of monoclonal antibodies (mAb), consist of three main types: (a) NKH-1+, Ti alpha/beta-, Ti gamma/delta-, and CD3- lymphocytes; (b) NKH-1+, Ti alpha/beta-, Ti gamma/delta+, and CD3+ lymphocytes and (c) NKH-1+, Ti alpha/beta+, Ti gamma/delta- and CD3+ lymphocytes. Northern blot analysis showed that all these cell populations express relatively high levels of perforin RNA, particularly cells exhibiting the first phenotype. This culture system may provide a tool for cellular and molecular studies on the mechanisms of antitumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced center.

Correlation of circular RNA abundance with proliferation – exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues

PubMed Central

Bachmayr-Heyda, Anna; Reiner, Agnes T.; Auer, Katharina; Sukhbaatar, Nyamdelger; Aust, Stefanie; Bachleitner-Hofmann, Thomas; Mesteri, Ildiko; Grunt, Thomas W.; Zeillinger, Robert; Pils, Dietmar

2015-01-01

Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells. PMID:25624062

CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice.

PubMed

Bernard-Valnet, Raphaël; Yshii, Lidia; Quériault, Clémence; Nguyen, Xuan-Hung; Arthaud, Sébastien; Rodrigues, Magda; Canivet, Astrid; Morel, Anne-Laure; Matthys, Arthur; Bauer, Jan; Pignolet, Béatrice; Dauvilliers, Yves; Peyron, Christelle; Liblau, Roland S

2016-09-27

Narcolepsy with cataplexy is a rare and severe sleep disorder caused by the destruction of orexinergic neurons in the lateral hypothalamus. The genetic and environmental factors associated with narcolepsy, together with serologic data, collectively point to an autoimmune origin. The current animal models of narcolepsy, based on either disruption of the orexinergic neurotransmission or neurons, do not allow study of the potential autoimmune etiology. Here, we sought to generate a mouse model that allows deciphering of the immune mechanisms leading to orexin(+) neuron loss and narcolepsy development. We generated mice expressing the hemagglutinin (HA) as a "neo-self-antigen" specifically in hypothalamic orexin(+) neurons (called Orex-HA), which were transferred with effector neo-self-antigen-specific T cells to assess whether an autoimmune process could be at play in narcolepsy. Given the tight association of narcolepsy with the human leukocyte antigen (HLA) HLA-DQB1*06:02 allele, we first tested the pathogenic contribution of CD4 Th1 cells. Although these T cells readily infiltrated the hypothalamus and triggered local inflammation, they did not elicit the loss of orexin(+) neurons or clinical manifestations of narcolepsy. In contrast, the transfer of cytotoxic CD8 T cells (CTLs) led to both T-cell infiltration and specific destruction of orexin(+) neurons. This phenotype was further aggravated upon repeated injections of CTLs. In situ, CTLs interacted directly with MHC class I-expressing orexin(+) neurons, resulting in cytolytic granule polarization toward neurons. Finally, drastic neuronal loss caused manifestations mimicking human narcolepsy, such as cataplexy and sleep attacks. This work demonstrates the potential role of CTLs as final effectors of the immunopathological process in narcolepsy.

CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice

PubMed Central

Bernard-Valnet, Raphaël; Yshii, Lidia; Quériault, Clémence; Nguyen, Xuan-Hung; Arthaud, Sébastien; Rodrigues, Magda; Canivet, Astrid; Morel, Anne-Laure; Matthys, Arthur; Bauer, Jan; Pignolet, Béatrice; Dauvilliers, Yves; Peyron, Christelle; Liblau, Roland S.

2016-01-01

Narcolepsy with cataplexy is a rare and severe sleep disorder caused by the destruction of orexinergic neurons in the lateral hypothalamus. The genetic and environmental factors associated with narcolepsy, together with serologic data, collectively point to an autoimmune origin. The current animal models of narcolepsy, based on either disruption of the orexinergic neurotransmission or neurons, do not allow study of the potential autoimmune etiology. Here, we sought to generate a mouse model that allows deciphering of the immune mechanisms leading to orexin+ neuron loss and narcolepsy development. We generated mice expressing the hemagglutinin (HA) as a “neo-self-antigen” specifically in hypothalamic orexin+ neurons (called Orex-HA), which were transferred with effector neo-self-antigen–specific T cells to assess whether an autoimmune process could be at play in narcolepsy. Given the tight association of narcolepsy with the human leukocyte antigen (HLA) HLA-DQB1*06:02 allele, we first tested the pathogenic contribution of CD4 Th1 cells. Although these T cells readily infiltrated the hypothalamus and triggered local inflammation, they did not elicit the loss of orexin+ neurons or clinical manifestations of narcolepsy. In contrast, the transfer of cytotoxic CD8 T cells (CTLs) led to both T-cell infiltration and specific destruction of orexin+ neurons. This phenotype was further aggravated upon repeated injections of CTLs. In situ, CTLs interacted directly with MHC class I-expressing orexin+ neurons, resulting in cytolytic granule polarization toward neurons. Finally, drastic neuronal loss caused manifestations mimicking human narcolepsy, such as cataplexy and sleep attacks. This work demonstrates the potential role of CTLs as final effectors of the immunopathological process in narcolepsy. PMID:27621438

ILK Induces Cardiomyogenesis in the Human Heart

PubMed Central

Traister, Alexandra; Aafaqi, Shabana; Masse, Stephane; Dai, Xiaojing; Li, Mark; Hinek, Aleksander; Nanthakumar, Kumaraswamy; Hannigan, Gregory; Coles, John G.

2012-01-01

Background Integrin-linked kinase (ILK) is a widely conserved serine/threonine kinase that regulates diverse signal transduction pathways implicated in cardiac hypertrophy and contractility. In this study we explored whether experimental overexpression of ILK would up-regulate morphogenesis in the human fetal heart. Methodology/Principal Findings Primary cultures of human fetal myocardial cells (19–22 weeks gestation) yielded scattered aggregates of cardioblasts positive for the early cardiac lineage marker nk×2.5 and containing nascent sarcomeres. Cardiac cells in colonies uniformly expressed the gap junction protein connexin 43 (C×43) and displayed a spectrum of differentiation with only a subset of cells exhibiting the late cardiomyogenic marker troponin T (cTnT) and evidence of electrical excitability. Adenovirus-mediated overexpression of ILK potently increased the number of new aggregates of primitive cardioblasts (p<0.001). The number of cardioblast colonies was significantly decreased (p<0.05) when ILK expression was knocked down with ILK targeted siRNA. Interestingly, overexpression of the activation resistant ILK mutant (ILKR211A) resulted in much greater increase in the number of new cell aggregates as compared to overexpression of wild-type ILK (ILKWT). The cardiomyogenic effects of ILKR211A and ILKWT were accompanied by concurrent activation of β-catenin (p<0.001) and increase expression of progenitor cell marker islet-1, which was also observed in lysates of transgenic mice with cardiac-specific over-expression of ILKR211A and ILKWT. Finally, endogenous ILK expression was shown to increase in concert with those of cardiomyogenic markers during directed cardiomyogenic differentiation in human embryonic stem cells (hESCs). Conclusions/Significance In the human fetal heart ILK activation is instructive to the specification of mesodermal precursor cells towards a cardiomyogenic lineage. Induction of cardiomyogenesis by ILK overexpression bypasses the requirement of proximal PI3K activation for transduction of growth factor- and β1-integrin-mediated differentiation signals. Altogether, our data indicate that ILK represents a novel regulatory checkpoint during human cardiomyogenesis. PMID:22666394

miRNA-205 affects infiltration and metastasis of breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhouquan; Department of Tumor, SenGong Hospital of Shaanxi, Xi’an 710300; Liao, Hehe

2013-11-08

Highlights: •We detected expression of miR-205 in breast cancer cell lines and tissue samples. •We suggest miR-205 is downregulated in human breast cancer tissues and MCF7 cells. •We suggest the lower expression of miR-205 play a role in breast cancer onset. •These data suggest that miR-205 directly targets HER3 in human breast cancer. -- Abstract: Background: An increasing number of studies have shown that miRNAs are commonly deregulated in human malignancies, but little is known about the function of miRNA-205 (miR-205) in human breast cancer. The present study investigated the influence of miR-205 on breast cancer malignancy. Methods: The expressionmore » level of miR-205 in the MCF7 breast cancer cell line was determined by quantitative (q)RT-PCR. We then analyzed the expression of miR-205 in breast cancer and paired non-tumor tissues. Finally, the roles of miR-205 in regulating tumor proliferation, apoptosis, migration, and target gene expression were studied by MTT assay, flow cytometry, qRT-PCR, Western blotting and luciferase assay. Results: miR-205 was downregulated in breast cancer cells or tissues compared with normal breast cell lines or non-tumor tissues. Overexpression of miR-205 reduced the growth and colony-formation capacity of MCF7 cells by inducing apoptosis. Overexpression of miR-205 inhibited MCF7 cell migration and invasiveness. By bioinformation analysis, miR-205 was predicted to bind to the 3′ untranslated regions of human epidermal growth factor receptor (HER)3 mRNA, and upregulation of miR-205 reduced HER3 protein expression. Conclusion: miR-205 is a tumor suppressor in human breast cancer by post-transcriptional inhibition of HER3 expression.« less

Micro-engineering a platform to reconstruct physiology and functionality of the human brain microvasculature in vitro

NASA Astrophysics Data System (ADS)

Daghighi, Yasaman; Heidari, Hossein; Taylor, Hayden

2018-02-01

A predominant unsolved challenge in tissue engineering is the need of a robust technique for producing vascular networks, particularly when modeling human brain tissue. The availability of reliable in vitro human brain microvasculature models would advance our understanding of its function and would provide a platform for highthroughput drug screening. Current strategies for modeling vascularized brain tissue suffer from limitations such as (1) culturing non-human cell lines, (2) limited multi-cell co-culture, and (3) the effects of neighboring physiologically unrealistic rigid polymeric surfaces, such as solid membranes. We demonstrate a new micro-engineered platform that can address these shortcomings. Specifically, we have designed and prototyped a molding system to enable the precise casting of 100μm-diameter coaxial hydrogel structures laden with the requisite cells to mimic a vascular lumen. Here we demonstrate that a fine wire with diameter 130 μm or a needle with outer diameter 300 μm can be used as a temporary mold insert, and agarose-collagen composite matrix can be cast around these inserts and thermally gelled. When the wire or needle is retracted under the precise positional control afforded by our system, a microchannel is formed which is then seeded with human microvascular endothelial cells. After seven days of culture these cells produce an apparently confluent monolayer on the channel walls. In principle, this platform could be used to create multilayered cellular structures. By arranging a fine wire and a hollow needle coaxially, three distinct zones could be defined in the model: first, the bulk gel surrounding the needle; then, after needle retraction, a cylindrical shell of matrix; and finally, after retraction of the wire, a lumen. Each zone could be independently cell-seeded. To this end, we have also successfully 3D cultured human astrocytes and SY5Y glial cells in our agarose-collagen matrix. Our approach ultimately promises scalable and repeatable production of vascular structures with physiologically realistic mechanical properties.

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

PubMed

Alfen, Johanna Sophie; Larghi, Paola; Facciotti, Federica; Gagliani, Nicola; Bosotti, Roberto; Paroni, Moira; Maglie, Stefano; Gruarin, Paola; Vasco, Chiara Maria; Ranzani, Valeria; Frusteri, Cristina; Iseppon, Andrea; Moro, Monica; Crosti, Maria Cristina; Gatti, Stefano; Pagani, Massimiliano; Caprioli, Flavio; Abrignani, Sergio; Flavell, Richard A; Geginat, Jens

2018-01-31

IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T R 1) cells but is also produced by CD25 + regulatory T (Treg) cells. We aimed to identify and characterize human intestinal T R 1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). CD4 + T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4 + T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed. Intestinal T R 1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5 + PD-1 + T R 1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ + T R 1 cells, but not IL-7 receptor-positive T H cells or CD25 + Treg cells, showed lower IL-10 expression in patients with IBDs. T R 1 cells were responsive to IL-23, and IFN-γ + T R 1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25 + Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. We provide the first ex vivo characterization of human intestinal T R 1 cells. Selective downregulation of IL-10 by IFN-γ + T R 1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

9-cis-Retinoic Acid Promotes Cell Adhesion Through Integrin Dependent and Independent Mechanisms Across Immune Lineages

PubMed Central

Whelan, Jarrett T.; Chen, Jianming; Miller, Jabin; Morrow, Rebekah L.; Lingo, Joshuah D.; Merrell, Kaitlin; Shaikh, Saame Raza; Bridges, Lance C.

