Cell division and endoreduplication: doubtful engines of vegetative growth.

PubMed

John, Peter C L; Qi, Ruhu

2008-03-01

Currently, there is little information to indicate whether plant cell division and development is the collective effect of individual cell programming (cell-based) or is determined by organ-wide growth (organismal). Modulation of cell division does not confirm cell autonomous programming of cell expansion; instead, final cell size seems to be determined by the balance between cells formed and subsequent tissue growth. Control of growth in regions of the plant therefore has great importance in determining cell, organ and plant development. Here, we question the view that formation of new cells and their programmed expansion is the driving force of growth. We believe there is evidence that division does not drive, but requires, cell growth and a similar requirement for growth is detected in the modified cycle termed endoreduplication.

A small molecule modulator of prion protein increases human mesenchymal stem cell lifespan, ex vivo expansion, and engraftment to bone marrow in NOD/SCID mice.

PubMed

Mohanty, Sindhu T; Cairney, Claire J; Chantry, Andrew D; Madan, Sanjeev; Fernandes, James A; Howe, Steven J; Moore, Harry D; Thompson, Mark J; Chen, Beining; Thrasher, Adrian; Keith, W Nicol; Bellantuono, Ilaria

2012-06-01

Human mesenchymal stem cells (hMSCs) have been shown to have potential in regenerative approaches in bone and blood. Most protocols rely on their in vitro expansion prior to clinical use. However, several groups including our own have shown that hMSCs lose proliferation and differentiation ability with serial passage in culture, limiting their clinical applications. Cellular prion protein (PrP) has been shown to enhance proliferation and promote self-renewal of hematopoietic, mammary gland, and neural stem cells. Here we show, for the first time, that expression of PrP decreased in hMSC following ex vivo expansion. When PrP expression was knocked down, hMSC showed significant reduction in proliferation and differentiation. In contrast, hMSC expanded in the presence of small molecule 3/689, a modulator of PrP expression, showed retention of PrP expression with ex vivo expansion and extended lifespan up to 10 population doublings. Moreover, cultures produced a 300-fold increase in the number of cells generated. These cells showed a 10-fold increase in engraftment levels in bone marrow 5 weeks post-transplant. hMSC treated with 3/689 showed enhanced protection from DNA damage and enhanced cell cycle progression, in line with data obtained by gene expression profiling. Moreover, upregulation of superoxide dismutase-2 (SOD2) was also observed in hMSC expanded in the presence of 3/689. The increase in SOD2 was dependent on PrP expression and suggests increased scavenging of reactive oxygen species as mechanism of action. These data point to PrP as a good target for chemical intervention in stem cell regenerative medicine. Copyright © 2012 AlphaMed Press.

Engineering Hydrogel Microenvironments to Recapitulate the Stem Cell Niche.

PubMed

Madl, Christopher M; Heilshorn, Sarah C

2018-06-04

Stem cells are a powerful resource for many applications including regenerative medicine, patient-specific disease modeling, and toxicology screening. However, eliciting the desired behavior from stem cells, such as expansion in a naïve state or differentiation into a particular mature lineage, remains challenging. Drawing inspiration from the native stem cell niche, hydrogel platforms have been developed to regulate stem cell fate by controlling microenvironmental parameters including matrix mechanics, degradability, cell-adhesive ligand presentation, local microstructure, and cell-cell interactions. We survey techniques for modulating hydrogel properties and review the effects of microenvironmental parameters on maintaining stemness and controlling differentiation for a variety of stem cell types. Looking forward, we envision future hydrogel designs spanning a spectrum of complexity, ranging from simple, fully defined materials for industrial expansion of stem cells to complex, biomimetic systems for organotypic cell culture models.

Promotion of chloroplast proliferation upon enhanced post-mitotic cell expansion in leaves.

PubMed

Kawade, Kensuke; Horiguchi, Gorou; Ishikawa, Naoko; Hirai, Masami Yokota; Tsukaya, Hirokazu

2013-09-28

Leaves are determinate organs; hence, precise control of cell proliferation and post-mitotic cell expansion is essential for their growth. A defect in cell proliferation often triggers enhanced post-mitotic cell expansion in leaves. This phenomenon is referred to as 'compensation'. Several lines of evidence from studies on compensation have shown that cell proliferation and post-mitotic cell expansion are coordinately regulated during leaf development. Therefore, compensation has attracted much attention to the mechanisms for leaf growth. However, our understanding of compensation at the subcellular level remains limited because studies of compensation have focused mainly on cellular-level phenotypes. Proper leaf growth requires quantitative control of subcellular components in association with cellular-level changes. To gain insight into the subcellular aspect of compensation, we investigated the well-known relationship between cell area and chloroplast number per cell in compensation-exhibiting lines, and asked whether chloroplast proliferation is modulated in response to the induction of compensation. We first established a convenient and reliable method for observation of chloroplasts in situ. Using this method, we analyzed Arabidopsis thaliana mutants fugu5 and angustifolia3 (an3), and a transgenic line KIP-RELATED PROTEIN2 overexpressor (KRP2 OE), which are known to exhibit typical features of compensation. We here showed that chloroplast number per cell increased in the subepidermal palisade tissue of these lines. We analyzed tetraploidized wild type, fugu5, an3 and KRP2 OE, and found that cell area itself, but not nuclear ploidy, is a key parameter that determines the activity of chloroplast proliferation. In particular, in the case of an3, we uncovered that promotion of chloroplast proliferation depends on the enhanced post-mitotic cell expansion. The expression levels of chloroplast proliferation-related genes are similar to or lower than that in the wild type during this process. This study demonstrates that chloroplast proliferation is promoted in compensation-exhibiting lines. This promotion of chloroplast proliferation takes place in response to cell-area increase in post-mitotic phase in an3. The expression of chloroplast proliferation-related genes were not promoted in compensation-exhibiting lines including an3, arguing that an as-yet-unknown mechanism is responsible for modulation of chloroplast proliferation in these lines.

Promotion of chloroplast proliferation upon enhanced post-mitotic cell expansion in leaves

PubMed Central

2013-01-01

Background Leaves are determinate organs; hence, precise control of cell proliferation and post-mitotic cell expansion is essential for their growth. A defect in cell proliferation often triggers enhanced post-mitotic cell expansion in leaves. This phenomenon is referred to as ‘compensation’. Several lines of evidence from studies on compensation have shown that cell proliferation and post-mitotic cell expansion are coordinately regulated during leaf development. Therefore, compensation has attracted much attention to the mechanisms for leaf growth. However, our understanding of compensation at the subcellular level remains limited because studies of compensation have focused mainly on cellular-level phenotypes. Proper leaf growth requires quantitative control of subcellular components in association with cellular-level changes. To gain insight into the subcellular aspect of compensation, we investigated the well-known relationship between cell area and chloroplast number per cell in compensation-exhibiting lines, and asked whether chloroplast proliferation is modulated in response to the induction of compensation. Results We first established a convenient and reliable method for observation of chloroplasts in situ. Using this method, we analyzed Arabidopsis thaliana mutants fugu5 and angustifolia3 (an3), and a transgenic line KIP-RELATED PROTEIN2 overexpressor (KRP2 OE), which are known to exhibit typical features of compensation. We here showed that chloroplast number per cell increased in the subepidermal palisade tissue of these lines. We analyzed tetraploidized wild type, fugu5, an3 and KRP2 OE, and found that cell area itself, but not nuclear ploidy, is a key parameter that determines the activity of chloroplast proliferation. In particular, in the case of an3, we uncovered that promotion of chloroplast proliferation depends on the enhanced post-mitotic cell expansion. The expression levels of chloroplast proliferation-related genes are similar to or lower than that in the wild type during this process. Conclusions This study demonstrates that chloroplast proliferation is promoted in compensation-exhibiting lines. This promotion of chloroplast proliferation takes place in response to cell-area increase in post-mitotic phase in an3. The expression of chloroplast proliferation-related genes were not promoted in compensation-exhibiting lines including an3, arguing that an as-yet-unknown mechanism is responsible for modulation of chloroplast proliferation in these lines. PMID:24074400

Grape seed procyanidin extract modulates proliferation and apoptosis of pancreatic beta-cells.

PubMed

Cedó, Lídia; Castell-Auví, Anna; Pallarès, Victor; Blay, Mayte; Ardévol, Anna; Arola, Lluís; Pinent, Montserrat

2013-05-01

Grape seed procyanidin extract (GSPE) modulates glucose homeostasis and insulinemia in several animal models. Under pathological conditions, insulin levels are dependent on pancreatic beta-cell functionality, as well as on the beta-cell mass expansion or apoptosis in the pancreas. In this study, we analysed the effects of GSPE on modulating apoptosis and proliferation in beta-cells. We tested the effects of GSPE in the INS-1E pancreatic beta-cell line, either under basal or altered conditions with high glucose, insulin or palmitate levels. GSPE enhanced the pro-apoptotic effect of high glucose and showed clear antiproliferative effects under high glucose, insulin and palmitate conditions. These antiproliferative effects are likely due to high molecular weight compounds contained in the extract. GSPE also modulated pro- and anti-apoptotic markers in the pancreas of rats fed a cafeteria diet, with the effect depending on the dose of GSPE and duration of treatment. Thus, GSPE is able to modulate apoptosis and proliferation of beta-cells under altered, but not basal, conditions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase.

PubMed

Chen, Shaolin; Ehrhardt, David W; Somerville, Chris R

2010-10-05

The CESA1 component of cellulose synthase is phosphorylated at sites clustered in two hypervariable regions of the protein. Mutations of the phosphorylated residues to Ala (A) or Glu (E) alter anisotropic cell expansion and cellulose synthesis in rapidly expanding roots and hypocotyls. Expression of T166E, S686E, or S688E mutants of CESA1 fully rescued the temperature sensitive cesA1-1 allele (rsw1) at a restrictive temperature whereas mutations to A at these positions caused defects in anisotropic cell expansion. However, mutations to E at residues surrounding T166 (i.e., S162, T165, and S167) caused opposite effects. Live-cell imaging of fluorescently labeled CESA showed close correlations between tissue or cell morphology and patterns of bidirectional motility of CESA complexes in the plasma membrane. In the WT, CESA complexes moved at similar velocities in both directions along microtubule tracks. By contrast, the rate of movement of CESA particles was directionally asymmetric in mutant lines that exhibited abnormal tissue or cell expansion, and the asymmetry was removed upon depolymerizing microtubules with oryzalin. This suggests that phosphorylation of CESA differentially affects a polar interaction with microtubules that may regulate the length or quantity of a subset of cellulose microfibrils and that this, in turn, alters microfibril structure in the primary cell wall resulting in or contributing to the observed defect in anisotropic cell expansion.

BMS 493 Modulates Retinoic Acid-Induced Differentiation During Expansion of Human Hematopoietic Progenitor Cells for Islet Regeneration.

PubMed

Elgamal, Ruth M; Bell, Gillian I; Krause, Sarah C T; Hess, David A

2018-06-06

Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood-derived hematopoietic progenitor cells (HPCs), isolated by high aldehyde dehydrogenase activity (ALDH hi ), have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a twofold increase in the number of ALDH hi cells available for transplantation compared with untreated controls. Newly expanded ALDH hi cells showed increased numbers of CD34 and CD133-positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony-forming capacity compared with untreated cells, with ALDH hi subpopulations producing more colonies than low aldehyde dehydrogenase activity subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of β-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating β-cells was increased after 3- or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDH hi cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce retinoic acid differentiation during HPC expansion is required to expand ALDH hi cells without the loss of islet regenerative functions.

Generator module architecture for a large solid oxide fuel cell power plant

DOEpatents

Gillett, James E.; Zafred, Paolo R.; Riggle, Matthew W.; Litzinger, Kevin P.

2013-06-11

A solid oxide fuel cell module contains a plurality of integral bundle assemblies, the module containing a top portion with an inlet fuel plenum and a bottom portion receiving air inlet feed and containing a base support, the base supports dense, ceramic exhaust manifolds which are below and connect to air feed tubes located in a recuperator zone, the air feed tubes passing into the center of inverted, tubular, elongated, hollow electrically connected solid oxide fuel cells having an open end above a combustion zone into which the air feed tubes pass and a closed end near the inlet fuel plenum, where the fuel cells comprise a fuel cell stack bundle all surrounded within an outer module enclosure having top power leads to provide electrical output from the stack bundle, where the fuel cells operate in the fuel cell mode and where the base support and bottom ceramic air exhaust manifolds carry from 85% to all 100% of the weight of the stack, and each bundle assembly has its own control for vertical and horizontal thermal expansion control.

β-Arrestin2 plays a key role in the modulation of the pancreatic beta cell mass in mice.

PubMed

Ravier, Magalie A; Leduc, Michele; Richard, Joy; Linck, Nathalie; Varrault, Annie; Pirot, Nelly; Roussel, Morgane M; Bockaert, Joël; Dalle, Stéphane; Bertrand, Gyslaine

2014-03-01

Beta cell failure due to progressive secretory dysfunction and limited expansion of beta cell mass is a key feature of type 2 diabetes. Beta cell function and mass are controlled by glucose and hormones/neurotransmitters that activate G protein-coupled receptors or receptor tyrosine kinases. We have investigated the role of β-arrestin (ARRB)2, a scaffold protein known to modulate such receptor signalling, in the modulation of beta cell function and mass, with a specific interest in glucagon-like peptide-1 (GLP-1), muscarinic and insulin receptors. β-arrestin2-knockout mice and their wild-type littermates were fed a normal or a high-fat diet (HFD). Glucose tolerance, insulin sensitivity and insulin secretion were assessed in vivo. Beta cell mass was evaluated in pancreatic sections. Free cytosolic [Ca(2+)] and insulin secretion were determined using perifused islets. The insulin signalling pathway was evaluated by western blotting. Arrb2-knockout mice exhibited impaired glucose tolerance and insulin secretion in vivo, but normal insulin sensitivity compared with wild type. Surprisingly, the absence of ARRB2 did not affect glucose-stimulated insulin secretion or GLP-1- and acetylcholine-mediated amplifications from perifused islets, but it decreased the islet insulin content and beta cell mass. Additionally, there was no compensatory beta cell mass expansion through proliferation in response to the HFD. Furthermore, Arrb2 deletion altered the islet insulin signalling pathway. ARRB2 is unlikely to be involved in the regulation of insulin secretion, but it is required for beta cell mass plasticity. Additionally, we provide new insights into the mechanisms involved in insulin signalling in beta cells.

Decoy receptor 3 attenuates collagen-induced arthritis by modulating T cell activation and B cell expansion.

PubMed

Cheng, Chia-Pi; Sytwu, Huey-Kang; Chang, Deh-Ming

2011-12-01

To investigate the immune-modulated effects of decoy receptor 3 (DCR3) in an experimental model of rheumatoid arthritis (RA). We delivered DCR3 plasmid into collagen-induced arthritis (CIA) mice using the hydrodynamic method and evaluated the serum level of DCR3 protein by ELISA. After immunization, we assessed disease severity of arthritis incidence, arthritis scores, paw thickness, and means of arthritic limbs, and used hematoxylin and eosin staining to observe synovial hyperplasia. We analyzed numbers of murine splenocytes and inguinal lymphocyte cells, cell populations, and serum proinflammatory cytokines by flow cytometry. We investigated B cell proliferation by carboxyfluorescein succinimidyl ester assay. We evaluated serum levels of total IgG2a and type II collagen-specific IgG and IgG2a using ELISA. DCR3 expression in sera significantly attenuated disease severity in CIA mice. We found that DCR3 inhibited the volume of inguinal lymph nodes, numbers of CD19+ B cells, and populations of interferon-γ, interleukin 4 (IL-4), IL-17A, and Foxp3-producing CD4+ T cell in vivo. We found that DCR3 inhibited Pam3CSK4 (Toll-like receptor 1/2 ligand)-induced B220+ B cell proliferation in vitro. DCR3 treatment reduced the serum level of IL-6, total IgG2a, and CII-specific IgG2a antibody. We postulated that the protective effects of DCR3 in CIA resulted from modulation of the immune system by maintaining the B/T cell balance and decreasing lymphocyte expansion. We suggest DCR3 as a prophylactic and potential therapeutic agent in the treatment of RA.

SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing

PubMed Central

Wightman, Raymond; Chomicki, Guillaume; Kumar, Manoj; Carr, Paul; Turner, Simon R.

2013-01-01

Summary One of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs. PMID:24055158

Response of Escherichia coli growth rate to osmotic shock.

PubMed

Rojas, Enrique; Theriot, Julie A; Huang, Kerwyn Casey

2014-05-27

It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper- and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless "stored" growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan.

Response of Escherichia coli growth rate to osmotic shock

PubMed Central

Rojas, Enrique; Theriot, Julie A.; Huang, Kerwyn Casey

2014-01-01

It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper- and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless “stored” growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan. PMID:24821776

Cellular expansion and gene expression in the developing grape (Vitis vinifera L.).

PubMed

Schlosser, J; Olsson, N; Weis, M; Reid, K; Peng, F; Lund, S; Bowen, P

2008-01-01

Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1-->3)-beta-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth.

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis[C][W

PubMed Central

Spartz, Angela K.; Ren, Hong; Park, Mee Yeon; Grandt, Kristin N.; Lee, Sang Ho; Murphy, Angus S.; Sussman, Michael R.; Overvoorde, Paul J.; Gray, William M.

2014-01-01

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H+-ATPases (PM H+-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H+-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H+-ATPase function. We find that SAUR19 stimulates PM H+-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H+-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H+-ATPases, and negatively regulate PM H+-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion. PMID:24858935

Granulocytic Myeloid Derived Suppressor Cells Expansion during Active Pulmonary Tuberculosis Is Associated with High Nitric Oxide Plasma Level

PubMed Central

El Daker, Sary; Sacchi, Alessandra; Tempestilli, Massimo; Carducci, Claudia; Goletti, Delia; Vanini, Valentina; Colizzi, Vittorio; Lauria, Francesco Nicola; Martini, Federico; Martino, Angelo

2015-01-01

Tuberculosis (TB) is still the principal cause of death caused by a single infectious agent, and the balance between the bacillus and host defense mechanisms reflects the different manifestations of the pathology. The aim of this work was to study the role of myeloid-derived suppressor cells (MDSCs) during active pulmonary tuberculosis at the site of infection. We observed an expansion of MDSCs in the lung and blood of patients with active TB, which are correlated with an enhanced amount of nitric oxide in the plasma. We also found that these cells have the remarkable ability to suppress T-cell response, suggesting an important role in the modulation of the immune response against TB. Interestingly, a trend in the diminution of MDSCs was found after an efficacious anti-TB therapy, suggesting that these cells may be used as a potential biomarker for monitoring anti-TB therapy efficacy. PMID:25879532

Stem cell mobilization with G-CSF analogs: a rational approach to separate GVHD and GVL?

PubMed

Morris, Edward S; MacDonald, Kelli P A; Hill, Geoffrey R

2006-05-01

The separation of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) remains the "holy grail" of allogeneic stem cell transplantation, and improvements are urgently needed to allow more effective therapy of malignant disease. The use of G-CSF-mobilized peripheral blood as a clinical stem cell source is associated with enhanced GVL effects without amplification of significant acute GVHD. Preclinical studies have demonstrated that G-CSF modulates donor T cell function before transplantation, promoting T(H)2 differentiation and regulatory T cell function. In addition, the expansion of immature antigen-presenting cells (APCs) and plasmacytoid dendritic cells (DCs) favors the maintenance of this pattern of T cell differentiation after transplantation. Although these patterns of T cell differentiation attenuate acute GVHD, they do not have an impact on the cytolytic pathways of the CD8(+) T cells that are critical for effective GVL. Recently, it has been demonstrated that modification of G-CSF, either by pegylation of the native cytokine or conjugation to Flt-3L, results in the expansion and activation of donor iNKT cells, which significantly augment CD8(+) T cell-mediated cytotoxicity and GVL effects after transplantation. Given that these cytokines also enhance the expansion of regulatory T cells and APCs, they further separate GVHD and GVL, offering potential clinical advantages for the transplant recipient.

Modulation of Memory T Cells to Control Acquired Bone Marrow Failure

DTIC Science & Technology

2016-01-01

Representative images show the tissues from one of 6 recipients in each group at day 7 after transplantation. Images were obtained with an OlympusBX41...alloreactive effector T cells capable of mediating host tissue injury and could be beneficial targets for improving the efficacy of allogeneic HSCT...leukemia (GVL) effect, but showed impaired expansion in local tissues .69-72 This nTEM pool might have less diverse T cell receptor (TCR) repertoire

Live-Attenuated Lentivirus Immunization Modulates Innate Immunity and Inflammation while Protecting Rhesus Macaques from Vaginal Simian Immunodeficiency Virus Challenge

PubMed Central

Genescà, Meritxell; Ma, Zhong-Min; Wang, Yichuan; Assaf, Basel; Qureshi, Huma; Fritts, Linda; Huang, Ying; McChesney, Michael B.

2012-01-01

Immunization with attenuated lentiviruses is the only reliable method of protecting rhesus macaques (RM) from vaginal challenge with pathogenic simian immunodeficiency virus (SIV). CD8+ lymphocyte depletion prior to SIVmac239 vaginal challenge demonstrated that a modest, Gag-specific CD8+ T cell response induced by immunization with simian-human immunodeficiency virus 89.6 (SHIV89.6) protects RM. Although CD8+ T cells are required for protection, there is no anamnestic expansion of SIV-specific CD8+ T cells in any tissues except the vagina after challenge. Further, SHIV immunization increased the number of viral target cells in the vagina and cervix, suggesting that the ratio of target cells to antiviral CD8+ T cells was not a determinant of protection. We hypothesized that persistent replication of the attenuated vaccine virus modulates inflammatory responses and limits T cell activation and expansion by inducing immunoregulatory T cell populations. We found that attenuated SHIV infection decreased the number of circulating plasmacytoid dendritic cells, suppressed T cell activation, decreased mRNA levels of proinflammatory mediators, and increased mRNA levels of immunoregulatory molecules. Three days after SIV vaginal challenge, SHIV-immunized RM had significantly more T regulatory cells in the vagina than the unimmunized RM. By day 14 postchallenge, immune activation and inflammation were characteristic of unimmunized RM but were minimal in SHIV-immunized RM. Thus, a modest vaccine-induced CD8+ T cell response in the context of immunoregulatory suppression of T cell activation may protect against vaginal HIV transmission. PMID:22696662

WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis

NASA Technical Reports Server (NTRS)

Yuen, Christen Y L.; Pearlman, Rebecca S.; Silo-Suh, Laura; Hilson, Pierre; Carroll, Kathleen L.; Masson, Patrick H.

2003-01-01

Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.

Enhanced proliferation and dopaminergic differentiation of ventral mesencephalic precursor cells by synergistic effect of FGF2 and reduced oxygen tension

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

Jensen, Pia; Department of Neurosurgery, University of Bern, CH-3010 Bern; Gramsbergen, Jan-Bert

Effective numerical expansion of dopaminergic precursors might overcome the limited availability of transplantable cells in replacement strategies for Parkinson's disease. Here we investigated the effect of fibroblast growth factor-2 (FGF2) and FGF8 on expansion and dopaminergic differentiation of rat embryonic ventral mesencephalic neuroblasts cultured at high (20%) and low (3%) oxygen tension. More cells incorporated bromodeoxyuridine in cultures expanded at low as compared to high oxygen tension, and after 6 days of differentiation there were significantly more neuronal cells in low than in high oxygen cultures. Low oxygen during FGF2-mediated expansion resulted also in a significant increase in tyrosine hydroxylase-immunoreactivemore » (TH-ir) dopaminergic neurons as compared to high oxygen tension, but no corresponding effect was observed for dopamine release into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last two days of differentiation significantly enhanced dopamine release and intracellular dopamine levels as compared to all other treatment groups. In addition, the short-term exposure to high oxygen enhanced in situ assessed TH enzyme activity, which may explain the elevated dopamine levels. Our findings demonstrate that modulation of oxygen tension is a recognizable factor for in vitro expansion and dopaminergic differentiation of rat embryonic midbrain precursor cells.« less

Potential Sabotage of Host Cell Physiology by Apicomplexan Parasites for Their Survival Benefits

PubMed Central

Chakraborty, Shalini; Roy, Sonti; Mistry, Hiral Uday; Murthy, Shweta; George, Neena; Bhandari, Vasundhra; Sharma, Paresh

2017-01-01

Plasmodium, Toxoplasma, Cryptosporidium, Babesia, and Theileria are the major apicomplexan parasites affecting humans or animals worldwide. These pathogens represent an excellent example of host manipulators who can overturn host signaling pathways for their survival. They infect different types of host cells and take charge of the host machinery to gain nutrients and prevent itself from host attack. The mechanisms by which these pathogens modulate the host signaling pathways are well studied for Plasmodium, Toxoplasma, Cryptosporidium, and Theileria, except for limited studies on Babesia. Theileria is a unique pathogen taking into account the way it modulates host cell transformation, resulting in its clonal expansion. These parasites majorly modulate similar host signaling pathways, however, the disease outcome and effect is different among them. In this review, we discuss the approaches of these apicomplexan to manipulate the host–parasite clearance pathways during infection, invasion, survival, and egress. PMID:29081773

Detection of CFTR function and modulation in primary human nasal cell spheroids.

PubMed

Brewington, John J; Filbrandt, Erin T; LaRosa, F J; Ostmann, Alicia J; Strecker, Lauren M; Szczesniak, Rhonda D; Clancy, John P

2018-01-01

Expansion of CFTR modulators to patients with rare/undescribed mutations will be facilitated by patient-derived models quantifying CFTR function and restoration. We aimed to generate a personalized model system of CFTR function and modulation using non-surgically obtained nasal epithelial cells (NECs). NECs obtained by curettage from healthy volunteers and CF patients were expanded and grown in 3-dimensional culture as spheroids, characterized, and stimulated with cAMP-inducing agents to activate CFTR. Spheroid swelling was quantified as a proxy for CFTR function. NEC spheroids recapitulated characteristics of pseudostratified respiratory epithelia. When stimulated with forskolin/IBMX, spheroids swelled in the presence of functional CFTR, and shrank in its absence. Spheroid swelling quantified mutant CFTR restoration in F508del homozygous cells using clinically available CFTR modulators. NEC spheroids hold promise for understanding rare CFTR mutations and personalized modulator testing to drive evaluation for CF patients with common, rare or undescribed mutations. Portions of this data have previously been presented in abstract form at the 2016 meetings of the American Thoracic Society and the 2016 North American Cystic Fibrosis Conference. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Carlecortemcel-l: an ex vivo expanded umbilical cord blood cell graft for allogeneic transplantation.

PubMed

Petropoulos, Demetrios; Chan, Ka Wah

2009-11-01

Success of umbilical cord blood transplantation (UCBT) is mostly affected by the cell dose infused and its application is limited by the size of the recipient. For most adults and older children it is not possible to find a single UCB unit large enough for reliable engraftment. One strategy to increase the number of progenitor cells available is ex vivo expansion of the unit. The main challenge of ex vivo expansion systems is how not to deplete the self-renewing cell population by driving them into differentiation into committed progenitors. Copper modulates basic cell functions, such as survival, proliferation, and differentiation. Reduction of cellular copper in ex vivo culture conditions enabled preferential proliferation of early progenitors and increased engraftment capabilities. The result of a Phase I study of carlecortemcel-l, a product derived from ex vivo expansion of UCB progenitors in the presence of a copper chelator and early-acting cytokines, and the study design for the current pivotal study are presented. A literature review using PubMed and the investigator's brochure from the manufacturer. Early results suggest that carlecortemcel-l infusion is safe and may be associated with favorable non-relapse mortality rates. A pivotal global study is currently being conducted to evaluate safety and efficacy of this product from centralized manufacturing facilities.

High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington’s Disease Model

PubMed Central

Titus, Steven A; Southall, Noel; Marugan, Juan; Austin, Christopher P; Zheng, Wei

2012-01-01

A hallmark of Huntington’s disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins. Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson’s, Alzheimer’s, and prion diseases. However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear. Identification of small molecule probes that modulate protein aggregation and cytotoxicity caused by aggregated proteins may greatly facilitate the studies on pathogenesis of these diseases and potentially lead to development of new therapies. Based on a detergent insoluble property of the Huntingtin protein aggregates, we have developed a homogenous assay to rapidly quantitate the levels of protein aggregates in a cellular model of Huntington’s disease. The protein aggregation assay has also been multiplexed with a protease release assay for the measurement of cytotoxicity resulting from aggregated proteins in the same cells. Through a testing screen of a compound library, we have demonstrated that this multiplexed cytotoxicity and protein aggregation assay has ability to identify active compounds that prevent cell death and/or modulate protein aggregation in cells of the Huntington’s disease model. Therefore, this multiplexed screening approach is also useful for development of high-throughput screening assays for other neurodegenerative diseases involving protein aggregation. PMID:23346268

Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation

PubMed Central

Procopio, Maria-Giuseppina; Laszlo, Csaba; Labban, Dania Al; Kim, Dong Eun; Bordignon, Pino; Jo, Seunghee; Goruppi, Sandro; Menietti, Elena; Ostano, Paola; Ala, Ugo; Provero, Paolo; Hoetzenecker, Wolfram; Neel, Victor; Kilarski, Witek; Swartz, Melody A.; Brisken, Cathrin; Lefort, Karine; Dotto, G. Paolo

2015-01-01

Stromal fibroblast senescence has been linked to aging-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAF) are frequently increased. Loss or down-modulation of the Notch effector CSL/RBP-Jκ in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumors. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is down-modulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas (SCC), while p53 expression and function is down-modulated only in the latter, with paracrine FGF signaling as likely culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation/stromal co-evolution model under convergent CSL/p53 control. PMID:26302407

A genomics approach to understanding the role of auxin in apple (Malus x domestica) fruit size control.

PubMed

Devoghalaere, Fanny; Doucen, Thomas; Guitton, Baptiste; Keeling, Jeannette; Payne, Wendy; Ling, Toby John; Ross, John James; Hallett, Ian Charles; Gunaseelan, Kularajathevan; Dayatilake, G A; Diak, Robert; Breen, Ken C; Tustin, D Stuart; Costes, Evelyne; Chagné, David; Schaffer, Robert James; David, Karine Myriam

2012-01-13

Auxin is an important phytohormone for fleshy fruit development, having been shown to be involved in the initial signal for fertilisation, fruit size through the control of cell division and cell expansion, and ripening related events. There is considerable knowledge of auxin-related genes, mostly from work in model species. With the apple genome now available, it is possible to carry out genomics studies on auxin-related genes to identify genes that may play roles in specific stages of apple fruit development. High amounts of auxin in the seed compared with the fruit cortex were observed in 'Royal Gala' apples, with amounts increasing through fruit development. Injection of exogenous auxin into developing apples at the start of cell expansion caused an increase in cell size. An expression analysis screen of auxin-related genes involved in auxin reception, homeostasis, and transcriptional regulation showed complex patterns of expression in each class of gene. Two mapping populations were phenotyped for fruit size over multiple seasons, and multiple quantitative trait loci (QTLs) were observed. One QTL mapped to a region containing an Auxin Response Factor (ARF106). This gene is expressed during cell division and cell expansion stages, consistent with a potential role in the control of fruit size. The application of exogenous auxin to apples increased cell expansion, suggesting that endogenous auxin concentrations are at least one of the limiting factors controlling fruit size. The expression analysis of ARF106 linked to a strong QTL for fruit weight suggests that the auxin signal regulating fruit size could partially be modulated through the function of this gene. One class of gene (GH3) removes free auxin by conjugation to amino acids. The lower expression of these GH3 genes during rapid fruit expansion is consistent with the apple maximising auxin concentrations at this point.

A genomics approach to understanding the role of auxin in apple (Malus x domestica) fruit size control

PubMed Central

2012-01-01

Background Auxin is an important phytohormone for fleshy fruit development, having been shown to be involved in the initial signal for fertilisation, fruit size through the control of cell division and cell expansion, and ripening related events. There is considerable knowledge of auxin-related genes, mostly from work in model species. With the apple genome now available, it is possible to carry out genomics studies on auxin-related genes to identify genes that may play roles in specific stages of apple fruit development. Results High amounts of auxin in the seed compared with the fruit cortex were observed in 'Royal Gala' apples, with amounts increasing through fruit development. Injection of exogenous auxin into developing apples at the start of cell expansion caused an increase in cell size. An expression analysis screen of auxin-related genes involved in auxin reception, homeostasis, and transcriptional regulation showed complex patterns of expression in each class of gene. Two mapping populations were phenotyped for fruit size over multiple seasons, and multiple quantitative trait loci (QTLs) were observed. One QTL mapped to a region containing an Auxin Response Factor (ARF106). This gene is expressed during cell division and cell expansion stages, consistent with a potential role in the control of fruit size. Conclusions The application of exogenous auxin to apples increased cell expansion, suggesting that endogenous auxin concentrations are at least one of the limiting factors controlling fruit size. The expression analysis of ARF106 linked to a strong QTL for fruit weight suggests that the auxin signal regulating fruit size could partially be modulated through the function of this gene. One class of gene (GH3) removes free auxin by conjugation to amino acids. The lower expression of these GH3 genes during rapid fruit expansion is consistent with the apple maximising auxin concentrations at this point. PMID:22243694

Morphology and dynamics of tumor cell colonies propagating in epidermal growth factor supplemented media

NASA Astrophysics Data System (ADS)

Muzzio, N. E.; Carballido, M.; Pasquale, M. A.; González, P. H.; Azzaroni, O.; Arvia, A. J.