2012-01-01

Retinoids are essential in the proper establishment and maintenance of immunity. Although retinoids are implicated in immune related processes, their role in immune cell adhesion has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) on human hematopoietic cell adhesion was investigated. 9-cis-RA treatment specifically induced cell adhesion of the human immune cell lines HuT-78, NB4, RPMI 8866, and U937. Due to the prominent role of integrin receptors in mediating immune cell adhesion, we sought to evaluate if cell adhesion was integrin-dependent. By employing a variety of integrin antagonist including function-blocking antibodies and EDTA, we establish that 9-cis-RA prompts immune cell adhesion through established integrin receptors in addition to a novel integrin-independent process. The novel integrin-independent adhesion required the presence of retinoid and was attenuated by treatment with synthetic corticosteroids. Finally, we demonstrate that 9-cis-RA treatment of primary murine B-cells induces ex vivo adhesion that persists in the absence of integrin function. Our study is the first to demonstrate that 9-cis-retinoic acid influences immune cell adhesion through at least two functionally distinct mechanisms. PMID:22925918

Phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat Pteropus alecto.

PubMed

Martínez Gómez, Julia María; Periasamy, Pravin; Dutertre, Charles-Antoine; Irving, Aaron Trent; Ng, Justin Han Jia; Crameri, Gary; Baker, Michelle L; Ginhoux, Florent; Wang, Lin-Fa; Alonso, Sylvie

2016-11-24

The unique ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat's immunity. Using cross-reactive antibodies, we report the phenotypic and functional characterization of T cell subsets, B and NK cells in the fruit-eating bat Pteropus alecto. Our findings indicate the predominance of CD8 + T cells in the spleen from wild-caught bats that may reflect either the presence of viruses in this organ or predominance of this cell subset at steady state. Instead majority of T cells in circulation, lymph nodes and bone marrow (BM) were CD4 + subsets. Interestingly, 40% of spleen T cells expressed constitutively IL-17, IL-22 and TGF-β mRNA, which may indicate a strong bias towards the Th17 and regulatory T cell subsets. Furthermore, the unexpected high number of T cells in bats BM could suggest an important role in T cell development. Finally, mitogenic stimulation induced proliferation and production of effector molecules by bats immune cells. This work contributes to a better understanding of bat's immunity, opening up new perspectives of therapeutic interventions for humans.

Phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat Pteropus alecto

PubMed Central

Martínez Gómez, Julia María; Periasamy, Pravin; Dutertre, Charles-Antoine; Irving, Aaron Trent; Ng, Justin Han Jia; Crameri, Gary; Baker, Michelle L.; Ginhoux, Florent; Wang, Lin-Fa; Alonso, Sylvie

2016-01-01

The unique ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat’s immunity. Using cross-reactive antibodies, we report the phenotypic and functional characterization of T cell subsets, B and NK cells in the fruit-eating bat Pteropus alecto. Our findings indicate the predominance of CD8+ T cells in the spleen from wild-caught bats that may reflect either the presence of viruses in this organ or predominance of this cell subset at steady state. Instead majority of T cells in circulation, lymph nodes and bone marrow (BM) were CD4+ subsets. Interestingly, 40% of spleen T cells expressed constitutively IL-17, IL-22 and TGF-β mRNA, which may indicate a strong bias towards the Th17 and regulatory T cell subsets. Furthermore, the unexpected high number of T cells in bats BM could suggest an important role in T cell development. Finally, mitogenic stimulation induced proliferation and production of effector molecules by bats immune cells. This work contributes to a better understanding of bat’s immunity, opening up new perspectives of therapeutic interventions for humans. PMID:27883085

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection.

PubMed

Nunes-Alves, Cláudio; Booty, Matthew G; Carpenter, Stephen M; Rothchild, Alissa C; Martin, Constance J; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M

2015-05-01

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.

Chemical UV Filters Mimic the Effect of Progesterone on Ca2+ Signaling in Human Sperm Cells.

PubMed

Rehfeld, A; Dissing, S; Skakkebæk, N E

2016-11-01

Progesterone released by cumulus cells surrounding the egg induces a Ca 2+ influx into human sperm cells via the cationic channel of sperm (CatSper) Ca 2+ channel and controls multiple Ca 2+ -dependent responses essential for fertilization. We hypothesized that chemical UV filters may mimic the physiological action of progesterone on CatSper, thus affecting Ca 2+ signaling in human sperm cells. We examined 29 UV filters allowed in sunscreens in the United States and/or the European Union for their ability to induce Ca 2+ signals in human sperm by applying measurements of the intracellular free Ca 2+ concentration. We found that 13 UV filters induced a significant Ca 2+ signal at 10 μM. Nine UV filters induced Ca 2+ signals primarily by activating the CatSper channel. The UV filters 3-benzylidene camphor (3-BC) and benzylidene camphor sulfonic acid competitively inhibited progesterone-induced Ca 2+ signals. Dose-response relations for the UV filters showed that the Ca 2+ signal-inducing effects began in the nanomolar-micromolar range. Single-cell Ca 2+ measurements showed a Ca 2+ signal-inducing effect of the most potent UV filter, 3-BC, at 10 nM. Finally, we demonstrated that the 13 UV filters acted additively in low-dose mixtures to induce Ca 2+ signals. In conclusion, 13 of 29 examined UV filters (44%) induced Ca 2+ signals in human sperm. Nine UV filters primarily activated CatSper and thereby mimicked the effect of progesterone. The UV filters 3-BC and benzylidene camphor sulfonic acid competitively inhibited progesterone-induced Ca 2+ signals. In vivo exposure studies are needed to investigate whether UV filter exposure affects human fertility.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

PubMed

Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

2016-02-01

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

The novel mTORC1/2 dual inhibitor INK-128 suppresses survival and proliferation of primary and transformed human pancreatic cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lou, Hai-zhou; Weng, Xiao-chuan; Pan, Hong-ming

Highlights: • INK-128 inhibits the survival and growth of human pancreatic cancer cells. • INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. • INK-128 blocks mTORC1/2 activation simultaneously in pancreatic cancer cells. • INK-128 down-regulates cyclin D1 and causes pancreatic cancer cell cycle arrest. • INK-128 significantly increases sensitivity of pancreatic cancer cells to gemcitabine. - Abstract: Pancreatic cancer has one of worst prognosis among all human malignancies around the world, the development of novel and more efficient anti-cancer agents against this disease is urgent. In the current study, we tested the potential effect of INK-128, a novel mammalianmore » target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells in vitro. Our results demonstrated that INK-128 concentration- and time-dependently inhibited the survival and growth of pancreatic cancer cells (both primary cells and transformed cells). INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. Further, INK-128 dramatically inhibited phosphorylation of 4E-binding protein 1 (4E-BP1), ribosomal S6 kinase 1 (S6K1) and Akt at Ser 473 in pancreatic cancer cells. Meanwhile, it downregulated cyclin D1 expression and caused cell cycle arrest. Finally, we found that a low concentration of INK-128 significantly increased the sensitivity of pancreatic cancer cells to gemcitabine. Together, our in vitro results suggest that INK-128 might be further investigated as a novel anti-cancer agent or chemo-adjuvant for pancreatic cancer treatment.« less

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target.

PubMed

Apps, John R; Carreno, Gabriela; Gonzalez-Meljem, Jose Mario; Haston, Scott; Guiho, Romain; Cooper, Julie E; Manshaei, Saba; Jani, Nital; Hölsken, Annett; Pettorini, Benedetta; Beynon, Robert J; Simpson, Deborah M; Fraser, Helen C; Hong, Ying; Hallang, Shirleen; Stone, Thomas J; Virasami, Alex; Donson, Andrew M; Jones, David; Aquilina, Kristian; Spoudeas, Helen; Joshi, Abhijit R; Grundy, Richard; Storer, Lisa C D; Korbonits, Márta; Hilton, David A; Tossell, Kyoko; Thavaraj, Selvam; Ungless, Mark A; Gil, Jesus; Buslei, Rolf; Hankinson, Todd; Hargrave, Darren; Goding, Colin; Andoniadou, Cynthia L; Brogan, Paul; Jacques, Thomas S; Williams, Hywel J; Martinez-Barbera, Juan Pedro

2018-05-01

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection

PubMed Central

2013-01-01

Background Chlamydia trachomatis infection results in reproductive damage in some women. The process and factors involved in this immunopathology are not well understood. This study aimed to investigate the role of primary human cellular responses to chlamydial stress response proteases and chlamydial infection to further identify the immune processes involved in serious disease sequelae. Results Laboratory cell cultures and primary human reproductive epithelial cultures produced IL-6 in response to chlamydial stress response proteases (CtHtrA and CtTsp), UV inactivated Chlamydia, and live Chlamydia. The magnitude of the IL-6 response varied considerably (up to 1000 pg ml-1) across different primary human reproductive cultures. Thus different levels of IL-6 production by reproductive epithelia may be a determinant in disease outcome. Interestingly, co-culture models with either THP-1 cells or autologous primary human PBMC generally resulted in increased levels of IL-6, except in the case of live Chlamydia where the level of IL-6 was decreased compared to the epithelial cell culture only, suggesting this pathway may be able to be modulated by live Chlamydia. PBMC responses to the stress response proteases (CtTsp and CtHtrA) did not significantly vary for the different participant cohorts. Therefore, these proteases may possess conserved innate PAMPs. MAP kinases appeared to be involved in this IL-6 induction from human cells. Finally, we also demonstrated that IL-6 was induced by these proteins and Chlamydia from mouse primary reproductive cell cultures (BALB/C mice) and mouse laboratory cell models. Conclusions We have demonstrated that IL-6 may be a key factor for the chlamydial disease outcome in humans, given that primary human reproductive epithelial cell culture showed considerable variation in IL-6 response to Chlamydia or chlamydial proteins, and that the presence of live Chlamydia (but not UV killed) during co-culture resulted in a reduced IL-6 response suggesting this response may be moderated by the presence of the organism. PMID:24238294

SIZE FRACTIONS OF AMBIENT PARTICULATE MATTER INDUCE GRANULOCYTE MACROPHAGE COLONY-STIMULATING FACTOR IN HUMAN BRONCHIAL EPITHELIAL CELLS BY MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS. (R827351C004)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Spindle neurons of the human anterior cingulate cortex

NASA Technical Reports Server (NTRS)

Nimchinsky, E. A.; Vogt, B. A.; Morrison, J. H.; Hof, P. R.; Bloom, F. E. (Principal Investigator)

1995-01-01

The human anterior cingulate cortex is distinguished by the presence of an unusual cell type, a large spindle neuron in layer Vb. This cell has been noted numerous times in the historical literature but has not been studied with modern neuroanatomic techniques. For instance, details regarding the neuronal class to which these cells belong and regarding their precise distribution along both ventrodorsal and anteroposterior axes of the cingulate gyrus are still lacking. In the present study, morphological features and the anatomic distribution of this cell type were studied using computer-assisted mapping and immunocytochemical techniques. Spindle neurons are restricted to the subfields of the anterior cingulate cortex (Brodmann's area 24), exhibiting a greater density in anterior portions of this area than in posterior portions, and tapering off in the transition zone between anterior and posterior cingulate cortex. Furthermore, a majority of the spindle cells at any level is located in subarea 24b on the gyral surface. Immunocytochemical analysis revealed that the neurofilament protein triple was present in a large percentage of these neurons and that they did not contain calcium-binding proteins. Injections of the carbocyanine dye DiI into the cingulum bundle revealed that these cells are projection neurons. Finally, spindle cells were consistently affected in Alzheimer's disease cases, with an overall loss of about 60%. Taken together, these observations indicate that the spindle cells of the human cingulate cortex represent a morphological subpopulation of pyramidal neurons whose restricted distribution may be associated with functionally distinct areas.

Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

PubMed

Al-Alem, Linah; Puttabyatappa, Muraly; Rosewell, Kathy; Brännström, Mats; Akin, James; Boldt, Jeffrey; Muse, Ken; Curry, Thomas E

2015-09-01

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12-18 h) and late ovulatory (18-34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women.

Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

PubMed Central

Al-Alem, Linah; Puttabyatappa, Muraly; Rosewell, Kathy; Brännström, Mats; Akin, James; Boldt, Jeffrey; Muse, Ken

2015-01-01

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12–18 h) and late ovulatory (18–34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women. PMID:26125463

Melatonin: an inhibitor of breast cancer.

PubMed

Hill, Steven M; Belancio, Victoria P; Dauchy, Robert T; Xiang, Shulin; Brimer, Samantha; Mao, Lulu; Hauch, Adam; Lundberg, Peter W; Summers, Whitney; Yuan, Lin; Frasch, Tripp; Blask, David E

2015-06-01

The present review discusses recent work on melatonin-mediated circadian regulation, the metabolic and molecular signaling mechanisms that are involved in human breast cancer growth, and the associated consequences of circadian disruption by exposure to light at night (LEN). The anti-cancer actions of the circadian melatonin signal in human breast cancer cell lines and xenografts heavily involve MT1 receptor-mediated mechanisms. In estrogen receptor alpha (ERα)-positive human breast cancer, melatonin suppresses ERα mRNA expression and ERα transcriptional activity via the MT1 receptor. Melatonin also regulates the transactivation of other members of the nuclear receptor superfamily, estrogen-metabolizing enzymes, and the expression of core clock and clock-related genes. Furthermore, melatonin also suppresses tumor aerobic metabolism (the Warburg effect) and, subsequently, cell-signaling pathways critical to cell proliferation, cell survival, metastasis, and drug resistance. Melatonin demonstrates both cytostatic and cytotoxic activity in breast cancer cells that appears to be cell type-specific. Melatonin also possesses anti-invasive/anti-metastatic actions that involve multiple pathways, including inhibition of p38 MAPK and repression of epithelial-mesenchymal transition (EMT). Studies have demonstrated that melatonin promotes genomic stability by inhibiting the expression of LINE-1 retrotransposons. Finally, research in animal and human models has indicated that LEN-induced disruption of the circadian nocturnal melatonin signal promotes the growth, metabolism, and signaling of human breast cancer and drives breast tumors to endocrine and chemotherapeutic resistance. These data provide the strongest understanding and support of the mechanisms that underpin the epidemiologic demonstration of elevated breast cancer risk in night-shift workers and other individuals who are increasingly exposed to LEN. © 2015 Society for Endocrinology.

Arterial and venous endothelia display differential functional fractalkine (CX3CL1) expression by angiotensin-II.

PubMed

Rius, Cristina; Piqueras, Laura; González-Navarro, Herminia; Albertos, Fernando; Company, Chantal; López-Ginés, Concha; Ludwig, Andreas; Blanes, Jose-Ignacio; Morcillo, Esteban J; Sanz, Maria-Jesus

2013-01-01

Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX(3)CL1) was explored. Enhanced CX(3)CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 μmol/L Ang-II increased CX(3)CL1 expression, yet neutralization of CX(3)CL1 activity only significantly inhibited Ang-II-induced mononuclear cell-human umbilical arterial endothelial cell interactions (73%) but not with human umbilical venous endothelial cells. The use of small interfering RNA revealed the involvement of tumor necrosis factor-α in Ang-II-induced CX(3)CL1 upregulation and mononuclear cell arrest. Nox5 knockdown with small interfering RNA or pharmacological inhibition of extracellular signal-regulated kinases1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB also abolished these responses. Finally, when human umbilical arterial endothelial cells were costimulated with Ang-II, tumor necrosis factor-α, and interferon-γ, CX(3)CL1 expression and mononuclear cell adhesiveness were more pronounced than when each stimulus was provided alone. These results suggest that Ang-II induces functional CX(3)CL1 expression in arterial but not in venous endothelia. Thus, targeting endothelial CX(3)CL1-mononuclear leukocyte CX(3)CR1 interactions may constitute a new therapeutic strategy in the treatment of Ang-II-associated cardiovascular diseases.

MicroRNA-320a suppresses human colon cancer cell proliferation by directly targeting {beta}-catenin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Jian-Yong; State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, 710032 Xi'an; Huang, Yi

2012-04-20

Highlights: Black-Right-Pointing-Pointer miR-320a is downregulated in human colorectal carcinoma. Black-Right-Pointing-Pointer Overexpression of miR-320a inhibits colon cancer cell proliferation. Black-Right-Pointing-Pointer {beta}-Catenin is a direct target of miR-320a in colon cancer cells. Black-Right-Pointing-Pointer miR-320a expression inversely correlates with mRNA expression of {beta}-catenin's target genes in human colon carcinoma. -- Abstract: Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-320a) in human colorectal carcinoma. However, its expression pattern and underlying mechanisms in the development and progression of colorectal carcinoma has not been elucidated clearly. Here, we performed real-time PCR to examine the expression levels of miR-320a in colonmore » cancer cell lines and tumor tissues. And then, we investigated its biological functions in colon cancer cells by a gain of functional strategy. Further more, by the combinational approaches of bioinformatics and experimental validation, we confirmed target associations of miR-320a in colorectal carcinoma. Our results showed that miR-320a was frequently downregulated in cancer cell lines and colon cancer tissues. And we demonstrated that miR-320a restoration inhibited colon cancer cell proliferation and {beta}-catenin, a functionally oncogenic molecule was a direct target gene of miR-320a. Finally, the data of real-time PCR showed the reciprocal relationship between miR-320a and {beta}-catenin's downstream genes in colon cancer tissues. These findings indicate that miR-320a suppresses the growth of colon cancer cells by directly targeting {beta}-catenin, suggesting its application in prognosis prediction and cancer treatment.« less

Genomic Distribution and Inter-Sample Variation of Non-CpG Methylation across Human Cell Types

PubMed Central

Liao, Jing; Zhang, Yingying; Gu, Hongcang; Bock, Christoph; Boyle, Patrick; Epstein, Charles B.; Bernstein, Bradley E.; Lengauer, Thomas; Gnirke, Andreas; Meissner, Alexander

2011-01-01

DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set. PMID:22174693

Long-term exposure of immortalized keratinocytes to arsenic induces EMT, impairs differentiation in organotypic skin models and mimics aspects of human skin derangements.

PubMed

Weinmuellner, R; Kryeziu, K; Zbiral, B; Tav, K; Schoenhacker-Alte, B; Groza, D; Wimmer, L; Schosserer, M; Nagelreiter, F; Rösinger, S; Mildner, M; Tschachler, E; Grusch, M; Grillari, J; Heffeter, P

2018-01-01

Arsenic is one of the most important human carcinogens and environmental pollutants. However, the evaluation of the underlying carcinogenic mechanisms is challenging due to the lack of suitable in vivo and in vitro models, as distinct interspecies differences in arsenic metabolism exist. Thus, it is of high interest to develop new experimental models of arsenic-induced skin tumorigenesis in humans. Consequently, aim of this study was to establish an advanced 3D model for the investigation of arsenic-induced skin derangements, namely skin equivalents, built from immortalized human keratinocytes (NHEK/SVTERT3-5). In contrast to spontaneously immortalized HACAT cells, NHEK/SVTERT3-5 cells more closely resembled the differentiation pattern of primary keratinocytes. With regard to arsenic, our results showed that while our new cell model was widely unaffected by short-time treatment (72 h) with low, non-toxic doses of ATO (0.05-0.25 µM), chronic exposure (6 months) resulted in distinct changes of several cell characteristics. Thus, we observed an increase in the G2 fraction of the cell cycle accompanied by increased nucleus size and uneven tubulin distribution. Moreover, cells showed strong signs of de-differentiation and upregulation of several epithelial-to-mesenchymal transition markers. In line with these effects, chronic contact to arsenic resulted in impaired skin-forming capacities as well as localization of ki67-positive (proliferating) cells at the upper layers of the epidermis; a condition termed Bowen's disease. Finally, chronically arsenic-exposed cells were characterized by an increased tumorigenicity in SCID mice. Taken together, our study presents a new model system for the investigation of mechanisms underlying the tumor-promoting effects of chronic arsenic exposure.

Trimeprazine increases IRS2 in human islets and promotes pancreatic β cell growth and function in mice

PubMed Central

Kuznetsova, Alexandra; Yu, Yue; Hollister-Lock, Jennifer; Opare-Addo, Lynn; Rozzo, Aldo; Sadagurski, Marianna; Norquay, Lisa; Reed, Jessica E.; El Khattabi, Ilham; Bonner-Weir, Susan; Weir, Gordon C.; Sharma, Arun

2016-01-01

The capacity of pancreatic β cells to maintain glucose homeostasis during chronic physiologic and immunologic stress is important for cellular and metabolic homeostasis. Insulin receptor substrate 2 (IRS2) is a regulated adapter protein that links the insulin and IGF1 receptors to downstream signaling cascades. Since strategies to maintain or increase IRS2 expression can promote β cell growth, function, and survival, we conducted a screen to find small molecules that can increase IRS2 mRNA in isolated human pancreatic islets. We identified 77 compounds, including 15 that contained a tricyclic core. To establish the efficacy of our approach, one of the tricyclic compounds, trimeprazine tartrate, was investigated in isolated human islets and in mouse models. Trimeprazine is a first-generation antihistamine that acts as a partial agonist against the histamine H1 receptor (H1R) and other GPCRs, some of which are expressed on human islets. Trimeprazine promoted CREB phosphorylation and increased the concentration of IRS2 in islets. IRS2 was required for trimeprazine to increase nuclear Pdx1, islet mass, β cell replication and function, and glucose tolerance in mice. Moreover, trimeprazine synergized with anti-CD3 Abs to reduce the progression of diabetes in NOD mice. Finally, it increased the function of human islet transplants in streptozotocin-induced (STZ-induced) diabetic mice. Thus, trimeprazine, its analogs, or possibly other compounds that increase IRS2 in islets and β cells without adverse systemic effects might provide mechanism-based strategies to prevent the progression of diabetes. PMID:27152363

Intraoperative Detection of Cell Injury and Cell Death with an 800 nm Near-Infrared Fluorescent Annexin V Derivative

PubMed Central

Ohnishi, Shunsuke; Vanderheyden, Jean-Luc; Tanaka, Eiichi; Patel, Bhavesh; De Grand, Alec; Laurence, Rita G.; Yamashita, Kenichiro; Frangioni, John V.

2008-01-01

The intraoperative detection of cell injury and cell death is fundamental to human surgeries such as organ transplantation and resection. Because of low autofluorescence background and relatively high tissue penetration, invisible light in the 800 nm region provides sensitive detection of disease pathology without changing the appearance of the surgical field. In order to provide surgeons with real-time intraoperative detection of cell injury and death after ischemia/reperfusion (I/R), we have developed a bioactive derivative of human annexin V (annexin800), which fluoresces at 800 nm. Total fluorescence yield, as a function of bioactivity, was optimized in vitro, and final performance was assessed in vivo. In liver, intestine and heart animal models of I/R, an optimal signal to background ratio was obtained 30 min after intravenous injection of annexin800, and histology confirmed concordance between planar reflectance images and actual deep tissue injury. In summary, annexin800 permits sensitive, real-time detection of cell injury and cell death after I/R in the intraoperative setting, and can be used during a variety of surgeries for rapid assessment of tissue and organ status. PMID:16869796

Emerging concepts and future challenges in innate lymphoid cell biology

PubMed Central

Artis, David

2016-01-01

Innate lymphoid cells (ILCs) are innate immune cells that are ubiquitously distributed in lymphoid and nonlymphoid tissues and enriched at mucosal and barrier surfaces. Three major ILC subsets are recognized in mice and humans. Each of these subsets interacts with innate and adaptive immune cells and integrates cues from the epithelium, the microbiota, and pathogens to regulate inflammation, immunity, tissue repair, and metabolic homeostasis. Although intense study has elucidated many aspects of ILC development, phenotype, and function, numerous challenges remain in the field of ILC biology. In particular, recent work has highlighted key new questions regarding how these cells communicate with their environment and other cell types during health and disease. This review summarizes new findings in this rapidly developing field that showcase the critical role ILCs play in directing immune responses through their ability to interact with a variety of hematopoietic and nonhematopoietic cells. In addition, we define remaining challenges and emerging questions facing the field. Finally, this review discusses the potential application of basic studies of ILC biology to the development of new treatments for human patients with inflammatory and infectious diseases in which ILCs play a role. PMID:27811053

Lentiviral vectors and cardiovascular diseases: a genetic tool for manipulating cardiomyocyte differentiation and function.