2018-07-01

The epidermal growth factor (EGF) plays a key role in physiological and pathological processes. This work reports on the influence of EGF concentration (c EGF) on the modulation of individual cell phenotype and cell colony kinetics with the aim of perturbing the colony front roughness fluctuations. For this purpose, HeLa cell colonies that remain confluent along the whole expansion process with initial quasi-radial geometry and different initial cell populations, as well as colonies with initial quasi-linear geometry and large cell population, are employed. Cell size and morphology as well as its adhesive characteristics depend on c EGF. Quasi-radial colonies (QRC) expansion kinetics in EGF-containing medium exhibits a complex behavior. Namely, at the first stages of growth, the average QRC radius evolution can be described by a t 1/2 diffusion term coupled with exponential growth kinetics up to a critical time, and afterwards a growth regime approaching constant velocity. The extension of each regime depends on c EGF and colony history. In the presence of EGF, the initial expansion of quasi-linear colonies (QLCs) also exhibits morphological changes at both the cell and the colony levels. In these cases, the cell density at the colony border region becomes smaller than in the absence of EGF and consequently, the extension of the effective rim where cell duplication and motility contribute to the colony expansion increases. QLC front displacement velocity increases with c EGF up to a maximum value in the 2–10 ng ml‑1 range. Individual cell velocity is increased by EGF, and an enhancement in both the persistence and the ballistic characteristics of cell trajectories can be distinguished. For an intermediate c EGF, collective cell displacements contribute to the roughening of the colony contours. This global dynamics becomes compatible with the standard Kardar–Parisi–Zhang growth model, although a faster colony roughness saturation in EGF-containing medium than in the control medium is observed.

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells

PubMed Central

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya

2015-01-01

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13+CD133+ cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13+CD133+ cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation. PMID:25808356

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells.

PubMed

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya; Kamiya, Akihide

2015-07-15

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13(+)CD133(+) cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13(+)CD133(+) cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation.

Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium.

PubMed

Teng, Xiang; Qin, Lei; Le Borgne, Roland; Toyama, Yusuke

2017-01-01

Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, contraction of actomyosin cables formed in apoptotic and neighboring cells drives cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. We propose a model in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion. © 2017. Published by The Company of Biologists Ltd.

Ability of γδ T cells to modulate the Foxp3 T cell response is dependent on adenosine.

PubMed

Liang, Dongchun; Woo, Jeong-Im; Shao, Hui; Born, Willi K; O'Brien, Rebecca L; Kaplan, Henry J; Sun, Deming

2018-01-01

Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases.

IL-7–dependent STAT1 activation limits homeostatic CD4+ T cell expansion

PubMed Central

Le Saout, Cecile; Luckey, Megan A.; Villarino, Alejandro V.; Smith, Mindy; Hasley, Rebecca B.; Myers, Timothy G.; Imamichi, Hiromi; Park, Jung-Hyun; O’Shea, John J.; Lane, H. Clifford

2017-01-01

IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that, under steady-state conditions, IL-7 signaling is principally mediated by activation of signal transducers and activators of transcription 5 (STAT5). In contrast, under lymphopenic conditions, there is a modulation of STAT1 expression resulting in an IL-7–dependent STAT1 and STAT5 activation. Consequently, the IL-7–induced transcriptome is altered with enrichment of IFN-stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4+ T cells undergoing lymphopenia-induced proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, “switching on” an alternate IL-7–dependent program. This mechanism could be a physiological process to regulate the expansion and size of the CD4+ T cell pool. During HIV infection, the virus could exploit this pathway, leading to the homeostatic dysregulation of the T cell pools observed in these patients. PMID:29202461

Overview of a HLA-Ig based "Lego-like system" for T cell monitoring, modulation and expansion.

PubMed

Oelke, Mathias; Schneck, Jonathan P

2010-07-01

Recent advances in molecular medicine have shown that soluble MHC-multimers can be valuable tools for both analysis and modulation of antigen-specific immune responses in vitro and in vivo. In this review, we describe the use of dimeric human and mouse major histocompatibility complexes, MHC-Ig, as part of an artificial Antigen-Presenting Cell (aAPC). MHC-Ig-based aAPC and its derivatives represent an exciting new platform technology for measuring and manipulating immune responses in vitro as well as in vivo. This new technology has the potential to help overcome many of the obstacles associated with limitations in current antigen-specific approaches of immunotherapy for the treatment of cancer, infectious diseases and autoimmunity.

Possible role of acetylcholine in regulating spatial novelty effects on theta rhythm and grid cells

PubMed Central

Barry, Caswell; Heys, James G.; Hasselmo, Michael E.

2012-01-01

Existing pharmacological and lesion data indicate that acetylcholine plays an important role in memory formation. For example, increased levels of acetylcholine in the hippocampal formation are known to be associated with successful encoding while disruption of the cholinergic system leads to impairments on a range of mnemonic tasks. However, cholinergic signaling from the medial septum also plays a central role in generating and pacing theta-band oscillations throughout the hippocampal formation. Recent experimental results suggest a potential link between these distinct phenomena. Environmental novelty, a condition associated with strong cholinergic drive, has been shown to induce an expansion in the firing pattern of entorhinal grid cells and a reduction in the frequency of theta measured from the LFP. Computational modeling suggests the spatial activity of grid cells is produced by interference between neuronal oscillators; scale being determined by theta-band oscillations impinging on entorhinal stellate cells, the frequency of which is modulated by acetylcholine. Here we propose that increased cholinergic signaling in response to environmental novelty triggers grid expansion by reducing the frequency of the oscillations. Furthermore, we argue that cholinergic induced grid expansion may enhance, or even induce, encoding by producing a mismatch between expanded grid cells and other spatial inputs to the hippocampus, such as boundary vector cells. Indeed, a further source of mismatch is likely to occur between grid cells of different native scales which may expand by different relative amounts. PMID:22363266

Honeycomb vs. Foam: Evaluating Potential Upgrades to ISS Module Shielding

NASA Technical Reports Server (NTRS)

Ryan, Shannon J.; Christiansen, Eric L.

2009-01-01

The presence of honeycomb cells in a dual-wall structure is advantageous for mechanical performance and low weight in spacecraft primary structures but detrimental for shielding against impact of micrometeoroid and orbital debris particles (MMOD). The presence of honeycomb cell walls acts to restrict the expansion of projectile and bumper fragments, resulting in the impact of a more concentrated (and thus lethal) fragment cloud upon the shield rear wall. The Multipurpose Laboratory Module (MLM) is a Russian research module scheduled for launch and ISS assembly in 2011 (currently under review). Baseline shielding of the MLM is expected to be predominantly similar to that of the existing Functional Energy Block (FGB), utilizing a baseline triple wall configuration with honeycomb sandwich panels for the dual bumpers and a thick monolithic aluminum pressure wall. The MLM module is to be docked to the nadir port of the Zvezda service module and, as such, is subject to higher debris flux than the FGB module (which is aligned along the ISS flight vector). Without upgrades to inherited shielding, the MLM penetration risk is expected to be significantly higher than that of the FGB module. Open-cell foam represents a promising alternative to honeycomb as a sandwich panel core material in spacecraft primary structures as it provides comparable mechanical performance with a minimal increase in weight while avoiding structural features (i.e. channeling cells) detrimental to MMOD shielding performance. In this study, the effect of replacing honeycomb sandwich panel structures with metallic open-cell foam structures on MMOD shielding performance is assessed for an MLM-representative configuration. A number of hypervelocity impact tests have been performed on both the baseline honeycomb configuration and upgraded foam configuration, and differences in target damage, failure limits, and derived ballistic limit equations are discussed.

Pten Cell Autonomously Modulates the Hematopoietic Stem Cell Response to Inflammatory Cytokines.

PubMed

Porter, Shaina N; Cluster, Andrew S; Signer, Robert A J; Voigtmann, Jenna; Monlish, Darlene A; Schuettpelz, Laura G; Magee, Jeffrey A

2016-06-14

Pten negatively regulates the phosphatidylinositol 3-kinase (PI3K) pathway and is required to maintain quiescent adult hematopoietic stem cells (HSCs). Pten has been proposed to regulate HSCs cell autonomously and non-cell autonomously, but the relative importance of each mechanism has not been directly tested. Furthermore, the cytokines that activate the PI3K pathway upstream of Pten are not well defined. We sought to clarify whether Pten cell autonomously or non-cell autonomously regulates HSC mobilization. We also tested whether Pten deficiency affects the HSC response to granulocyte colony-stimulating factor (G-CSF) and interferon-α (IFNα) since these cytokines induce HSC mobilization or proliferation, respectively. We show that Pten regulates HSC mobilization and expansion in the spleen primarily via cell-autonomous mechanisms. Pten-deficient HSCs do not require G-CSF to mobilize, although they are hyper-sensitized to even low doses of exogenous G-CSF. Pten-deficient HSCs are similarly sensitized to IFNα. Pten therefore modulates the HSC response to inflammatory cytokines. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Modulated wave formation in myocardial cells under electromagnetic radiation

NASA Astrophysics Data System (ADS)

Takembo, Clovis N.; Mvogo, A.; Ekobena Fouda, H. P.; Kofané, T. C.

2018-06-01

We exclusively analyze the onset and condition of formation of modulated waves in a diffusive FitzHugh-Nagumo model for myocardial cell excitations. The cells are connected through gap junction coupling. An additive magnetic flux variable is used to describe the effect of electromagnetic induction, while electromagnetic radiation is imposed on the magnetic flux variable as a periodic forcing. We used the discrete multiple scale expansion and obtained, from the model equations, a single differential-difference amplitude nonlinear equation. We performed the linear stability analysis of this equation and found that instability features are importantly influenced by the induced electromagnetic gain. We present the unstable and stable regions of modulational instability (MI). The resulting analytic predictions are confirmed by numerical experiments of the generic equations. The results reveal that due to MI, an initial steady state that consisted of a plane wave with low amplitude evolves into a modulated localized wave patterns, soliton-like in shape, with features of synchronization. Furthermore, the formation of periodic pulse train with breathing motion presents a disappearing pattern in the presence of electromagnetic radiation. This could provide guidance and better understanding of sudden heart failure exposed to heavily electromagnetic radiation.

Modulation of CD4(+) T cell-dependent specific cytotoxic CD8(+) T cells differentiation and proliferation by the timing of increase in the pathogen load.

PubMed

Tzelepis, Fanny; Persechini, Pedro M; Rodrigues, Mauricio M

2007-04-25

Following infection with viruses, bacteria or protozoan parasites, naïve antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. In contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. The evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. The differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.

Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling

PubMed Central

Mace, Thomas A.; King, Samantha A.; Ameen, Zeenath; Elnaggar, Omar; Young, Gregory; Riedl, Kenneth M.; Schwartz, Steven J.; Clinton, Steven K.; Knobloch, Thomas J.; Weghorst, Christopher M.; Lesinski, Gregory B.

2014-01-01

Bioactive phyotochemicals from natural products, such as black raspberries (BRB; Rubus occidentalis) have direct anti-cancer properties on malignant cells in culture and in xenograft models. BRB components inhibit cancer progression in more complex rodent carcinogenesis models. Although mechanistic targets for BRB phytochemicals in cancer cells are beginning to emerge, the potential role in modulating host immune processes impacting cancer have not been systematically examined. We hypothesized that BRB contain compounds capable of eliciting potent immunomodulatory properties that impact cellular mediators relevant to chronic inflammation and tumor progression. We studied both an ethanol extract from black raspberries (BRB-E) containing a diverse mixture of phytochemicals and two abundant phytochemical metabolites of BRB produced upon ingestion (Cyanidin-3-Rutinoside, C3R; Quercitin-3-Rutinoside, Q3R). BRB-E inhibited proliferation and viability of CD3/CD28 activated human CD4+ and CD8+ T lymphocytes. BRB-E also limited in vitro expansion of myeloid-derived suppressor cells (MDSC) and their suppressive capacity. Pre-treatment of immune cells with BRB-E attenuated IL-6-mediated phosphorylation of signal transducer and activator of transcription-3 (STAT3) and IL-2 induced STAT5 phosphorylation. In contrast, pre-treatment of immune cells with the C3R and Q3R metabolites inhibited MDSC expansion, IL-6-mediated STAT3 signaling, but not IL-2 induced STAT5 phosphorylation and were less potent inhibitors of T cell viability. Together these data indicate that BRB extracts and their physiologically-relevant metabolites contain phytochemicals that affect immune processes relevant to carcinogenesis and immunotherapy. Furthermore, specific BRB components and their metabolites may be a source of lead compounds for drug development that exhibit targeted immunological outcomes or inhibition of specific STAT-regulated signaling pathways. PMID:24893859

Symbiotic Fungi Control Plant Root Cortex Development through the Novel GRAS Transcription Factor MIG1.

PubMed

Heck, Carolin; Kuhn, Hannah; Heidt, Sven; Walter, Stefanie; Rieger, Nina; Requena, Natalia

2016-10-24

In an approaching scenario of soil nutrient depletion, root association with soil microorganisms can be key for plant health and sustainability [1-3]. Symbiotic arbuscular mycorrhizal (AM) fungi are major players in helping plants growing under nutrient starvation conditions. They provide plants with minerals like phosphate and, furthermore, act as modulators of plant growth altering the root developmental program [4, 5]. However, the precise mechanisms involved in this latter process are not well understood. Here, we show that AM fungi are able to modulate root cortex development in Medicago truncatula by activating a novel GRAS-domain transcription factor, MIG1, that determines the size of cortical root cells. MIG1 expression peaks in arbuscule-containing cells, suggesting a role in cell remodeling during fungal accommodation. Roots ectopically expressing MIG1 become thicker due to an increase in the number and width of cortical cells. This phenotype is fully counteracted by gibberellin (GA) and phenocopied with a GA biosynthesis inhibitor or by expression of a dominant DELLA (Δ18DELLA1) protein. MIG1 downregulation leads to malformed arbuscules, a phenotype rescued by Δ18DELLA1, suggesting that MIG1 intersects with the GA signaling to control cell morphogenesis through DELLA1. DELLA1 was shown to be a central node controlling arbuscule branching [6-8]. Now we provide evidence that, together with MIG1, DELLA1 is responsible for radial cortical cell expansion during arbuscule development. Our data point toward DELLA proteins being not only longitudinal root growth repressors [9] but also positive regulators of cortical radial cell expansion, extending the knowledge of how DELLAs control root growth. Copyright © 2016 Elsevier Ltd. All rights reserved.

Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells

PubMed Central

Kobayashi, Hideki; Butler, Jason M.; O'Donnell, Rebekah; Kobayashi, Mariko; Ding, Bi-Sen; Bonner, Bryant; Chiu, Vi K.; Nolan, Daniel J.; Shido, Koji; Benjamin, Laura; Rafii, Shahin

2010-01-01

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34−Flt3− KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34−Flt3− KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the upregulation of angiocrine factors, with Akt–mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs. PMID:20972423

ADF Proteins Are Involved in the Control of Flowering and Regulate F-Actin Organization, Cell Expansion, and Organ Growth in Arabidopsis

PubMed Central

Dong, Chun-Hai; Xia, Gui-Xian; Hong, Yan; Ramachandran, Srinivasan; Kost, Benedikt; Chua, Nam-Hai

2001-01-01

Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis. PMID:11402164

A spatial model of the efficiency of T cell search in the influenza-infected lung.

PubMed

Levin, Drew; Forrest, Stephanie; Banerjee, Soumya; Clay, Candice; Cannon, Judy; Moses, Melanie; Koster, Frederick

2016-06-07

Emerging strains of influenza, such as avian H5N1 and 2009 pandemic H1N1, are more virulent than seasonal H1N1 influenza, yet the underlying mechanisms for these differences are not well understood. Subtle differences in how a given strain interacts with the immune system are likely a key factor in determining virulence. One aspect of the interaction is the ability of T cells to locate the foci of the infection in time to prevent uncontrolled expansion. Here, we develop an agent based spatial model to focus on T cell migration from lymph nodes through the vascular system to sites of infection. We use our model to investigate whether different strains of influenza modulate this process. We calibrate the model using viral and chemokine secretion rates we measure in vitro together with values taken from literature. The spatial nature of the model reveals unique challenges for T cell recruitment that are not apparent in standard differential equation models. In this model comparing three influenza viruses, plaque expansion is governed primarily by the replication rate of the virus strain, and the efficiency of the T cell search-and-kill is limited by the density of infected epithelial cells in each plaque. Thus for each virus there is a different threshold of T cell search time above which recruited T cells are unable to control further expansion. Future models could use this relationship to more accurately predict control of the infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Method and apparatus for assembling solid oxide fuel cells

DOEpatents

Szreders, Bernard E.; Campanella, Nicholas

1989-01-01

A plurality of jet air tubes are supported and maintained in a spaced matrix array by a positioning/insertion assembly for insertion in respective tubes of a solid oxide fuel cell (SOFC) in the assembly of an SOFC module. The positioning/insertion assembly includes a plurality of generally planar, elongated, linear vanes which are pivotally mounted at each end thereof to a support frame. The vanes, which each include a plurality of spaced slots along the facing edges thereof, may be pivotally displaced from a generally vertical orientation, wherein each jet air tube is positioned within and engaged by the aligned slots of a plurality of paired upper and lower vanes to facilitate their insertion in respective aligned SOFC tubes arranged in a matrix array, to an inclined orientation, wherein the jet air tubes may be removed from the positioning/insertion assembly after being inserted in the SOFC tubes. A rectangular compression assembly of adjustable size is adapted to receive and squeeze a matrix of SOFC tubes so as to compress the inter-tube nickel felt conductive pads which provide series/parallel electrical connection between adjacent SOFCs, with a series of increasingly larger retainer frames used to maintain larger matrices of SOFC tubes in position. Expansion of the SOFC module housing at the high operating temperatures of the SOFC is accommodated by conductive, flexible, resilient expansion, connector bars which provide support and electrical coupling at the top and bottom of the SOFC module housing.

Embryo growth, testa permeability, and endosperm weakening are major targets for the environmentally regulated inhibition of Lepidium sativum seed germination by myrigalone A

PubMed Central

2012-01-01

Myrigalone A (MyA) is a rare flavonoid in fruit leachates of Myrica gale, a deciduous shrub adapted to flood-prone habitats. As a putative allelochemical it inhibits seed germination and seedling growth. Using Lepidium sativum as a model target species, experiments were conducted to investigate how environmental cues modulate MyA’s interference with key processes of seed germination. Time course analyses of L. sativum testa and endosperm rupture under different light conditions and water potentials were combined with quantifying testa permeability, endosperm weakening, tissue-specific gibberellin (GA) and abscisic acid (ABA) contents, as well as embryo growth and apoplastic superoxide production important for cell expansion growth. Lepidium sativum testa permeability and early water uptake by imbibition is enhanced by MyA. During late germination, MyA inhibits endosperm weakening and embryo growth, both processes required for endosperm rupture. Inhibition of embryo cell expansion by MyA depends on environmental cues, which is evident from the light-modulated severity of the MyA-mediated inhibition of apoplastic superoxide accumulation. Several important key weakening and growth processes during early and late germination are targets for MyA. These effects are modulated by light conditions and ambient water potential. It is speculated that MyA is a soil seed bank-destroying allelochemical that secures the persistence of M. gale in its flood-prone environment. PMID:22821938

A mobile information management system used in textile enterprises

NASA Astrophysics Data System (ADS)

Huang, C.-R.; Yu, W.-D.

2008-02-01

The mobile information management system (MIMS) for textile enterprises is based on Microsoft Visual Studios. NET2003 Server, Microsoft SQL Server 2000, C++ language and wireless application protocol (WAP) and wireless markup language (WML) technology. The portable MIMS is composed of three-layer structures, i.e. showing layer; operating layer; and data visiting layer corresponding to the port-link module; processing module; and database module. By using the MIMS, not only the information exchanges become more convenient and easier, but also the compatible between the giant information capacity and a micro-cell phone and functional expansion nature in operating and designing can be realized by means of build-in units. The development of MIMS is suitable for the utilization in textile enterprises.

Pectin Methylesterification Impacts the Relationship between Photosynthesis and Plant Growth1[OPEN

PubMed Central

Kim, Sang-Jin; Renna, Luciana; Brandizzi, Federica

2016-01-01

Photosynthesis occurs in mesophyll cells of specialized organs such as leaves. The rigid cell wall encapsulating photosynthetic cells controls the expansion and distribution of cells within photosynthetic tissues. The relationship between photosynthesis and plant growth is affected by leaf area. However, the underlying genetic mechanisms affecting carbon partitioning to different aspects of leaf growth are not known. To fill this gap, we analyzed Arabidopsis plants with altered levels of pectin methylesterification, which is known to modulate cell wall plasticity and plant growth. Pectin methylesterification levels were varied through manipulation of cotton Golgi-related (CGR) 2 or 3 genes encoding two functionally redundant pectin methyltransferases. Increased levels of methylesterification in a line over-expressing CGR2 (CGR2OX) resulted in highly expanded leaves with enhanced intercellular air spaces; reduced methylesterification in a mutant lacking both CGR-genes 2 and 3 (cgr2/3) resulted in thin but dense leaf mesophyll that limited CO2 diffusion to chloroplasts. Leaf, root, and plant dry weight were enhanced in CGR2OX but decreased in cgr2/3. Differences in growth between wild type and the CGR-mutants can be explained by carbon partitioning but not by variations in area-based photosynthesis. Therefore, photosynthesis drives growth through alterations in carbon partitioning to new leaf area growth and leaf mass per unit leaf area; however, CGR-mediated pectin methylesterification acts as a primary factor in this relationship through modulation of the expansion and positioning of the cells in leaves, which in turn drive carbon partitioning by generating dynamic carbon demands in leaf area growth and leaf mass per unit leaf area. PMID:27208234

Pectin Methylesterification Impacts the Relationship between Photosynthesis and Plant Growth.

PubMed

M Weraduwage, Sarathi; Kim, Sang-Jin; Renna, Luciana; C Anozie, Fransisca; D Sharkey, Thomas; Brandizzi, Federica

2016-06-01

Photosynthesis occurs in mesophyll cells of specialized organs such as leaves. The rigid cell wall encapsulating photosynthetic cells controls the expansion and distribution of cells within photosynthetic tissues. The relationship between photosynthesis and plant growth is affected by leaf area. However, the underlying genetic mechanisms affecting carbon partitioning to different aspects of leaf growth are not known. To fill this gap, we analyzed Arabidopsis plants with altered levels of pectin methylesterification, which is known to modulate cell wall plasticity and plant growth. Pectin methylesterification levels were varied through manipulation of cotton Golgi-related (CGR) 2 or 3 genes encoding two functionally redundant pectin methyltransferases. Increased levels of methylesterification in a line over-expressing CGR2 (CGR2OX) resulted in highly expanded leaves with enhanced intercellular air spaces; reduced methylesterification in a mutant lacking both CGR-genes 2 and 3 (cgr2/3) resulted in thin but dense leaf mesophyll that limited CO2 diffusion to chloroplasts. Leaf, root, and plant dry weight were enhanced in CGR2OX but decreased in cgr2/3. Differences in growth between wild type and the CGR-mutants can be explained by carbon partitioning but not by variations in area-based photosynthesis. Therefore, photosynthesis drives growth through alterations in carbon partitioning to new leaf area growth and leaf mass per unit leaf area; however, CGR-mediated pectin methylesterification acts as a primary factor in this relationship through modulation of the expansion and positioning of the cells in leaves, which in turn drive carbon partitioning by generating dynamic carbon demands in leaf area growth and leaf mass per unit leaf area. © 2016 American Society of Plant Biologists. All Rights Reserved.

Zoledronic acid modulates maturation of human monocyte-derived dendritic cells.

PubMed

Orsini, Giulia; Failli, Alessandra; Legitimo, Annalisa; Adinolfi, Barbara; Romanini, Antonella; Consolini, Rita

2011-12-01

Zoledronic acid (ZA) is a drug of the bisphosphonate class, which is widely used for the treatment of both osteoporosis and skeletal metastasis. Besides its main bone antiresorptive activity, ZA displays antitumor properties, by triggering the expansion and activation of γδ T-cells, which exert an antitumor effect through dendritic cells (DCs). Several studies have reported the interaction between ZA and γδ T-cells, but the potential immunoregulatory activity of this drug on DCs has scarcely been investigated. Therefore, in this paper, we evaluated the effects of a therapeutic dose of ZA on the in vitro generation and maturation of DCs derived from peripheral blood monocytes of healthy adult donors. We demonstrate that ZA treatment did not affect DC differentiation, but inhibited DC maturation on lipopolysaccharide activation, as shown by the impaired expression of maturation surface markers and reduced ability to induce allogeneic T-cell proliferation. Interestingly, IL-10 secretion by mature DCs was significantly lower in ZA-treated cells than in controls. We conclude that ZA exerts its immunological in vitro activity also by modulating the maturation of DCs.

Regulation of endoplasmic reticulum turnover by selective autophagy.

PubMed

Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

2015-06-18

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues

PubMed Central

Rhee, Ki-Jong; Jasper, Paul J.; Sethupathi, Periannan; Shanmugam, Malathy; Lanning, Dennis; Knight, Katherine L.

2005-01-01

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express VHn allotype immunoglobulin (Ig)M. Within weeks, the number of VHn B cells decreases, whereas VHa allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from VHn to VHa B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the VHn to VHa repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of VHa B cells. By comparing amino acid sequences of VHn and VHa Ig, we identified a putative VH ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with VHa B cells results in their selective expansion. PMID:15623575

Isolation and Expansion of Hepatic Stem-like Cells from a Healthy Rat Liver and their Efficient Hepatic Differentiation of under Well-defined Vivo Hepatic like Microenvironment in a Multiwell Bioreactor

PubMed Central

Giri, Shibashish; Acikgöz, Ali; Bader, Augustinus

2015-01-01

Background Currently, undifferentiated cells are found in all tissue and term as local stem cells which are quiescent in nature and less in number under normal healthy conditions but activate upon injury and repair the tissue or organs via automated activating mechanism. Due to very scanty presence of local resident somatic local stem cells in healthy organs, isolation and expansion of these adult stems is an immense challenge for medical research and cell based therapy. Particularly organ like liver, there is an ongoing controversy about existence of liver stem cells. Methods Herein, Hepatic stem cells population was identified during culture of primary hepatocyte cells upon immediate isolation of primary hepatocyte cells. These liver stem cells has been expanded extensively and differentiated into primary hepatocytes under defined culture conditions in a nanostructured self assembling peptides modular bioreactor that mimic the state of art of liver microenvironment and compared with Matrigel as a positive control. Nanostructured self assembling peptides were used a defined extracellular matrix and Matrigel was used for undefined extracellular matrix. Proliferation of hepatic stem cells was investigated by two strategies. First strategy is to provide high concentration of hepatocyte growth factor (HGF) and second strategy is to evaluate the role of recombinant human erythropoietin (rHuEPO) in presence of trauma/ischemia cytokines (IL-6, TNF-α). Expansion to hepatic differentiation is observed by morphological analysis and was evaluated for the expression of hepatocyte-specific genes using RT-PCR and biochemical methods. Results Hepatocyte-specific genes are well expressed at final stage (day 21) of differentiation period. The differentiated hepatocytes exhibited functional hepatic characteristics such as albumin secretion, urea secretion and cytochrome P450 expression. Additionally, immunofluorescence analysis revealed that hepatic stem cells derived hepatocytes exhibited mature hepatocyte markers (albumin, CK-19, CPY3A1, alpha 1-antitrypsin). Expansion and hepatic differentiation was efficiently in nanostructured self assembling peptides without such batch to batch variation while there was much variation in Matrigel coated bioreactor. In conclusion, the results of the study suggest that the nanostructured self assembling peptides coated bioreactor supports expansion as well as hepatic differentiation of liver stem cells which is superior than Matrigel. Conclusion This defined microenvironment conditions in bioreactor module can be useful for research involving bioartificial liver system, stem cell research and engineered liver tissue which could contribute to regenerative cell therapies or drug discovery and development. PMID:26155038

The prolyl isomerase Pin1 modulates development of CD8+ cDC in mice.

PubMed

Barberi, Theresa J; Dunkle, Alexis; He, You-Wen; Racioppi, Luigi; Means, Anthony R

2012-01-01

Pin1 has previously been described to regulate cells that participate in both innate and adaptive immunity. Thus far, however, no role for Pin1 has been described in modulating conventional dendritic cells, innate antigen presenting cells that potently activate naïve T cells, thereby bridging innate and adaptive immune responses. When challenged with LPS, Pin1-null mice failed to accumulate spleen conventional dendritic cells (cDC). Analysis of steady-state spleen DC populations revealed that Pin1-null mice had fewer CD8+ cDC. This defect was recapitulated by culturing Pin1-null bone marrow with the DC-instructive cytokine Flt3 Ligand. Additionally, injection of Flt3 Ligand for 9 days failed to induce robust expansion of CD8+ cDC in Pin1-null mice. Upon infection with Listeria monocytogenes, Pin1-null mice were defective in stimulating proliferation of adoptively transferred WT CD8+ T cells, suggesting that decreases in Pin1 null CD8+ cDC may affect T cell responses to infection in vivo. Finally, upon analyzing expression of proteins involved in DC development, elevated expression of PU.1 was detected in Pin1-null cells, which resulted from an increase in PU.1 protein half-life. We have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity.

Modulation of Regulatory T Cell Activity by TNF Receptor Type II-Targeting Pharmacological Agents

PubMed Central

Zou, Huimin; Li, Ruixin; Hu, Hao; Hu, Yuanjia; Chen, Xin

2018-01-01

There is now compelling evidence that tumor necrosis factor (TNF)–TNF receptor type II (TNFR2) interaction plays a decisive role in the activation, expansion, and phenotypical stability of suppressive CD4+Foxp3+ regulatory T cells (Tregs). In an effort to translate this basic research finding into a therapeutic benefit, a number of agonistic or antagonistic TNFR2-targeting biological agents with the capacity to activate or inhibit Treg activity have been developed and studied. Recent studies also show that thalidomide analogs, cyclophosphamide, and other small molecules are able to act on TNFR2, resulting in the elimination of TNFR2-expressing Tregs. In contrast, pharmacological agents, such as vitamin D3 and adalimumab, were reported to induce the expansion of Tregs by promoting the interaction of transmembrane TNF (tmTNF) with TNFR2. These studies clearly show that TNFR2-targeting pharmacological agents represent an effective approach to modulating the function of Tregs and thus may be useful in the treatment of major human diseases such as autoimmune disorders, graft-versus-host disease (GVHD), and cancer. In this review, we will summarize and discuss the latest progress in the study of TNFR2-targeting pharmacological agents and their therapeutic potential based on upregulation or downregulation of Treg activity. PMID:29632537

Genetically re-engineered K562 cells significantly expand and functionally activate cord blood natural killer cells: Potential for adoptive cellular immunotherapy.

PubMed

Ayello, Janet; Hochberg, Jessica; Flower, Allyson; Chu, Yaya; Baxi, Laxmi V; Quish, William; van de Ven, Carmella; Cairo, Mitchell S

2017-02-01

Natural killer (NK) cells play a significant role in reducing relapse in patients with hematological malignancies after allogeneic stem cell transplantation, but NK cell number and naturally occurring inhibitory signals limit their capability. Interleukin-15 (IL-15) and 4-1BBL are important modulators of NK expansion and functional activation. To overcome these limitations, cord blood mononuclear cells (CB MNCs) were ex vivo expanded for 7 days with genetically modified K562-mbIL15-41BBL (MODK562) or wild-type K562 (WTK562). NK cell expansion; expression of lysosome-associated membrane protein-1 (LAMP-1), granzyme B, and perforin; and in vitro and in vivo cytotoxicity against B-cell non-Hodgkin lymphoma (B-NHL) were evaluated. In vivo tumor growth in B-NHL-xenografted nonobese diabetic severe combined immune deficient (NOD-scid) gamma (NSG) mice was monitored by tumor volume, cell number, and survival. CB MNCs cultured with MODK562 compared with WTK562 demonstrated significantly increased NK expansion (thirty-fivefold, p < 0.05); LAMP-1 (p < 0.05), granzyme B, and perforin expression (p < 0.001); and in vitro cytotoxicity against B-NHL (p < 0.01). Xenografted mice treated with MODK562 CB experienced significantly decreased B-NHL tumor volume (p = 0.0086) and B-NHL cell numbers (p < 0.01) at 5 weeks and significantly increased survival (p < 0.001) at 10 weeks compared with WTK562. In summary, MODK562 significantly enhanced CB NK expansion and cytotoxicity, enhanced survival in a human Burkitt's lymphoma xenograft NSG model, and could be used in the future as adoptive cellular immunotherapy after umbilical CB transplantation. Future directions include expanding anti-CD20 chimeric receptor-modified CB NK cells to enhance B-NHL targeting in vitro and in vivo. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

OCIAD1 Controls Electron Transport Chain Complex I Activity to Regulate Energy Metabolism in Human Pluripotent Stem Cells.