PubMed

Di Pasquale, E; Latronico, M V G; Jotti, G S; Condorelli, G

2012-06-01

Engineered recombinant viral vectors are a powerful tool for vehiculating genetic information into mammalian cells. Because of their ability to infect both dividing and non-dividing cells with high efficiency, lentiviral vectors have gained particular interest for basic research and preclinical studies in the cardiovascular field. We review here the major applications for lentiviral-vector technology in the cardiovascular field: we will discuss their use in trailing gene expression during the induction of differentiation, in protocols for the isolation of cardiac cells and in the tracking of cardiac cells after transplantation in vivo; we will also describe lentivirally-mediated gene delivery uses, such as the induction of a phenotype of interest in a target cell or the treatment of cardiovascular diseases. In addition, a section of the review will be dedicated to reprogramming approaches, focusing attention on the generation of pluripotent stem cells and on transdifferentiation, two emerging strategies for the production of cardiac myocytes from human cells and for the investigation of human diseases. Finally, in order to give a perspective on their future clinical use we will critically discuss advantages and disadvantages of lentivirus-based strategies for the treatment of cardiovascular diseases.

Inhibition of Intracellular ROS Accumulation by Formononetin Attenuates Cisplatin-Mediated Apoptosis in LLC-PK1 Cells

PubMed Central

Lee, Haesol; Lee, Dahae; Kang, Ki Sung; Song, Ji Hoon; Choi, You-Kyoung

2018-01-01

Cisplatin is a well-known anticancer drug frequently used for treating solid tumors, including ovarian, testicular, bladder, and cervical tumors. However, usage of cisplatin has been limited because of its adverse effects, particularly nephrotoxicity. Therefore, the present study sought to investigate the protective effect of formononetin against cisplatin-induced cytotoxicity in LLC-PK1 pig kidney epithelial cells as well as the anticancer effect of cisplatin in three different human cervical cancer cell lines, including HeLa, SiHa, and CaSKi cells. We first demonstrated that formononetin strongly prevented cisplatin-induced LLC-PK1 cell death. Although formononetin had no anticancer effect, it did not interrupt the anticancer effect of cisplatin in human cervical carcinoma cell lines. Furthermore, the treatment with formononetin reduced reactive oxygen species (ROS) accumulation and chromatin condensation. The percentage of Annexin V-positive cells also increased following cisplatin treatment. Finally, formononetin-inhibited c-Jun N-terminal kinase (JNK) phosphorylation, cleavage of caspase-8 and caspase-3, and the ratio of Bax to Bcl-2 increased with cisplatin. Taken together, these findings suggest that formononetin may be a possible option to prevent nephrotoxicity induced by cisplatin during treatment for cervical cancer. PMID:29534504

Inhibition of Intracellular ROS Accumulation by Formononetin Attenuates Cisplatin-Mediated Apoptosis in LLC-PK1 Cells.

PubMed

Lee, Haesol; Lee, Dahae; Kang, Ki Sung; Song, Ji Hoon; Choi, You-Kyoung

2018-03-12

Cisplatin is a well-known anticancer drug frequently used for treating solid tumors, including ovarian, testicular, bladder, and cervical tumors. However, usage of cisplatin has been limited because of its adverse effects, particularly nephrotoxicity. Therefore, the present study sought to investigate the protective effect of formononetin against cisplatin-induced cytotoxicity in LLC-PK1 pig kidney epithelial cells as well as the anticancer effect of cisplatin in three different human cervical cancer cell lines, including HeLa, SiHa, and CaSKi cells. We first demonstrated that formononetin strongly prevented cisplatin-induced LLC-PK1 cell death. Although formononetin had no anticancer effect, it did not interrupt the anticancer effect of cisplatin in human cervical carcinoma cell lines. Furthermore, the treatment with formononetin reduced reactive oxygen species (ROS) accumulation and chromatin condensation. The percentage of Annexin V-positive cells also increased following cisplatin treatment. Finally, formononetin-inhibited c-Jun N-terminal kinase (JNK) phosphorylation, cleavage of caspase-8 and caspase-3, and the ratio of Bax to Bcl-2 increased with cisplatin. Taken together, these findings suggest that formononetin may be a possible option to prevent nephrotoxicity induced by cisplatin during treatment for cervical cancer.

In vitro control of human bone marrow stromal cells for bone tissue engineering.

PubMed

Anselme, Karine; Broux, Odile; Noel, Benoit; Bouxin, Bertrand; Bascoulergue, Gerard; Dudermel, Anne-France; Bianchi, Fabien; Jeanfils, Joseph; Hardouin, Pierre

2002-12-01

For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.

Anaerobic Probiotics: The Key Microbes for Human Health.

PubMed

El Enshasy, Hesham; Malik, Khairuddin; Malek, Roslinda Abd; Othman, Nor Zalina; Elsayed, Elsayed Ahmed; Wadaan, Mohammad

Human gastrointestinal microbiota (HGIM) incorporate a large number of microbes from different species. Anaerobic bacteria are the dominant organisms in this microbial consortium and play a crucial role in human health. In addition to their functional role as the main source of many essential metabolites for human health, they are considered as biotherapeutic agents in the regulation of different human metabolites. They are also important in the prevention and in the treatment of different physical and mental diseases. Bifidobacteria are the dominant anaerobic bacteria in HGIM and are widely used in the development of probiotic products for infants, children and adults. To develop bifidobacteria-based bioproducts, therefore, it is necessary to develop a large-scale biomass production platform based on a good understanding of the ideal medium and bioprocessing parameters for their growth and viability. In addition, high cell viability should be maintained during downstream processing and storage of probiotic cell powder or the final formulated product. In this work we review the latest information about the biology, therapeutic activities, cultivation and industrial production of bifidobacteria.

Trace elements during primordial plexiform network formation in human cerebral organoids

PubMed Central

Sartore, Rafaela C.; Cardoso, Simone C.; Lages, Yury V.M.; Paraguassu, Julia M.; Stelling, Mariana P.; Madeiro da Costa, Rodrigo F.; Guimaraes, Marilia Z.; Pérez, Carlos A.

2017-01-01

Systematic studies of micronutrients during brain formation are hindered by restrictions to animal models and adult post-mortem tissues. Recently, advances in stem cell biology have enabled recapitulation of the early stages of human telencephalon development in vitro. In the present work, we analyzed cerebral organoids derived from human pluripotent stem cells by synchrotron radiation X-ray fluorescence in order to measure biologically valuable micronutrients incorporated and distributed into the exogenously developing brain. Our findings indicate that elemental inclusion in organoids is consistent with human brain tissue and involves P, S, K, Ca, Fe and Zn. Occurrence of different concentration gradients also suggests active regulation of elemental transmembrane transport. Finally, the analysis of pairs of elements shows interesting elemental interaction patterns that change from 30 to 45 days of development, suggesting short- or long-term associations, such as storage in similar compartments or relevance for time-dependent biological processes. These findings shed light on which trace elements are important during human brain development and will support studies aimed to unravel the consequences of disrupted metal homeostasis for neurodevelopmental diseases, including those manifested in adulthood. PMID:28194309

Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology

PubMed Central

Saxena, Kapil; Blutt, Sarah E.; Ettayebi, Khalil; Zeng, Xi-Lei; Broughman, James R.; Crawford, Sue E.; Karandikar, Umesh C.; Sastri, Narayan P.; Conner, Margaret E.; Opekun, Antone R.; Graham, David Y.; Qureshi, Waqar; Sherman, Vadim; Foulke-Abel, Jennifer; In, Julie; Kovbasnjuk, Olga; Zachos, Nicholas C.; Donowitz, Mark

2015-01-01

ABSTRACT Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion. IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤5 years of age. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, cellularly diverse, and physiologically relevant epithelial cell cultures that recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions; host and cell type restriction; virus-induced fluid secretion; cell-cell communication within the epithelium; and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures. PMID:26446608

Autophagic flux without a block differentiates varicella-zoster virus infection from herpes simplex virus infection.

PubMed

Buckingham, Erin M; Carpenter, John E; Jackson, Wallen; Zerboni, Leigh; Arvin, Ann M; Grose, Charles

2015-01-06

Autophagy is a process by which misfolded and damaged proteins are sequestered into autophagosomes, before degradation in and recycling from lysosomes. We have extensively studied the role of autophagy in varicella-zoster virus (VZV) infection, and have observed that vesicular cells are filled with >100 autophagosomes that are easily detectable after immunolabeling for the LC3 protein. To confirm our hypothesis that increased autophagosome formation was not secondary to a block, we examined all conditions of VZV infection as well as carrying out two assessments of autophagic flux. We first investigated autophagy in human skin xenografts in the severe combined immunodeficiency (SCID) mouse model of VZV pathogenesis, and observed that autophagosomes were abundant in infected human skin tissues. We next investigated autophagy following infection with sonically prepared cell-free virus in cultured cells. Under these conditions, autophagy was detected in a majority of infected cells, but was much less than that seen after an infected-cell inoculum. In other words, inoculation with lower-titered cell-free virus did not reflect the level of stress to the VZV-infected cell that was seen after inoculation of human skin in the SCID mouse model or monolayers with higher-titered infected cells. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid); both showed no evidence of a block in autophagy. Overall, therefore, autophagy within a VZV-infected cell was remarkably different from autophagy within an HSV-infected cell, whose genome contains two modifiers of autophagy, ICP34.5 and US11, not present in VZV.

Hippo signaling promotes JNK-dependent cell migration.

PubMed

Ma, Xianjue; Wang, Hongxiang; Ji, Jiansong; Xu, Wenyan; Sun, Yihao; Li, Wenzhe; Zhang, Xiaoping; Chen, Juxiang; Xue, Lei

2017-02-21

Overwhelming studies show that dysregulation of the Hippo pathway is positively correlated with cell proliferation, growth, and tumorigenesis. Paradoxically, the detailed molecular roles of the Hippo pathway in cell invasion remain debatable. Using a Drosophila invasion model in wing epithelium, we show herein that activated Hippo signaling promotes cell invasion and epithelial-mesenchymal transition through JNK, as inhibition of JNK signaling dramatically blocked Hippo pathway activation-induced matrix metalloproteinase 1 expression and cell invasion. Furthermore, we identify bantam -Rox8 modules as essential components downstream of Yorkie in mediating JNK-dependent cell invasion. Finally, we confirm that YAP (Yes-associated protein) expression negatively regulates TIA1 (Rox8 ortholog) expression and cell invasion in human cancer cells. Together, these findings provide molecular insights into Hippo pathway-mediated cell invasion and also raise a noteworthy concern in therapeutic interventions of Hippo-related cancers, as simply inhibiting Yorkie or YAP activity might paradoxically accelerate cell invasion and metastasis.

The levels and kinetics of oxygen tension detectable at the surface of human dermal fibroblast cultures.

PubMed

Tokuda, Y; Crane, S; Yamaguchi, Y; Zhou, L; Falanga, V

2000-03-01

Low oxygen tension has recently been shown to stimulate cell growth and clonal expansion, as well as synthesis and transcription of certain growth factors and extracellular matrix components. These results have been obtained by exposing cell cultures to a hypoxic environment. Using an oxygen probe, we have now studied how experimental conditions affect the oxygen tension detectable at the cell surface. Dissolved oxygen tension was directly related to the height of the medium above the cell surface (r = 0.8793, P = 0.021), but was constant when no cells were present in the flask (r = -0. 9732, P = 0.001). In both human dermal fibroblasts and NIH/3T3 cultures, oxygen tension decreased linearly as cell density increased (r = -0.835, P < 0.0001; r = -0.916, P < 0.0001, respectively). When human dermal fibroblasts were exposed to 2% O(2), maximum hypoxic levels (0 mmHg) were achieved within approximately 15 min, and the recovery time was within a similar time frame. The addition of rotenone, an inhibitor of cellular respiration, blocked this decrease in oxygen tension at the cell surface, suggesting that cellular consumption of oxygen is responsible for the decline. Finally, we examined the cell-surface oxygen tension in control and acutely wounded human skin equivalents (HSE), consisting of a keratinocyte layer over a type I collagen matrix containing fibroblasts. We found that oxygen tension dropped significantly (P < 0.0001) in acutely wounded areas of HSE as compared to unwounded areas of HSE and that this drop was prevented by the addition of mitomycin C. These results indicate that cell-surface oxygen tension is indirectly related to cell density, and that the amount of detectable oxygen at the cell surface is a function of cell density, the oxygen tension in the incubator, and increased cellular activity, as occurs after injury. Copyright 2000 Wiley-Liss, Inc.

Establishment and characterization of human osteosarcoma cells resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy.

PubMed

Tao, Yong; Ou, Yunsheng; Yin, Hang; Chen, Yanyang; Zhong, Shenxi; Gao, Yongjian; Zhao, Zenghui; He, Bin; Huang, Qiu; Deng, Qianxing

2017-11-01

The present study was performed to establish and characterize new human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester‑mediated photodynamic therapy (MPPa-PDT). MPPa-PDT-resistant cells are isolated from the human osteosarcoma MG63 and HOS cell lines and two resistant populations were finally acquired, including MG63/PDT and HOS/PDT. Cell Counting Kit-8 (CCK-8) assay was used to determine the MPPa-PDT, cisplatin (CDDP) resistance and proliferation of MG63, MG63/PDT, HOS and HOS/PDT cells. The intracellular ROS were analyzed using DCFH-DA staining. The colony formation, invasion and migration of parental and resistant cells were compared. FCM was employed to examine the cell cycle distribution, the apoptosis rate and the proportion of CD133+ cells. The fluorescence intensity of intracellular MPPa was observed by fluorescence microscopy and quantified using microplate reader. The protein levels were assessed by western blotting (WB). Compared with two parental cells, MG63/PDT and HOS/PDT were 1.67- and 1.61-fold resistant to MPPa-PDT, respectively, and also exhibited the resistance to CDDP. FCM assays confirmed that both MG63/PDT and HOS/PDT cells treated with MPPa-PDT displayed a significantly lower apoptosis rate in comparison with their corresponding parental cells. The expression of apoptosis-related proteins (i.e. cleaved-caspase 3 and cleaved‑PARP), intracellular ROS and the antioxidant proteins (HO-1 and SOD1) in MG63/PDT and HOS/PDT cells was also lower than that in parental cells. Both MG63/PDT and HOS/PDT cells exhibited changes in proliferation, photosensitizer absorption, colony formation, invasion, migration and the cell cycle distribution as compared to MG63 and HOS cells, respectively. Compared to MG63 and HOS cells, both resistant cell lines had a higher expression of CD133, survivin, Bcl-xL, Bcl-2, MRP1, MDR1 and ABCG2, but a lower expression of Bax. The present study successfully established two resistant human osteosarcoma cell lines which are valuable to explore the resistance-related mechanisms and the approaches to overcome resistance.

Establishment and characterization of human osteosarcoma cells resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy

PubMed Central

Tao, Yong; Ou, Yunsheng; Yin, Hang; Chen, Yanyang; Zhong, Shenxi; Gao, Yongjian; Zhao, Zenghui; He, Bin; Huang, Qiu; Deng, Qianxing

2017-01-01

The present study was performed to establish and characterize new human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT). MPPa-PDT-resistant cells are isolated from the human osteosarcoma MG63 and HOS cell lines and two resistant populations were finally acquired, including MG63/PDT and HOS/PDT. Cell Counting Kit-8 (CCK-8) assay was used to determine the MPPa-PDT, cisplatin (CDDP) resistance and proliferation of MG63, MG63/PDT, HOS and HOS/PDT cells. The intracellular ROS were analyzed using DCFH-DA staining. The colony formation, invasion and migration of parental and resistant cells were compared. FCM was employed to examine the cell cycle distribution, the apoptosis rate and the proportion of CD133+ cells. The fluorescence intensity of intracellular MPPa was observed by fluorescence microscopy and quantified using microplate reader. The protein levels were assessed by western blotting (WB). Compared with two parental cells, MG63/PDT and HOS/PDT were 1.67- and 1.61-fold resistant to MPPa-PDT, respectively, and also exhibited the resistance to CDDP. FCM assays confirmed that both MG63/PDT and HOS/PDT cells treated with MPPa-PDT displayed a significantly lower apoptosis rate in comparison with their corresponding parental cells. The expression of apoptosis-related proteins (i.e. cleaved-caspase 3 and cleaved-PARP), intracellular ROS and the antioxidant proteins (HO-1 and SOD1) in MG63/PDT and HOS/PDT cells was also lower than that in parental cells. Both MG63/PDT and HOS/PDT cells exhibited changes in proliferation, photosensitizer absorption, colony formation, invasion, migration and the cell cycle distribution as compared to MG63 and HOS cells, respectively. Compared to MG63 and HOS cells, both resistant cell lines had a higher expression of CD133, survivin, Bcl-xL, Bcl-2, MRP1, MDR1 and ABCG2, but a lower expression of Bax. The present study successfully established two resistant human osteosarcoma cell lines which are valuable to explore the resistance-related mechanisms and the approaches to overcome resistance. PMID:29048645

A cell transportation solution that preserves live circulating tumor cells in patient blood samples.

PubMed

Stefansson, Steingrimur; Adams, Daniel L; Ershler, William B; Le, Huyen; Ho, David H

2016-05-06

Circulating tumor cells (CTCs) are typically collected into CellSave fixative tubes, which kills the cells, but preserves their morphology. Currently, the clinical utility of CTCs is mostly limited to their enumeration. More detailed investigation of CTC biology can be performed on live cells, but obtaining live CTCs is technically challenging, requiring blood collection into biocompatible solutions and rapid isolation which limits transportation options. To overcome the instability of CTCs, we formulated a sugar based cell transportation solution (SBTS) that stabilizes cell viability at ambient temperature. In this study we examined the long term viability of human cancer cell lines, primary cells and CTCs in human blood samples in the SBTS for transportation purposes. Four cell lines, 5 primary human cells and purified human PBMCs were tested to determine the viability of cells stored in the transportation solution at ambient temperature for up to 7 days. We then demonstrated viability of MCF-7 cells spiked into normal blood with SBTS and stored for up to 7 days. A pilot study was then run on blood samples from 3 patients with metastatic malignancies stored with or without SBTS for 6 days. CTCs were then purified by Ficoll separation/microfilter isolation and identified using CTC markers. Cell viability was assessed using trypan blue or CellTracker™ live cell stain. Our results suggest that primary/immortalized cell lines stored in SBTS remain ~90% viable for > 72 h. Further, MCF-7 cells spiked into whole blood remain viable when stored with SBTS for up to 7 days. Finally, live CTCs were isolated from cancer patient blood samples kept in SBTS at ambient temperature for 6 days. No CTCs were isolated from blood samples stored without SBTS. In this proof of principle pilot study we show that viability of cell lines is preserved for days using SBTS. Further, this solution can be used to store patient derived blood samples for eventual isolation of viable CTCs after days of storage. Therefore, we suggest an effective and economical transportation of cancer patient blood samples containing live CTCs can be achieved.

Organ-On-A-Chip Platforms: A Convergence of Advanced Materials, Cells, and Microscale Technologies.

PubMed

Ahadian, Samad; Civitarese, Robert; Bannerman, Dawn; Mohammadi, Mohammad Hossein; Lu, Rick; Wang, Erika; Davenport-Huyer, Locke; Lai, Ben; Zhang, Boyang; Zhao, Yimu; Mandla, Serena; Korolj, Anastasia; Radisic, Milica

2018-01-01

Significant advances in biomaterials, stem cell biology, and microscale technologies have enabled the fabrication of biologically relevant tissues and organs. Such tissues and organs, referred to as organ-on-a-chip (OOC) platforms, have emerged as a powerful tool in tissue analysis and disease modeling for biological and pharmacological applications. A variety of biomaterials are used in tissue fabrication providing multiple biological, structural, and mechanical cues in the regulation of cell behavior and tissue morphogenesis. Cells derived from humans enable the fabrication of personalized OOC platforms. Microscale technologies are specifically helpful in providing physiological microenvironments for tissues and organs. In this review, biomaterials, cells, and microscale technologies are described as essential components to construct OOC platforms. The latest developments in OOC platforms (e.g., liver, skeletal muscle, cardiac, cancer, lung, skin, bone, and brain) are then discussed as functional tools in simulating human physiology and metabolism. Future perspectives and major challenges in the development of OOC platforms toward accelerating clinical studies of drug discovery are finally highlighted. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Suppression of intestinal carcinogenesis in Apc-mutant mice by limonin.

PubMed

Shimizu, Satomi; Miyamoto, Shingo; Fujii, Gen; Nakanishi, Ruri; Onuma, Wakana; Ozaki, Yoshihiko; Fujimoto, Kyoko; Yano, Tomohiro; Mutoh, Michihiro

2015-07-01

Limonoids in citrus fruits are known to possess multiple biological functions, such as anti-proliferative functions in human cancer cell lines. Therefore, we aimed to investigate the suppressive effect of limonin on intestinal polyp development in Apc-mutant Min mice. Five-week-old female Min mice were fed a basal diet or a diet containing 250 or 500 ppm limonin for 8 weeks. The total number of polyps in mice treated with 500 ppm limonin decreased to 74% of the untreated control value. Neoplastic cell proliferation in the polyp parts was assessed by counting PCNA positive cells, and a tendency of reduction was obtained by limonin treatment. Moreover, expression levels of c-Myc and MCP-1 mRNA in the polyp part were reduced by administration of limonin. We finally confirmed the effects of limonin on β-catenin signaling, and found limonin significantly inhibited T-cell factor/lymphocyte enhancer factor-dependent transcriptional activity in a dose-dependent manner in the Caco-2 human colon cancer cell line. Our results suggest that limonin might be a candidate chemopreventive agent against intestinal carcinogenesis.

Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems.

PubMed

Badenes, Sara M; Fernandes, Tiago G; Rodrigues, Carlos A V; Diogo, Maria Margarida; Cabral, Joaquim M S

2016-09-20

Human pluripotent stem cells (hPSC) have attracted a great attention as an unlimited source of cells for cell therapies and other in vitro biomedical applications such as drug screening, toxicology assays and disease modeling. The implementation of scalable culture platforms for the large-scale production of hPSC and their derivatives is mandatory to fulfill the requirement of obtaining large numbers of cells for these applications. Microcarrier technology has been emerging as an effective approach for the large scale ex vivo hPSC expansion and differentiation. This review presents recent achievements in hPSC microcarrier-based culture systems and discusses the crucial aspects that influence the performance of these culture platforms. Recent progress includes addressing chemically-defined culture conditions for manufacturing of hPSC and their derivatives, with the development of xeno-free media and microcarrier coatings to meet good manufacturing practice (GMP) quality requirements. Finally, examples of integrated platforms including hPSC expansion and directed differentiation to specific lineages are also presented in this review. Copyright © 2016 Elsevier B.V. All rights reserved.

Human rotavirus strain Wa downregulates NHE1 and NHE6 expressions in rotavirus-infected Caco-2 cells.