PubMed

Shetty, Deeti K; Kalamkar, Kaustubh P; Inamdar, Maneesha S

2018-06-14

Pluripotent stem cells (PSCs) derive energy predominantly from glycolysis and not the energy-efficient oxidative phosphorylation (OXPHOS). Differentiation is initiated with energy metabolic shift from glycolysis to OXPHOS. We investigated the role of mitochondrial energy metabolism in human PSCs using molecular, biochemical, genetic, and pharmacological approaches. We show that the carcinoma protein OCIAD1 interacts with and regulates mitochondrial complex I activity. Energy metabolic assays on live pluripotent cells showed that OCIAD1-depleted cells have increased OXPHOS and may be poised for differentiation. OCIAD1 maintains human embryonic stem cells, and its depletion by CRISPR/Cas9-mediated knockout leads to rapid and increased differentiation upon induction, whereas OCIAD1 overexpression has the opposite effect. Pharmacological alteration of complex I activity was able to rescue the defects of OCIAD1 modulation. Thus, hPSCs can exist in energy metabolic substates. OCIAD1 provides a target to screen for additional modulators of mitochondrial activity to promote transient multipotent precursor expansion or enhance differentiation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer.

PubMed

Singh, Sandeep; Trevino, Jose; Bora-Singhal, Namrata; Coppola, Domenico; Haura, Eric; Altiok, Soner; Chellappan, Srikumar P

2012-09-25

Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self-renew; however the mechanistic correlation between oncogenic pathways and self-renewal of cancer stem-like cells has remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self-renewal of SP cells is dependent on EGFR mediated signaling. SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to self-renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic inhibitors suppresses the self-renewal growth and expansion of SP-cells and resulted in specific downregulation of Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its self-renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in regulating self-renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples. Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates self-renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs.

Anisotropic Cell Expansion Is Affected through the Bidirectional Mobility of Cellulose Synthase Complexes and Phosphorylation at Two Critical Residues on CESA31[OPEN

PubMed Central

Liu, Yanmei; Bauer, Stefan

2016-01-01

Here we report that phosphorylation status of S211 and T212 of the CESA3 component of Arabidopsis (Arabidopsis thaliana) cellulose synthase impacts the regulation of anisotropic cell expansion as well as cellulose synthesis and deposition and microtubule-dependent bidirectional mobility of CESA complexes. Mutation of S211 to Ala caused a significant decrease in the length of etiolated hypocotyls and primary roots, while root hairs were not significantly affected. By contrast, the S211E mutation stunted the growth of root hairs, but primary roots were not significantly affected. Similarly, T212E caused a decrease in the length of root hairs but not root length. However, T212E stunted the growth of etiolated hypocotyls. Live-cell imaging of fluorescently labeled CESA showed that the rate of movement of CESA particles was directionally asymmetric in etiolated hypocotyls of S211A and T212E mutants, while similar bidirectional velocities were observed with the wild-type control and S211E and T212A mutant lines. Analysis of cell wall composition and the innermost layer of cell wall suggests a role for phosphorylation of CESA3 S211 and T212 in cellulose aggregation into fibrillar bundles. These results suggest that microtubule-guided bidirectional mobility of CESA complexes is fine-tuned by phosphorylation of CESA3 S211 and T212, which may, in turn, modulate cellulose synthesis and organization, resulting in or contributing to the observed defects of anisotropic cell expansion. PMID:26969722

Thermal Expansion of Vitrified Blood Vessels Permeated with DP6 and Synthetic Ice Modulators

PubMed Central

Eisenberg, David P.; Taylor, Michael J.; Jimenez-Rios, Jorge L.; Rabin, Yoed

2014-01-01

This study provides thermal expansion data for blood vessels permeated with the cryoprotective cocktail DP6, when combined with selected synthetic ice modulators (SIMs): 12% polyethylene glycol 400, 6% 1,3-cyclohexanediol, and 6% 2,3-butanediol. The general classification of SIMs includes molecules that modulate ice nucleation and growth, or possess properties of stabilizing the amorphous state, by virtue of their chemical structure and at concentrations that are not explained on a purely colligative basis. The current study is part of an ongoing effort to characterize thermo-mechanical effects on structural integrity of cryopreserved materials, where thermal expansion is the driving mechanism to thermo-mechanical stress. This study focuses on the lower part of the cryogenic temperature range, where the cryoprotective agent (CPA) behaves as a solid for all practical applications. By combining results obtained in the current study with literature data on the thermal expansion in the upper part of the cryogenic temperature range, unified thermal expansion curves are presented. PMID:24769313

The antagonistic modulation of Arp2/3 activity by N-WASP, WAVE2 and PICK1 defines dynamic changes in astrocyte morphology.

PubMed

Murk, Kai; Blanco Suarez, Elena M; Cockbill, Louisa M R; Banks, Paul; Hanley, Jonathan G

2013-09-01

Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult.

Immune modulation of CD4+CD25+ regulatory T cells by zoledronic acid.

PubMed

Liu, Hsien; Wang, Shih-Han; Chen, Shin-Cheh; Chen, Ching-Ying; Lo, Jo-Lin; Lin, Tsun-Mei

2016-11-25

CD4 + CD25 + regulatory T (Treg) cells suppress tumor immunity by inhibiting immune cells. Manipulation of Treg cells represents a new strategy for cancer treatment. Zoledronic acid (ZA), a nitrogen-containing bisphosphonate, inhibits the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) on osteoblasts to inhibit osteoclastogenesis. In a mouse model of bisphosphonate-related osteonecrosis of the jaw, administration of ZA suppressed Treg-cell activity and activated inflammatory Th17 cells. However, the interaction between ZA and Treg cells remained unclear. This study investigated the immune modulation of Treg cells by ZA. Flow cytometry was used to analyze the phenotypic and immunosuppressive characteristics of Treg cells treated with ZA. Chemotactic migration was evaluated using transwell assays. Quantitative real-time PCR (qRT-PCR) was used to investigate the effect of ZA on the expression of suppressive molecules by Treg cells. Proliferation of isolated Treg cells in culture was inhibited by ZA, although ZA did not induce apoptosis. qRT-PCR and flow cytometry showed that ZA significantly downregulated the expression of CCR4, CTLA4, PD-1 and RANKL on Treg cells. Chemotactic migration and immunosuppressive functions were also significantly attenuated in Treg cells pretreated with ZA, and these effects were dose-dependent. Co-culture with Treg cells significantly increased the migration rate of breast cancer cells, while pretreatment of Treg cells with ZA attenuated this effect. Our findings demonstrated that ZA acted as an immune modulator by significantly inhibiting the expansion, migration, immunosuppressive function and pro-metastatic ability of Treg cells. Immunomodulation of Treg cells by ZA represents a new strategy for cancer therapy.

Prevention strategies differentially modulate the impact of cytomegalovirus replication on CD8(+) T-cell differentiation in high-risk solid organ transplant patients.

PubMed

Cantisán, Sara; Páez-Vega, Aurora; Pérez-Romero, Pilar; Montejo, Miguel; Cordero, Elisa; Gracia-Ahufinger, Irene; Martín-Gandul, Cecilia; Maruri, Naroa; Aguado, Rocío; Solana, Rafael; Torre-Cisneros, Julián

2016-08-01

The present study aimed to determine whether antiviral prevention strategies against cytomegalovirus (CMV) infection used in high-risk D+R- solid organ transplanted patients can modulate the impact of CMV replication on CD8(+) T-cell differentiation. The different CD8(+) T-cell subpopulations were measured at a single point when at least one year had elapsed since transplantation. A total of 68 D+R- patients were included, of which 33 underwent pre-emptive therapy and 35 received prophylaxis. Multivariate analysis showed that CMV replication was associated with the expansion of CD28־ EMRA CD8(+) T cells in patients managed pre-emptively but not in patients under prophylaxis (21.4% vs. 3.6%). This finding is likely related to the higher frequency of CMV recurrence observed in patients under pre-emptive therapy compared to those under prophylaxis (75% vs. 14.3%; p < 0.001). In fact, multivariate analysis showed that having more than one replication episode was associated with a 17.2% increase (p = 0.001) in the percentage of CD28־ EMRA CD8(+) T cells compared to "no episode" and with a 10.9% increase with respect to "single episodes" (p = 0.025). Additionally, patients with IFNγ response to CMV (QuantiFERON-CMV Reactive) had a higher percentage of late-differentiated CD8(+) T cells than patients lacking this response. In summary, recurrent CMV replication in D+R- patients under pre-emptive therapy was associated with the expansion of CD28־ EMRA CD8(+) T cells, which might have a short-term beneficial effect related to the high functionality of this T-cell subpopulation. Nevertheless, we cannot rule out that this accumulation might have a long-term detrimental effect related to immunosenescence and inflammation. Copyright © 2016 Elsevier B.V. All rights reserved.

Brain injury expands the numbers of neural stem cells and progenitors in the SVZ by enhancing their responsiveness to EGF

PubMed Central

Alagappan, Dhivyaa; Lazzarino, Deborah A; Felling, Ryan J; Balan, Murugabaskar; Kotenko, Sergei V; Levison, Steven W

2009-01-01

There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries. PMID:19570028

The interruption of PKC-ι signaling and TRAIL combination therapy against glioblastoma cells.

PubMed

McCray, Andrea N; Desai, Shraddha; Acevedo-Duncan, Mildred

2014-09-01

Glioblastoma is a highly aggressive type of brain cancer which currently has limited options for treatment. It is imperative to develop combination therapies that could cause apoptosis in glioblastoma. The aim of this study was to characterize the affect of modified ICA-1, a PKC-iota inhibitor, on the growth pattern of various glioblastoma cell lines. T98G and U87 glioblastoma cells were treated with ICA-1 alone and the absolute cell numbers of each group were determined for cell growth expansion analysis, cell viability analysis, and cell death analysis. Low dose ICA-1 treatment alone significantly inhibited cell growth expansion of high density glioblastoma cells without inducing cell death. However, the high dose ICA-1 treatment regimen provided significant apoptosis for glioblastoma cells. Furthermore, this study was conducted to use a two layer molecular level approach for treating glioblastoma cells with ICA-1 plus an apoptosis agent, tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL), to induce apoptosis in such chemo-refractory cancer cells. Following ICA-1 plus TRAIL treatment, apoptosis was detected in glioblastoma cells via the TUNEL assay and via flow cytometric analysis using Annexin-V FITC/PI. This study offers the first evidence for ICA-1 alone to inhibit glioblastoma cell proliferation as well as the novel combination of ICA-1 with TRAIL to cause robust apoptosis in a caspase-3 mediated mechanism. Furthermore, ICA-1 plus TRAIL simultaneously modulates down-regulation of PKC-iota and c-Jun.

Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis.

PubMed

Liu, Peng; Stajich, Jason E

2015-04-01

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians. Copyright © 2015 Elsevier Inc. All rights reserved.

Stem Cell Microencapsulation for Phenotypic Control, Bioprocessing, and Transplantation

PubMed Central

Wilson, Jenna L.

2014-01-01

Cell microencapsulation has been utilized for decades as a means to shield cells from the external environment while simultaneously permitting transport of oxygen, nutrients, and secretory molecules. In designing cell therapies, donor primary cells are often difficult to obtain and expand to appropriate numbers, rendering stem cells an attractive alternative due to their capacities for self-renewal, differentiation, and trophic factor secretion. Microencapsulation of stem cells offers several benefits, namely the creation of a defined microenvironment which can be designed to modulate stem cell phenotype, protection from hydrodynamic forces and prevention of agglomeration during expansion in suspension bioreactors, and a means to transplant cells behind a semi-permeable barrier, allowing for molecular secretion while avoiding immune reaction. This review will provide an overview of relevant microencapsulation processes and characterization in the context of maintaining stem cell potency, directing differentiation, investigating scalable production methods, and transplanting stem cells for clinically relevant disorders. PMID:23239279

Thermoelectric converter for SP-100 space reactor power system

NASA Technical Reports Server (NTRS)

Terrill, W. R.; Haley, V. F.

1986-01-01

Conductively coupling the thermoelectric converter to the heat source and the radiator maximizes the utilization of the reactor and radiator temperatures and thereby minimizes the power system weight. This paper presents the design for the converter and the individual thermoelectric cells that are the building block modules for the converter. It also summarizes progress on the fabrication of initial cells and the results obtained from the preparation of a manufacturing plan. The design developed for the SP-100 system utilizes thermally conductive compliant pads that can absorb the displacement and distortion caused by the combinations of temperatures and thermal expansion coefficients. The converter and cell designs provided a 100 kWe system which met the system requirements. Initial cells were fabricated and tested.

Concatenated Coding Using Trellis-Coded Modulation

NASA Technical Reports Server (NTRS)

Thompson, Michael W.

1997-01-01

In the late seventies and early eighties a technique known as Trellis Coded Modulation (TCM) was developed for providing spectrally efficient error correction coding. Instead of adding redundant information in the form of parity bits, redundancy is added at the modulation stage thereby increasing bandwidth efficiency. A digital communications system can be designed to use bandwidth-efficient multilevel/phase modulation such as Amplitude Shift Keying (ASK), Phase Shift Keying (PSK), Differential Phase Shift Keying (DPSK) or Quadrature Amplitude Modulation (QAM). Performance gain can be achieved by increasing the number of signals over the corresponding uncoded system to compensate for the redundancy introduced by the code. A considerable amount of research and development has been devoted toward developing good TCM codes for severely bandlimited applications. More recently, the use of TCM for satellite and deep space communications applications has received increased attention. This report describes the general approach of using a concatenated coding scheme that features TCM and RS coding. Results have indicated that substantial (6-10 dB) performance gains can be achieved with this approach with comparatively little bandwidth expansion. Since all of the bandwidth expansion is due to the RS code we see that TCM based concatenated coding results in roughly 10-50% bandwidth expansion compared to 70-150% expansion for similar concatenated scheme which use convolution code. We stress that combined coding and modulation optimization is important for achieving performance gains while maintaining spectral efficiency.

Immunomodulatory Nature and Site Specific Affinity of Mesenchymal Stem Cells: a Hope in Cell Therapy

PubMed Central

Lotfinegad, Parisa; Shamsasenjan, karim; Movassaghpour, Aliakbar; Majidi, Jafar; Baradaran, Behzad

2014-01-01

Immunosuppressive ability of mesenchymal stem cells (MSCs), their differentiation properties to various specialized tissue types, ease of in vitro and in vivo expansion and specific migration capacity, make them to be tested in different clinical trials for the treatment of various diseases. The immunomodulatory effects of MSCs are less identified which probably has high clinically significance. The clinical trials based on primary research will cause better understanding the ability of MSCs in immunomodulatory applications and site specific migration in the optimization of therapy. So, this review focus on MSCs functional role in modulating immune responses, their ability in homing to tumor, their potency as delivery vehicle and their medical importance. PMID:24409403

Positive selection of the peripheral B cell repertoire in gut-associated lymphoid tissues.

PubMed

Rhee, Ki-Jong; Jasper, Paul J; Sethupathi, Periannan; Shanmugam, Malathy; Lanning, Dennis; Knight, Katherine L

2005-01-03

Gut-associated lymphoid tissues (GALTs) interact with intestinal microflora to drive GALT development and diversify the primary antibody repertoire; however, the molecular mechanisms that link these events remain elusive. Alicia rabbits provide an excellent model to investigate the relationship between GALT, intestinal microflora, and modulation of the antibody repertoire. Most B cells in neonatal Alicia rabbits express V(H)n allotype immunoglobulin (Ig)M. Within weeks, the number of V(H)n B cells decreases, whereas V(H)a allotype B cells increase in number and become predominant. We hypothesized that the repertoire shift from V(H)n to V(H)a B cells results from interactions between GALT and intestinal microflora. To test this hypothesis, we surgically removed organized GALT from newborn Alicia pups and ligated the appendix to sequester it from intestinal microflora. Flow cytometry and nucleotide sequence analyses revealed that the V(H)n to V(H)a repertoire shift did not occur, demonstrating the requirement for interactions between GALT and intestinal microflora in the selective expansion of V(H)a B cells. By comparing amino acid sequences of V(H)n and V(H)a Ig, we identified a putative V(H) ligand binding site for a bacterial or endogenous B cell superantigen. We propose that interaction of such a superantigen with V(H)a B cells results in their selective expansion.

Thermal expansion of vitrified blood vessels permeated with DP6 and synthetic ice modulators.

PubMed

Eisenberg, David P; Taylor, Michael J; Jimenez-Rios, Jorge L; Rabin, Yoed

2014-06-01

This study provides thermal expansion data for blood vessels permeated with the cryoprotective cocktail DP6, when combined with selected synthetic ice modulators (SIMs): 12% polyethylene glycol 400, 6% 1,3-cyclohexanediol, and 6% 2,3-butanediol. The general classification of SIMs includes molecules that modulate ice nucleation and growth, or possess properties of stabilizing the amorphous state, by virtue of their chemical structure and at concentrations that are not explained on a purely colligative basis. The current study is part of an ongoing effort to characterize thermo-mechanical effects on structural integrity of cryopreserved materials, where thermal expansion is the driving mechanism to thermo-mechanical stress. This study focuses on the lower part of the cryogenic temperature range, where the cryoprotective agent (CPA) behaves as a solid for all practical applications. By combining results obtained in the current study with literature data on the thermal expansion in the upper part of the cryogenic temperature range, unified thermal expansion curves are presented. Copyright © 2014 Elsevier Inc. All rights reserved.

Heparan Sulfate Proteoglycans as Drivers of Neural Progenitors Derived From Human Mesenchymal Stem Cells.

PubMed

Okolicsanyi, Rachel K; Oikari, Lotta E; Yu, Chieh; Griffiths, Lyn R; Haupt, Larisa M

2018-01-01

Background: Due to their relative ease of isolation and their high ex vivo and in vitro expansive potential, human mesenchymal stem cells (hMSCs) are an attractive candidate for therapeutic applications in the treatment of brain injury and neurological diseases. Heparan sulfate proteoglycans (HSPGs) are a family of ubiquitous proteins involved in a number of vital cellular processes including proliferation and stem cell lineage differentiation. Methods: Following the determination that hMSCs maintain neural potential throughout extended in vitro expansion, we examined the role of HSPGs in mediating the neural potential of hMSCs. hMSCs cultured in basal conditions (undifferentiated monolayer cultures) were found to co-express neural markers and HSPGs throughout expansion with modulation of the in vitro niche through the addition of exogenous HS influencing cellular HSPG and neural marker expression. Results: Conversion of hMSCs into hMSC Induced Neurospheres (hMSC IN) identified distinctly localized HSPG staining within the spheres along with altered gene expression of HSPG core protein and biosynthetic enzymes when compared to undifferentiated hMSCs. Conclusion: Comparison of markers of pluripotency, neural self-renewal and neural lineage specification between hMSC IN, hMSC and human neural stem cell (hNSC H9) cultures suggest that in vitro generated hMSC IN may represent an intermediary neurogenic cell type, similar to a common neural progenitor cell. In addition, this data demonstrates HSPGs and their biosynthesis machinery, are associated with hMSC IN formation. The identification of specific HSPGs driving hMSC lineage-specification will likely provide new markers to allow better use of hMSCs in therapeutic applications and improve our understanding of human neurogenesis.

Canine and Equine Mesenchymal Stem Cells Grown in Serum Free Media Have Altered Immunophenotype.

PubMed

Clark, Kaitlin C; Kol, Amir; Shahbenderian, Salpi; Granick, Jennifer L; Walker, Naomi J; Borjesson, Dori L

2016-04-01

Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE2, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE2. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions.

Concise Review: Amniotic Fluid Stem Cells: The Known, the Unknown, and Potential Regenerative Medicine Applications.

PubMed

Loukogeorgakis, Stavros P; De Coppi, Paolo

2017-07-01

The amniotic fluid has been identified as an untapped source of cells with broad potential, which possess immunomodulatory properties and do not have the ethical and legal limitations of embryonic stem cells. CD117(c-Kit)+ cells selected from amniotic fluid have been shown to differentiate into cell lineages representing all three embryonic germ layers without generating tumors, making them ideal candidates for regenerative medicine applications. Moreover, their ability to engraft in injured organs and modulate immune and repair responses of host tissues, suggest that transplantation of such cells may be useful for the treatment of various degenerative and inflammatory diseases. Although significant questions remain regarding the origin, heterogeneous phenotype, and expansion potential of amniotic fluid stem cells, evidence to date supports their potential role as a valuable stem cell source for the field of regenerative medicine. Stem Cells 2017;35:1663-1673. © 2016 AlphaMed Press.

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

PubMed

Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Novel Therapy for Glioblastoma Multiforme by Restoring LRRC4 in Tumor Cells: LRRC4 Inhibits Tumor-Infitrating Regulatory T Cells by Cytokine and Programmed Cell Death 1-Containing Exosomes

PubMed Central

Li, Peiyao; Feng, Jianbo; Liu, Yang; Liu, Qiang; Fan, Li; Liu, Qing; She, Xiaoling; Liu, Changhong; Liu, Tao; Zhao, Chunhua; Wang, Wei; Li, Guiyuan; Wu, Minghua

2017-01-01

Glioblastoma multiforme (GBM) is a heterogeneous malignant brain tumor, the pathological incidence of which induces the accumulation of tumor-infiltrating lymphocytes (TILs). As a tumor suppressor gene, LRRC4 is absent in GBM cells. Here, we report that the recovery of LRRC4 in GBM cells inhibited the infiltration of tumor-infiltrating regulatory T cells (Ti-Treg), promoted the expansion of tumor-infiltrating effector T (Ti-Teff) cells and CD4+CCR4+ T cells, and enhanced the chemotaxis of CD4+CCR4+ T cells in the GBM immune microenvironment. LRRC4 was not transferred into TILs from GBM cells through exosomes but mainly exerted its inhibiting function on Ti-Treg cell expansion by directly promoting cytokine secretion. GBM cell-derived exosomes (cytokine-free and programmed cell death 1 containing) also contributed to the modulation of LRRC4 on Ti-Treg, Ti-Teff, and CD4+CCR4+ T cells. In GBM cells, LRRC4 directly bound to phosphoinositide-dependent protein kinase 1 (PDPK1), phosphorylated IKKβser181, facilitated NF-κB activation, and promoted the secretion of interleukin-6 (IL-6), CCL2, and interferon gamma. In addition, HSP90 was required to maintain the interaction between LRRC4 and PDPK1. However, the inhibition of Ti-Treg cell expansion and promotion of CD4+CCR4+ T cell chemotaxis by LRRC4 could be blocked by anti-IL-6 antibody or anti-CCL2 antibody, respectively. miR-101 is a suppressor gene in GBM. Our previous studies have shown that EZH2, EED, and DNMT3A are direct targets of miR-101. Here, we showed that miR-101 reversed the hypermethylation of the LRRC4 promoter and induced the re-expression of LRRC4 in GBM cells by directly targeting EZH2, EED, and DNMT3A. Our results reveal a novel mechanism underlying GBM microenvironment and provide a new therapeutic strategy using re-expression of LRRC4 in GBM cells to create a permissive intratumoral environment. PMID:29312296

Regeneration of spine disc and joint cartilages under temporal and space modulated laser radiation

NASA Astrophysics Data System (ADS)

Sobol, E.; Shekhter, A.; Baskov, A.; Baskov, V.; Baum, O.; Borchshenko, I.; Golubev, V.; Guller, A.; Kolyshev, I.; Omeltchenko, A.; Sviridov, A.; Zakharkina, O.

2009-02-01

The effect of laser radiation on the generation of hyaline cartilage in spine disc and joints has been demonstrated. The paper considers physical processes and mechanisms of laser regeneration, presents results of investigations aimed to optimize laser settings and to develop feedback control system for laser reconstruction of spine discs. Possible mechanisms of laser-induced regeneration include: (1) Space and temporary modulated laser beam induces nonhomogeneous and pulse repetitive thermal expansion and stress in the irradiated zone of cartilage. Mechanical effect due to controllable thermal expansion of the tissue and micro and nano gas bubbles formation in the course of the moderate (up to 45-50 oC) heating of the NP activate biological cells (chondrocytes) and promote cartilage regeneration. (2) Nondestructive laser radiation leads to the formation of nano and micro-pores in cartilage matrix. That promotes water permeability and increases the feeding of biological cells. Results provide the scientific and engineering basis for the novel low-invasive laser procedures to be used in orthopedics for the treatment cartilages of spine and joints. The technology and equipment for laser reconstruction of spine discs have been tested first on animals, and then in a clinical trial. Since 2001 the laser reconstruction of intervertebral discs have been performed for 340 patients with chronic symptoms of low back or neck pain who failed to improve with non-operative care. Substantial relief of back pain was obtained in 90% of patients treated who returned to their daily activities. The experiments on reparation of the defects in articular cartilage of the porcine joints under temporal and spase modulated laser radiation have shown promising results.

DJ-1 deficiency attenuates expansion of liver progenitor cells through modulating the inflammatory and fibrogenic niches

PubMed Central

Chen, L; Luo, M; Sun, X; Qin, J; Yu, C; Wen, Y; Zhang, Q; Gu, J; Xia, Q; Kong, X

2016-01-01

Our previous study suggested that DJ-1 has a critical role in initiating an inflammatory response, but its role in the liver progenitor cell (LPC) expansion, a process highly dependent on the inflammatory niche, remains elusive. The objective of this study is to determine the role of DJ-1 in LPC expansion. The correlation of DJ-1 expression with LPC markers was examined in the liver of patients with hepatitis B or hepatitis C virus (HBV and HCV, respectively) infection, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), nonalcoholic fatty liver disease (NAFLD), cirrhosis or hepatocellular carcinoma (HCC), respectively. The role of DJ-1 in LPC expansion and the formation of LPC-associated fibrosis and inflammation was examined in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced liver injury murine model. We also determined the ability of hepatic stellate cells (HSCs) in recruiting macrophages in DJ-1 knockout (KO) mice. The expression levels of DJ-1 were upregulated in the liver of HBV, HCV, PBC and PSC patients and DDC-fed mice. Additionally, DJ-1 expression was positively correlated with LPC proliferation in patients with liver injury and mice with DDC exposure. DJ-1 has no direct effect on LPC proliferation. Reduced activation of HSCs and collagen deposition were observed in DJ-1 KO mice. Furthermore, infiltrated CD11b+Gr-1low macrophages and pro-inflammatory factors (IL-6, TNF-α) were attenuated in DJ-1 KO mice. Mechanistically, we found that HSCs isolated from DJ-1 KO mice had decreased secretion of macrophage-mobilizing chemokines, such as CCL2 and CX3CL1, resulting in impaired macrophage infiltration. DJ-1 positively correlates with LPC expansion during liver injury. DJ-1 deficiency negatively regulates LPC proliferation by impairing the formation of LPC-associated fibrosis and inflammatory niches. PMID:27277679

NK T Cells Contribute to Expansion of CD8+ T Cells and Amplification of Antiviral Immune Responses to Respiratory Syncytial Virus

PubMed Central

Johnson, Teresa R.; Hong, Seokmann; Van Kaer, Luc; Koezuka, Yasuhiko; Graham, Barney S.

2002-01-01

CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack Vα14+ natural killer T cells. Respiratory syncytial virus (RSV) immunopathogenesis was evaluated in 129×C57BL/6, C57BL/6, and BALB/c CD1d−/− mice. CD8+ T lymphocytes were reduced in CD1d−/− mice of all strains, as shown by cell surface staining and major histocompatibility complex class I tetramer analysis, and resulted in strain-specific alterations in illness, viral clearance, and gamma interferon (IFN-γ) production. Transient activation of NK T cells in CD1d+/+ mice by α-GalCer resulted in reduced illness and delayed viral clearance. These data suggest that early IFN-γ production and efficient induction of CD8+-T-cell responses during primary RSV infection require CD1d-dependent events. We also tested the ability of α-GalCer as an adjuvant to modulate the type 2 immune responses induced by RSV glycoprotein G or formalin-inactivated RSV immunization. However, immunized CD1-deficient or α-GalCer-treated wild-type mice did not exhibit diminished disease following RSV challenge. Rather, some disease parameters, including cytokine production, eosinophilia, and viral clearance, were increased. These findings indicate that CD1d-dependent NK T cells play a role in expansion of CD8+ T cells and amplification of antiviral responses to RSV. PMID:11932395

Broncho-Vaxom attenuates allergic airway inflammation by restoring GSK3β-related T regulatory cell insufficiency.

PubMed

Fu, Ran; Li, Jian; Zhong, Hua; Yu, Dehong; Zeng, Xianping; Deng, Mengxia; Sun, Yueqi; Wen, Weiping; Li, Huabin

2014-01-01

Oral administration of bacterial extracts (eg, Broncho-Vaxom (BV)) has been proposed to attenuate asthma through modulating Treg cells. However, the underlying mechanism has not been fully characterized. This study sought to assess the effects of oral administration of BV on GSK-3β expression and Treg cells in ovalbumin (OVA)-induced asthmatic mice models. Asthmatic mice models were established with OVA challenge and treated with oral administration of BV. Next, infiltration of inflammatory cells including eosinophil and neutrophils, mucous metaplasia, levels of Th1/Th2/Treg-typed cytokines and expression of GSK3β and Foxp3 were examined in asthmatic mice models by histological analysis, Bio-Plex and western blot, respectively. Moreover, the frequencies of Treg cells were evaluated in cultured splenocytes by flow cytometry in the presence of BV or GSK3β siRNA interference. We found significant decrease of infiltrated inflammatory cells in bronchoalveolar lavage fluid (BALF) in asthmatic mice models after oral administration of BV. Oral administration of BV was shown to significantly suppress mucus metaplasia, Th2-typed cytokine levels and GSK3β expression while increasing Foxp3 production in asthmatic mice models. Moreover, BV significantly enhanced GSK3β-related expansion of Treg cells in cultured spleen cells in vitro. Our findings provide evidence that oral administration of BV is capable of attenuating airway inflammation in asthmatic mice models, which may be associated with GSK3β-related expansion of Treg cells.

Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis1[OPEN

PubMed Central

Cao, Lingyan; Blanchoin, Laurent; Staiger, Christopher J.

2016-01-01

Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion. PMID:26574597

Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells

PubMed Central

Wells, Alexandria C; Daniels, Keith A; Angelou, Constance C; Fagerberg, Eric; Burnside, Amy S; Markstein, Michele; Alfandari, Dominique; Welsh, Raymond M; Pobezinskaya, Elena L; Pobezinsky, Leonid A

2017-01-01

The differentiation of naive CD8 T cells into effector cytotoxic T lymphocytes upon antigen stimulation is necessary for successful antiviral, and antitumor immune responses. Here, using a mouse model, we describe a dual role for the let-7 microRNAs in the regulation of CD8 T cell responses, where maintenance of the naive phenotype in CD8 T cells requires high levels of let-7 expression, while generation of cytotoxic T lymphocytes depends upon T cell receptor-mediated let-7 downregulation. Decrease of let-7 expression in activated T cells enhances clonal expansion and the acquisition of effector function through derepression of the let-7 targets, including Myc and Eomesodermin. Ultimately, we have identified a novel let-7-mediated mechanism, which acts as a molecular brake controlling the magnitude of CD8 T cell responses. DOI: http://dx.doi.org/10.7554/eLife.26398.001 PMID:28737488

Wnt signal transduction pathways: modules, development and evolution.

PubMed

Nayak, Losiana; Bhattacharyya, Nitai P; De, Rajat K

2016-08-01

Wnt signal transduction pathway (Wnt STP) is a crucial intracellular pathway mainly due to its participation in important biological processes, functions, and diseases, i.e., embryonic development, stem-cell management, and human cancers among others. This is why Wnt STP is one of the highest researched signal transduction pathways. Study and analysis of its origin, expansion and gradual development to the present state as found in humans is one aspect of Wnt research. The pattern of development and evolution of the Wnt STP among various species is not clear till date. A phylogenetic tree created from Wnt STPs of multiple species may address this issue. In this respect, we construct a phylogenetic tree from modules of Wnt STPs of diverse species. We term it as the 'Module Tree'. A module is nothing but a self-sufficient minimally-dependent subset of the original Wnt STP. Authenticity of the module tree is tested by comparing it with the two reference trees. The module tree performs better than an alternative phylogenetic tree constructed from pathway topology of Wnt STPs. Moreover, an evolutionary emergence pattern of the Wnt gene family is created and the module tree is tallied with it to showcase the significant resemblances.