PubMed

Chen, Honglang; Song, Lijun; Li, Guixian; Chen, Wenfeng; Zhao, Shumin; Zhou, Ruoxia; Shi, Xiaoying; Peng, Zhenying; Zhao, Wenchang

2017-06-01

Rotavirus (RV) is the most common cause of severe gastroenteritis and fatal dehydration in human infants and neonates of different species. However, the pathogenesis of rotavirus-induced diarrhea is poorly understood. Secretory diarrhea caused by rotavirus may lead to a combination of excessive secretion of fluid and electrolytes into the intestinal lumen. Fluid absorption in the small intestine is driven by Na + -coupled transport mechanisms at the luminal membrane, including Na + /H + exchanger (NHE). Here, we performed qRT-PCR to detect the transcription of NHEs. Western blotting was employed for protein detection. Furthermore, immunocytochemistry was used to validate the NHE's protein expression. Finally, intracellular Ca 2+ concentration was detected by confocal laser scanning microscopy. The results demonstrated that the NHE6 mRNA and protein expressed in the human colon adenocarcinoma cell line (Caco-2). Furthermore, RV-Wa induced decreased expression of the NHE1 and NHE6 in Caco-2 cell in a time-dependent manner. In addition, intracellular Ca 2+ concentration in RV-Wa-infected Caco-2 cells was higher than that in the mock-infected cells. Furthermore, RV-Wa also can downregulate the expression of calmodulin (CaM) and calmodulin kinase II (CaMKII) in Caco-2 cells. These findings provides important insights into the mechanisms of rotavirus-induced diarrhea. Further studies on the underlying pathophysiological mechanisms that downregulate NHEs in RV-induced diarrhea are required.

Targeting IL11 Receptor in Leukemia and Lymphoma: A Functional Ligand-Directed Study and Hematopathology Analysis of Patient-Derived Specimens.

PubMed

Karjalainen, Katja; Jaalouk, Diana E; Bueso-Ramos, Carlos; Bover, Laura; Sun, Yan; Kuniyasu, Akihiko; Driessen, Wouter H P; Cardó-Vila, Marina; Rietz, Cecilia; Zurita, Amado J; O'Brien, Susan; Kantarjian, Hagop M; Cortes, Jorge E; Calin, George A; Koivunen, Erkki; Arap, Wadih; Pasqualini, Renata

2015-07-01

The IL11 receptor (IL11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors, such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here, we evaluated the IL11R as a candidate therapeutic target in human leukemia and lymphoma. First, we show that the IL11R protein is expressed in a variety of human leukemia- and lymphoma-derived cell lines and in a large panel of bone marrow samples from leukemia and lymphoma patients, whereas expression is absent from nonmalignant control bone marrow. Moreover, a targeted peptidomimetic prototype (termed BMTP-11), specifically bound to leukemia and lymphoma cell membranes, induced ligand-receptor internalization mediated by the IL11R, and resulted in a specific dose-dependent cell death induction in these cells. Finally, a pilot drug lead-optimization program yielded a new myristoylated BMTP-11 analogue with an apparent improved antileukemia cell profile. These results indicate (i) that the IL11R is a suitable cell surface target for ligand-directed applications in human leukemia and lymphoma and (ii) that BMTP-11 and its derivatives have translational potential against this group of malignant diseases. ©2015 American Association for Cancer Research.

Targeting interleukin-11 receptor in leukemia and lymphoma: A functional ligand-directed study and hematopathology analysis of patient-derived specimens

PubMed Central

Karjalainen, Katja; Jaalouk, Diana E.; Bueso-Ramos, Carlos; Bover, Laura; Sun, Yan; Kuniyasu, Akihiko; Driessen, Wouter H. P.; Cardó-Vila, Marina; Rietz, Cecilia; Zurita, Amado J.; O’Brien, Susan; Kantarjian, Hagop M.; Cortes, Jorge E.; Calin, George A.; Koivunen, Erkki; Arap, Wadih; Pasqualini, Renata

2015-01-01

Purpose The interleukin-11 receptor (IL-11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here we evaluated the IL-11R as a candidate therapeutic target in human leukemia and lymphoma. Experimental Design and Results First, we show that the IL-11R protein is expressed in a variety of human leukemia- and lymphoma derived cell lines and in a large panel of bone marrow samples from leukemia and lymphoma patients, while expression is absent from non-malignant control bone marrow. Moreover, a targeted peptidomimetic prototype (termed BMTP-11) specifically bound to leukemia and lymphoma cell membranes, induced ligand-receptor internalization mediated by the IL-11R, and resulted in a specific dose-dependent cell death induction in these cells. Finally, a pilot drug lead-optimization program yielded a new myristoylated BMTP-11 analog with an apparent improved anti-leukemia cell profile. Conclusion These results indicate (i) that the IL-11R is a suitable cell surface target for ligand-directed applications in human leukemia and lymphoma and (ii) that BMTP-11 and its derivatives have translational potential against this group of malignant diseases. PMID:25779950

Immunomodulating and anti-allergic effects of Negroamaro and Koshu Vitis vinifera fermented grape marc (FGM).

PubMed

Marzulli, Giuseppe; Magrone, Thea; Vonghia, Luisa; Kaneko, Masahiro; Takimoto, Hiroaki; Kumazawa, Yoshio; Jirillo, Emilio

2014-01-01

Polyphenols contained in FGM from Negroamaro (N) and Koshu (K) Vitis vinifera have been shown to exhibit several immunomodulating activities. For instance, mice affected by experimental colitis when administered with K-FGM showed an attenuation of the inflammatory process. In murine asthma, K-FGM reduced IgE production and eosinophil number in bronchial alveolar lavage fluid. In vitro, both N- and K-FGM were able to induce T regulatory cells in terms of Foxp-3 molecule expression and release of interleukin-10. In another set of experiments both N- and K-FGM were able to balance rate of proliferation/apoptosis/necrosis of normal human peripheral lymphocytes, thus indicating the property of these compounds to maintain immune homeostatic mechanisms in the host. On the other hand, N- and K-FGM inhibited human basophil degranulation, thus, confirming our previous results obtained with rat basophilic leukemia cells. Finally, N- and K-FGM also decreased oxidative burst of human polymorphonuclear cells and monocytes.Taken together, these findings imply the potential clinical usefulness of FGM administration in inflammatory/allergic conditions, such as chronic asthma.

Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

PubMed

Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

2011-04-01

The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

D-tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity.

PubMed

Park, Jisu; Jung, Hyejung; Kim, Kyuri; Lim, Kyung-Min; Kim, Ji-Young; Jho, Eek-Hoon; Oh, Eok-Soo

2018-05-01

Although L-tyrosine is well known for its melanogenic effect, the contribution of D-tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L-tyrosine, D-tyrosine dose-dependently reduced the melanin contents of human MNT-1 melanoma cells and primary human melanocytes. In addition, 500 μM of D-tyrosine completely inhibited 10 μM L-tyrosine-induced melanogenesis, and both in vitro assays and L-DOPA staining MNT-1 cells showed that tyrosinase activity is reduced by D-tyrosine treatment. Thus, D-tyrosine appears to inhibit L-tyrosine-mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D-tyrosine inhibited melanogenesis induced by α-MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D-tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D-tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte-derived cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Grb2-SH3 ligand inhibits the growth of HER2+ cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel.

PubMed

Gril, Brunilde; Vidal, Michel; Assayag, Franck; Poupon, Marie-France; Liu, Wang-Qing; Garbay, Christiane

2007-07-15

HER2 represents an important signaling pathway in breast and other cancers. Herceptin has demonstrated clinical activity, but resistance is common. Thus, new therapeutic approaches to the HER2 pathway are needed. Grb2 is an adaptor protein involved in Ras-dependent signaling induced by HER2 receptors. A specific Grb2-SH3 ligand, designed and synthesized in our laboratory, called peptidimer-c, inhibited colony formation in HER2 overexpressing SKBr3 cancer cells. Combined treatment of peptidimer-c with docetaxel further inhibited both colony formation and tumor cell survival compared to docetaxel treatment alone. Efficacy of this combined treatment was correlated with a reduction in the phosphorylation of MAPK and AKT. Finally, peptidimer-c reduced the growth of a HER2(+) human breast cancer (BK111) xenograft in nude mice and potentiated the antitumor effect of docetaxel in a HER2+ hormone-independent human prostate adenocarcinoma (PAC120 HID28) xenograft. These results validate Grb2 as a new target for the HER2 pathway. (c) 2007 Wiley-Liss, Inc.

Grb2-SH3 ligand inhibits the growth of HER2+ cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel

PubMed Central

Gril, Brunilde; Vidal, Michel; Assayag, Franck; Poupon, Marie-France; Liu, Wang-Qing; Garbay, Christiane

2007-01-01

HER2 represents an important signaling pathway in breast and other cancers. Herceptin has demonstrated clinical activity, but resistance is common. Thus, new therapeutic approaches to the HER2 pathway are needed. Grb2 is an adaptor protein involved in Ras-dependent signaling induced by HER2 receptors. A specific Grb2-SH3 ligand, designed and synthesized in our laboratory, called peptidimer-c, inhibited colony formation in HER2 over-expressing SKBr3 cancer cells. Combined treatment of peptidimer-c with docetaxel further inhibited both colony formation and tumor cell survival compared to docetaxel treatment alone. Efficacy of this combined treatment was correlated with a reduction in the phosphorylation of MAPK and AKT. Finally, peptidimer-c reduced the growth of a HER2+ human breast cancer (BK111) xenograft in nude mice and potentiated the anti-tumor effect of docetaxel in a HER2+ hormone-independent human prostate adenocarcinoma (PAC120 HID28) xenograft. These results validate Grb2 as a new target for the HER2 pathway. PMID:17372910

Establishment and characterization of Asian oral cancer cell lines as in vitro models to study a disease prevalent in Asia.

PubMed

Hamid, Sharifah; Lim, Kue Peng; Zain, Rosnah Binti; Ismail, Siti Mazlipah; Lau, Shin Hin; Mustafa, Wan Mahadzir Wan; Abraham, M Thomas; Nam, Noor Akmar; Teo, Soo-Hwang; Cheong, Sok Ching

2007-03-01

We have established 3 cell lines ORL-48, -115 and -136 from surgically resected specimens obtained from untreated primary human oral squamous cell carcinomas of the oral cavity. The in vitro growth characteristics, epithelial origin, in vitro anchorage independency, human papilloma-virus (HPV) infection, microsatellite instability status, karyotype and the status of various cell cycle regulators and gatekeepers of these cell lines were investigated. All 3 cell lines grew as monolayers with doubling times ranging between 26.4 and 40.8 h and were immortal. Karyotyping confirmed that these cell lines were of human origin with multiple random losses and gains of entire chromosomes and regions of chromosomes. Immunohistochemistry staining of cytokeratins confirmed the epithelial origin of these cell lines, and the low degree of anchorage independency expressed by these cell lines suggests non-transformed phenotypes. Genetic analysis identified mutations in the p53 gene in all cell lines and hypermethylation of p16INK4a in ORL-48 and -136. Analysis of MDM2 and EGFR expression indicated MDM2 overexpression in ORL-48 and EGFR overexpression in ORL-136 in comparison to the protein levels in normal oral keratinocytes. Analysis of the BAT-26 polyadenine repeat sequence and MLH-1 and MSH-2 repair enzymes demonstrated that all 3 cell lines were microsatellite stable. The role of HPV in driving carcinogenesis in these tumours was negated by the absence of HPV. Finally, analysis of the tissues from which these cell lines were derived indicated that the cell lines were genetically representative of the tumours, and, therefore, are useful tools in the understanding of the molecular changes associated with oral cancers.

Irradiated and activated autologous PBMCs induce expansion of highly cytotoxic human NK cells in vitro.