With respect to coefficient of linear thermal expansion, bacterial vegetative cells and spores resemble plastics and metals, respectively.

PubMed

Nakanishi, Koichi; Kogure, Akinori; Fujii, Takenao; Kokawa, Ryohei; Deuchi, Keiji; Kuwana, Ritsuko; Takamatsu, Hiromu

2013-10-09

If a fixed stress is applied to the three-dimensional z-axis of a solid material, followed by heating, the amount of thermal expansion increases according to a fixed coefficient of thermal expansion. When expansion is plotted against temperature, the transition temperature at which the physical properties of the material change is at the apex of the curve. The composition of a microbial cell depends on the species and condition of the cell; consequently, the rate of thermal expansion and the transition temperature also depend on the species and condition of the cell. We have developed a method for measuring the coefficient of thermal expansion and the transition temperature of cells using a nano thermal analysis system in order to study the physical nature of the cells. The tendency was seen that among vegetative cells, the Gram-negative Escherichia coli and Pseudomonas aeruginosa have higher coefficients of linear expansion and lower transition temperatures than the Gram-positive Staphylococcus aureus and Bacillus subtilis. On the other hand, spores, which have low water content, overall showed lower coefficients of linear expansion and higher transition temperatures than vegetative cells. Comparing these trends to non-microbial materials, vegetative cells showed phenomenon similar to plastics and spores showed behaviour similar to metals with regards to the coefficient of liner thermal expansion. We show that vegetative cells occur phenomenon of similar to plastics and spores to metals with regard to the coefficient of liner thermal expansion. Cells may be characterized by the coefficient of linear expansion as a physical index; the coefficient of linear expansion may also characterize cells structurally since it relates to volumetric changes, surface area changes, the degree of expansion of water contained within the cell, and the intensity of the internal stress on the cellular membrane. The coefficient of linear expansion holds promise as a new index for furthering the understanding of the characteristics of cells. It is likely to be a powerful tool for investigating changes in the rate of expansion and also in understanding the physical properties of cells.

With respect to coefficient of linear thermal expansion, bacterial vegetative cells and spores resemble plastics and metals, respectively

PubMed Central

2013-01-01

Background If a fixed stress is applied to the three-dimensional z-axis of a solid material, followed by heating, the amount of thermal expansion increases according to a fixed coefficient of thermal expansion. When expansion is plotted against temperature, the transition temperature at which the physical properties of the material change is at the apex of the curve. The composition of a microbial cell depends on the species and condition of the cell; consequently, the rate of thermal expansion and the transition temperature also depend on the species and condition of the cell. We have developed a method for measuring the coefficient of thermal expansion and the transition temperature of cells using a nano thermal analysis system in order to study the physical nature of the cells. Results The tendency was seen that among vegetative cells, the Gram-negative Escherichia coli and Pseudomonas aeruginosa have higher coefficients of linear expansion and lower transition temperatures than the Gram-positive Staphylococcus aureus and Bacillus subtilis. On the other hand, spores, which have low water content, overall showed lower coefficients of linear expansion and higher transition temperatures than vegetative cells. Comparing these trends to non-microbial materials, vegetative cells showed phenomenon similar to plastics and spores showed behaviour similar to metals with regards to the coefficient of liner thermal expansion. Conclusions We show that vegetative cells occur phenomenon of similar to plastics and spores to metals with regard to the coefficient of liner thermal expansion. Cells may be characterized by the coefficient of linear expansion as a physical index; the coefficient of linear expansion may also characterize cells structurally since it relates to volumetric changes, surface area changes, the degree of expansion of water contained within the cell, and the intensity of the internal stress on the cellular membrane. The coefficient of linear expansion holds promise as a new index for furthering the understanding of the characteristics of cells. It is likely to be a powerful tool for investigating changes in the rate of expansion and also in understanding the physical properties of cells. PMID:24107328

A proteomic network approach across the ALS-FTD disease spectrum resolves clinical phenotypes and genetic vulnerability in human brain.

PubMed

Umoh, Mfon E; Dammer, Eric B; Dai, Jingting; Duong, Duc M; Lah, James J; Levey, Allan I; Gearing, Marla; Glass, Jonathan D; Seyfried, Nicholas T

2018-01-01

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with overlap in clinical presentation, neuropathology, and genetic underpinnings. The molecular basis for the overlap of these disorders is not well established. We performed a comparative unbiased mass spectrometry-based proteomic analysis of frontal cortical tissues from postmortem cases clinically defined as ALS, FTD, ALS and FTD (ALS/FTD), and controls. We also included a subset of patients with the C9orf72 expansion mutation, the most common genetic cause of both ALS and FTD Our systems-level analysis of the brain proteome integrated both differential expression and co-expression approaches to assess the relationship of these differences to clinical and pathological phenotypes. Weighted co-expression network analysis revealed 15 modules of co-expressed proteins, eight of which were significantly different across the ALS-FTD disease spectrum. These included modules associated with RNA binding proteins, synaptic transmission, and inflammation with cell-type specificity that showed correlation with TDP-43 pathology and cognitive dysfunction. Modules were also examined for their overlap with TDP-43 protein-protein interactions, revealing one module enriched with RNA-binding proteins and other causal ALS genes that increased in FTD/ALS and FTD cases. A module enriched with astrocyte and microglia proteins was significantly increased in ALS cases carrying the C9orf72 mutation compared to sporadic ALS cases, suggesting that the genetic expansion is associated with inflammation in the brain even without clinical evidence of dementia. Together, these findings highlight the utility of integrative systems-level proteomic approaches to resolve clinical phenotypes and genetic mechanisms underlying the ALS-FTD disease spectrum in human brain. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Severity of Acute Infectious Mononucleosis Correlates with Cross-Reactive Influenza CD8 T-Cell Receptor Repertoires

PubMed Central

Aslan, Nuray; Watkin, Levi B.; Gil, Anna; Mishra, Rabinarayan; Clark, Fransenio G.; Welsh, Raymond M.; Ghersi, Dario; Luzuriaga, Katherine

2017-01-01

ABSTRACT Fifty years after the discovery of Epstein-Barr virus (EBV), it remains unclear how primary infection with this virus leads to massive CD8 T-cell expansion and acute infectious mononucleosis (AIM) in young adults. AIM can vary greatly in severity, from a mild transient influenza-like illness to a prolonged severe syndrome. We questioned whether expansion of a unique HLA-A2.01-restricted, cross-reactive CD8 T-cell response between influenza virus A-M158 (IAV-M1) and EBV BMLF1280 (EBV-BM) could modulate the immune response to EBV and play a role in determining the severity of AIM in 32 college students. Only ex vivo total IAV-M1 and IAV-M1+EBV-BM cross-reactive tetramer+ frequencies directly correlated with AIM severity and were predictive of severe disease. Expansion of specific cross-reactive memory IAV-M1 T-cell receptor (TCR) Vβ repertoires correlated with levels of disease severity. There were unique profiles of qualitatively different functional responses in the cross-reactive and EBV-specific CD8 T-cell responses in each of the three groups studied, severe-AIM patients, mild-AIM patients, and seropositive persistently EBV-infected healthy donors, that may result from differences in TCR repertoire use. IAV-M1 tetramer+ cells were functionally cross-reactive in short-term cultures, were associated with the highest disease severity in AIM, and displayed enhanced production of gamma interferon, a cytokine that greatly amplifies immune responses, thus frequently contributing to induction of immunopathology. Altogether, these data link heterologous immunity via CD8 T-cell cross-reactivity to CD8 T-cell repertoire selection, function, and resultant disease severity in a common and important human infection. In particular, it highlights for the first time a direct link between the TCR repertoire with pathogenesis and the diversity of outcomes upon pathogen encounter. PMID:29208744

Small molecule modulator of protein disulfide isomerase attenuates mutant huntingtin toxicity and inhibits endoplasmic reticulum stress in a mouse model of Huntington's disease.

PubMed

Zhou, Xiao; Li, Gang; Kaplan, Anna; Gaschler, Michael M; Zhang, Xiaoyan; Hou, Zhipeng; Jiang, Mali; Zott, Roseann; Cremers, Serge; Stockwell, Brent R; Duan, Wenzhen

2018-05-01

Huntington's disease (HD) is caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat expansion in the huntingtin (HTT) gene encoding an elongated polyglutamine tract within the N-terminal of the huntingtin protein (Htt) and leads to Htt misfolding, aberrant protein aggregation, and progressive appearance of disease symptoms. Chronic activation of endoplasmic reticulum (ER) stress by mutant Htt (mHtt) results in cellular dysfunction and ultimately cell death. Protein disulfide isomerase (PDI) is a chaperone protein located in the ER. Our previous studies demonstrated that mHtt caused PDI to accumulate at mitochondria-associated ER membranes and triggered cell death, and that modulating PDI activity using small molecules protected cells again mHtt toxicity in cell and brain slice models of HD. In this study, we demonstrated that PDI is upregulated in the HD human brain, in cell and mouse models. Chronic administration of a reversible, brain penetrable small molecule PDI modulator, LOC14 (20 mg/kg/day), significantly improved motor function, attenuated brain atrophy and extended survival in the N171-82Q HD mice. Moreover, LOC14 preserved medium spiny neuronal marker dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32 000 (DARPP32) levels in the striatum of HD mice. Mechanistic study revealed that LOC14 suppressed mHtt-induced ER stress, indicated by repressing the abnormally upregulated ER stress proteins in HD models. These findings suggest that LOC14 is promising to be further optimized for clinical trials of HD, and modulation of signaling pathways coping with ER stress may constitute an attractive approach to reduce mHtt toxicity and identify new therapeutic targets for treatment of HD.

Crustal fingering: solidification on a viscously unstable interface

NASA Astrophysics Data System (ADS)

Fu, Xiaojing; Jimenez-Martinez, Joaquin; Cueto-Felgueroso, Luis; Porter, Mark; Juanes, Ruben

2017-11-01

Motivated by the formation of gas hydrates in seafloor sediments, here we study the volumetric expansion of a less viscous gas pocket into a more viscous liquid when the gas-liquid interfaces readily solidify due to hydrate formation. We first present a high-pressure microfluidic experiment to study the depressurization-controlled expansion of a Xenon gas pocket in a water-filled Hele-Shaw cell. The evolution of the pocket is controlled by three processes: (1) volumetric expansion of the gas; (2) rupturing of existing hydrate films on the gas-liquid interface; and (3) formation of new hydrate films. These result in gas fingering leading to a complex labyrinth pattern. To reproduce these observations, we propose a phase-field model that describes the formation of hydrate shell on viscously unstable interfaces. We design the free energy of the three-phase system to rigorously account for interfacial effects, gas compressibility and phase transitions. We model the hydrate shell as a highly viscous fluid with shear-thinning rheology to reproduce shell-rupturing behavior. We present high-resolution numerical simulations of the model, which illustrate the emergence of complex crustal fingering patterns as a result of gas expansion dynamics modulated by hydrate growth at the interface.

An intermittent rocking platform for integrated expansion and differentiation of human pluripotent stem cells to cardiomyocytes in suspended microcarrier cultures.

PubMed

Ting, Sherwin; Chen, Allen; Reuveny, Shaul; Oh, Steve

2014-09-01

The development of novel platforms for large scale production of human embryonic stem cells (hESC) derived cardiomyocytes (CM) becomes more crucial as the demand for CMs in preclinical trials, high throughput cardio toxicity assays and future regenerative therapeutics rises. To this end, we have designed a microcarrier (MC) suspension agitated platform that integrates pluripotent hESC expansion followed by CM differentiation in a continuous, homogenous process. Hydrodynamic shear stresses applied during the hESC expansion and CM differentiation steps drastically reduced the capability of the cells to differentiate into CMs. Applying vigorous stirring during pluripotent hESC expansion on Cytodex 1 MC in spinner cultures resulted in low CM yields in the following differentiation step (cardiac troponin-T (cTnT): 22.83±2.56%; myosin heavy chain (MHC): 19.30±5.31%). Whereas the lower shear experienced in side to side rocker (wave type) platform resulted in higher CM yields (cTNT: 47.50±7.35%; MHC: 42.85±2.64%). The efficiency of CM differentiation is also affected by the hydrodynamic shear stress applied during the first 3days of the differentiation stage. Even low shear applied continuously by side to side rocker agitation resulted in very low CM differentiation efficiency (cTnT<5%; MHC<2%). Simply by applying intermittent agitation during these 3days followed by continuous agitation for the subsequent 9days, CM differentiation efficiency can be substantially increased (cTNT: 65.73±10.73%; MHC: 59.73±9.17%). These yields are 38.3% and 39.3% higher (for cTnT and MHC respectively) than static culture control. During the hESC expansion phase, cells grew on continuously agitated rocker platform as pluripotent cell/MC aggregates (166±88×10(5)μm(2)) achieving a cell concentration of 3.74±0.55×10(6)cells/mL (18.89±2.82 fold expansion) in 7days. These aggregates were further differentiated into CMs using a WNT modulation differentiation protocol for the subsequent 12days on a rocking platform with an intermittent agitation regime during the first 3days. Collectively, the integrated MC rocker platform produced 190.5±58.8×10(6) CMs per run (31.75±9.74 CM/hESC seeded). The robustness of the system was demonstrated by using 2 cells lines, hESC (HES-3) and human induced pluripotent stem cell (hiPSC) IMR-90. The CM/MC aggregates formed extensive sarcomeres that exhibited cross-striations confirming cardiac ontogeny. Functionality of the CMs was demonstrated by monitoring the effect of inotropic drug, Isoproterenol on beating frequency. In conclusion, we have developed a simple robust and scalable platform that integrates both hESC expansion and CM differentiation in one unit process which is capable of meeting the need for large amounts of CMs. Copyright © 2014. Published by Elsevier B.V.

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

PubMed Central

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M.; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2018-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4+ FoxP3+ regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3+-Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance. PMID:29422893

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo.

PubMed

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2017-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4 + FoxP3 + regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3 + -Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo . They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

The antagonistic modulation of Arp2/3 activity by N-WASP, WAVE2 and PICK1 defines dynamic changes in astrocyte morphology

PubMed Central

Murk, Kai; Blanco Suarez, Elena M.; Cockbill, Louisa M. R.; Banks, Paul; Hanley, Jonathan G.

2013-01-01

Summary Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult. PMID:23843614

Electrochemical-mechanical coupled modeling and parameterization of swelling and ionic transport in lithium-ion batteries

NASA Astrophysics Data System (ADS)

Sauerteig, Daniel; Hanselmann, Nina; Arzberger, Arno; Reinshagen, Holger; Ivanov, Svetlozar; Bund, Andreas

2018-02-01

The intercalation and aging induced volume changes of lithium-ion battery electrodes lead to significant mechanical pressure or volume changes on cell and module level. As the correlation between electrochemical and mechanical performance of lithium ion batteries at nano and macro scale requires a comprehensive and multidisciplinary approach, physical modeling accounting for chemical and mechanical phenomena during operation is very useful for the battery design. Since the introduced fully-coupled physical model requires proper parameterization, this work also focuses on identifying appropriate mathematical representation of compressibility as well as the ionic transport in the porous electrodes and the separator. The ionic transport is characterized by electrochemical impedance spectroscopy (EIS) using symmetric pouch cells comprising LiNi1/3Mn1/3Co1/3O2 (NMC) cathode, graphite anode and polyethylene separator. The EIS measurements are carried out at various mechanical loads. The observed decrease of the ionic conductivity reveals a significant transport limitation at high pressures. The experimentally obtained data are applied as input to the electrochemical-mechanical model of a prismatic 10 Ah cell. Our computational approach accounts intercalation induced electrode expansion, stress generation caused by mechanical boundaries, compression of the electrodes and the separator, outer expansion of the cell and finally the influence of the ionic transport within the electrolyte.

Pathogenesis of chronic active Epstein-Barr virus infection: is this an infectious disease, lymphoproliferative disorder, or immunodeficiency?

PubMed

Kimura, Hiroshi

2006-01-01

Chronic active Epstein-Barr virus infection (CAEBV) is characterised by chronic or recurrent infectious mononucleosis-like symptoms, such as fever, hepatosplenomegaly, persistent hepatitis and extensive lymphadenopathy. Patients with CAEBV have high viral loads in their peripheral blood and/or an unusual pattern of EBV-related antibodies. This disease is rare but severe with high morbidity and mortality. Nearly three decades have passed since this disease was first identified, and recent advances in technology have increased our understanding of CAEBV pathophysiology. There is accumulating evidence that the clonal expansion of EBV-infected T or natural killer (NK) cells plays a central role in the pathogenesis of CAEBV. However, it remains unclear whether CAEBV is truly a monoclonal lymphoproliferative disorder. EBV-infected T or NK cells are able to evade the host cellular immune system due to the limited expression of viral proteins of reduced antigenicity. Recent studies suggest that infection of T or NK cells is a common event during primary EBV infection. A defect or single nucleotide polymorphism in host immune-modulating genes may allow for the expansion of virus infected cells giving rise to CAEBV. In this review, I summarise our current understanding of the pathogenesis of CAEBV and propose a model of CAEBV pathogenicity.

Excess fructose intake-induced hypertrophic visceral adipose tissue results from unbalanced precursor cell adipogenic signals.

PubMed

Zubiría, María G; Fariña, Juan P; Moreno, Griselda; Gagliardino, Juan J; Spinedi, Eduardo; Giovambattista, Andrés

2013-11-01

We studied the effect of feeding normal adult male rats with a commercial diet supplemented with fructose added to the drinking water (10% w/v; fructose-rich diet, FRD) on the adipogenic capacity of stromal-vascular fraction (SVF) cells isolated from visceral adipose tissue (VAT) pads. Animals received either the commercial diet or FRD ad libitum for 3 weeks; thereafter, we evaluated the in vitro proliferative and adipogenic capacities of their VAT SVF cells. FRD significantly increased plasma insulin, triglyceride and leptin levels, VAT mass/cell size, and the in vitro adipogenic capacity of SVF cells. Flow cytometry studies indicated that the VAT precursor cell population number did not differ between groups; however, the accelerated adipogenic process could result from an imbalance between endogenous pro- and anti-adipogenic SVF cell signals, which are clearly shifted towards the former. The increased insulin milieu and its intracellular mediator (insulin receptor substrate-1) in VAT pads, as well as the enhanced SVF cell expression of Zpf423 and peroxisome proliferator receptor-γ2 (all pro-adipogenic modulators), together with a decreased SVF cell concentration of anti-adipogenic factors (pre-adipocyte factor-1 and wingless-type MMTV-10b), strongly supports this assumption. We hypothesize that the VAT mass expansion recorded in FRD rats results from the combination of initial accelerated adipogenesis and final cell hypertrophy. It remains to be determined whether FRD administration over longer periods could perpetuate both processes, or whether cell hypertrophy itself remains responsible for a further VAT mass expansion, as observed in advanced/morbid obesity. © 2013 FEBS.

Modulating the stem cell niche for tissue regeneration

PubMed Central

Lane, Steven W; Williams, David A; Watt, Fiona M

2015-01-01

The field of regenerative medicine holds considerable promise for treating diseases that are currently intractable. Although many researchers are adopting the strategy of cell transplantation for tissue repair, an alternative approach to therapy is to manipulate the stem cell microenvironment, or niche, to facilitate repair by endogenous stem cells. The niche is highly dynamic, with multiple opportunities for intervention. These include administration of small molecules, biologics or biomaterials that target specific aspects of the niche, such as cell-cell and cell–extracellular matrix interactions, to stimulate expansion or differentiation of stem cells, or to cause reversion of differentiated cells to stem cells. Nevertheless, there are several challenges in targeting the niche therapeutically, not least that of achieving specificity of delivery and responses. We envisage that successful treatments in regenerative medicine will involve different combinations of factors to target stem cells and niche cells, applied at different times to effect recovery according to the dynamics of stem cell–niche interactions. PMID:25093887

Objectively Quantifying Radiation Esophagitis With Novel Computed Tomography–Based Metrics

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

Niedzielski, Joshua S., E-mail: jsniedzielski@mdanderson.org; University of Texas Houston Graduate School of Biomedical Science, Houston, Texas; Yang, Jinzhong

Purpose: To study radiation-induced esophageal expansion as an objective measure of radiation esophagitis in patients with non-small cell lung cancer (NSCLC) treated with intensity modulated radiation therapy. Methods and Materials: Eighty-five patients had weekly intra-treatment CT imaging and esophagitis scoring according to Common Terminlogy Criteria for Adverse Events 4.0, (24 Grade 0, 45 Grade 2, and 16 Grade 3). Nineteen esophageal expansion metrics based on mean, maximum, spatial length, and volume of expansion were calculated as voxel-based relative volume change, using the Jacobian determinant from deformable image registration between the planning and weekly CTs. An anatomic variability correction method wasmore » validated and applied to these metrics to reduce uncertainty. An analysis of expansion metrics and radiation esophagitis grade was conducted using normal tissue complication probability from univariate logistic regression and Spearman rank for grade 2 and grade 3 esophagitis endpoints, as well as the timing of expansion and esophagitis grade. Metrics' performance in classifying esophagitis was tested with receiver operating characteristic analysis. Results: Expansion increased with esophagitis grade. Thirteen of 19 expansion metrics had receiver operating characteristic area under the curve values >0.80 for both grade 2 and grade 3 esophagitis endpoints, with the highest performance from maximum axial expansion (MaxExp1) and esophageal length with axial expansion ≥30% (LenExp30%) with area under the curve values of 0.93 and 0.91 for grade 2, 0.90 and 0.90 for grade 3 esophagitis, respectively. Conclusions: Esophageal expansion may be a suitable objective measure of esophagitis, particularly maximum axial esophageal expansion and esophageal length with axial expansion ≥30%, with 2.1 Jacobian value and 98.6 mm as the metric value for 50% probability of grade 3 esophagitis. The uncertainty in esophageal Jacobian calculations can be reduced with anatomic correction methods.« less

The mechanics of surface expansion anisotropy in Medicago truncatula root hairs.

PubMed

Dumais, Jacques; Long, Sharon R; Shaw, Sidney L

2004-10-01

Wall expansion in tip-growing cells shows variations according to position and direction. In Medicago truncatula root hairs, wall expansion exhibits a strong meridional gradient with a maximum near the pole of the cell. Root hair cells also show a striking expansion anisotropy, i.e. over most of the dome surface the rate of circumferential wall expansion exceeds the rate of meridional expansion. Concomitant measurements of expansion rates and wall stresses reveal that the extensibility of the cell wall must vary abruptly along the meridian of the cell to maintain the gradient of wall expansion. To determine the mechanical basis of expansion anisotropy, we compared measurements of wall expansion with expansion patterns predicted from wall structural models that were either fully isotropic, transversely isotropic, or fully anisotropic. Our results indicate that a model based on a transversely isotropic wall structure can provide a good fit of the data although a fully anisotropic model offers the best fit overall. We discuss how such mechanical properties could be controlled at the microstructural level.

Intermediate progenitors are increased by lengthening of the cell cycle through calcium signaling and p53 expression in human neural progenitors

PubMed Central

García-García, Elisa; Pino-Barrio, María José; López-Medina, Laura; Martínez-Serrano, Alberto

2012-01-01

During development, neurons can be generated directly from a multipotent progenitor or indirectly through an intermediate progenitor (IP). This last mode of division amplifies the progeny of neurons. The mechanisms governing the generation and behavior of IPs are not well understood. In this work, we demonstrate that the lengthening of the cell cycle enhances the generation of neurons in a human neural progenitor cell system in vitro and also the generation and expansion of IPs. These IPs are insulinoma-associated 1 (Insm1)+/BTG family member 2 (Btg2)−, which suggests an increase in a self-amplifying IP population. Later the cultures express neurogenin 2 (Ngn2) and become neurogenic. The signaling responsible for this cell cycle modulation is investigated. It is found that the release of calcium from the endoplasmic reticulum to the cytosol in response to B cell lymphoma-extra large overexpression or ATP addition lengths the cell cycle and increases the number of IPs and, in turn, the final neuron outcome. Moreover, data suggest that the p53–p21 pathway is responsible for the changes in cell cycle. In agreement with this, increased p53 levels are necessary for a calcium-induced increase in neurons. Our findings contribute to understand how calcium signaling can modulate cell cycle length during neurogenesis. PMID:22323293

FamNet: A Framework to Identify Multiplied Modules Driving Pathway Expansion in Plants1

PubMed Central

Tohge, Takayuki; Klie, Sebastian; Fernie, Alisdair R.

2016-01-01

Gene duplications generate new genes that can acquire similar but often diversified functions. Recent studies of gene coexpression networks have indicated that, not only genes, but also pathways can be multiplied and diversified to perform related functions in different parts of an organism. Identification of such diversified pathways, or modules, is needed to expand our knowledge of biological processes in plants and to understand how biological functions evolve. However, systematic explorations of modules remain scarce, and no user-friendly platform to identify them exists. We have established a statistical framework to identify modules and show that approximately one-third of the genes of a plant’s genome participate in hundreds of multiplied modules. Using this framework as a basis, we implemented a platform that can explore and visualize multiplied modules in coexpression networks of eight plant species. To validate the usefulness of the platform, we identified and functionally characterized pollen- and root-specific cell wall modules that multiplied to confer tip growth in pollen tubes and root hairs, respectively. Furthermore, we identified multiplied modules involved in secondary metabolite synthesis and corroborated them by metabolite profiling of tobacco (Nicotiana tabacum) tissues. The interactive platform, referred to as FamNet, is available at http://www.gene2function.de/famnet.html. PMID:26754669

[Ex vivo expansion and clonal variation of CD34(+)CD59(+) cells from bone marrow in children with paroxysmal nocturnal hemoglobinuria].

PubMed

Xiao, Juan; Wu, Yong-Ji; Han, Bing; Dong, Hong-Yan; Chen, Shi-Ping

2013-08-01

To investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH. CD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted. The optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01). The CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.

Stem cells as delivery vehicles for regenerative medicine-challenges and perspectives

PubMed Central

Labusca, Luminita; Herea, Dumitru Daniel; Mashayekhi, Kaveh

2018-01-01

The use of stem cells as carriers for therapeutic agents is an appealing modality for targeting tissues or organs of interest. Combined delivery of cells together with various information molecules as therapeutic agents has the potential to enhance, modulate or even initiate local or systemic repair processes, increasing stem cell efficiency for regenerative medicine applications. Stem-cell-mediated delivery of genes, proteins or small molecules takes advantage of the innate capability of stem cells to migrate and home to injury sites. As the native migratory properties are affected by in vitro expansion, the existent methods for enhancing stem cell targeting capabilities (modified culture methods, genetic modification, cell surface engineering) are described. The role of various nanoparticles in equipping stem cells with therapeutic small molecules is revised together with their class-specific advantages and shortcomings. Modalities to circumvent common challenges when designing a stem-cell-mediated targeted delivery system are described as well as future prospects in using this approach for regenerative medicine applications. PMID:29849930

Three-dimensional patterns of cell division and expansion throughout the development of Arabidopsis thaliana leaves.

PubMed

Kalve, Shweta; Fotschki, Joanna; Beeckman, Tom; Vissenberg, Kris; Beemster, Gerrit T S

2014-12-01

Variations in size and shape of multicellular organs depend on spatio-temporal regulation of cell division and expansion. Here, cell division and expansion rates were quantified relative to the three spatial axes in the first leaf pair of Arabidopsis thaliana. The results show striking differences in expansion rates: the expansion rate in the petiole is higher than in the leaf blade; expansion rates in the lateral direction are higher than longitudinal rates between 5 and 10 days after stratification, but become equal at later stages of leaf blade development; and anticlinal expansion co-occurs with, but is an order of magnitude slower than periclinal expansion. Anticlinal expansion rates also differed greatly between tissues: the highest rates occurred in the spongy mesophyll and the lowest in the epidermis. Cell division rates were higher and continued for longer in the epidermis compared with the palisade mesophyll, causing a larger increase of palisade than epidermal cell area over the course of leaf development. The cellular dynamics underlying the effect of shading on petiole length and leaf thickness were then investigated. Low light reduced leaf expansion rates, which was partly compensated by increased duration of the growth phase. Inversely, shading enhanced expansion rates in the petiole, so that the blade to petiole ratio was reduced by 50%. Low light reduced leaf thickness by inhibiting anticlinal cell expansion rates. This effect on cell expansion was preceded by an effect on cell division, leading to one less layer of palisade cells. The two effects could be uncoupled by shifting plants to contrasting light conditions immediately after germination. This extended kinematic analysis maps the spatial and temporal heterogeneity of cell division and expansion, providing a framework for further research to understand the molecular regulatory mechanisms involved. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A Rab5 GTPase module is important for autophagosome closure

PubMed Central

Lipatova, Zhanna; Sun, Dan; Zhu, Xiaolong; Li, Rui; Wu, Zulin; You, Weiming; Cong, Xiaoxia; Zhou, Yiting; Gyurkovska, Valeriya; Liu, Yutao; Li, Qunli; Li, Wenjing; Cheng, Jie; Segev, Nava

2017-01-01

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure. PMID:28934205

The contribution of B-cell proliferation to spleen enlargement in Babesia microti-infected mice.

PubMed Central

Inchley, C J

1987-01-01

Flow cytofluorimetric analysis showed that B-cell proliferation makes a major contribution to the enlargement and increased cellularity of the spleen, which are characteristic of Babesia microti infections in mice. Expansion of the B-cell population was accompanied by modulation of the cell surface, which affected most B lymphocytes, and which was detected as a reduction in the density of surface immunoglobulin. This effect was noted as early as Day 7, shortly after the appearance of parasites in the circulation and the onset of gross spleen changes. In contrast to the results for B cells, the frequency of splenic T cells declined, and when the data were transformed into absolute numbers it became clear that only limited T-cell proliferation had occurred. There was no evidence to suggest that the balance of T-cell subsets was shifted in favour of suppressor T cells. The relationships of these results to reports of immunosuppression by this parasite are discussed. Images Figure 2 Figure 5 PMID:3493207

Aryl hydrocarbon receptor activation impairs the priming but not the recall of influenza virus-specific CD8+ T cells in the lung.

PubMed

Lawrence, B Paige; Roberts, Alan D; Neumiller, Joshua J; Cundiff, Jennifer A; Woodland, David L

2006-11-01

The response of CD8+ T cells to influenza virus is very sensitive to modulation by aryl hydrocarbon receptor (AhR) agonists; however, the mechanism underlying AhR-mediated alterations in CD8+ T cell function remains unclear. Moreover, very little is known regarding how AhR activation affects anamnestic CD8+ T cell responses. In this study, we analyzed how AhR activation by the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the in vivo distribution and frequency of CD8+ T cells specific for three different influenza A virus epitopes during and after the resolution of a primary infection. We then determined the effects of TCDD on the expansion of virus-specific memory CD8+ T cells during recall challenge. Adoptive transfer of AhR-null CD8+ T cells into congenic AhR(+/+) recipients, and the generation of CD45.2AhR(-/-)-->CD45.1AhR(+/+) chimeric mice demonstrate that AhR-regulated events within hemopoietic cells, but not directly within CD8+ T cells, underlie suppressed expansion of virus-specific CD8+ T cells during primary infection. Using a dual-adoptive transfer approach, we directly compared the responsiveness of virus-specific memory CD8+ T cells created in the presence or absence of TCDD, which revealed that despite profound suppression of the primary response to influenza virus, the recall response of virus-specific CD8+ T cells that form in the presence of TCDD is only mildly impaired. Thus, the delayed kinetics of the recall response in TCDD-treated mice reflects the fact that there are fewer memory cells at the time of reinfection rather than an inherent defect in the responsive capacity of virus-specific memory CD8+ cells.

Enhanced hepatic differentiation of human amniotic epithelial cells on polyethylene glycol-linked multiwalled carbon nanotube-coated hydrogels.

PubMed

Zhao, Chunyan; Lin, Jamie Siqi; Choolani, Mahesh; Dan, Yock Young; Pastorin, Giorgia; Ho, Han Kiat

2018-04-26

Polyethylene glycol-linked multiwalled carbon nanotube-coated poly-acrylamide hydrogel (CNT-PA) was customized to mimic human liver stiffness and nanostructured surface in liver cells for modulating differentiation of human amniotic epithelial cells (hAECs) into functional hepatocyte-like cells (HLCs) in vitro. This composite of CNT-PA matrix enhanced the hepatic differentiation of hAECs into HLCs with suppression of pluripotent markers and up-regulation of hepatic markers at both transcript and protein levels. Furthermore, the HLCs on CNT-PA demonstrated hepatocytic functions in terms of albumin secretion, higher uptake of indocyanine green, and comparable CYP3A4 enzymatic function and inducibility when matched against HepG2 cells. Taken together, CNT-PA provides an efficient and scalable platform for the expansion of HLCs from hAECs and could be explored further for downstream development. Copyright © 2018 John Wiley & Sons, Ltd.

Adoptive immunotherapy for the treatment of glioblastoma: progress and possibilities.

PubMed

Kuramitsu, Shunichiro; Yamamichi, Akane; Ohka, Fumiharu; Motomura, Kazuya; Hara, Masahito; Natsume, Atsushi

2016-12-01

Patients with glioblastoma have a very poor prognosis. Adoptive cellular therapy (ACT) is defined as the collection of circulating or tumor-infiltrating lymphocytes, their selection, modification, expansion and activation, and their re-administration to patients in order to induce antitumor activity. Although various ACTs have been attempted, most failed to improve the outcome. Immune checkpoint blockade antibodies and T cell engineering with tumor-specific chimeric antigen receptors suggest the emergence of a new era of immunotherapy. Here, we summarize approaches with ACTs using genetically modified T cells, which have been improved by enhancing their antitumor activity, and discuss strategies to develop these therapies. The mechanisms by which gliomas modulate and evade the immune system are also discussed.

Attention Induced Gain Stabilization in Broad and Narrow-Spiking Cells in the Frontal Eye-Field of Macaque Monkeys

PubMed Central

Brandt, Christian; Dasilva, Miguel; Gotthardt, Sascha; Chicharro, Daniel; Panzeri, Stefano; Distler, Claudia

2016-01-01

Top-down attention increases coding abilities by altering firing rates and rate variability. In the frontal eye field (FEF), a key area enabling top-down attention, attention induced firing rate changes are profound, but its effect on different cell types is unknown. Moreover, FEF is the only cortical area investigated in which attention does not affect rate variability, as assessed by the Fano factor, suggesting that task engagement affects cortical state nonuniformly. We show that putative interneurons in FEF of Macaca mulatta show stronger attentional rate modulation than putative pyramidal cells. Partitioning rate variability reveals that both cell types reduce rate variability with attention, but more strongly so in narrow-spiking cells. The effects are captured by a model in which attention stabilizes neuronal excitability, thereby reducing the expansive nonlinearity that links firing rate and variance. These results show that the effect of attention on different cell classes and different coding properties are consistent across the cortical hierarchy, acting through increased and stabilized neuronal excitability. SIGNIFICANCE STATEMENT Cortical processing is critically modulated by attention. A key feature of this influence is a modulation of “cortical state,” resulting in increased neuronal excitability and resilience of the network against perturbations, lower rate variability, and an increased signal-to-noise ratio. In the frontal eye field (FEF), an area assumed to control spatial attention in human and nonhuman primates, firing rate changes with attention occur, but rate variability, quantified by the Fano factor, appears to be unaffected by attention. Using recently developed analysis tools and models to quantify attention effects on narrow- and broad-spiking cell activity, we show that attention alters cortical state strongly in the FEF, demonstrating that its effect on the neuronal network is consistent across the cortical hierarchy. PMID:27445139

Broncho-Vaxom Attenuates Allergic Airway Inflammation by Restoring GSK3β-Related T Regulatory Cell Insufficiency

PubMed Central

Zhong, Hua; Yu, Dehong; Zeng, Xianping; Deng, Mengxia; Sun, Yueqi; Wen, Weiping; Li, Huabin

2014-01-01

Background Oral administration of bacterial extracts (eg, Broncho-Vaxom (BV)) has been proposed to attenuate asthma through modulating Treg cells. However, the underlying mechanism has not been fully characterized. This study sought to assess the effects of oral administration of BV on GSK-3β expression and Treg cells in ovalbumin (OVA)-induced asthmatic mice models. Method Asthmatic mice models were established with OVA challenge and treated with oral administration of BV. Next, infiltration of inflammatory cells including eosinophil and neutrophils, mucous metaplasia, levels of Th1/Th2/Treg-typed cytokines and expression of GSK3β and Foxp3 were examined in asthmatic mice models by histological analysis, Bio-Plex and western blot, respectively. Moreover, the frequencies of Treg cells were evaluated in cultured splenocytes by flow cytometry in the presence of BV or GSK3β siRNA interference. Results We found significant decrease of infiltrated inflammatory cells in bronchoalveolar lavage fluid (BALF) in asthmatic mice models after oral administration of BV. Oral administration of BV was shown to significantly suppress mucus metaplasia, Th2-typed cytokine levels and GSK3β expression while increasing Foxp3 production in asthmatic mice models. Moreover, BV significantly enhanced GSK3β-related expansion of Treg cells in cultured spleen cells in vitro. Conclusion Our findings provide evidence that oral administration of BV is capable of attenuating airway inflammation in asthmatic mice models, which may be associated with GSK3β-related expansion of Treg cells. PMID:24667347

Induction of cell expansion of goldfish melanocytoma cells (GMM-1) by epinephrine and dexamethasone requires external calcium.

PubMed

Shih, Y L; Lo, S J

1993-05-01

Treatment of GMM-1 (a goldfish melanocytoma cell line) cells with epinephrine induced a rapid cell expansion (flattening of cells, extension and broadening of cellular processes) similar to the effect of dexamethasone reported previously (Shih et al., 1990). Studies on the possible involvement of secondary messengers in cell expansion indicated that (i) both 8-bromo-CAMP and forskolin caused cell shrinking (the opposite of cell expansion); (ii) TPA also caused cell shrinking; (iii) phospholipid derivatives, such as 1,2-dioctanoyl-sn-glycerol, lysophosphatidic acid, and arachidonic acid caused cell expansion; and (iv) EGTA (calcium chelator) and nifedipine (calcium channel blocker) inhibited the effect of epinephrine. Together with the previous findings, these observations indicate that epinephrine and dexamethasone may share a common pathway in triggering an external calcium influx to cause cell expansion. The results of the effects of epinephrine agonists and antagonists, together with those of other workers, also show that there are multiple isoforms of adrenoceptor in the goldfish.

Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures

PubMed Central

Olmos, Enrique; García De La Garma, Jesús; Gomez-Jimenez, Maria C.; Fernandez-Garcia, Nieves

2017-01-01

Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations. PMID:28676820

Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures.

PubMed

Olmos, Enrique; García De La Garma, Jesús; Gomez-Jimenez, Maria C; Fernandez-Garcia, Nieves

2017-01-01

Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

SHIP deficiency enhances HSC proliferation and survival but compromises homing and repopulation

PubMed Central

Desponts, Caroline; Hazen, Amy L.; Paraiso, Kim H. T.; Kerr, William G.

2006-01-01

The SH2 domain–containing inositol 5′-phosphatase-1 (SHIP) has the potential to modulate multiple signaling pathways downstream of receptors that impact hematopoietic stem cell (HSC) biology. Therefore, we postulated that SHIP might play an important role in HSC homeostasis and function. Consistent with this hypothesis, HSC proliferation and numbers are increased in SHIP–/– mice. Despite expansion of the compartment, SHIP–/– HSCs exhibit reduced capacity for long-term repopulation. Interestingly, we observe that SHIP–/– stem/progenitor cells home inefficiently to bone marrow (BM), and consistent with this finding, have reduced surface levels of both CXCR4 and vascular cell adhesion marker-1 (VCAM-1). These studies demonstrate that SHIP is critical for normal HSC function, homeostasis, and homing. PMID:16467196

A phase I open-label dose-escalation study of the anti-HER3 monoclonal antibody LJM716 in patients with advanced squamous cell carcinoma of the esophagus or head and neck and HER2-overexpressing breast or gastric cancer.

PubMed

Reynolds, Kerry Lynn; Bedard, Philippe L; Lee, Se-Hoon; Lin, Chia-Chi; Tabernero, Josep; Alsina, Maria; Cohen, Ezra; Baselga, José; Blumenschein, George; Graham, Donna M; Garrido-Laguna, Ignacio; Juric, Dejan; Sharma, Sunil; Salgia, Ravi; Seroutou, Abdelkader; Tian, Xianbin; Fernandez, Rose; Morozov, Alex; Sheng, Qing; Ramkumar, Thiruvamoor; Zubel, Angela; Bang, Yung-Jue

2017-09-12

Human epidermal growth factor receptor 3 (HER3) is important in maintaining epidermal growth factor receptor-driven cancers and mediating resistance to targeted therapy. A phase I study of anti-HER3 monoclonal antibody LJM716 was conducted with the primary objective to identify the maximum tolerated dose (MTD) and/or recommended dose for expansion (RDE), and dosing schedule. Secondary objectives were to characterize safety/tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity. This open-label, dose-finding study comprised dose escalation, followed by expansion in patients with squamous cell carcinoma of the head and neck or esophagus, and HER2-overexpressing metastatic breast cancer or gastric cancer. During dose escalation, patients received LJM716 intravenous once weekly (QW) or every two weeks (Q2W), in 28-day cycles. An adaptive Bayesian logistic regression model was used to guide dose escalation and establish the RDE. Exploratory pharmacodynamic tumor studies evaluated modulation of HER3 signaling. Patients received LJM716 3-40 mg/kg QW and 20 mg/kg Q2W (54 patients; 36 patients at 40 mg/kg QW). No dose-limiting toxicities (DLTs) were reported during dose-escalation. One patient experienced two DLTs (diarrhea, hypokalemia [both grade 3]) in the expansion phase. The RDE was 40 mg/kg QW, providing drug levels above the preclinical minimum effective concentration. One patient with gastric cancer had an unconfirmed partial response; 17/54 patients had stable disease, two lasting >30 weeks. Down-modulation of phospho-HER3 was observed in paired tumor samples. LJM716 was well tolerated; the MTD was not reached, and the RDE was 40 mg/kg QW. Further development of LJM716 is ongoing. Clinicaltrials.gov registry number NCT01598077 (registered on 4 May, 2012).

Investigation of a thiazolidinone derivative as an allosteric modulator of follicle stimulating hormone receptor: evidence for its ability to support follicular development and ovulation.

PubMed

Sriraman, Venkataraman; Denis, Deborah; de Matos, Daniel; Yu, Henry; Palmer, Stephen; Nataraja, Selva

2014-05-15

FSH signalling through its cognate receptor is critical for follicular development and ovulation. An earlier study had documented thiazolidinone derivatives to activate FSH receptor expressed in CHO cells and rat granulosa cells; however development of this compound for clinical use was halted for unobvious reasons. The objective of the current study is to extend the previous investigations in detail on the ability of thiazolidinone derivative (henceforth referred to as Compound 5) to activate FSH signalling and learn the barriers that preclude development of this derivative for clinical purposes. Our results demonstrate that the Compound 5 in a dose-dependent manner stimulated cAMP production, activated AKT and ERK signalling pathways and induced estradiol production in cultured rat granulosa cells. Compound 5 also caused dose-dependent increase in estradiol production from human granulosa cells. In increasingly more complex in vitro systems, Compound 5 was able to induce the expansion of mouse cumulus-oocyte-complex and support in vitro development of mouse preantral follicle to preovulatory stage and release of oocyte from the follicle. In vivo, the compound stimulated preovulatory follicular development and ovulation in immature rats. Pharmacokinetic and safety investigations reveal poor oral availability and genotoxicity. Together, our results document Compound 5 to act as a FSHR allosteric modulator but have poor pharmacological properties for development of an oral FSH receptor modulator. Copyright © 2014 Elsevier Inc. All rights reserved.

Vocational-Technical Physics Project. Thermometers: I. Temperature and Heat, II. Expansion Thermometers, III. Electrical Thermometers. Field Test Edition.

ERIC Educational Resources Information Center

Forsyth Technical Inst., Winston-Salem, NC.

This vocational physics individualized student instructional module on thermometers consists of the three units: Temperature and heat, expansion thermometers, and electrical thermometers. Designed with a laboratory orientation, experiments are included on linear expansion; making a bimetallic thermometer, a liquid-in-gas thermometer, and a gas…

Human bone marrow-derived mesenchymal cells differentiate and mature into endocrine pancreatic lineage in vivo.

PubMed

Phadnis, Smruti M; Joglekar, Mugdha V; Dalvi, Maithili P; Muthyala, Sudhakar; Nair, Prabha D; Ghaskadbi, Surendra M; Bhonde, Ramesh R; Hardikar, Anandwardhan A

2011-03-01

The scarcity of human islets for transplantation remains a major limitation of cell replacement therapy for diabetes. Bone marrow-derived progenitor cells are of interest because they can be isolated, expanded and offered for such therapy under autologous/allogeneic settings. We characterized and compared human bone marrow-derived mesenchymal cells (hBMC) obtained from (second trimester), young (1-24 years) and adult (34-81 years) donors. We propose a novel protocol that involves assessment of paracrine factors from regenerating pancreas in differentiation and maturation of hBMC into endocrine pancreatic lineage in vivo. We observed that donor age was inversely related to growth potential of hBMC. Following in vitro expansion and exposure to specific growth factors involved in pancreatic development, hBMC migrated and formed islet-like cell aggregates (ICA). ICA show increased abundance of pancreatic transcription factors (Ngn3, Brn4, Nkx6.1, Pax6 and Isl1). Although efficient differentiation was not achieved in vitro, we observed significant maturation and secretion of human c-peptide (insulin) upon transplantation into pancreactomized and Streptozotocin (STZ)-induced diabetic mice. Transplanted ICA responded to glucose and maintained normoglycemia in diabetic mice. Our data demonstrate that hBMC have tremendous in vitro expansion potential and can be differentiated into multiple lineages, including the endocrine pancreatic lineage. Paracrine factors secreted from regenerating pancreas help in efficient differentiation and maturation of hBMC, possibly via recruiting chromatin modulators, to generate glucose-responsive insulin-secreting cells.

Filarial infection modulates the immune response to Mycobacterium tuberculosis through expansion of CD4+ IL-4 memory T cells

PubMed Central

Chatterjee, Soumya; Clark, Carolyn E.; Lugli, Enrico; Roederer, Mario; Nutman, Thomas B.

2015-01-01

Exaggerated CD4+T helper 2-specific cytokine producing memory T cell responses developing concomitantly with a T helper1 response might have a detrimental role in immunity to infection caused by Mycobacterium tuberculosis (Mtb). To assess the dynamics of antigen (Ag)-specific memory T cell compartments in the context of filarial infection we used multiparameter flow cytometry on PBMCs from 25 microfilaremic filarial -infected (Inf) and 14 filarial-uninfected (Uninf) subjects following stimulation with filarial (BmA) or with the Mycobacterium tuberculosis (Mtb)-specific Ag CFP10. Our data demonstrated that the Inf group not only had a marked increase in BmA-specific CD4+IL-4+ cells (Median net frequency compared to baseline (Fo)=0.09% vs. 0.01%, p=0.038) but also to CFP10 (Fo =0.16% vs. 0.007%, p=0.04) and Staphylococcal Enterotoxin B (SEB) (Fo =0.49% vs. 0.26%, p=0.04). The Inf subjects showed a BmA-specific expansion of CD4+CD45RO+IL-4+ producing central memory (TCM, CD45RO+CCR7+CD27+) (Fo =1.1% vs. 0.5%, p=0.04) as well as effector memory (TEM CD45RO+CCR7-CD27-) (Fo =1.5% vs. 0.2%, p=0.03) with a similar but non-significant response to CFP10. In addition, there was expansion of CD4+ IL-4+ CD45RA+ CCR7+CD27+ (naïve-like) in Inf individuals compared to Uninf subjects. Among Inf subjects with definitive latent tuberculosis , there were no differences in frequencies of IL-4 producing cells within any of the memory compartments compared to the Uninf group. Our data suggest that filarial infection induces antigen-specific, exaggerated IL-4 responses in distinct T cell memory compartments to Mtb-specific antigens, which are attenuated in subjects who are able to mount a delayed type hypersensitivity reaction to Mtb. PMID:25667413

Ex vivo expansion of highly cytotoxic human NK cells by cocultivation with irradiated tumor cells for adoptive immunotherapy.

PubMed

Lim, Seon Ah; Kim, Tae-Jin; Lee, Jung Eun; Sonn, Chung Hee; Kim, Kwanghee; Kim, Jiyoung; Choi, Jong Gwon; Choi, Il-Kyu; Yun, Chae-Ok; Kim, Jae-Hong; Yee, Cassian; Kumar, Vinay; Lee, Kyung-Mi

2013-04-15

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I-negative or -mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1-mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1-lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation. ©2012 AACR.

Low-dose radiation modulates human mesenchymal stem cell proliferation through regulating CDK and Rb.

PubMed

Yang, Lei; Liu, Ziling; Chen, Chen; Cong, Xiaofeng; Li, Zhi; Zhao, Shasha; Ren, Meng

2017-01-01

Low-dose radiation (LDR) has been known to stimulate cell proliferation. The effect of LDR on human bone marrow mesenchymal stem cells (BMSCs), however, remains to be determined. The current study, therefore, aimed to investigate the effect of LDR on human BMSC proliferation and its mechanisms. To accomplish this, human BMSCs were isolated from ribs and cultured with or without exposition to LDR (75 mGy) for 24 h. Cell proliferation was assessed by MTT assay, the cytokines secreted by the BMSCs were quantified by ELISA, and the proteins associated with cell proliferation and cell cycle were evaluated by immunoblot analysis. BMSCs isolated from human ribs were capable of differentiating into osteoblasts and adipocytes. LDR stimulated human BMSC proliferation (0.580 ± 0.106 vs 0.419 ± 0.026 on day 4, P < 0.05; 0.794 ± 0.025 vs 0.689 ± 0.047 on day 7, P < 0.05) and increased S-phase proportion. LDR significantly enhanced the production of SCF, GM-CSF, and IL-11. Moreover, BMSCs modulated T-cell proliferation, and LDR further augmented the modulatory effect of BMSCs on T-cell proliferation. Cell cycle-associated proteins, such as Rb, CDK1, and CDC25B, appeared to mediate the stimulatory effect of LDR on BMSC proliferation. The findings of the current study indicate that physical stimulants, such as LDR, could be used for the large-scale expansion of human BMSCs, and thus may be used for MSC cellular therapy in clinic.

The cell fate determinant Scribble is required for maintenance of hematopoietic stem cell function.

PubMed

Mohr, Juliane; Dash, Banaja P; Schnoeder, Tina M; Wolleschak, Denise; Herzog, Carolin; Tubio Santamaria, Nuria; Weinert, Sönke; Godavarthy, Sonika; Zanetti, Costanza; Naumann, Michael; Hartleben, Björn; Huber, Tobias B; Krause, Daniela S; Kähne, Thilo; Bullinger, Lars; Heidel, Florian H

2018-05-01

Cell fate determinants influence self-renewal potential of hematopoietic stem cells. Scribble and Llgl1 belong to the Scribble polarity complex and reveal tumor-suppressor function in drosophila. In hematopoietic cells, genetic inactivation of Llgl1 leads to expansion of the stem cell pool and increases self-renewal capacity without conferring malignant transformation. Here we show that genetic inactivation of its putative complex partner Scribble results in functional impairment of hematopoietic stem cells (HSC) over serial transplantation and during stress. Although loss of Scribble deregulates transcriptional downstream effectors involved in stem cell proliferation, cell signaling, and cell motility, these effectors do not overlap with transcriptional targets of Llgl1. Binding partner analysis of Scribble in hematopoietic cells using affinity purification followed by mass spectometry confirms its role in cell signaling and motility but not for binding to polarity modules described in drosophila. Finally, requirement of Scribble for self-renewal capacity also affects leukemia stem cell function. Thus, Scribble is a regulator of adult HSCs, essential for maintenance of HSCs during phases of cell stress.

Cryopreservation of cord blood CD34+ cells before or after thrombopoietin expansion differentially affects early platelet recovery in NOD SCID mice.

PubMed

van Hensbergen, Yvette; van der Garde, Mark; Brand, Anneke; Slot, Manon C; de Graaf-Dijkstra, Alice; Watt, Suzanne; Zwaginga, Jaap Jan

2015-07-01

Expansion of human cord blood (CB) CD34+ cells with thrombopoietin (TPO) can accelerate delayed platelet (PLT) recovery after transplantation into immunodeficient mice. Clinical implementation, however, will depend on practical and effective protocols. The best timing of TPO expansion in relation to cryopreservation in this respect is unknown. In this study, we evaluated whether the order of cryopreservation and TPO expansion affected the expansion rate and numbers of clonogenic hematopoietic progenitor cells in vitro or PLT and longer-term hematopoietic repopulation in NOD SCID mice in vivo. Our results demonstrate higher expansion rates and the generation of higher numbers of multilineage and megakaryocytic progenitors (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units and megakaryocyte colony-forming units) in vitro when freshly isolated CB CD34+ cells are first cultured with TPO and then cryopreserved and thawed as compared to TPO expansion after CD34+ cell cryopreservation. In contrast, the cells produced with the latter strategy showed higher expression of CD62L and a superior stromal cell-derived factor-1α-mediated migration. This might play a role in an also observed superior early PLT recovery after transplantation of these cells into NOD SCID mice. The hematopoietic engraftment in the marrow 6 weeks after transplantation was not different between the two strategies. Although TPO expansion before cryopreservation would yield higher nucleated cell and clonogenic myeloid and megakaryocyte cell numbers and enable earlier availability, CB TPO expansion after cryopreservation is likely to be clinically more effective, despite the lower number of cells obtained after expansion. Moreover, the latter strategy is logistically more feasible. © 2015 AABB.

Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation.

PubMed

Teixeira, Fábio G; Panchalingam, Krishna M; Assunção-Silva, Rita; Serra, Sofia C; Mendes-Pinheiro, Bárbara; Patrício, Patrícia; Jung, Sunghoon; Anjo, Sandra I; Manadas, Bruno; Pinto, Luísa; Sousa, Nuno; Behie, Leo A; Salgado, António J

2016-06-15

In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome.

Uterine Natural Killer cells regulate endometrial bleeding in women and are suppressed by the progesterone receptor modulator asoprisnil

PubMed Central

Wilkens, Julia; Male, Victoria; Ghazal, Peter; Forster, Thorsten; Gibson, Douglas A.; Williams, Alistair RW; Brito-Mutunayagam, Savita L; Craigon, Marie; Lourenco, Paula; Cameron, Iain T; Chwalisz, Kristof; Moffett, Ashley; Critchley, Hilary OD

2013-01-01

Uterine NK cells (uNK) play a role in the regulation of placentation but their functions in non-pregnant endometrium are not understood. We have previously reported suppression of endometrial bleeding and alteration of spiral artery morphology in women exposed to asoprisnil, a progesterone receptor modulator. We now compare global endometrial gene expression in asoprisnil-treated versus control women, and we demonstrate a statistically significant reduction of genes in the IL-15 pathway, known to play a key role in uNK development and function. Suppression of IL-15 by asoprisnil was also observed at mRNA level (p<0.05), and immunostaining for NK cell marker CD56 revealed a striking reduction of uNK in asoprisnil-treated endometrium (p<0.001). IL-15 levels in normal endometrium are progesterone-responsive. Progesterone receptor (PR) positive stromal cells transcribe both IL-15 and IL-15RA. Thus, the response of stromal cells to progesterone will be to increase IL-15 trans-presentation to uNK, supporting their expansion and differentiation. In asoprisnil-treated endometrium, there is a marked down-regulation of stromal PR expression and virtual absence of uNK. These novel findings indicate that the IL-15 pathway provides a missing link in the complex interplay between endometrial stromal cells, uNK and spiral arteries affecting physiological and pathological endometrial bleeding. PMID:23913972

Dissection of enhanced cell expansion processes in leaves triggered by a defect in cell proliferation, with reference to roles of endoreduplication.

PubMed

Fujikura, Ushio; Horiguchi, Gorou; Tsukaya, Hirokazu

2007-02-01

Leaf development relies on cell proliferation, post-mitotic cell expansion and the coordination of these processes. In several Arabidopsis thaliana mutants impaired in cell proliferation, such as angustifolia3 (an3), leaf cells are larger than normal at their maturity. This phenomenon, which we call compensated cell enlargement, suggests the presence of such coordination in leaf development. To dissect genetically the cell expansion system(s) underlying this compensation seen in the an3 mutant, we isolated and utilized 10 extra-small sisters (xs) mutant lines that show decreased cell size but normal cell numbers in leaves. In the xs single mutants, the palisade cell sizes in mature leaves are about 20-50% smaller than those of wild-type cells. Phenotypes of the palisade cell sizes in all combinations of xs an3 double mutants fall into three classes. In the first class, the compensated cell enlargement was significantly suppressed. Conversely, in the second class, the defective cell expansion conferred by the xs mutations was significantly suppressed by the an3 mutation. The residual xs mutations had effects additive to those of the an3 mutation on cell expansion. The endopolyploidy levels in the first class of mutants were decreased, unaffected or increased, as compared with those in wild-type, suggesting that the abnormally enhanced cell expansion observed in an3 could be mediated, at least in part, by ploidy-independent mechanisms. Altogether, these results clearly showed that a defect in cell proliferation in leaf primordia enhances a part of the network that regulates cell expansion, which is required for normal leaf expansion.

The development of alignment turning system for precision len cells

NASA Astrophysics Data System (ADS)

Huang, Chien-Yao; Ho, Cheng-Fang; Wang, Jung-Hsing; Chung, Chien-Kai; Chen, Jun-Cheng; Chang, Keng-Shou; Kuo, Ching-Hsiang; Hsu, Wei-Yao; Chen, Fong-Zhi

2017-08-01

In general, the drop-in and cell-mounted assembly are used for standard and high performance optical system respectively. The optical performance is limited by the residual centration error and position accuracy of the conventional assembly. Recently, the poker chip assembly with high precision lens barrels that can overcome the limitation of conventional assembly is widely applied to ultra-high performance optical system. ITRC also develops the poker chip assembly solution for high numerical aperture objective lenses and lithography projection lenses. In order to achieve high precision lens cell for poker chip assembly, an alignment turning system (ATS) is developed. The ATS includes measurement, alignment and turning modules. The measurement module including a non-contact displacement sensor and an autocollimator can measure centration errors of the top and the bottom surface of a lens respectively. The alignment module comprising tilt and translation stages can align the optical axis of the lens to the rotating axis of the vertical lathe. The key specifications of the ATS are maximum lens diameter, 400mm, and radial and axial runout of the rotary table < 2 μm. The cutting performances of the ATS are surface roughness Ra < 1 μm, flatness < 2 μm, and parallelism < 5 μm. After measurement, alignment and turning processes on our ATS, the centration error of a lens cell with 200mm in diameter can be controlled in 10 arcsec. This paper also presents the thermal expansion of the hydrostatic rotating table. A poker chip assembly lens cell with three sub-cells is accomplished with average transmission centration error in 12.45 arcsec by fresh technicians. The results show that ATS can achieve high assembly efficiency for precision optical systems.

Lattice-structures and constructs with designed thermal expansion coefficients

DOEpatents

Spadaccini, Christopher; Hopkins, Jonathan

2014-10-28

A thermal expansion-managed lattice structure having a plurality of unit cells each having flexure bearing-mounted tabs supported on a base and actuated by thermal expansion of an actuator having a thermal expansion coefficient greater than the base and arranged so that the tab is inwardly displaced into a base cavity. The flexure bearing-mounted tabs are connected to other flexure-bearing-mounted tabs of adjacent unit cells so that the adjacent unit cells are spaced from each other to accommodate thermal expansion of individual unit cells while maintaining a desired bulk thermal expansion coefficient of the lattice structure as a whole.

Architectural and Mechanical Cues Direct Mesenchymal Stem Cell Interactions with Crosslinked Gelatin Scaffolds

PubMed Central

McAndrews, Kathleen M.; Kim, Min Jeong; Lam, Tuyet Y.; McGrail, Daniel J.

2014-01-01

Naturally derived biomaterials have emerged as modulators of cell function and tissue substitutes. Here, we developed crosslinked glutaraldehyde (GTA) scaffolds for the expansion and differentiation of mesenchymal stem cells (MSCs). The mechanical and architectural properties of the scaffolds were altered by varying the concentration of gelatin and GTA. Higher GTA concentrations were associated with an increase in more confined pores and osteogenic differentiation. In addition, myogenic potential varied with crosslinking degree, although bulk mechanical properties were unaltered. Correlation analysis revealed that ALP activity of differentiated MSCs on higher gelatin concentration scaffolds was dependent on traditional effectors, including environment elasticity and spread area. In contrast, the differentiation capacity of cells cultured on lower gelatin concentration scaffolds did not correlate with these factors, instead it was dependent on the hydrated pore structure. These results suggest that scaffold composition can determine what factors direct differentiation and may have critical implications for biomaterial design. PMID:24873687

Large-Scale Culture and Genetic Modification of Human Natural Killer Cells for Cellular Therapy.

PubMed

Lapteva, Natalia; Parihar, Robin; Rollins, Lisa A; Gee, Adrian P; Rooney, Cliona M

2016-01-01

Recent advances in methods for the ex vivo expansion of human natural killer (NK) cells have facilitated the use of these powerful immune cells in clinical protocols. Further, the ability to genetically modify primary human NK cells following rapid expansion allows targeting and enhancement of their immune function. We have successfully adapted an expansion method for primary NK cells from peripheral blood mononuclear cells or from apheresis products in gas permeable rapid expansion devices (G-Rexes). Here, we describe an optimized protocol for rapid and robust NK cell expansion as well as a method for highly efficient retroviral transduction of these ex vivo expanded cells. These methodologies are good manufacturing practice (GMP) compliant and could be used for clinical-grade product manufacturing.

The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis.

PubMed

Kobayashi, Masatoshi; Ohsugi, Mitsuru; Sasako, Takayoshi; Awazawa, Motoharu; Umehara, Toshihiro; Iwane, Aya; Kobayashi, Naoki; Okazaki, Yukiko; Kubota, Naoto; Suzuki, Ryo; Waki, Hironori; Horiuchi, Keiko; Hamakubo, Takao; Kodama, Tatsuhiko; Aoe, Seiichiro; Tobe, Kazuyuki; Kadowaki, Takashi; Ueki, Kohjiro

2018-06-04

Adipocyte differentiation is regulated by various mechanisms, of which the mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by the cell cycle modulators, the precise mechanism remains unclear. N 6 -methyladenosine (m 6 A) post-transcriptional RNA modification, whose methylation and demethylation is performed by respective enzymal molecules, has recently been suggested to be involved in the regulation of adipogenesis. Here, we show that an RNA N 6 -adenosine methyltransferase complex consisting of Wilms' tumor 1-associating protein (WTAP), methyltransferase like (METTL) 3 and METTL14 positively control adipogenesis, by promoting cell cycle transition in MCE during adipogenesis. WTAP, coupled with METTL3 and METTL14, is increased and distributed in nucleus by the induction of adipogenesis dependently on RNA in vitro Knockdown of each of these three proteins leads to cell cycle arrest and impaired adipogenesis associated with suppression of Cyclin A2 upregulation during MCE, whose knockdown also impairs adipogenesis. Consistently, Wtap heterozygous knockout mice are protected from diet-induced obesity with smaller size and number of adipocytes, leading to improved insulin sensitivity. These data provide a mechanism for adipogenesis through WTAP-METTL3-METTL14 complex and a potential strategy for treatment of obesity and associated disorders. Copyright © 2018 Kobayashi et al.

Numerical investigation of multi-beam laser heterodyne measurement with ultra-precision for linear expansion coefficient of metal based on oscillating mirror modulation

NASA Astrophysics Data System (ADS)

Li, Yan-Chao; Wang, Chun-Hui; Qu, Yang; Gao, Long; Cong, Hai-Fang; Yang, Yan-Ling; Gao, Jie; Wang, Ao-You

2011-01-01

This paper proposes a novel method of multi-beam laser heterodyne measurement for metal linear expansion coefficient. Based on the Doppler effect and heterodyne technology, the information is loaded of length variation to the frequency difference of the multi-beam laser heterodyne signal by the frequency modulation of the oscillating mirror, this method can obtain many values of length variation caused by temperature variation after the multi-beam laser heterodyne signal demodulation simultaneously. Processing these values by weighted-average, it can obtain length variation accurately, and eventually obtain the value of linear expansion coefficient of metal by the calculation. This novel method is used to simulate measurement for linear expansion coefficient of metal rod under different temperatures by MATLAB, the obtained result shows that the relative measurement error of this method is just 0.4%.

Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion.