PubMed

Ahn, Yong-Oon; Kim, Saerom; Kim, Tae Min; Song, Eun Young; Park, Myoung Hee; Heo, Dae Seog

2013-09-01

Adoptive cell transfer of ex vivo-activated natural killer (NK) cells is a promising therapy for cancer treatment. Because of inhibitory signaling through killer immunoglobulin-like receptor (KIR)-KIR ligands, KIR-mismatched allogeneic NK cell transfer is considered to be a more effective strategy than is autologous transfer. However, purified NK cells do not expand well enough in vitro with good manufacturing practice-compliant components for clinical use. Some investigators have developed selective expansion of NK cells from peripheral blood mononuclear cells, but these cells have the risk of graft-versus-host disease in allogeneic settings because of T cells contamination. In this study, we developed a novel method for NK cell activation and expansion. Using only good manufacturing practice-compliant components and autologous feeder cells, once purified NK cells were effectively expanded (2500-fold at day 17). The expanded cells were highly purified NK cells, and the use of these cells is suitable for allogeneic transfer without the risk of graft-versus-host disease induction. Importantly, the expanded NK cells also showed enhanced cytotoxicity compared with NK cells conventionally expanded by recombinant human interleukin 2. Finally, induction of NKG2D ligand expression on feeder cells implies that the NKG2D-NKG2DL interaction may play a role in NK cell expansion. In conclusion, this method can be used to obtain NK cells for more successful allogeneic NK cell adoptive transfer for use in antitumor immune therapy.

Phenotype of NK-Like CD8(+) T Cells with Innate Features in Humans and Their Relevance in Cancer Diseases

PubMed Central

Barbarin, Alice; Cayssials, Emilie; Jacomet, Florence; Nunez, Nicolas Gonzalo; Basbous, Sara; Lefèvre, Lucie; Abdallah, Myriam; Piccirilli, Nathalie; Morin, Benjamin; Lavoue, Vincent; Catros, Véronique; Piaggio, Eliane; Herbelin, André; Gombert, Jean-Marc

2017-01-01

Unconventional T cells are defined by their capacity to respond to signals other than the well-known complex of peptides and major histocompatibility complex proteins. Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells in the mouse were discovered in the early 2000s. This subset of CD8(+) T cells bears a memory phenotype without having encountered a foreign antigen and can respond to innate-like IL-12 + IL-18 stimulation. Although the concept of innate memory CD8(+) T cells is now well established in mice, whether an equivalent memory NK-like T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells responding to innate-like IL-12 + IL-18 stimulation and co-expressing the transcription factor Eomesodermin (Eomes) and KIR/NKG2A membrane receptors with a memory/EMRA phenotype may represent a new, functionally distinct innate T cell subset in humans. In this review, after a summary on the known innate CD8(+) T-cell features in the mouse, we propose Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Next, we discuss IL-4 and IL-15 involvement in the generation of innate CD8(+) T cells and particularly its possible dependency on the promyelocytic leukemia zinc-finger factor expressing iNKT cells, an innate T cell subset well documented for its susceptibility to tumor immune subversion. After that, focusing on cancer diseases, we provide new insights into the potential role of these innate CD8(+) T cells in a physiopathological context in humans. Based on empirical data obtained in cases of chronic myeloid leukemia, a myeloproliferative syndrome controlled by the immune system, and in solid tumors, we observe both the possible contribution of innate CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we note that during tumor progression, innate CD8(+) T lymphocytes could be controlled by immune checkpoints. This study significantly contributes to understanding of the role of NK-like CD8(+) T cells and raises the question of the possible involvement of an iNKT/innate CD8(+) T cell axis in cancer. PMID:28396661

Unique and shared inflammatory profiles of human brain endothelia and pericytes.

PubMed

Smyth, Leon C D; Rustenhoven, Justin; Park, Thomas I-H; Schweder, Patrick; Jansson, Deidre; Heppner, Peter A; O'Carroll, Simon J; Mee, Edward W; Faull, Richard L M; Curtis, Maurice; Dragunow, Mike

2018-05-11

Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed. To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1β, TNFα, LPS, IFN-γ, TGF-β 1 , IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1β. Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1β. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1β, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro. Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.

Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoyer, Friedrich Felix; Paul, Kathrin; Heiermann, Nadine; Becher, Marc Ulrich; Abu Hussein, Nebal; Kebschull, Moritz; Bedorf, Jörg; Franklin, Bernardo S; Latz, Eicke; Nickenig, Georg; Werner, Nikos

2012-08-01

Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I-downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor-dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.

Human Papillomavirus Infections and Cancer Stem Cells of Tumors from the Uterine Cervix

PubMed Central

López, Jacqueline; Ruíz, Graciela; Organista-Nava, Jorge; Gariglio, Patricio; García-Carrancá, Alejandro

2012-01-01

Different rate of development of productive infections (as low grade cervical intraepithelial neoplasias), or high grade lesions and cervical malignant tumors associated with infections of the Transformation zone (TZ) by High-Risk Human Papillomavirus (HR-HPV), could suggest that different epithelial host target cells could exist. If there is more than one target cell, their differential infection by HR-HPV may play a central role in the development of cervical cancer. Recently, the concept that cancer might arise from a rare population of cells with stem cell-like properties has received support in several solid tumors, including cervical cancer (CC). According to the cancer stem cell (CSC) hypothesis, CC can now be considered a disease in which stem cells of the TZ are converted to cervical cancer stem cells by the interplay between HR-HPV viral oncogenes and cellular alterations that are thought to be finally responsible for tumor initiation and maintenance. Current studies of CSC could provide novel insights regarding tumor initiation and progression, their relation with viral proteins and interplay with the tumor micro-environment. This review will focus on the biology of cervical cancer stem cells, which might contribute to our understanding of the mechanisms responsible for cervical tumor development. PMID:23341858

Regulation of hematogenous tumor metastasis by acid sphingomyelinase

PubMed Central

Carpinteiro, Alexander; Becker, Katrin Anne; Japtok, Lukasz; Hessler, Gabriele; Keitsch, Simone; Požgajovà, Miroslava; Schmid, Kurt W; Adams, Constantin; Müller, Stefan; Kleuser, Burkhard; Edwards, Michael J; Grassmé, Heike; Helfrich, Iris; Gulbins, Erich

2015-01-01

Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1−/− mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of α5β1 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing β1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis. PMID:25851537

Immunohistochemical Analysis of Connexin43 Expression in Infertile Human Testes

PubMed Central

Matsuo, Yuzo; Nomata, Koichiro; Eguchi, Jiro; Aoki, Daiyu; Hayashi, Tomayoshi; Hishikawa, Yoshitaka; Kanetake, Hiroshi; Shibata, Yoshisada; Koji, Takehiko

2007-01-01

Connexin43 (Cx43) is abundantly expressed in mammalian testes and implicated in the regulation of cell-to-cell interaction between germ cells and Sertoli cells, which is essential to the normal process of spermatogenesis. In the present study, we investigated the relation between Cx43 expression and the degree of spermatogenesis in infertile human testes. Immunohistochemical analysis of Cx43 was performed on testicular biopsies from 29 patients with azoospermia (n=23) and severe oligospermia (n=6), who gave informed consent to this experiment. The degree of testicular spermatogenesis was evaluated by Johnsen score. In the interstitium, immunostaining for Cx43 was localized to some focal parts of plasma membrane between neighboring Leydig cells. In seminiferous tubules with normal spermatogenesis, Cx43 expression was found between Sertoli cells and germ cells. However, Cx43 expression in maturation arrest was decreased and located mainly in the basal compartment of seminiferous tubules. Finally, there was a significant positive correlation between histological score of spermatogenesis and intensity of Cx43 (p=0.0294). These data suggest that the alteration of Cx43 expression may be involved in spermatogenic impairment, and that the communication between Sertoli cells and germ cells through Cx43 may be important for maturation of spermatogenesis. PMID:17653298

Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy.

PubMed

Estrada, J C; Torres, Y; Benguría, A; Dopazo, A; Roche, E; Carrera-Quintanar, L; Pérez, R A; Enríquez, J A; Torres, R; Ramírez, J C; Samper, E; Bernad, A

2013-06-27

In most clinical trials, human mesenchymal stem cells (hMSCs) are expanded in vitro before implantation. The genetic stability of human stem cells is critical for their clinical use. However, the relationship between stem-cell expansion and genetic stability is poorly understood. Here, we demonstrate that within the normal expansion period, hMSC cultures show a high percentage of aneuploid cells that progressively increases until senescence. Despite this accumulation, we show that in a heterogeneous culture the senescence-prone hMSC subpopulation has a lower proliferation potential and a higher incidence of aneuploidy than the non-senescent subpopulation. We further show that senescence is linked to a novel transcriptional signature that includes a set of genes implicated in ploidy control. Overexpression of the telomerase catalytic subunit (human telomerase reverse transcriptase, hTERT) inhibited senescence, markedly reducing the levels of aneuploidy and preventing the dysregulation of ploidy-controlling genes. hMSC-replicative senescence was accompanied by an increase in oxygen consumption rate (OCR) and oxidative stress, but in long-term cultures that overexpress hTERT, these parameters were maintained at basal levels, comparable to unmodified hMSCs at initial passages. We therefore propose that hTERT contributes to genetic stability through its classical telomere maintenance function and also by reducing the levels of oxidative stress, possibly, by controlling mitochondrial physiology. Finally, we propose that aneuploidy is a relevant factor in the induction of senescence and should be assessed in hMSCs before their clinical use.

A comprehensive gene expression analysis at sequential stages of in vitro cardiac differentiation from isolated MESP1-expressing-mesoderm progenitors

PubMed Central

den Hartogh, Sabine C.; Wolstencroft, Katherine; Mummery, Christine L.; Passier, Robert

2016-01-01

In vitro cardiac differentiation of human pluripotent stem cells (hPSCs) closely recapitulates in vivo embryonic heart development, and therefore, provides an excellent model to study human cardiac development. We recently generated the dual cardiac fluorescent reporter MESP1mCherry/wNKX2-5eGFP/w line in human embryonic stem cells (hESCs), allowing the visualization of pre-cardiac MESP1+ mesoderm and their further commitment towards the cardiac lineage, marked by activation of the cardiac transcription factor NKX2-5. Here, we performed a comprehensive whole genome based transcriptome analysis of MESP1-mCherry derived cardiac-committed cells. In addition to previously described cardiac-inducing signalling pathways, we identified novel transcriptional and signalling networks indicated by transient activation and interactive network analysis. Furthermore, we found a highly dynamic regulation of extracellular matrix components, suggesting the importance to create a versatile niche, adjusting to various stages of cardiac differentiation. Finally, we identified cell surface markers for cardiac progenitors, such as the Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4), belonging to the same subfamily of LGR5, and LGR6, established tissue/cancer stem cells markers. We provide a comprehensive gene expression analysis of cardiac derivatives from pre-cardiac MESP1-progenitors that will contribute to a better understanding of the key regulators, pathways and markers involved in human cardiac differentiation and development. PMID:26783251

Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks.

PubMed

Sorkio, Anni; Koch, Lothar; Koivusalo, Laura; Deiwick, Andrea; Miettinen, Susanna; Chichkov, Boris; Skottman, Heli

2018-07-01

There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Plasmid fermentation process for DNA immunization applications.

PubMed

Carnes, Aaron E; Williams, James A

2014-01-01

Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.

Imaging cells and sub-cellular structures with ultrahigh resolution full-field X-ray microscopy.

PubMed

Chien, C C; Tseng, P Y; Chen, H H; Hua, T E; Chen, S T; Chen, Y Y; Leng, W H; Wang, C H; Hwu, Y; Yin, G C; Liang, K S; Chen, F R; Chu, Y S; Yeh, H I; Yang, Y C; Yang, C S; Zhang, G L; Je, J H; Margaritondo, G

2013-01-01

Our experimental results demonstrate that full-field hard-X-ray microscopy is finally able to investigate the internal structure of cells in tissues. This result was made possible by three main factors: the use of a coherent (synchrotron) source of X-rays, the exploitation of contrast mechanisms based on the real part of the refractive index and the magnification provided by high-resolution Fresnel zone-plate objectives. We specifically obtained high-quality microradiographs of human and mouse cells with 29 nm Rayleigh spatial resolution and verified that tomographic reconstruction could be implemented with a final resolution level suitable for subcellular features. We also demonstrated that a phase retrieval method based on a wave propagation algorithm could yield good subcellular images starting from a series of defocused microradiographs. The concluding discussion compares cellular and subcellular hard-X-ray microradiology with other techniques and evaluates its potential impact on biomedical research. Copyright © 2012 Elsevier Inc. All rights reserved.