PubMed

Kaartinen, Tanja; Luostarinen, Annu; Maliniemi, Pilvi; Keto, Joni; Arvas, Mikko; Belt, Heini; Koponen, Jonna; Loskog, Angelica; Mustjoki, Satu; Porkka, Kimmo; Ylä-Herttuala, Seppo; Korhonen, Matti

2017-06-01

Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (T SCM , CD95 + CD45RO - CD45RA + CD27 + ) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Long-range intercellular Ca2+ wave patterns

NASA Astrophysics Data System (ADS)

Tabi, C. B.; Maïna, I.; Mohamadou, A.; Ekobena, H. P. F.; Kofané, T. C.

2015-10-01

Modulational instability is utilized to investigate intercellular Ca2+ wave propagation in an array of diffusively coupled cells. Cells are supposed to be connected via paracrine signaling, where long-range effects, due to the presence of extracellular messengers, are included. The multiple-scale expansion is used to show that the whole dynamics of Ca2+ waves, from the endoplasmic reticulum to the cytosol, can be reduced to a single differential-difference nonlinear equation whose solutions are assumed to be plane waves. Their linear stability analysis is studied, with emphasis on the impact of long-range coupling, via the range parameter s. It is shown that s, as well as the number of interacting cells, importantly modifies the features of modulational instability, as small values of s imply a strong coupling, and increasing its value rather reduces the problem to a first-neighbor one. Our theoretical findings are numerically tested, as the generic equations are fully integrated, leading to the emergence of nonlinear patterns of Ca2+ waves. Strong long-range coupling is pictured by extended trains of breather-like structures whose frequency decreases with increasing s. We also show numerically that the number of interacting cells plays on the spatio-temporal formation of Ca2+ patterns, whilst the quasi-perfect intercellular communication depends on the paracrine coupling parameter.

Galectin-3 Shapes Antitumor Immune Responses by Suppressing CD8+ T Cells via LAG-3 and Inhibiting Expansion of Plasmacytoid Dendritic Cells.

PubMed

Kouo, Theodore; Huang, Lanqing; Pucsek, Alexandra B; Cao, Minwei; Solt, Sara; Armstrong, Todd; Jaffee, Elizabeth

2015-04-01

Galectin-3 is a 31-kDa lectin that modulates T-cell responses through several mechanisms, including apoptosis, T-cell receptor (TCR) cross-linking, and TCR downregulation. We found that patients with pancreatic ductal adenocarcinoma (PDA) who responded to a granulocyte-macrophage colony-stimulating factor-secreting allogeneic PDA vaccine developed neutralizing antibodies to galectin-3 after immunization. We show that galectin-3 binds activated antigen-committed CD8(+) T cells only in the tumor microenvironment. Galectin-3-deficient mice exhibit improved CD8(+) T-cell effector function and increased expression of several inflammatory genes. Galectin-3 binds to LAG-3, and LAG-3 expression is necessary for galectin-3-mediated suppression of CD8(+) T cells in vitro. Lastly, galectin-3-deficient mice have elevated levels of circulating plasmacytoid dendritic cells, which are superior to conventional dendritic cells in activating CD8(+) T cells. Thus, inhibiting galectin-3 in conjunction with CD8(+) T-cell-directed immunotherapies should enhance the tumor-specific immune response. ©2015 American Association for Cancer Research.

FTY720 ameliorates murine sclerodermatous chronic graft-versus-host disease by promoting expansion of splenic regulatory cells and inhibiting immune cell infiltration into skin.

PubMed

Huu, Doanh Le; Matsushita, Takashi; Jin, Guihua; Hamaguchi, Yasuhito; Hasegawa, Minoru; Takehara, Kazuhiko; Fujimoto, Manabu

2013-06-01

Sphingosine 1-phosphate (S1P) exerts a variety of activities in immune, inflammatory, and vascular systems. S1P plays an important role in systemic sclerosis (SSc) pathogenesis. Regulation of S1P in fibrotic diseases as well as in SSc was recently reported. FTY720, an oral S1P receptor modulator, has been shown to be a useful agent for the prevention of transplant rejection and autoimmune diseases. Murine sclerodermatous chronic graft-versus-host disease (GVHD) is a model for human sclerodermatous chronic GVHD and SSc. We undertook this study to investigate the effects of FTY720 in murine sclerodermatous chronic GVHD. FTY720 was orally administered to allogeneic recipient mice from day 0 to day 20 (short-term, early-treatment group), from day 0 to day 42 (full-term, early-treatment group), or from day 22 to day 42 (delayed-treatment group) after bone marrow transplantation. Delayed administration of FTY720 attenuated, and early administration of FTY720 inhibited, the severity and fibrosis in murine sclerodermatous chronic GVHD. With early treatment, FTY720 induced expansion of splenic myeloid-derived suppressor cells, Treg cells, and Breg cells. Vascular damage in chronic GVHD was inhibited by FTY720 through down-regulating serum levels of S1P and soluble E-selectin. FTY720 inhibited infiltration of immune cells into skin. Moreover, FTY720 diminished the expression of messenger RNA for monocyte chemotactic protein 1, macrophage inflammatory protein 1α, RANTES, tumor necrosis factor α, interferon-γ, interleukin-6 (IL-6), IL-10, IL-17A, and transforming growth factor β1 in the skin. FTY720 suppressed the immune response by promoting the expansion of regulatory cells and reducing vascular damage and infiltration of immune cells into the skin. Taken together, these results have important implications for the potential use of FTY720 in the treatment of sclerodermatous chronic GVHD and SSc in humans. Copyright © 2013 by the American College of Rheumatology.

Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage.

PubMed

Raju, Ravali; Chau, David; Cho, Dong Seong; Park, Yonsil; Verfaillie, Catherine M; Hu, Wei-Shou

2017-02-15

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 10 9 -10 10 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications.

Human Platelet Lysate as a Xeno Free Alternative of Fetal Bovine Serum for the In Vitro Expansion of Human Mesenchymal Stromal Cells.

PubMed

Mohammadi, Saeed; Nikbakht, Mohsen; Malek Mohammadi, Ashraf; Zahed Panah, Mahdi; Ostadali, Mohammad Reza; Nasiri, Hajar; Ghavamzadeh, Ardeshir

2016-07-01

Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives. Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR. We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement. We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability.

Human Platelet Lysate as a Xeno Free Alternative of Fetal Bovine Serum for the In Vitro Expansion of Human Mesenchymal Stromal Cells

PubMed Central

Mohammadi, Saeed; Nikbakht, Mohsen; Malek Mohammadi, Ashraf; Zahed Panah, Mahdi; Ostadali, Mohammad Reza; Nasiri, Hajar; Ghavamzadeh, Ardeshir

2016-01-01

Background: Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives. Materials and Methods: Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR. Results: We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement. Conclusions: We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability. PMID:27489592

Local and systemic immune mechanisms underlying the anti-colitis effects of the dairy bacterium Lactobacillus delbrueckii.

PubMed

Santos Rocha, Clarissa; Gomes-Santos, Ana Cristina; Garcias Moreira, Thais; de Azevedo, Marcela; Diniz Luerce, Tessalia; Mariadassou, Mahendra; Longaray Delamare, Ana Paula; Langella, Philippe; Maguin, Emmanuelle; Azevedo, Vasco; Caetano de Faria, Ana Maria; Miyoshi, Anderson; van de Guchte, Maarten

2014-01-01

Several probiotic bacteria have been proposed for treatment or prevention of inflammatory bowel diseases (IBD), showing a protective effect in animal models of experimental colitis and for some of them also in human clinical trials. While most of these probiotic bacteria are isolated from the digestive tract, we recently reported that a Lactobacillus strain isolated from cheese, L. delbrueckii subsp. lactis CNRZ327 (Lb CNRZ327), also possesses anti-inflammatory effects in vitro and in vivo, demonstrating that common dairy bacteria may be useful in the treatment or prevention of IBD. Here, we studied the mechanisms underlying the protective effects of Lb CNRZ327 in vivo, in a mouse dextran sodium sulfate (DSS) colitis model. During colitis, Lb CNRZ327 modulated the production of TGF-β, IL-6, and IL-12 in colonic tissue and of TGF-β and IL-6 in the spleen, and caused an expansion of CD4+Foxp3+ regulatory T cells in the cecal lymph nodes. Moreover, a strong tendency to CD4+Foxp3+ expansion was also observed in the spleen. The results of this study for the first time show that orally administered dairy lactobacilli can not only modulate mucosal but also systemic immune responses and constitute an effective treatment of IBD.

Modulation of hematopoietic progenitor cell fate in vitro by varying collagen oligomer matrix stiffness in the presence or absence of osteoblasts.

PubMed

Chitteti, Brahmananda Reddy; Kacena, Melissa A; Voytik-Harbin, Sherry L; Srour, Edward F

2015-10-01

To recreate the in vivo hematopoietic cell microenvironment or niche and to study the impact of extracellular matrix (ECM) biophysical properties on hematopoietic progenitor cell (HPC) proliferation and function, mouse bone-marrow derived HPC (Lin-Sca1+cKit+/(LSK) were cultured within three-dimensional (3D) type I collagen oligomer matrices. To generate a more physiologic milieu, 3D cultures were established in both the presence and absence of calvariae-derived osteoblasts (OB). Collagen oligomers were polymerized at varying concentration to give rise to matrices of different fibril densities and therefore matrix stiffness (shear storage modulus, 50-800 Pa). Decreased proliferation and increased clonogenicity of LSK cells was associated with increase of matrix stiffness regardless of whether OB were present or absent from the 3D culture system. Also, regardless of whether OB were or were not added to the 3D co-culture system, LSK within 800 Pa collagen oligomer matrices maintained the highest percentage of Lin-Sca1+ cells as well as higher percentage of cells in quiescent state (G0/G1) compared to 50 Pa or 200Pa matrices. Collectively, these data illustrate that biophysical features of collagen oligomer matrices, specifically fibril density-induced modulation of matrix stiffness, provide important guidance cues in terms of LSK expansion and differentiation and therefore maintenance of progenitor cell function. Copyright © 2015. Published by Elsevier B.V.

XTH31, Encoding an in Vitro XEH/XET-Active Enzyme, Regulates Aluminum Sensitivity by Modulating in Vivo XET Action, Cell Wall Xyloglucan Content, and Aluminum Binding Capacity in Arabidopsis[W

PubMed Central

Zhu, Xiao Fang; Shi, Yuan Zhi; Lei, Gui Jie; Fry, Stephen C.; Zhang, Bao Cai; Zhou, Yi Hua; Braam, Janet; Jiang, Tao; Xu, Xiao Yan; Mao, Chuan Zao; Pan, Yuan Jiang; Yang, Jian Li; Wu, Ping; Zheng, Shao Jian

2012-01-01

Xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET) activities, encoded by xyloglucan endotransglucosylase-hydrolase (XTH) genes, are involved in cell wall extension by cutting or cutting and rejoining xyloglucan chains, respectively. However, the physiological significance of this biochemical activity remains incompletely understood. Here, we find that an XTH31 T-DNA insertion mutant, xth31, is more Al resistant than the wild type. XTH31 is bound to the plasma membrane and the encoding gene is expressed in the root elongation zone and in nascent leaves, suggesting a role in cell expansion. XTH31 transcript accumulation is strongly downregulated by Al treatment. XTH31 expression in yeast yields a protein with an in vitro XEH:XET activity ratio of >5000:1. xth31 accumulates significantly less Al in the root apex and cell wall, shows remarkably lower in vivo XET action and extractable XET activity, has a lower xyloglucan content, and exhibits slower elongation. An exogenous supply of xyloglucan significantly ameliorates Al toxicity by reducing Al accumulation in the roots, owing to the formation of an Al-xyloglucan complex in the medium, as verified by an obvious change in chemical shift of 27Al-NMR. Taken together, the data indicate that XTH31 affects Al sensitivity by modulating cell wall xyloglucan content and Al binding capacity. PMID:23204407

Characterization of bone marrow-derived mesenchymal stem cells in aging.

PubMed

Baker, Natasha; Boyette, Lisa B; Tuan, Rocky S

2015-01-01

Adult mesenchymal stem cells are a resource for autologous and allogeneic cell therapies for immune-modulation and regenerative medicine. However, patients most in need of such therapies are often of advanced age. Therefore, the effects of the aged milieu on these cells and their intrinsic aging in vivo are important considerations. Furthermore, these cells may require expansion in vitro before use as well as for future research. Their aging in vitro is thus also an important consideration. Here, we focus on bone marrow mesenchymal stem cells (BMSCs), which are unique compared to other stem cells due to their support of hematopoietic cells in addition to contributing to bone formation. BMSCs may be sensitive to age-related diseases and could perpetuate degenerative diseases in which bone remodeling is a contributory factor. Here, we review (1) the characterization of BMSCs, (2) the characterization of in vivo-aged BMSCs, (3) the characterization of in vitro-aged BMSCs, and (4) potential approaches to optimize the performance of aged BMSCs. This article is part of a Special Issue entitled "Stem Cells and Bone". Copyright © 2014 Elsevier Inc. All rights reserved.

Stem Cell Metabolism in Cancer and Healthy Tissues: Pyruvate in the Limelight

PubMed Central

Corbet, Cyril

2018-01-01

Normal and cancer stem cells (CSCs) share the remarkable potential to self-renew and differentiate into many distinct cell types. Although most of the stem cells remain under quiescence to maintain their undifferentiated state, they can also undergo cell divisions as required to regulate tissue homeostasis. There is now a growing evidence that cell fate determination from stem cells implies a fine-tuned regulation of their energy balance and metabolic status. Stem cells can shift their metabolic substrate utilization, between glycolysis and mitochondrial oxidative metabolism, during specification and/or differentiation, as well as in order to adapt their microenvironmental niche. Pyruvate appears as a key metabolite since it is at the crossroads of cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. This Review describes how metabolic reprogramming, focusing on pyruvate utilization, drives the fate of normal and CSCs by modulating their capacity for self-renewal, clonal expansion/differentiation, as well as metastatic potential and treatment resistance in cancer. This Review also explores potential therapeutic strategies to restore or manipulate stem cell function through the use of small molecules targeting the pyruvate metabolism. PMID:29403375

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells

PubMed Central

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A.; de Boer, Jan; Watt, Fiona M.

2016-01-01

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation. PMID:26757610

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells.

PubMed

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A; de Boer, Jan; Watt, Fiona M

2016-01-13

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation.

Thermal Expansion of Polyurethane Foam

NASA Technical Reports Server (NTRS)

Lerch, Bradley A.; Sullivan, Roy M.

2006-01-01

Closed cell foams are often used for thermal insulation. In the case of the Space Shuttle, the External Tank uses several thermal protection systems to maintain the temperature of the cryogenic fuels. A few of these systems are polyurethane, closed cell foams. In an attempt to better understand the foam behavior on the tank, we are in the process of developing and improving thermal-mechanical models for the foams. These models will start at the microstructural level and progress to the overall structural behavior of the foams on the tank. One of the key properties for model characterization and verification is thermal expansion. Since the foam is not a material, but a structure, the modeling of the expansion is complex. It is also exacerbated by the anisoptropy of the material. During the spraying and foaming process, the cells become elongated in the rise direction and this imparts different properties in the rise direction than in the transverse directions. Our approach is to treat the foam as a two part structure consisting of the polymeric cell structure and the gas inside the cells. The polymeric skeleton has a thermal expansion of its own which is derived from the basic polymer chemistry. However, a major contributor to the thermal expansion is the volume change associated with the gas inside of the closed cells. As this gas expands it exerts pressure on the cell walls and changes the shape and size of the cells. The amount that this occurs depends on the elastic and viscoplastic properties of the polymer skeleton. The more compliant the polymeric skeleton, the more influence the gas pressure has on the expansion. An additional influence on the expansion process is that the polymeric skeleton begins to breakdown at elevated temperatures and releases additional gas species into the cell interiors, adding to the gas pressure. The fact that this is such a complex process makes thermal expansion ideal for testing the models. This report focuses on the thermal expansion tests and the response of the microstructure. A novel optical method is described which is appropriate for measuring thermal expansion at high temperatures without influencing the thermal expansion measurement. Detailed microstructural investigations will also be described which show cell expansion as a function of temperature. Finally, a phenomenological model on thermal expansion will be described.

Cell division and turgor mediate enhanced plant growth in Arabidopsis plants treated with the bacterial signalling molecule lumichrome.

PubMed

Pholo, Motlalepula; Coetzee, Beatrix; Maree, Hans J; Young, Philip R; Lloyd, James R; Kossmann, Jens; Hills, Paul N

2018-05-17

Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement. Mitotic CYCD3.3, CYCA1.1, SP1L3, RSW7 and PDF1 transcripts were increased in lumichrome-treated Arabidopsis thaliana plants, suggesting enhanced growth was underpinned by increased cell differentiation and expansion with a consequential increase in biomass. Synergistic ethylene-auxin cross-talk was also observed through reciprocal over-expression of ACO1 and SAUR54, in which ethylene activates the auxin signalling pathway and regulates Arabidopsis growth by both stimulating auxin biosynthesis and modulating the auxin transport machinery to the leaves. Decreased transcription of jasmonate biosynthesis and responsive-related transcripts (LOX2; LOX3; LOX6; JAL34; JR1) might contribute towards suppression of the negative effects of methyl jasmonate (MeJa) such as chlorophyll loss and decreases in RuBisCO and photosynthesis. This work contributes towards a deeper understanding of how lumichrome enhances plant growth and development.

Systematically Studying Kinase Inhibitor Induced Signaling Network Signatures by Integrating Both Therapeutic and Side Effects

PubMed Central

Shao, Hongwei; Peng, Tao; Ji, Zhiwei; Su, Jing; Zhou, Xiaobo

2013-01-01

Substantial effort in recent years has been devoted to analyzing data based large-scale biological networks, which provide valuable insight into the topologies of complex biological networks but are rarely context specific and cannot be used to predict the responses of cell signaling proteins to specific ligands or compounds. In this work, we proposed a novel strategy to investigate kinase inhibitor induced pathway signatures by integrating multiplex data in Library of Integrated Network-based Cellular Signatures (LINCS), e.g. KINOMEscan data and cell proliferation/mitosis imaging data. Using this strategy, we first established a PC9 cell line specific pathway model to investigate the pathway signatures in PC9 cell line when perturbed by a small molecule kinase inhibitor GW843682. This specific pathway revealed the role of PI3K/AKT in modulating the cell proliferation process and the absence of two anti-proliferation links, which indicated a potential mechanism of abnormal expansion in PC9 cell number. Incorporating the pathway model for side effects on primary human hepatocytes, it was used to screen 27 kinase inhibitors in LINCS database and PF02341066, known as Crizotinib, was finally suggested with an optimal concentration 4.6 uM to suppress PC9 cancer cell expansion while avoiding severe damage to primary human hepatocytes. Drug combination analysis revealed that the synergistic effect region can be predicted straightforwardly based on a threshold which is an inherent property of each kinase inhibitor. Furthermore, this integration strategy can be easily extended to other specific cell lines to be a powerful tool for drug screen before clinical trials. PMID:24339888

Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage

PubMed Central

Raju, Ravali; Chau, David; Cho, Dong Seong; Park, Yonsil; Verfaillie, Catherine M.

2017-01-01

The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 109–1010 cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications. PMID:27806669

The grape berry-specific basic helix-loop-helix transcription factor VvCEB1 affects cell size.

PubMed

Nicolas, Philippe; Lecourieux, David; Gomès, Eric; Delrot, Serge; Lecourieux, Fatma

2013-02-01

The development of fleshy fruits involves complex physiological and biochemical changes. After fertilization, fruit growth usually begins with cell division, continues with both cell division and expansion, allowing fruit set to occur, and ends with cell expansion only. In spite of the economical importance of grapevine, the molecular mechanisms controlling berry growth are not fully understood. The present work identified and characterized Vitis vinifera cell elongation bHLH protein (VvCEB1), a basic helix-loop-helix (bHLH) transcription factor controlling cell expansion in grape. VvCEB1 was expressed specifically in berry-expanding tissues with a maximum around veraison. The study of VvCEB1 promoter activity in tomato confirmed its specific fruit expression during the expansion phase. Overexpression of VvCEB1 in grape embryos showed that this protein stimulates cell expansion and affects the expression of genes involved in cell expansion, including genes of auxin metabolism and signalling. Taken together, these data show that VvCEB1 is a fruit-specific bHLH transcription factor involved in grape berry development.

The grape berry-specific basic helix–loop–helix transcription factor VvCEB1 affects cell size

PubMed Central

Lecourieux, Fatma

2013-01-01

The development of fleshy fruits involves complex physiological and biochemical changes. After fertilization, fruit growth usually begins with cell division, continues with both cell division and expansion, allowing fruit set to occur, and ends with cell expansion only. In spite of the economical importance of grapevine, the molecular mechanisms controlling berry growth are not fully understood. The present work identified and characterized Vitis vinifera cell elongation bHLH protein (VvCEB1), a basic helix–loop–helix (bHLH) transcription factor controlling cell expansion in grape. VvCEB1 was expressed specifically in berry-expanding tissues with a maximum around veraison. The study of VvCEB1 promoter activity in tomato confirmed its specific fruit expression during the expansion phase. Overexpression of VvCEB1 in grape embryos showed that this protein stimulates cell expansion and affects the expression of genes involved in cell expansion, including genes of auxin metabolism and signalling. Taken together, these data show that VvCEB1 is a fruit-specific bHLH transcription factor involved in grape berry development. PMID:23314819

Colony Expansion of Socially Motile Myxococcus xanthus Cells Is Driven by Growth, Motility, and Exopolysaccharide Production

PubMed Central

Patra, Pintu; Kissoon, Kimberley; Cornejo, Isabel; Kaplan, Heidi B.; Igoshin, Oleg A.

2016-01-01

Myxococcus xanthus, a model organism for studies of multicellular behavior in bacteria, moves exclusively on solid surfaces using two distinct but coordinated motility mechanisms. One of these, social (S) motility is powered by the extension and retraction of type IV pili and requires the presence of exopolysaccharides (EPS) produced by neighboring cells. As a result, S motility requires close cell-to-cell proximity and isolated cells do not translocate. Previous studies measuring S motility by observing the colony expansion of cells deposited on agar have shown that the expansion rate increases with initial cell density, but the biophysical mechanisms involved remain largely unknown. To understand the dynamics of S motility-driven colony expansion, we developed a reaction-diffusion model describing the effects of cell density, EPS deposition and nutrient exposure on the expansion rate. Our results show that at steady state the population expands as a traveling wave with a speed determined by the interplay of cell motility and growth, a well-known characteristic of Fisher’s equation. The model explains the density-dependence of the colony expansion by demonstrating the presence of a lag phase–a transient period of very slow expansion with a duration dependent on the initial cell density. We propose that at a low initial density, more time is required for the cells to accumulate enough EPS to activate S-motility resulting in a longer lag period. Furthermore, our model makes the novel prediction that following the lag phase the population expands at a constant rate independent of the cell density. These predictions were confirmed by S motility experiments capturing long-term expansion dynamics. PMID:27362260

Colony Expansion of Socially Motile Myxococcus xanthus Cells Is Driven by Growth, Motility, and Exopolysaccharide Production.

PubMed

Patra, Pintu; Kissoon, Kimberley; Cornejo, Isabel; Kaplan, Heidi B; Igoshin, Oleg A

2016-06-01

Myxococcus xanthus, a model organism for studies of multicellular behavior in bacteria, moves exclusively on solid surfaces using two distinct but coordinated motility mechanisms. One of these, social (S) motility is powered by the extension and retraction of type IV pili and requires the presence of exopolysaccharides (EPS) produced by neighboring cells. As a result, S motility requires close cell-to-cell proximity and isolated cells do not translocate. Previous studies measuring S motility by observing the colony expansion of cells deposited on agar have shown that the expansion rate increases with initial cell density, but the biophysical mechanisms involved remain largely unknown. To understand the dynamics of S motility-driven colony expansion, we developed a reaction-diffusion model describing the effects of cell density, EPS deposition and nutrient exposure on the expansion rate. Our results show that at steady state the population expands as a traveling wave with a speed determined by the interplay of cell motility and growth, a well-known characteristic of Fisher's equation. The model explains the density-dependence of the colony expansion by demonstrating the presence of a lag phase-a transient period of very slow expansion with a duration dependent on the initial cell density. We propose that at a low initial density, more time is required for the cells to accumulate enough EPS to activate S-motility resulting in a longer lag period. Furthermore, our model makes the novel prediction that following the lag phase the population expands at a constant rate independent of the cell density. These predictions were confirmed by S motility experiments capturing long-term expansion dynamics.

Novel polyglutamine model uncouples proteotoxicity from aging.

PubMed

Christie, Nakeirah T M; Lee, Amy L; Fay, Hannah G; Gray, Amelia A; Kikis, Elise A

2014-01-01

Polyglutamine expansions in certain proteins are the genetic determinants for nine distinct progressive neurodegenerative disorders and resultant age-related dementia. In these cases, neurodegeneration is due to the aggregation propensity and resultant toxic properties of the polyglutamine-containing proteins. We are interested in elucidating the underlying mechanisms of toxicity of the protein ataxin-3, in which a polyglutamine expansion is the genetic determinant for Machado-Joseph Disease (MJD), also referred to as spinocerebellar ataxia 3 (SCA3). To this end, we have developed a novel model for ataxin-3 protein aggregation, by expressing a disease-related polyglutamine-containing fragment of ataxin-3 in the genetically tractable body wall muscle cells of the model system C. elegans. Here, we demonstrate that this ataxin-3 fragment aggregates in a polyQ length-dependent manner in C. elegans muscle cells and that this aggregation is associated with cellular dysfunction. However, surprisingly, this aggregation and resultant toxicity was not influenced by aging. This is in contrast to polyglutamine peptides alone whose aggregation/toxicity is highly dependent on age. Thus, the data presented here not only describe a new polyglutamine model, but also suggest that protein context likely influences the cellular interactions of the polyglutamine-containing protein and thereby modulates its toxic properties.

CXCR4(+) dendritic cells promote angiogenesis during embryo implantation in mice.

PubMed

Barrientos, Gabriela; Tirado-González, Irene; Freitag, Nancy; Kobelt, Peter; Moschansky, Petra; Klapp, Burghard F; Thijssen, Victor L J L; Blois, Sandra M

2013-04-01

Early pregnancy is characterized by decidual adaption to the developing embryo involving angiogenesis and vascular growth. Failure of decidual vascular expansion is linked to diseases of pregnancy. Dendritic cells (DC) have been associated with vascular growth during early gestation, though it is unknown whether their capacity to modulate angiogenesis is ubiquitous to all DC subsets. Here, we show that DC normally found associated with the decidual vasculature co-express the C-X-C chemokine receptor type 4 (CXCR4). In addition, we demonstrate that impaired homing of CXCR4(+)DC during early gestation provoked a disorganized decidual vasculature with impaired spiral artery remodeling later in gestation. In contrast, adoptive transfer experiments provided evidence that CXCR4(+)DC are able to rescue early pregnancy by normalizing decidual vascular growth and delivery of pro-angiogenic factors, which results in adequate remodeling of the spiral arteries during placental development. Taken together, our results indicate an important role of CXCR4(+)DC in the regulation of decidual angiogenesis and highlight the importance of the CXCL12/CXCR4 pathway during this process, suggesting that this may represent a key pathway to evaluate during pregnancy pathologies associated with impaired vascular expansion.

Anion exchanger 2 is critical for CD8(+) T cells to maintain pHi homeostasis and modulate immune responses.

PubMed

Concepcion, Axel R; Salas, January T; Sarvide, Sarai; Sáez, Elena; Ferrer, Alex; López, María; Portu, Ainhoa; Banales, Jesús M; Hervás-Stubbs, Sandra; Oude Elferink, Ronald P J; Prieto, Jesús; Medina, Juan F

2014-05-01

Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pHi ). Subsequent pHi regulation may involve HCO3 (-) extrusion through Cl(-) /HCO3 (-) exchangers and/or Na(+) -HCO3 (-) co-transporters with acid-loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8(+) T-cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na(+) -independent Cl(-) /HCO3 (-) anion-exchange activity). Here we report that CD8(+) T cells but not CD4(+) T cells or other lymphocyte populations, are crucially dependent on Ae2 for pHi regulation. While total lymphocytes (including isolated CD4(+) T cells) exhibit Ae1 expression and Na(+) -HCO3 (-) co-transport with acidifying potential, CD8(+) T cells lack these acid-loading mechanisms. In Ae2-KO mice, CD4(+) but not CD8(+) T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2-KO CD8(+) T cells exhibit alkalinized pHi , and dramatically increase their pHi upon CD3 stimulation. Moreover, stimulated Ae2-deficient CD8(+) T cells show enhanced intracellular production of IL-2 and membrane expression of its receptor IL-2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8(+) T cells are critically dependent on Ae2 for pHi homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8(+) T-cell responses. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of umbilical cord blood CD34+ hematopoietic stem cells expansion with inhibition of TGF-β receptorII in co-culture with bone marrow mesenchymal stromal cells.

PubMed

Sohrabi Akhkand, Saman; Amirizadeh, Naser; Nikougoftar, Mahin; Alizadeh, Javad; Zaker, Farhad; Sarveazad, Arash; Joghataei, Mohammad Taghi; Faramarzi, Mahmood

2016-08-01

Umbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs). However, low number of HSCs in UCB has been an obstacle for adult hematopoietic stem cell transplantation. The expansion of HSCs in culture is one approach to overcome this problem. In this study, we investigated the expansion of UCB-HSCs by using human bone marrow mesenchymal stromal cells (MSCs) as feeder layer as well as inhibiting the TGF-β signaling pathway through reduction of TGF-βRII expression. CD34(+) cells were isolated from UCB and transfected by SiRNA targeting TGF-βRII mRNA. CD34(+) cells were expanded in four culture media with different conditions, including 1) expansion of CD34(+) cells in serum free medium containing growth factors, 2) expansion of cells transfected with SiRNA targeting TGF-βRII in medium containing growth factors, 3) expansion of cells in presence of growth factors and MSCs, 4) expansion of cells transfected with SiRNA targeting TGF-βRII on MSCs feeder layer in medium containing growth factors. These culture conditions were evaluated for the number of total nucleated cells (TNCs), CD34 surface marker as well as using CFU assay on 8th day after culture. The fold increase in CD34(+) cells, TNCs, and colony numbers (71.8±6.9, 93.2±10.2 and 128±10, respectively) was observed to be highest in fourth culture medium compared to other culture conditions. The difference between number of cells in four culture media in 8th day compared to unexpanded cells (0day) before expansion was statistically significant (P<0.05). The results showed that transfection of CD34(+) cells with SiRNA targeting TGF-βRII and their co-culture with MSCs could considerably increase the number of progenitors. Therefore, this method could be useful for UCB-HSCs expansion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma: a prerequisite for adoptive cell transfer.

PubMed

Junker, Niels; Andersen, Mads Hald; Wenandy, Lynn; Dombernowsky, Sarah Louise; Kiss, Katalin; Sørensen, Christian Hjort; Therkildsen, Marianne Hamilton; Von Buchwald, Christian; Andersen, Elo; Straten, Per Thor; Svane, Inge Marie

2011-08-01

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies. In a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr(51) release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion. TIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8(+) and CD4(+) subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells. The procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.

IL-12-producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion.

PubMed

Rölle, Alexander; Pollmann, Julia; Ewen, Eva-Maria; Le, Vu Thuy Khanh; Halenius, Anne; Hengel, Hartmut; Cerwenka, Adelheid

2014-12-01

Human cytomegalovirus (HCMV) infection is the most common cause of congenital viral infections and a major source of morbidity and mortality after organ transplantation. NK cells are pivotal effector cells in the innate defense against CMV. Recently, hallmarks of adaptive responses, such as memory-like features, have been recognized in NK cells. HCMV infection elicits the expansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C), a response that resembles the clonal expansion of adaptive immune cells. Here, we determined that expansion of this NKG2C(+) subset and general NK cell recovery rely on signals derived from CD14(+) monocytes. In a coculture system, a subset of CD14(+) cells with inflammatory monocyte features produced IL-12 in response to HCMV-infected fibroblasts, and neutralization of IL-12 in this model substantially reduced CD25 upregulation and NKG2C(+) subset expansion. Finally, blockade of CD94/NKG2C on NK cells or silencing of the cognate ligand HLA-E in infected fibroblasts greatly impaired expansion of NKG2C(+) NK cells. Together, our results reveal that IL-12, CD14(+) cells, and the CD94/NKG2C/HLA-E axis are critical for the expansion of NKG2C(+) NK cells in response to HCMV infection. Moreover, strategies targeting the NKG2C(+) NK cell subset have the potential to be exploited in NK cell-based intervention strategies against viral infections and cancer.

Noncanonical Wnt signaling promotes obesity-induced adipose tissue inflammation and metabolic dysfunction independent of adipose tissue expansion.