Soybean extracts increase cell surface ZIP4 abundance and cellular zinc levels: a potential novel strategy to enhance zinc absorption by ZIP4 targeting.

PubMed

Hashimoto, Ayako; Ohkura, Katsuma; Takahashi, Masakazu; Kizu, Kumiko; Narita, Hiroshi; Enomoto, Shuichi; Miyamae, Yusaku; Masuda, Seiji; Nagao, Masaya; Irie, Kazuhiro; Ohigashi, Hajime; Andrews, Glen K; Kambe, Taiho

2015-12-01

Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans. © 2015 Authors; published by Portland Press Limited.

Adenoid basal hyperplasia of the uterine cervix: a lesion of reserve cell type, distinct from adenoid basal carcinoma.

PubMed

Kerdraon, Olivier; Cornélius, Aurélie; Farine, Marie-Odile; Boulanger, Loïc; Wacrenier, Agnès

2012-12-01

Adenoid basal hyperplasia is an underrecognized cervical lesion, resembling adenoid basal carcinoma, except the absence of deep invasion into the stroma. We report a series of 10 cases, all extending less than 1 mm from the basement membrane. Our results support the hypothesis that adenoid basal hyperplasia arises from reserve cells of the cervix. Lesions were found close to the squamocolumnar junction, in continuity with the nearby subcolumnar reserve cells. They shared the same morphology and immunoprofile using a panel of 4 antibodies (keratin 5/6, keratin 14, keratin 7 and p63) designed to differentiate reserve cells from mature squamous cells and endocervical columnar cells. We detected no human papillomavirus infection by in situ hybridization targeting high-risk human papillomavirus, which was concordant with the absence of immunohistochemical p16 expression. We demonstrated human papillomavirus infection in 4 (80%) of 5 adenoid basal carcinoma, which is in the same range as previous studies (88%). Thus, adenoid basal hyperplasia should be distinguished from adenoid basal carcinoma because they imply different risk of human papillomavirus infection and of subsequent association with high-grade invasive carcinoma. In our series, the most reliable morphological parameters to differentiate adenoid basal hyperplasia from adenoid basal carcinoma were the depth of the lesion and the size of the lesion nests. Furthermore, squamous differentiation was rare in adenoid basal hyperplasia and constant in adenoid basal carcinoma. Finally, any mitotic activity and/or an increase of Ki67 labeling index should raise the hypothesis of adenoid basal carcinoma. Copyright © 2012 Elsevier Inc. All rights reserved.

Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

PubMed Central

Balagué, Cristina; Noya, Francisco; Alemany, Ramon; Chow, Louise T.; Curiel, David T.

2001-01-01

Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells. PMID:11462032

Bioinformatics Symposium of the Analytical Division of the American Chemical Society Meeting. Final Technical Report from 03/15/2000 to 03/14/2001 [sample pages of agenda, abstracts, index

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kennedy, Robert T.

Sparked by the Human Genome Project, biological and biomedical research has become an information science. Information tools are now being generated for proteins, cell modeling, and genomics. The opportunity for analytical chemistry in this new environment is profound. New analytical techniques that can provide the information on genes, SNPs, proteins, protein modifications, cells, and cell chemistry are required. In this symposium, we brought together both informatics experts and leading analytical chemists to discuss this interface. Over 200 people attended this highly successful symposium.

Apoptosis triggered by pyropheophorbide-α methyl ester-mediated photodynamic therapy in a giant cell tumor in bone

NASA Astrophysics Data System (ADS)

Li, K.-T.; Zhang, J.; Duan, Q.-Q.; Bi, Y.; Bai, D.-Q.; Ou, Y.-S.

2014-06-01

A giant cell tumor in bone is the common primary bone tumor with aggressive features, occurring mainly in young adults. Photodynamic therapy is a new therapeutic technique for tumors. In this study, we investigated the effects of Pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy on the proliferation of giant cell tumor cells and its mechanism of action. Cell proliferation was evaluated using an MTT assay. Cellular apoptosis was detected by Hoechst nuclear staining, and flow cytometric assay. Mitochondrial membrane potential changes and cytochrome c, caspase-9, caspase-3, and Bcl-2 expression was assessed. Finally, we found that MPPa-mediated photodynamic therapy could effectively suppress the proliferation of human giant cell tumor cells and induce apoptosis. The mitochondrial pathway was involved in the MPPa-photodynamic therapy-induced apoptosis.

Cell nuclei segmentation in fluorescence microscopy images using inter- and intra-region discriminative information.

PubMed

Song, Yang; Cai, Weidong; Feng, David Dagan; Chen, Mei

2013-01-01

Automated segmentation of cell nuclei in microscopic images is critical to high throughput analysis of the ever increasing amount of data. Although cell nuclei are generally visually distinguishable for human, automated segmentation faces challenges when there is significant intensity inhomogeneity among cell nuclei or in the background. In this paper, we propose an effective method for automated cell nucleus segmentation using a three-step approach. It first obtains an initial segmentation by extracting salient regions in the image, then reduces false positives using inter-region feature discrimination, and finally refines the boundary of the cell nuclei using intra-region contrast information. This method has been evaluated on two publicly available datasets of fluorescence microscopic images with 4009 cells, and has achieved superior performance compared to popular state of the art methods using established metrics.

Embryonic stem cells and the next generation of developmental toxicity testing.

PubMed

Kugler, Josephine; Huhse, Bettina; Tralau, Tewes; Luch, Andreas

2017-08-01

The advent of stem cell technology has seen the establishment of embryonic stem cells (ESCs) as molecular model systems and screening tools. Although ESCs are nowadays widely used in research, regulatory implementation for developmental toxicity testing is pending. Areas Covered: This review evaluates the performance of current ESC, including human (h)ESC testing systems, trying to elucidate their potential for developmental toxicity testing. It shall discuss defining parameters and mechanisms, their relevance and contemplate what can realistically be expected. Crucially this includes the question of how to ascertain the quality of currently employed cell lines and tests based thereon. Finally, the use of hESCs will raise ethical concerns which should be addressed early on. Expert Opinion: While the suitability of (h)ESCs as tools for research and development goes undisputed, any routine use for developmental toxicity testing currently still seems premature. The reasons for this comprise inherent biological deficiencies as well as cell line quality and system validation. Overcoming these issues will require collaboration of scientists, test developers and regulators. Also, validation needs to be made worthwhile for academia. Finally we have to continuously rethink existing strategies, making room for improved testing and innovative approaches.

A combination of biomolecules enhances expression of E-cadherin and peroxisome proliferator-activated receptor gene leading to increased cell proliferation in primary human meniscal cells: an in vitro study.

PubMed

Pillai, Mamatha M; Elakkiya, V; Gopinathan, J; Sabarinath, C; Shanthakumari, S; Sahanand, K Santosh; Dinakar Rai, B K; Bhattacharyya, Amitava; Selvakumar, R

2016-10-01

The present study investigates the impact of biomolecules (biotin, glucose, chondroitin sulphate, proline) as supplement, (individual and in combination) on primary human meniscus cell proliferation. Primary human meniscus cells isolated from patients undergoing meniscectomy were maintained in Dulbecco's Modified Eagle's Medium (DMEM). The isolated cells were treated with above mentioned biomolecules as individual (0-100 µg/ml) and in combinations, as a supplement to DMEM. Based on the individual biomolecule study, a unique combination of biomolecules (UCM) was finalized using one way ANOVA analysis. With the addition of UCM as supplement to DMEM, meniscal cells reached 100 % confluency within 4 days in 60 mm culture plate; whereas the cells in medium devoid of UCM, required 36 days for reaching confluency. The impact of UCM on cell viability, doubling time, histology, gene expression, biomarkers expression, extra cellular matrix synthesis, meniscus cell proliferation with respect to passages and donor's age were investigated. The gene expression studies for E-cadherin and peroxisome proliferator-activated receptor (PPAR∆) using RT-qPCR and immunohistochemical analysis for Ki67, CD34 and Vimentin confirmed that UCM has significant impact on cell proliferation. The extracellular collagen and glycosaminoglycan secretion in cells supplemented with UCM were found to increase by 31 and 37 fold respectively, when compared to control on the 4th day. The cell doubling time was reduced significantly when supplemented with UCM. The addition of UCM showed positive influence on different passages and age groups. Hence, this optimized UCM can be used as an effective supplement for meniscal tissue engineering.

Identification of NY-BR-1-specific CD4(+) T cell epitopes using HLA-transgenic mice.

PubMed

Gardyan, Adriane; Osen, Wolfram; Zörnig, Inka; Podola, Lilli; Agarwal, Maria; Aulmann, Sebastian; Ruggiero, Eliana; Schmidt, Manfred; Halama, Niels; Leuchs, Barbara; von Kalle, Christof; Beckhove, Philipp; Schneeweiss, Andreas; Jäger, Dirk; Eichmüller, Stefan B

2015-06-01

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer-related deaths among women. The differentiation antigen NY-BR-1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen-specific CD4(+) effector T cells, NY-BR-1 was screened for the presence of HLA-restricted CD4(+) T cell epitopes that could be included in immunological treatment approaches. Upon NY-BR-1-specific DNA immunization of HLA-transgenic mice and functional ex vivo analysis, a panel of NY-BR-1-derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY-BR-1-specific, HLA-DRB1*0301- or HLA-DRB1*0401-restricted CD4(+) T cell lines from splenocytes of peptide immunized HLA-transgenic mice. Notably, all four CD4(+) T cell lines recognized human HLA-DR-matched dendritic cells (DC) pulsed with lysates of NY-BR-1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4(+) T cells specific for all four CD4(+) T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4(+) T cell responses against the new epitopes are not deleted nor inactivated by self-tolerance mechanisms. Our results present the first NY-BR-1-specific HLA-DRB1*0301- and HLA-DRB1*0401-restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer. © 2014 UICC.

Insulin Exhibits an Antiproliferative and Hypertrophic Effect in First Trimester Human Extravillous Trophoblasts.

PubMed

Silva, Cláudia; Nunes, Catarina; Correia-Branco, Ana; Araújo, João R; Martel, Fátima

2017-04-01

Our aim was to investigate the effect of high levels of glucose, insulin, leptin, and tumor necrosis factor alpha, biomarkers of diabetes in pregnancy, in the process of placentation, using as a cell model a first trimester extravillous human trophoblast cell line (HTR8/SVneo cells). Exposure of HTR8/SVneo cells for 24 hours to either glucose (20 mmol/L) or leptin (25-100 ng/mL) did not cause significant changes in cell proliferation and viability. Tumor necrosis factor alpha (24 hours; 10-100 ng/L) caused a small decrease (10%) in cell proliferation and an increase (9%) in cell viability; however, both effects disappeared when exposure time was increased. Insulin (24 hours; 1-10 nmol/L) caused a concentration- and time-dependent decrease (10%-20%) in cell proliferation; the effect of insulin (10 nmol/L) was more pronounced after a 48 hours exposure (35%). In contrast, exposure to insulin (10 nmol/L; 48 hours) showed no significant effect on cell viability, apoptosis, and migration capacity. Insulin appears to cause hypertrophy of HTR8/SVneo cells as it reduces the cell mitotic index while increasing the culture protein content. The antiproliferative effect of insulin seems to involve activation of mammalian target of rapamycin, phosphoinositide 3-kinase, and p38 mitogen-activated protein kinase. Finally, simvastatin and the polyphenol quercetin potentiated the antiproliferative effect of insulin; on the contrary, the polyphenol resveratrol, the polyunsaturated fatty acids eicosapentaenoic and docosahexaenoic acids, and folic acid were not able to change it. In conclusion, we show that insulin has an antiproliferative and hypertrophic effect on a first trimester extravillous human trophoblast cell line. So insulin might affect the process of placentation.