PubMed

Fuster, José J; Zuriaga, María A; Ngo, Doan Thi-Minh; Farb, Melissa G; Aprahamian, Tamar; Yamaguchi, Terry P; Gokce, Noyan; Walsh, Kenneth

2015-04-01

Adipose tissue dysfunction plays a pivotal role in the development of insulin resistance in obese individuals. Cell culture studies and gain-of-function mouse models suggest that canonical Wnt proteins modulate adipose tissue expansion. However, no genetic evidence supports a role for endogenous Wnt proteins in adipose tissue dysfunction, and the role of noncanonical Wnt signaling remains largely unexplored. Here we provide evidence from human, mouse, and cell culture studies showing that Wnt5a-mediated, noncanonical Wnt signaling contributes to obesity-associated metabolic dysfunction by increasing adipose tissue inflammation. Wnt5a expression is significantly upregulated in human visceral fat compared with subcutaneous fat in obese individuals. In obese mice, Wnt5a ablation ameliorates insulin resistance, in parallel with reductions in adipose tissue inflammation. Conversely, Wnt5a overexpression in myeloid cells augments adipose tissue inflammation and leads to greater impairments in glucose homeostasis. Wnt5a ablation or overexpression did not affect fat mass or adipocyte size. Mechanistically, Wnt5a promotes the expression of proinflammatory cytokines by macrophages in a Jun NH2-terminal kinase-dependent manner, leading to defective insulin signaling in adipocytes. Exogenous interleukin-6 administration restores insulin resistance in obese Wnt5a-deficient mice, suggesting a central role for this cytokine in Wnt5a-mediated metabolic dysfunction. Taken together, these results demonstrate that noncanonical Wnt signaling contributes to obesity-induced insulin resistance independent of adipose tissue expansion. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Melanopsin Phototransduction Contributes to Light-Evoked Choroidal Expansion and Rod L-Type Calcium Channel Function In Vivo.

PubMed

Berkowitz, Bruce A; Schmidt, Tiffany; Podolsky, Robert H; Roberts, Robin

2016-10-01

In humans, rodents, and pigeons, the dark → light transition signals nonretinal brain tissue to increase choroidal thickness, a major control element of choroidal blood flow, and thus of photoreceptor and retinal pigment epithelium function. However, it is unclear which photopigments in the retina relay the light signal to the brain. Here, we test the hypothesis that melanopsin (Opn4)-regulated phototransduction modulates light-evoked choroidal thickness expansion in mice. Two-month-old C57Bl/6 wild-type (B6), 4- to 5-month-old C57Bl/6/129S6 wild-type (B6 + S6), and 2-month-old melanopsin knockout (Opn4-/-) on a B6 + S6 background were studied. Retinal anatomy was evaluated in vivo by optical coherence tomography and MRI. Choroidal thickness in dark and light were measured by diffusion-weighted MRI. Rod cell L-type calcium channel (LTCC) function in dark and light (manganese-enhanced MRI [MEMRI]) was also measured. Opn4-/- mice did not show the light-evoked expansion of choroidal thickness observed in B6 and B6 + S6 controls. Additionally, Opn4-/- mice had lower than normal rod cell and inner retinal LTCC function in the dark but not in the light. These deficits were not due to structural abnormalities because retinal laminar architecture and thickness, and choroidal thickness in the Opn4-/- mice were similar to controls. First time evidence is provided that melanopsin phototransduction contributes to dark → light control of murine choroidal thickness. The data also highlight a contribution in vivo of melanopsin phototransduction to rod cell and inner retinal depolarization in the dark.

Mixed-linker UiO-66: structure-property relationships revealed by a combination of high-resolution powder X-ray diffraction and density functional theory calculations.

PubMed

Taddei, Marco; Tiana, Davide; Casati, Nicola; van Bokhoven, Jeroen A; Smit, Berend; Ranocchiari, Marco

2017-01-04

The use of mixed-linker metal-organic frameworks (MIXMOFs) is one of the most effective strategies to modulate the physical-chemical properties of MOFs without affecting the overall crystal structure. In many instances, MIXMOFs have been recognized as solid solutions, with random distribution of ligands, in agreement with the empirical rule known as Vegard's law. In this work, we have undertaken a study combining high-resolution powder X-ray diffraction (HR-PXRD) and density functional theory (DFT) calculations with the aim of understanding the reasons why UiO-66-based amino- and bromo-functionalized MIXMOFs (MIXUiO-66) undergo cell expansion obeying Vegard's law and how this behaviour is related to their physical-chemical properties. DFT calculations predict that the unit cell in amino-functionalized UiO-66 experiences only minor expansion as a result of steric effects, whereas major modification to the electronic features of the framework leads to weaker metal-linker interaction and consequently to the loss of stability at higher degrees of functionalization. For bromo-functionalized UiO-66, steric repulsion due to the size of bromine yields a large cell expansion, but the electronic features remain very similar to pristine UiO-66, preserving the stability of the framework upon functionalization. MIXUiO-66 obtained by either direct synthesis or by post-synthetic exchange shows Vegard-like behaviour, suggesting that both preparation methods yield solid solutions, but the thermal stability and the textural properties of the post-synthetic exchanged materials do not display a clear dependence on the chemical composition, as observed for the MOFs obtained by direct synthesis.

PAF-Myc-Controlled Cell Stemness Is Required for Intestinal Regeneration and Tumorigenesis.

PubMed

Kim, Moon Jong; Xia, Bo; Suh, Han Na; Lee, Sung Ho; Jun, Sohee; Lien, Esther M; Zhang, Jie; Chen, Kaifu; Park, Jae-Il

2018-03-12

The underlying mechanisms of how self-renewing cells are controlled in regenerating tissues and cancer remain ambiguous. PCNA-associated factor (PAF) modulates DNA repair via PCNA. Also, PAF hyperactivates Wnt/β-catenin signaling independently of PCNA interaction. We found that PAF is expressed in intestinal stem and progenitor cells (ISCs and IPCs) and markedly upregulated during intestinal regeneration and tumorigenesis. Whereas PAF is dispensable for intestinal homeostasis, upon radiation injury, genetic ablation of PAF impairs intestinal regeneration along with the severe loss of ISCs and Myc expression. Mechanistically, PAF conditionally occupies and transactivates the c-Myc promoter, which induces the expansion of ISCs/IPCs during intestinal regeneration. In mouse models, PAF knockout inhibits Apc inactivation-driven intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/β-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that the PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Possible role of hemichannels in cancer

PubMed Central

Schalper, Kurt A.; Carvajal-Hausdorf, Daniel; Oyarzo, Mauricio P.

2014-01-01

In humans, connexins (Cxs) and pannexins (Panxs) are the building blocks of hemichannels. These proteins are frequently altered in neoplastic cells and have traditionally been considered as tumor suppressors. Alteration of Cxs and Panxs in cancer cells can be due to genetic, epigenetic and post-transcriptional/post-translational events. Activated hemichannels mediate the diffusional membrane transport of ions and small signaling molecules. In the last decade hemichannels have been shown to participate in diverse cell processes including the modulation of cell proliferation and survival. However, their possible role in tumor growth and expansion remains largely unexplored. Herein, we hypothesize about the possible role of hemichannels in carcinogenesis and tumor progression. To support this theory, we summarize the evidence regarding the involvement of hemichannels in cell proliferation and migration, as well as their possible role in the anti-tumor immune responses. In addition, we discuss the evidence linking hemichannels with cancer in diverse models and comment on the current technical limitations for their study. PMID:25018732

Hepatic carcinoma-associated fibroblasts induce IDO-producing regulatory dendritic cells through IL-6-mediated STAT3 activation

PubMed Central

Cheng, J-t; Deng, Y-n; Yi, H-m; Wang, G-y; Fu, B-s; Chen, W-j; Liu, W; Tai, Y; Peng, Y-w; Zhang, Q

2016-01-01

Although carcinoma-associated fibroblasts (CAFs) in tumor microenvironments have a critical role in immune cell modulation, their effects on the generation of regulatory dendritic cells (DCs) are still unclear. In this study, we initially show that CAFs derived from hepatocellular carcinoma (HCC) tumors facilitate the generation of regulatory DCs, which are characterized by low expression of costimulatory molecules, high suppressive cytokines production and enhanced regulation of immune responses, including T-cell proliferation impairment and promotion of regulatory T-cell (Treg) expansion via indoleamine 2,3-dioxygenase (IDO) upregulation. Our findings also indicate that STAT3 activation in DCs, as mediated by CAF-derived interleukin (IL)-6, is essential to IDO production. Moreover, IDO inhibitor, STAT3 and IL-6 blocking antibodies can reverse this hepatic CAF-DC regulatory function. Therefore, our results provide new insights into the mechanisms by which CAFs induce tumor immune escape as well as a novel cancer immunotherapeutic approach (for example, targeting CAFs, IDO or IL-6). PMID:26900950

Large-scale expansion of γδ T cells and peptide-specific cytotoxic T cells using zoledronate for adoptive immunotherapy.

PubMed

Yoshikawa, Toshiaki; Takahara, Masashi; Tomiyama, Mai; Nieda, Mie; Maekawa, Ryuji; Nakatsura, Tetsuya

2014-11-01

Specific cellular immunotherapy for cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated antigens. However, it is difficult to isolate and expand functionally active T-cells ex vivo. In this study, we investigated the efficacy of a new method to induce expansion of antigen-specific CTLs for adoptive immunotherapy. We used tumor-associated antigen glypican-3 (GPC3)-derived peptide and cytomegalovirus (CMV)-derived peptide as antigens. Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T-cell expansion. To induce expansion of γδ T cells and antigen-specific CTLs, the PBMCs of healthy volunteers or patients vaccinated with GPC3 peptide were cultured with both peptide and zoledronate for 14 days. The expansion of γδ T cells and peptide-specific CTLs from a few PBMCs using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3‑specific CTLs was approximately 24- to 170,000-fold. These CD8(+) cells, including CTLs, showed GPC3-specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with human immunodeficiency virus peptide. On the other hand, CD8(-) cells, including γδ T cells, showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec, but did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8(+) cells, CD8(-) cells, and total cells after expansion significantly inhibited tumor growth in an NOD/SCID mouse model. This study indicates that simultaneous expansion of γδ T cells and peptide-specific CTLs using zoledronate is useful for adoptive immunotherapy.

Friends not foes: CTLA-4 blockade and mTOR inhibition cooperate during CD8+ T cell priming to promote memory formation and metabolic readiness.

PubMed

Pedicord, Virginia A; Cross, Justin R; Montalvo-Ortiz, Welby; Miller, Martin L; Allison, James P

2015-03-01

During primary Ag encounter, T cells receive numerous positive and negative signals that control their proliferation, function, and differentiation, but how these signals are integrated to modulate T cell memory has not been fully characterized. In these studies, we demonstrate that combining seemingly opposite signals, CTLA-4 blockade and rapamycin-mediated mammalian target of rapamycin inhibition, during in vivo T cell priming leads to both an increase in the frequency of memory CD8(+) T cells and improved memory responses to tumors and bacterial challenges. This enhanced efficacy corresponds to increased early expansion and memory precursor differentiation of CD8(+) T cells and increased mitochondrial biogenesis and spare respiratory capacity in memory CD8(+) T cells in mice treated with anti-CTLA-4 and rapamycin during immunization. Collectively, these results reveal that mammalian target of rapamycin inhibition cooperates with rather than antagonizes blockade of CTLA-4, promoting unrestrained effector function and proliferation, and an optimal metabolic program for CD8(+) T cell memory. Copyright © 2015 by The American Association of Immunologists, Inc.

Is There Any Reason to Prefer Cord Blood Instead of Adult Donors for Hematopoietic Stem Cell Transplants?

PubMed

Beksac, Meral

2015-01-01

As cord blood (CB) enables rapid access and tolerance to HLA mismatches, a number of unrelated CB transplants have reached 30,000. Such transplant activity has been the result of international accreditation programs maintaining highly qualified cord blood units (CBUs) reaching more than 600,000 CBUs stored worldwide. Efforts to increase stem cell content or engraftment rate of the graft by ex vivo expansion, modulation by molecules such as fucose, prostaglandin E2 derivative, complement CD26 inhibitors, or CXCR4/CXCL12 axis have been able to accelerate engraftment speed and rate. Furthermore, introduction of reduced intensity conditioning protocols, better HLA matching, and recognition of the importance of HLA-C have improved CB transplants success by decreasing transplant-related mortality. CB progenitor/stem cell content has been compared with adult stem cells revealing higher long-term repopulating capacity compared to bone marrow-mesenchymal stromal cells and lesser oncogenic potential than progenitor-induced stem cells. This chapter summarizes the advantages and disadvantages of CB compared to adult stem cells within the context of stem cell biology and transplantation.

A Unified Method of Finding Laplace Transforms, Fourier Transforms, and Fourier Series. [and] An Inversion Method for Laplace Transforms, Fourier Transforms, and Fourier Series. Integral Transforms and Series Expansions. Modules and Monographs in Undergraduate Mathematics and Its Applications Project. UMAP Units 324 and 325.

ERIC Educational Resources Information Center

Grimm, C. A.

This document contains two units that examine integral transforms and series expansions. In the first module, the user is expected to learn how to use the unified method presented to obtain Laplace transforms, Fourier transforms, complex Fourier series, real Fourier series, and half-range sine series for given piecewise continuous functions. In…

Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists

PubMed Central

Petersen, Christopher T.; Hassan, Mojibade; Morris, Anna B.; Jeffery, Jasmin; Lee, Kunhee; Jagirdar, Neera; Staton, Ashley D.; Raikar, Sunil S.; Spencer, Harold T.; Sulchek, Todd; Flowers, Christopher R.

2018-01-01

Adoptive therapy with ex vivo–expanded genetically modified antigen-specific T cells can induce remissions in patients with relapsed/refractory cancer. The clinical success of this therapy depends upon efficient transduction and expansion of T cells ex vivo and their homing, persistence and cytotoxicity following reinfusion. Lower rates of ex vivo expansion and clinical response using anti-CD19 chimeric antigen receptor (CAR) T cells have been seen in heavily pretreated lymphoma patients compared with B-cell acute lymphoblastic leukemia patients and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo. We demonstrate that inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) and antagonism of vasoactive intestinal peptide (VIP) signaling partially inhibits the terminal differentiation of T cells during anti-CD3/CD28 bead-mediated expansion (mean, 54.4% CD27+CD28+ T cells vs 27.4% in control cultures; P < .05). This strategy results in a mean of 83.7% more T cells cultured from lymphoma patients in the presence of PI3Kδ and VIP antagonists, increased survival of human T cells from a lymphoma patient in a murine xenograft model, enhanced cytotoxic activity of antigen-specific human CAR T cells and murine T cells against lymphoma, and increased transduction and expansion of anti-CD5 human CAR T cells. PI3Kδ and VIP antagonist-expanded T cells from lymphoma patients show reduced terminal differentiation, enhanced polyfunctional cytokine expression, and preservation of costimulatory molecule expression. Taken together, synergistic blockade of these pathways is an attractive strategy to enhance the expansion and functional capacity of ex vivo–expanded cancer-specific T cells. PMID:29386194

Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells.

PubMed

Ikeda, Kazuhiro; Nagata, Shogo; Okitsu, Teru; Takeuchi, Shoji

2017-06-06

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

IL-12–producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion

PubMed Central

Rölle, Alexander; Pollmann, Julia; Ewen, Eva-Maria; Le, Vu Thuy Khanh; Halenius, Anne; Hengel, Hartmut; Cerwenka, Adelheid

2014-01-01

Human cytomegalovirus (HCMV) infection is the most common cause of congenital viral infections and a major source of morbidity and mortality after organ transplantation. NK cells are pivotal effector cells in the innate defense against CMV. Recently, hallmarks of adaptive responses, such as memory-like features, have been recognized in NK cells. HCMV infection elicits the expansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C), a response that resembles the clonal expansion of adaptive immune cells. Here, we determined that expansion of this NKG2C+ subset and general NK cell recovery rely on signals derived from CD14+ monocytes. In a coculture system, a subset of CD14+ cells with inflammatory monocyte features produced IL-12 in response to HCMV-infected fibroblasts, and neutralization of IL-12 in this model substantially reduced CD25 upregulation and NKG2C+ subset expansion. Finally, blockade of CD94/NKG2C on NK cells or silencing of the cognate ligand HLA-E in infected fibroblasts greatly impaired expansion of NKG2C+ NK cells. Together, our results reveal that IL-12, CD14+ cells, and the CD94/NKG2C/HLA-E axis are critical for the expansion of NKG2C+ NK cells in response to HCMV infection. Moreover, strategies targeting the NKG2C+ NK cell subset have the potential to be exploited in NK cell–based intervention strategies against viral infections and cancer. PMID:25384219

The Cdk4-E2f1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors

PubMed Central

Kim, So Yoon; Rane, Sushil G.

2011-01-01

Cell division and cell differentiation are intricately regulated processes vital to organ development. Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle that orchestrate the cell division and differentiation programs. Cdk1 is essential to drive cell division and is required for the first embryonic divisions, whereas Cdks 2, 4 and 6 are dispensable for organogenesis but vital for tissue-specific cell development. Here, we illustrate an important role for Cdk4 in regulating early pancreas development. Pancreatic development involves extensive morphogenesis, proliferation and differentiation of the epithelium to give rise to the distinct cell lineages of the adult pancreas. The cell cycle molecules that specify lineage commitment within the early pancreas are unknown. We show that Cdk4 and its downstream transcription factor E2f1 regulate mouse pancreas development prior to and during the secondary transition. Cdk4 deficiency reduces embryonic pancreas size owing to impaired mesenchyme development and fewer Pdx1+ pancreatic progenitor cells. Expression of activated Cdk4R24C kinase leads to increased Nkx2.2+ and Nkx6.1+ cells and a rise in the number and proliferation of Ngn3+ endocrine precursors, resulting in expansion of the β cell lineage. We show that E2f1 binds and activates the Ngn3 promoter to modulate Ngn3 expression levels in the embryonic pancreas in a Cdk4-dependent manner. These results suggest that Cdk4 promotes β cell development by directing E2f1-mediated activation of Ngn3 and increasing the pool of endocrine precursors, and identify Cdk4 as an important regulator of early pancreas development that modulates the proliferation potential of pancreatic progenitors and endocrine precursors. PMID:21490060

HIV-1 Tat affects the programming and functionality of human CD8⁺ T cells by modulating the expression of T-box transcription factors.

PubMed

Sforza, Fabio; Nicoli, Francesco; Gallerani, Eleonora; Finessi, Valentina; Reali, Eva; Cafaro, Aurelio; Caputo, Antonella; Ensoli, Barbara; Gavioli, Riccardo

2014-07-31

HIV infection is characterized by several immune dysfunctions of both CD8⁺ and CD4⁺ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8⁺ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4⁺ T cells; however, its effects on CD8⁺ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8⁺ T-cell functionality and programming. CD8⁺ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Tat favors the secretion of interleukin-2, interferon-γ and granzyme B in CD8⁺ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8⁺ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Tat deeply alters the programming and functionality of CD8⁺ T lymphocytes.

Intra-hydrogel culture prevents transformation of mesenchymal stem cells induced by monolayer expansion.

PubMed

Jiang, Tongmeng; Liu, Junting; Ouyang, Yiqiang; Wu, Huayu; Zheng, Li; Zhao, Jinmin; Zhang, Xingdong

2018-05-01

In this study, we report that the intra-hydrogel culture system mitigates the transformation of mesenchymal stem cells (MSCs) induced by two-dimensional (2D) expansion. MSCs expanded in monolayer culture prior to encapsulation in collagen hydrogels (group eMSCs-CH) featured impaired stemness in chondrogenesis, comparing with the freshly isolated bone marrow mononuclear cells seeded directly in collagen hydrogels (group fMSCs-CH). The molecular mechanism of the in vitro expansion-triggered damage to MSCs was detected through genome-wide microarray analysis. Results indicated that pathways such as proteoglycans in cancer and pathways in cancer expansion were highly enriched in eMSCs-CH. And multiple up-regulated oncoma-associated genes were verified in eMSCs-CH compared with fMSCs-CH, indicating that expansion in vitro triggered cellular transformation was associated with signaling pathways related to tumorigenicity. Besides, focal adhesion (FA) and mitogen-activated protein kinase (MAPK) signaling pathways were also involved in in vitro expansion, indicating restructuring of the cell architecture. Thus, monolayer expansion in vitro may contribute to vulnerability of MSCs through the regulation of FA and MAPK. This study indicates that intra-hydrogel culture can mitigate the monolayer expansion induced transformation of MSCs and maintain the uniformity of the stem cells, which is a viable in vitro culture system for stem cell therapy.

Penium margaritaceum as a model organism for cell wall analysis of expanding plant cells.

PubMed

Rydahl, Maja G; Fangel, Jonatan U; Mikkelsen, Maria Dalgaard; Johansen, I Elisabeth; Andreas, Amanda; Harholt, Jesper; Ulvskov, Peter; Jørgensen, Bodil; Domozych, David S; Willats, William G T

2015-01-01

The growth of a plant cell encompasses a complex set of subcellular components interacting in a highly coordinated fashion. Ultimately, these activities create specific cell wall structural domains that regulate the prime force of expansion, internally generated turgor pressure. The precise organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying mechanics of the cell wall in a single plant cell.

A Novel Cutaneous Fatty Acid–Binding Protein-Related Signaling Pathway Leading to Malignant Progression in Prostate Cancer Cells

PubMed Central

Bao, Zhengzheng; Malki, Mohammad I.; Forootan, Shiva S.; Adamson, Janet; Forootan, Farzad S.; Chen, Danqing; Foster, Christopher S.; Rudland, Philip S.

2013-01-01

Cutaneous fatty acid–binding protein (C-FABP), a cancer promoter and metastasis inducer, is overexpressed in the majority of prostatic carcinomas. Investigation of molecular mechanisms involved in tumor-promoting activity of C-FABP has established that there is a fatty acid–initiated signaling pathway leading to malignant progression of prostatic cancer cells. Increased C-FABP expression plays an important role in this novel signaling pathway. Thus, when C-FABP expression is increased, excessive amounts of fatty acids are transported into the nucleus where they act as signaling molecules to stimulate their nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). The activated PPARγ then modulates the expression of its downstream target regulatory genes, which eventually lead to enhanced tumor expansion and aggressiveness caused by an overgrowth of cells with reduced apoptosis and an increased angiogenesis. PMID:24167657

A discrete method for modal analysis of overhead line conductor bundles

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

Migdalovici, M.A.; Sireteanu, T.D.; Albrecht, A.A.

The paper presents a mathematical model and a semi-analytical procedure to calculate the vibration modes and eigenfrequencies of single or bundled conductors with spacers which are needed for evaluation of the wind induced vibration of conductors and for optimization of spacer-dampers placement. The method consists in decomposition of conductors in modules and the expansion by polynomial series of unknown displacements on each module. A complete system of polynomials are deduced for this by Legendre polynomials. Each module is considered either boundary conditions at the extremity of the module or the continuity conditions between the modules and also a number ofmore » projections of module equilibrium equation on the polynomials from the expansion series of unknown displacement. The global system of the eigenmodes and eigenfrequencies is of the matrix form: A X + {omega}{sup 2} M X = 0. The theoretical considerations are exemplified on one conductor and on bundle of two conductors with spacers. From this, a method for forced vibration calculus of a single or bundled conductors is also presented.« less

Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

PubMed

Jin, Jianjian; Gkitsas, Nikolaos; Fellowes, Vicki S; Ren, Jiaqiang; Feldman, Steven A; Hinrichs, Christian S; Stroncek, David F; Highfill, Steven L

2018-01-24

Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

Low-dose controlled release of mTOR inhibitors maintains T cell plasticity and promotes central memory T cells.

PubMed

Gammon, Joshua M; Gosselin, Emily A; Tostanoski, Lisa H; Chiu, Yu-Chieh; Zeng, Xiangbin; Zeng, Qin; Jewell, Christopher M

2017-10-10

An important goal for improving vaccine and immunotherapy technologies is the ability to provide further control over the specific phenotypes of T cells arising from these agents. Along these lines, frequent administration of rapamycin (Rapa), a small molecule inhibitor of the mammalian target of rapamycin (mTOR), exhibits a striking ability to polarize T cells toward central memory phenotypes (T CM ), or to suppress immune function, depending on the concentrations and other signals present during administration. T CM exhibit greater plasticity and proliferative capacity than effector memory T cells (T EFF ) and, therefore, polarizing vaccine-induced T cells toward T CM is an intriguing strategy to enhance T cell expansion and function against pathogens or tumors. Here we combined biodegradable microparticles encapsulating Rapa (Rapa MPs) with vaccines composed of soluble peptide antigens and molecular adjuvants to test if this approach allows polarization of differentiating T cells toward T CM . We show Rapa MPs modulate DC function, enhancing secretion of inflammatory cytokines at very low doses, and suppressing function at high doses. While Rapa MP treatment reduced - but did not stop - T cell proliferation in both CD4 + and CD8 + transgenic T cell co-cultures, the expanding CD8 + T cells differentiated to higher frequencies of T CM at low doses of MP Rapa MPs. Lastly, we show in mice that local delivery of Rapa MPs to lymph nodes during vaccination either suppresses or enhances T cell function in response to melanoma antigens, depending on the dose of drug in the depots. In particular, at low Rapa MP doses, vaccines increased antigen-specific T CM , resulting in enhanced T cell expansion measured during subsequent booster injections over at least 100days. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

PubMed Central

2011-01-01

Background Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs) with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. Methods To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs) for the direct and rapid expansion of TILs isolated from primary cancer specimens. Results TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. Conclusion Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy. PMID:21827675

Coupling of solute transport and cell expansion in pea stems

NASA Technical Reports Server (NTRS)

Schmalstig, J. G.; Cosgrove, D. J.

1990-01-01

As cells expand and are displaced through the elongation zone of the epicotyl of etiolated pea (Pisum sativum L. var Alaska) seedlings, there is little net dilution of the cell sap, implying a coordination between cell expansion and solute uptake from the phloem. Using [14C] sucrose as a phloem tracer (applied to the hypogeous cotyledons), the pattern of label accumulation along the stem closely matched the growth rate pattern: high accumulation in the growing zone, little accumulation in nongrowing regions. Several results suggest that a major portion of phloem contents enters elongating cells through the symplast. We propose that the coordination between phloem transport and cell expansion is accomplished via regulatory pathways affecting both plasmodesmata conductivity and cell expansion.

Sonic hedgehog (Shh)/Gli modulates the spatial organization of neuroepithelial cell proliferation in the developing chick optic tectum.

PubMed

Rapacioli, Melina; Botelho, Joao; Cerda, Gustavo; Duarte, Santiago; Elliot, Matías; Palma, Verónica; Flores, Vladimir

2012-10-02

Sonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis. In ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods. The present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and modelling. This effect is probably mediated by a differential mitogenic effect that increases the NEc proliferation and modulates the spatial organization of the NEc proliferation activity.

Histone methylation mediates plasticity of human FOXP3(+) regulatory T cells by modulating signature gene expressions.

PubMed

He, Haiqi; Ni, Bing; Tian, Yi; Tian, Zhiqiang; Chen, Yanke; Liu, Zhengwen; Yang, Xiaomei; Lv, Yi; Zhang, Yong

2014-03-01

CD4(+) FOXP3(+) regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4(+) CD25(high) CD127(low/-) Treg cells convert to two subpopulations with distinctive FOXP3(+) and FOXP3(-) phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events. © 2013 John Wiley & Sons Ltd.

Bus electrode having same thermal expansion coefficient as crystalline silicon solar cell

NASA Astrophysics Data System (ADS)

Kato, T.; Morita, H.; Nakano, H.; Washida, H.; Onoe, A.; Inomata, K.; Mori, F.; Sugai, S.

1982-01-01

It is well known that the bus electrode plays a main role in series resistance of solar cells. Bus electrodes composed of bare leads, were investigated for which thermal expansion coefficients are less than those of the cell and which are coated with highly conducting metals. These leads exhibited the lower expansion coefficient than expected by empirical law, and the origins of these phenomena were explained. Work hardening effect on the expansion coefficient was then measured. Solar cell fabrication with these leads and rigid solders rationalized assembly processing. Cell characteristics proved to be excellent compared with conventional ones. Finally, lead costs were compared for various materials.

Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells

PubMed Central

2018-01-01

Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38−, and CD133+CD38−) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems. PMID:29681947

Reduction in CD4 central memory T-cell subset in costimulation modulator abatacept-treated patients with recent-onset type 1 diabetes is associated with slower C-peptide decline.

PubMed

Orban, Tihamer; Beam, Craig A; Xu, Ping; Moore, Keith; Jiang, Qi; Deng, Jun; Muller, Sarah; Gottlieb, Peter; Spain, Lisa; Peakman, Mark

2014-10-01

We previously reported that continuous 24-month costimulation blockade by abatacept significantly slows the decline of β-cell function after diagnosis of type 1 diabetes. In a mechanistic extension of that study, we evaluated peripheral blood immune cell subsets (CD4, CD8-naive, memory and activated subsets, myeloid and plasmacytoid dendritic cells, monocytes, B lymphocytes, CD4(+)CD25(high) regulatory T cells, and invariant NK T cells) by flow cytometry at baseline and 3, 6, 12, 24, and 30 months after treatment initiation to discover biomarkers of therapeutic effect. Using multivariable analysis and lagging of longitudinally measured variables, we made the novel observation in the placebo group that an increase in central memory (CM) CD4 T cells (CD4(+)CD45R0(+)CD62L(+)) during a preceding visit was significantly associated with C-peptide decline at the subsequent visit. These changes were significantly affected by abatacept treatment, which drove the peripheral contraction of CM CD4 T cells and the expansion of naive (CD45R0(-)CD62L(+)) CD4 T cells in association with a significantly slower rate of C-peptide decline. The findings show that the quantification of CM CD4 T cells can provide a surrogate immune marker for C-peptide decline after the diagnosis of type 1 diabetes and that costimulation blockade may exert its beneficial therapeutic effect via modulation of this subset. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Chromosomal aberrations and deoxyribonucleic acid single-strand breaks in adipose-derived stem cells during long-term expansion in vitro.

PubMed

Froelich, Katrin; Mickler, Johannes; Steusloff, Gudrun; Technau, Antje; Ramos Tirado, Mario; Scherzed, Agmal; Hackenberg, Stephan; Radeloff, Andreas; Hagen, Rudolf; Kleinsasser, Norbert

2013-07-01

Adipose-derived stem cells (ASCs) are a promising mesenchymal cell source for tissue engineering approaches. To obtain an adequate cell amount, in vitro expansion of the cells may be required in some cases. To monitor potential contraindications for therapeutic applications in humans, DNA strand breaks and chromosomal aberrations in ASCs during in vitro expansion were examined. After isolation of ASC from human lipoaspirates of seven patients, in vitro expansion over 10 passages was performed. Cells from passages 1, 2, 3, 5 and 10 were used for the alkaline single-cell microgel electrophoresis (comet) assay to detect DNA single-strand breaks and alkali labile as well as incomplete excision repair sites. Chromosomal changes were examined by means of the chromosomal aberration test. During in vitro expansion, ASC showed no DNA single-strand breaks in the comet assay. With the chromosomal aberration test, however, a significant increase in chromosomal aberrations were detected. The study showed that although no DNA fragmentation could be determined, the safety of ASC cannot be ensured with respect to chromosome stability during in vitro expansion. Thus, reliable analyses for detecting ASC populations, which accumulate chromosomal aberrations or even undergo malignant transformation during extensive in vitro expansion, must be implemented as part of the safety evaluation of these cells for stem cell-based therapy. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Utilizing T-cell Activation Signals 1, 2, and 3 for Tumor-infiltrating Lymphocytes (TIL) Expansion: The Advantage Over the Sole Use of Interleukin-2 in Cutaneous and Uveal Melanoma.

PubMed

Tavera, René J; Forget, Marie-Andrée; Kim, Young Uk; Sakellariou-Thompson, Donastas; Creasy, Caitlin A; Bhatta, Ankit; Fulbright, Orenthial J; Ramachandran, Renjith; Thorsen, Shawne T; Flores, Esteban; Wahl, Arely; Gonzalez, Audrey M; Toth, Christopher; Wardell, Seth; Mansaray, Rahmatu; Radvanyi, Laszlo G; Gombos, Dan S; Patel, Sapna P; Hwu, Patrick; Amaria, Rodabe N; Bernatchez, Chantale; Haymaker, Cara

2018-05-11

In this study, we address one of the major critiques for tumor-infiltrating lymphocyte (TIL) therapy-the time needed for proper expansion of a suitable product. We postulated that T-cell receptor activation in the first phase of expansion combined with an agonistic stimulation of CD137/4-1BB and interleukin-2 would favor preferential expansion of CD8 TIL. Indeed, this novel 3-signal approach for optimal T-cell activation resulted in faster and more consistent expansion of CD8CD3 TIL. This new method allowed for successful expansion of TIL from cutaneous and uveal melanoma tumors in 100% of the cultures in <3 weeks. Finally, providing the 3 signals attributed to optimal T-cell activation led to expansion of TIL capable of recognizing their tumor counterpart in cutaneous and uveal melanoma. This new methodology for the initial phase of TIL expansion brings a new opportunity for translation of TIL therapy in challenging malignancies such as uveal melanoma.

Atmospheric Oxygen Inhibits Growth and Differentiation of Marrow-Derived Mouse Mesenchymal Stem Cells via a p53 Dependent Mechanism: Implications for Long-Term Culture Expansion

PubMed Central

Boregowda, Siddaraju; Krishnappa, Veena; Chambers, Jeremy; LoGrasso, Phillip V.; Lai, Wen-Tzu; Ortiz, Luis A.; Phinney, Donald G.

2013-01-01

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A and BAX expression and mitochondrial ROS generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immuno-depletion, and supported sustained MSC growth resulting in a 2300-fold increase in cumulative cell yield by 4th passage. MSCs cultured in 5% oxygen also exhibited enhanced tri-lineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA mediated knockdown of p53 in wild type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro. PMID:22367737

Xeno-Free Strategies for Safe Human Mesenchymal Stem/Stromal Cell Expansion: Supplements and Coatings

PubMed Central

Gonçalves, R. M.; Barrias, C. C.

2017-01-01

Human mesenchymal stem/stromal cells (hMSCs) have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiring in vitro expansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS) as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way. PMID:29158740

Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement

PubMed Central

Friedman, Kevin M; DeVillier, Laura E; Feldman, Steven A; Rosenberg, Steven A; Dudley, Mark E

2011-01-01

Treatment of patients with adoptive T cell therapy requires expansion of unique tumor-infiltrating lymphocyte (TIL) cultures from single cell suspensions processed from melanoma biopsies. Strategies which increase the expansion and reliability of TIL generation from tumor digests are necessary to improve access to TIL therapy. Prior work evaluated artificial antigen presenting cells (aAPCs) for their antigen-specific and costimulatory properties. We investigated engineered cells for co-stimulatory enhancement (ECCE) consisting of K562 cells which express 4-1BBL in the absence of artificial antigen stimulation. ECCE accelerated TIL expansion and significantly improved TIL numbers (p=0.001) from single cell melanoma suspensions. TIL generated with ECCE contain significantly more CD8+CD62L+ and CD8+CD27+ T cells then comparable IL-2-expanded TIL and maintained anti-tumor reactivity. Moreover, ECCE improved TIL expansion from non-melanoma cell suspensions similar to that seen with melanoma tumors. These data demonstrate that ECCE addition to TIL production will enable treatment of patients ineligible using current methods. PMID:21989413

Hepatocyte-derived exosomes promote T follicular regulatory cell expansion during hepatitis C virus infection.

PubMed

Cobb, Dustin A; Kim, Ok-Kyung; Golden-Mason, Lucy; Rosen, Hugo R; Hahn, Young S

2018-01-01

Hepatitis C virus (HCV) is a global health concern that can cause severe liver disease, such as cirrhosis and hepatocellular carcinoma. Control of HCV requires vigorous T-cell responses, yet CD4 + T cells in chronic HCV patients are dysfunctional. T follicular regulatory (Tfr) cells are a subset of regulatory T cells that suppress T follicular helper (Tfh) cells and the generation of high affinity antibody-producing B cells. In this study, we examined the accumulation of Tfr cells in the liver compartment during chronic HCV infection and defined the cellular and molecular mechanisms underlying their expansion. Our analysis revealed a substantial population of Tfr cells in livers of chronic HCV patients that is absent in liver tissues from nonviral hepatitis or healthy subjects. Coculture of PBMCs from healthy subjects with HCV-infected hepatoma cells resulted in preferential expansion of circulating Tfr cells, leading to suppression of Tfh cells. Additionally, coculture of tonsillar cells with infected hepatoma cells lead to an expansion of germinal center Tfr. Notably, expansion was mediated by transforming growth factor beta (TGF-β)-containing exosomes released from HCV-infected hepatocytes given that blockade of exosome-associated TGF-β or inhibition of exosome release abrogated Tfr expansion. These results show that liver-derived exosomes play a pivotal role in the accumulation of Tfr cells, likely leading to suppression of Tfh responses in HCV-infected patients. Our study identifies a novel pathway in which HCV infection in hepatocytes exacerbates Tfr cell responses to subvert antiviral immunity. (Hepatology 2018;67:71-85). © 2017 by the American Association for the Study of Liver Diseases.

Dimensionless number is central to stress relaxation and expansive growth of the cell wall.

PubMed

Ortega, Joseph K E

2017-06-07

Experiments demonstrate that both plastic and elastic deformation of the cell wall are necessary for wall stress relaxation and expansive growth of walled cells. A biophysical equation (Augmented Growth Equation) was previously shown to accurately model the experimentally observed wall stress relaxation and expansive growth rate. Here, dimensional analysis is used to obtain a dimensionless Augmented Growth Equation with dimensionless coefficients (groups of variables, or Π parameters). It is shown that a single Π parameter controls the wall stress relaxation rate. The Π parameter represents the ratio of plastic and elastic deformation rates, and provides an explicit relationship between expansive growth rate and the wall's mechanical properties. Values for Π are calculated for plant, algal, and fungal cells from previously reported experimental results. It is found that the Π values for each cell species are large and very different from each other. Expansive growth rates are calculated using the calculated Π values and are compared to those measured for plant and fungal cells during different growth conditions, after treatment with IAA, and in different developmental stages. The comparison shows good agreement and supports the claim that the Π parameter is central to expansive growth rate of walled cells.

Characteristics of an axisymmetric sudden expansion flow

NASA Technical Reports Server (NTRS)

Stevenson, W. H.; Thompson, H. D.

1985-01-01

A two-color, two component Laser Doppler Velocimeter (LDV) system operating in forward scatter has been developed in order to make simultaneous measurements of the axial and radial velocity components in an axisymmetric sudden expansion flow with and without combustion. The LDV system includes Bragg cell modulators in the four beam paths to allow a net frequency shift of 5MHz in both the green and blue beams. This permits an unambiguous measurement of negative velocities and also eliminates incomplete signal bias. The green beam probe volume has a waist diameter of 0.200 mm and is approximately 2mm long. The blue beam has a probe volume waist of 0.250 mm and is approximately 1 mm long. The scattered light from the probe volume is separated so that approximately 80% of each color passes to its respective photomultiplier tube by using a dichroic filter. Narrow bandpass filters are used to further filter unwanted signals before they are detected. A schematic diagram of the LDV system is shown.

Effects of GABAA receptor inhibition on response properties of barrel cortical neurons in C-fiber-depleted rats.

PubMed

Farazifard, Rasoul; Kiani, Roozbeh; Esteky, Hossein

2005-07-19

C-fiber depletion results in expansion of low threshold somatosensory mechanoreceptive fields. In this study, we investigated the role of intact C-fibers in GABAA-mediated inhibition in barrel cortical neurons. We used electronically controlled mechanical stimulation of whiskers to quantitatively examine the responses of barrel cells to whisker displacements. After systemic injection of picrotoxin neuronal responses were recorded at 5 min intervals for 20 min and then at 10 min intervals for 100 min. Picrotoxin injection caused a 3-fold increase in response magnitude of adjacent whisker stimulation and 1.4-fold increase in response magnitude of principal whisker stimulation with a maximum enhancement 50 min after the injection. There was no significant change in spontaneous activity following picrotoxin injection. The response enhancement and receptive field expansion observed in normal rats were completely absent in the C-fiber-depleted rats. These results suggest that the GABAA-mediated inhibition that modulates the receptive field functional organization of the barrel cortex depends on intact C-fibers.

The role of spatial dynamics in modulating metabolic interactions in biofilm development

NASA Astrophysics Data System (ADS)

Bocci, Federico; Lu, Mingyang; Suzuki, Yoko; Onuchic, Jose

Cell phenotypic expression is substantially affected by the presence of environmental stresses and cell-cell communication mechanisms. We study the metabolic interactions of the glutamate synthesis pathway to explain the oscillation of growth rate observed in a B. Subtilis colony. Previous modelling schemes had failed in fully reproducing quantitative experimental observations as they did not explicitly address neither the diffusion of small metabolites nor the spatial distribution of phenotypically distinct bacteria inside the colony. We introduce a continuous space-temporal framework to explain how biofilm development dynamics is influenced by the metabolic interplay between two bacterial phenotypes composing the interior and the peripheral layer of the biofilm. Growth oscillations endorse the preservation of a high level of nutrients in the interior through diffusion and colony expansion in the periphery altogether. Our findings point out that perturbations of environmental conditions can result in the interruption of the interplay between cell populations and advocate alternative approaches to biofilm control strategies.

Injury-activated glial cells promote wound healing of the adult skin in mice.

PubMed

Parfejevs, Vadims; Debbache, Julien; Shakhova, Olga; Schaefer, Simon M; Glausch, Mareen; Wegner, Michael; Suter, Ueli; Riekstina, Una; Werner, Sabine; Sommer, Lukas

2018-01-16

Cutaneous wound healing is a complex process that aims to re-establish the original structure of the skin and its functions. Among other disorders, peripheral neuropathies are known to severely impair wound healing capabilities of the skin, revealing the importance of skin innervation for proper repair. Here, we report that peripheral glia are crucially involved in this process. Using a mouse model of wound healing, combined with in vivo fate mapping, we show that injury activates peripheral glia by promoting de-differentiation, cell-cycle re-entry and dissemination of the cells into the wound bed. Moreover, injury-activated glia upregulate the expression of many secreted factors previously associated with wound healing and promote myofibroblast differentiation by paracrine modulation of TGF-β signalling. Accordingly, depletion of these cells impairs epithelial proliferation and wound closure through contraction, while their expansion promotes myofibroblast formation. Thus, injury-activated glia and/or their secretome might have therapeutic potential in human wound healing disorders.

Uncontrolled angiogenic precursor expansion causes coronary artery anomalies in mice lacking Pofut1.

PubMed

Wang, Yidong; Wu, Bingruo; Lu, Pengfei; Zhang, Donghong; Wu, Brian; Varshney, Shweta; Del Monte-Nieto, Gonzalo; Zhuang, Zhenwu; Charafeddine, Rabab; Kramer, Adam H; Sibinga, Nicolas E; Frangogiannis, Nikolaos G; Kitsis, Richard N; Adams, Ralf H; Alitalo, Kari; Sharp, David J; Harvey, Richard P; Stanley, Pamela; Zhou, Bin

2017-09-18

Coronary artery anomalies may cause life-threatening cardiac complications; however, developmental mechanisms underpinning coronary artery formation remain ill-defined. Here we identify an angiogenic cell population for coronary artery formation in mice. Regulated by a DLL4/NOTCH1/VEGFA/VEGFR2 signaling axis, these angiogenic cells generate mature coronary arteries. The NOTCH modulator POFUT1 critically regulates this signaling axis. POFUT1 inactivation disrupts signaling events and results in excessive angiogenic cell proliferation and plexus formation, leading to anomalous coronary arteries, myocardial infarction and heart failure. Simultaneous VEGFR2 inactivation fully rescues these defects. These findings show that dysregulated angiogenic precursors link coronary anomalies to ischemic heart disease.Though coronary arteries are crucial for heart function, the mechanisms guiding their formation are largely unknown. Here, Wang et al. identify a unique, endocardially-derived angiogenic precursor cell population for coronary artery formation in mice and show that a DLL4/NOTCH1/VEGFA/VEGFR2 signaling axis is key for coronary artery development.

Wavefront reconstruction from non-modulated pyramid wavefront sensor data using a singular value type expansion

NASA Astrophysics Data System (ADS)

Hutterer, Victoria; Ramlau, Ronny

2018-03-01

The new generation of extremely large telescopes includes adaptive optics systems to correct for atmospheric blurring. In this paper, we present a new method of wavefront reconstruction from non-modulated pyramid wavefront sensor data. The approach is based on a simplified sensor model represented as the finite Hilbert transform of the incoming phase. Due to the non-compactness of the finite Hilbert transform operator the classical theory for singular systems is not applicable. Nevertheless, we can express the Moore-Penrose inverse as a singular value type expansion with weighted Chebychev polynomials.

A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes.

PubMed

Mueller, A J; Tew, S R; Vasieva, O; Clegg, P D; Canty-Laird, E G

2016-09-27

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

Discerning the role of mechanosensors in regulating proximal tubule function

PubMed Central

Weisz, Ora A.

2015-01-01

All cells in the body experience external mechanical forces such as shear stress and stretch. These forces are sensed by specialized structures in the cell known as mechanosensors. Cells lining the proximal tubule (PT) of the kidney are continuously exposed to variations in flow rates of the glomerular ultrafiltrate, which manifest as changes in axial shear stress and radial stretch. Studies suggest that these cells respond acutely to variations in flow by modulating their ion transport and endocytic functions to maintain glomerulotubular balance. Conceptually, changes in the axial shear stress in the PT could be sensed by three known structures, namely, the microvilli, the glycocalyx, and primary cilia. The orthogonal component of the force produced by flow exhibits as radial stretch and can cause expansion of the tubule. Forces of stretch are transduced by integrins, by stretch-activated channels, and by cell-cell contacts. This review summarizes our current understanding of flow sensing in PT epithelia, discusses challenges in dissecting the role of individual flow sensors in the mechanosensitive responses, and identifies potential areas of opportunity for new study. PMID:26662200

3D printed lattices as an activation and expansion platform for T cell therapy.

PubMed

Delalat, Bahman; Harding, Frances; Gundsambuu, Batjargal; De-Juan-Pardo, Elena M; Wunner, Felix M; Wille, Marie-Luise; Jasieniak, Marek; Malatesta, Kristen A L; Griesser, Hans J; Simula, Antonio; Hutmacher, Dietmar W; Voelcker, Nicolas H; Barry, Simon C

2017-09-01

One of the most significant hurdles to the affordable, accessible delivery of cell therapy is the cost and difficulty of expanding cells to clinically relevant numbers. Immunotherapy to prevent autoimmune disease, tolerate organ transplants or target cancer critically relies on the expansion of specialized T cell populations. We have designed 3D-printed cell culture lattices with highly organized micron-scale architectures, functionalized via plasma polymerization to bind monoclonal antibodies that trigger cell proliferation. This 3D technology platform facilitate the expansion of therapeutic human T cell subsets, including regulatory, effector, and cytotoxic T cells while maintaining the correct phenotype. Lentiviral gene delivery to T cells is enhanced in the presence of the lattices. Incorporation of the lattice format into existing cell culture vessels such as the G-Rex system is feasible. This cell expansion platform is user-friendly and expedites cell recovery and scale-up, making it ideal for translating T cell therapies from bench to bedside. Copyright © 2017 Elsevier Ltd. All rights reserved.

JAK2V617F-mutant megakaryocytes contribute to hematopoietic stem/progenitor cell expansion in a model of murine myeloproliferation

PubMed Central

Zhan, H; Ma, Y; Lin, CHS; Kaushansky, K

2016-01-01

The myeloproliferative neoplasms (MPNs) are characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs, but the mechanism(s) responsible for MPN stem cell expansion remain incomplete. One hallmark feature of the marrow in patients with MPNs is megakaryocyte (MK) hyperplasia. We report here that mice bearing a human JAK2V617F gene restricted exclusively to the MK lineage develop many of the features of a MPN. Specifically, these mice exhibit thrombocytosis, splenomegaly, increased numbers of marrow and splenic hematopoietic progenitors and a substantial expansion of HSPCs. In addition, wild-type mice transplanted with cells from JAK2V617F-bearing MK marrow develop a myeloproliferative syndrome with thrombocytosis and erythrocytosis as well as pan-hematopoietic progenitor and stem cell expansion. As marrow histology in this murine model of myeloproliferation reveals a preferentially perivascular localization of JAK2V617F-mutant MKs and an increased marrow sinusoid vascular density, it adds to accumulating data that MKs are an important component of the marrow HSPC niche, and that MK expansion might indirectly contribute to the critical role of the thrombopoietin/c-Mpl signaling pathway in HSPC maintenance and expansion. PMID:27133820

Hematopoietic Stem Cells: Transcriptional Regulation, Ex Vivo Expansion and Clinical Application

PubMed Central

Aggarwal, R.; Lu, J.; Pompili, V.J.; Das, H.

2012-01-01

Maintenance of ex vivo hematopoietic stem cells (HSC) pool and its differentiated progeny is regulated by complex network of transcriptional factors, cell cycle proteins, extracellular matrix, and their microenvironment through an orchestrated fashion. Strides have been made to understand the mechanisms regulating in vivo quiescence and proliferation of HSCs to develop strategies for ex vivo expansion. Ex vivo expansion of HSCs is important to procure sufficient number of stem cells and as easily available source for HSC transplants for patients suffering from hematological disorders and malignancies. Our lab has established a nanofiber-based ex vivo expansion strategy for HSCs, while preserving their stem cell characteristics. Ex vivo expanded cells were also found biologically functional in various disease models. However, the therapeutic potential of expanded stem cells at clinical level still needs to be verified. This review outlines transcriptional factors that regulate development of HSCs and their commitment, genes that regulate cell cycle status, studies that attempt to develop an effective and efficient protocol for ex vivo expansion of HSCs and application of HSC in various non-malignant and malignant disorders. Overall the goal of the current review is to deliver an understanding of factors that are critical in resolving the challenges that limit the expansion of HSCs in vivo and ex vivo. PMID:22082480

Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

PubMed

LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M

1997-09-05

Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment. While many of the culture bag materials gave satisfactory results, all of the tubing materials severely inhibited total cell and CFU-GM expansion. Taken together, our results show that many materials approved for blood contact or considered biocompatible are not suitable for use with hematopoietic cells cultured in serum-free medium. As hematopoietic cultures are scaled up for a variety of clinical applications, it will be essential to carefully examine the biocompatibility of all materials involved.

The C. elegans engrailed homolog ceh-16 regulates the self-renewal expansion division of stem cell-like seam cells.

PubMed

Huang, Xinxin; Tian, E; Xu, Yanhua; Zhang, Hong

2009-09-15

Stem cells undergo symmetric and asymmetric division to maintain the dynamic equilibrium of the stem cell pool and also to generate a variety of differentiated cells. The homeostatic mechanism controlling the choice between self-renewal and differentiation of stem cells is poorly understood. We show here that ceh-16, encoding the C. elegans ortholog of the transcription factor Engrailed, controls symmetric and asymmetric division of stem cell-like seam cells. Loss of function of ceh-16 causes certain seam cells, which normally undergo symmetric self-renewal expansion division with both daughters adopting the seam cell fate, to divide asymmetrically with only one daughter retaining the seam cell fate. The human engrailed homolog En2 functionally substitutes the role of ceh-16 in promoting self-renewal expansion division of seam cells. Loss of function of apr-1, encoding the C. elegans homolog of the Wnt signaling component APC, results in transformation of self-renewal maintenance seam cell division to self-renewal expansion division, leading to seam cell hyperplasia. The apr-1 mutation suppresses the seam cell division defect in ceh-16 mutants. Our study reveals that ceh-16 interacts with the Wnt signaling pathway to control the choice between self-renewal expansion and maintenance division and also demonstrates an evolutionarily conserved function of engrailed in promoting cell proliferation.

MiR-17 Partly Promotes Hematopoietic Cell Expansion through Augmenting HIF-1α in Osteoblasts

PubMed Central

Yang, Yuxia; Ma, Wei; Wu, Dan; Huang, Yu; Li, Hongge; Zou, Junhua; Zhang, Yanju; Feng, Meifu; Luo, Jianyuan

2013-01-01

Background Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment, of which osteogenic cells play a crucial role. While evidence is accumulating for an important role of intrinsic miR-17 in regulating HSCs and HPCs, whether miR-17 signaling pathways are also necessary in the cell-extrinsic control of hematopoiesis hereto remains poorly understood. Methodology/Principal Findings Using the immortalized clone with the characteristics of osteoblasts, FBMOB-hTERT, in vitro expansion, long-term culture initiating cell (LTC-IC) and non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice repopulating cell (SRC) assay revealed that the ectopic expression of miR-17 partly promoted the ability of FBMOB-hTERT to support human cord blood (CB) CD34+ cell expansion and maintain their multipotency. It also seemed that osteoblastic miR-17 was prone to cause a specific expansion of the erythroid lineage. Conversely, deficient expression of miR-17 partly inhibited the hematopoietic supporting ability of FBMOB-hTERT. We further identified that HIF-1α is responsible for, at least in part, the promoted hematopoietic supporting ability of FBMOB-hTERT caused by miR-17. HIF-1α expression is markedly enhanced in miR-17 overexpressed FBMOB-hTERT upon interaction with CB CD34+ cells compared to other niche associated factors. More interestingly, the specific erythroid lineage expansion of CB CD34+ cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down. Conclusion/Significance Our data demonstrated that CB CD34+ cell expansion can be partly promoted by osteoblastic miR-17, and in particular, ectopic miR-17 can cause a specific expansion of the erythroid lineage through augmenting HIF-1α in osteoblasts. PMID:23936170

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

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

Pham, Son; CSIRO Australian Animal Health Laboratory, Victoria 3220; Tabarin, Thibault

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the ‘lynchpin’ for size expansion is highly specific. We demonstratemore » that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection. - Highlights: • Cell free viruses are able to receive external trigger that leads to apparent size expansion. • Virus envelope and CD4 receptor engagement is the lynchpin of virus size expansion. • Internal capsid organisation can influence receptor mediated virus size expansion. • Pre-existing virus-associated lipid membrane in cell free virus can accommodate the receptor mediated virus size expansion.« less

Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

PubMed

Flaishman, Moshe A; Peles, Yuval; Dahan, Yardena; Milo-Cochavi, Shira; Frieman, Aviad; Naor, Amos

2015-04-01

Temperature is one of the most significant factors affecting physiological and biochemical aspects of fruit development. Current and progressing global warming is expected to change climate in the traditional deciduous fruit tree cultivation regions. In this study, 'Golden Delicious' trees, grown in a controlled environment or commercial orchard, were exposed to different periods of heat treatment. Early fruitlet development was documented by evaluating cell number, cell size and fruit diameter for 5-70 days after full bloom. Normal activities of molecular developmental and growth processes in apple fruitlets were disrupted under daytime air temperatures of 29°C and higher as a result of significant temporary declines in cell-production and cell-expansion rates, respectively. Expression screening of selected cell cycle and cell expansion genes revealed the influence of high temperature on genetic regulation of apple fruitlet development. Several core cell-cycle and cell-expansion genes were differentially expressed under high temperatures. While expression levels of B-type cyclin-dependent kinases and A- and B-type cyclins declined moderately in response to elevated temperatures, expression of several cell-cycle inhibitors, such as Mdwee1, Mdrbr and Mdkrps was sharply enhanced as the temperature rose, blocking the cell-cycle cascade at the G1/S and G2/M transition points. Moreover, expression of several expansin genes was associated with high temperatures, making them potentially useful as molecular platforms to enhance cell-expansion processes under high-temperature regimes. Understanding the molecular mechanisms of heat tolerance associated with genes controlling cell cycle and cell expansion may lead to the development of novel strategies for improving apple fruit productivity under global warming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures

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

Lam, Alan Tin-Lun, E-mail: alan_lam@bti.a-star.edu.sg; Chen, Allen Kuan-Liang; Ting, Sherwin Qi-Peng

Current methods for human pluripotent stem cells (hPSC) expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are expanded as cell/MC aggregates and then directly differentiated as embryoid bodies (EBs) in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. This review describes the principles ofmore » such microcarriers based integrated hPSC expansion and differentiation process, and parameters that can affect its efficiency (such as MC type and extracellular matrix proteins coatings, cell/MC aggregates size, and agitation). Finally examples of integrated process for generation cardiomyocytes (CM) and neural progenitor cells (NPC) as well as challenges to be solved are described. - Highlights: • Expansion of hPSC on microcarriers. • Differentiation of hPSC on microcarriers. • Parameters that can affect the expansion and differentiation of hPSC on microcarriers. • Integration of expansion and differentiation of hPSC on microcarriers in one unit operation.« less

Expansion of microvascular networks in vivo by phthalimide neovascular factor 1 (PNF1)

PubMed Central

Wieghaus, Kristen A.; Nickerson, Meghan M.; Aronin, Caren E. Petrie; Sefcik, Lauren S.; Price, Richard J.; Paige, Mikell A.; Brown, Milton L.; Botchwey, Edward A.

2010-01-01

Phthalimide neovascular factor (PNF1, formerly SC-3-149) is a potent stimulator of proangiogenic signaling pathways in endothelial cells. In this study, we evaluated the in vivo effects of sustained PNF1 release to promote ingrowth and expansion of microvascular networks surrounding biomaterial implants. The dorsal skinfold window chamber was used to evaluate the structural remodeling response of the local microvasculature. PNF1 was released from poly(lactic-co-glycolic acid) (PLAGA) films, and a transport model was utilized to predict PNF1 penetration into the surrounding tissue. PNF1 significantly expanded microvascular networks within a 2 mm radius from implants after 3 and 7 days by increasing microvessel length density and lumenal diameter of local arterioles and venules. Staining of histological sections with CD11b showed enhanced recruitment of circulating white blood cells, including monocytes, which are critical for the process of vessel enlargement through arteriogenesis. As PNF1 has been shown to modulate MT1-MMP, a facilitator of CCL2 dependent leukocyte transmigration, aspects of window chamber experiments were repeated in CCR2−/− (CCL2 receptor) mouse chimeras to more fully explore the critical nature of monocyte recruitment on the therapeutic benefits of PNF1 function in vivo. PMID:18804278

Expansion of microvascular networks in vivo by phthalimide neovascular factor 1 (PNF1).

PubMed

Wieghaus, Kristen A; Nickerson, Meghan M; Petrie Aronin, Caren E; Sefcik, Lauren S; Price, Richard J; Paige, Mikell A; Brown, Milton L; Botchwey, Edward A

2008-12-01

Phthalimide neovascular factor (PNF1, formerly SC-3-149) is a potent stimulator of proangiogenic signaling pathways in endothelial cells. In this study, we evaluated the in vivo effects of sustained PNF1 release to promote ingrowth and expansion of microvascular networks surrounding biomaterial implants. The dorsal skinfold window chamber was used to evaluate the structural remodeling response of the local microvasculature. PNF1 was released from poly(lactic-co-glycolic acid) (PLAGA) films, and a transport model was utilized to predict PNF1 penetration into the surrounding tissue. PNF1 significantly expanded microvascular networks within a 2mm radius from implants after 3 and 7 days by increasing microvessel length density and lumenal diameter of local arterioles and venules. Staining of histological sections with CD11b showed enhanced recruitment of circulating white blood cells, including monocytes, which are critical for the process of vessel enlargement through arteriogenesis. As PNF1 has been shown to modulate MT1-MMP, a facilitator of CCL2 dependent leukocyte transmigration, aspects of window chamber experiments were repeated in CCR2(-/-) (CCL2 receptor) mouse chimeras to more fully explore the critical nature of monocyte recruitment on the therapeutic benefits of PNF1 function in vivo.

The mechanisms of the SAMS experiment flown on Nimbus 7 with particular reference to the 2 axis scanning mirror. [infrared radiometer for stratospheric and mesospheric investigations

NASA Technical Reports Server (NTRS)

Hadley, H.

1980-01-01

The stratospheric and mesospheric sounder (SAMS) experiment on Nimbus 7 includes a 2 axis scanning mirror and 7 pressure modulator cells. The SAMS experiment is a limb sounding instrument to measure the temperature profile and minor constituents of the atmosphere. The limb scan requires small mirror steps over a 3 deg range, while the scan in azimuth is in larger steps over a 15 deg range. The mirror is plane, 20 cm in diameter, and of zero expansion glass-ceramic. It is supported on two tilt tables, fitted one on the other, with the axes at right angles. The angle of tilt is adjusted by means of recirculating ball screws which are ion plated with lead for lubrication and driven by stepper motors. The seven gas filled cells are each pressure modulated by a 3 cm diameter, 0.3 cm stroke piston which is supported by diaphragm springs and driven electromagnetically at the system's mechanical resonant frequency. The mean pressure of the filling gas, which is the atmospheric constituent being measured, is changed by varying the temperature of a suitable molecular sieve.

A phase cell cluster expansion for Euclidean field theories

NASA Astrophysics Data System (ADS)

Battle, Guy A., III; Federbush, Paul

1982-08-01

We adapt the cluster expansion first used to treat infrared problems for lattice models (a mass zero cluster expansion) to the usual field theory situation. The field is expanded in terms of special block spin functions and the cluster expansion given in terms of the expansion coefficients (phase cell variables); the cluster expansion expresses correlation functions in terms of contributions from finite coupled subsets of these variables. Most of the present work is carried through in d space time dimensions (for φ24 the details of the cluster expansion are pursued and convergence is proven). Thus most of the results in the present work will apply to a treatment of φ34 to which we hope to return in a succeeding paper. Of particular interest in this paper is a substitute for the stability of the vacuum bound appropriate to this cluster expansion (for d = 2 and d = 3), and a new method for performing estimates with tree graphs. The phase cell cluster expansions have the renormalization group incorporated intimately into their structure. We hope they will be useful ultimately in treating four dimensional field theories.

Intracellular Pressure Dynamics in Blebbing Cells

PubMed Central

Strychalski, Wanda; Guy, Robert D.

2016-01-01

Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results. PMID:26958893

Pasteurella multocida Toxin Manipulates T Cell Differentiation

PubMed Central

Hildebrand, Dagmar; Heeg, Klaus; Kubatzky, Katharina F.

2015-01-01

Pasteurella multocida causes various diseases in a broad range of wild and domestic animals. Toxigenic strains of the serotypes A and D produce an AB protein toxin named Pasteurella multocida toxin (PMT). PMT constitutively activates the heterotrimeric G protein subunits Gαq, Gα13, and Gαi through deamidation of a glutamine residue, which results in cytoskeletal rearrangements as well as increased proliferation and survival of the host cell. In human monocytes, PMT alters the lipopolysaccharide (LPS)-induced activation toward a phenotype that suppresses T cell activation. Here we describe that the toxin also modulates CD4-positive T helper (Th) cells directly. PMT amplifies the expansion of Th cells through enhanced cell cycle progression and suppression of apoptosis and manipulates the differentiation of Th subclasses through activation of Signal Transducers and Activators of Transcription (STAT) family members and induction of subtype-specific master transcription factors. A large population of toxin-treated T cells is double-positive for Foxp3 and RORγt, the transcription factors expressed by Treg and Th17 cells, respectively. This suggests that these cells could have the potential to turn into Th17 cells or suppressive Treg cells. However, in terms of function, the PMT-differentiated cells behave as inflammatory Th17 cells that produce IL-17 and trigger T cell proliferation. PMID:26635744

Palmitate induces cisternal ER expansion via the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

PubMed

Kim, Seong Keun; Oh, Eunhye; Yun, Mihee; Lee, Seong-Beom; Chae, Gue Tae

2015-07-16

Endoplasmic reticulum (ER) stress induces ER expansion. The expansion of the intracisternal space of the ER was found in macrophages associated with human atherosclerotic lesions. We also previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. In this study, we report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells. RAW 264.7 cells were pre-treated with the designated inhibitor or siRNA, followed by treatment with palmitate. Changes in the ER structure were examined by transmission electron microscopy. The induction of ER stress was confirmed by an increase in the extent of phosphorylation of PERK, the expression of BiP and CHOP, and the splicing of XBP-1 mRNA. Phospholipid staining was performed with the LipidTOX Red phospholipidosis detection reagent. Related gene expressions were detected by quantitative real time-RT-PCR or RT-PCR. Palmitate was found to induce ER stress and cisternal ER expansion. In addition, palmitate-induced cisternal ER expansion was attenuated by ER stress inhibitors, such as 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA). The findings also show that palmitate induced-mRNA expression of CCTα, which increases phospholipid synthesis, was attenuated by the down-regulation of XBP-1, a part of ER stress. Furthermore, palmitate-induced phospholipid accumulation and cisternal ER expansion were attenuated by the down-regulation of XBP-1 or CCTα. The findings reported herein indicate that palmitate-induced cisternal ER expansion is dependent on the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

Response of γδ T cells to plant-derived tannins

PubMed Central

Holderness, Jeff; Hedges, Jodi F.; Daughenbaugh, Katie; Kimmel, Emily; Graff, Jill; Freedman, Brett; Jutila, Mark A.

2008-01-01

Many pharmaceutical drugs are isolated from plants used in traditional medicines. Through screening plant extracts, both traditional medicines and compound libraries, new pharmaceutical drugs continue to be identified. Currently, two plant-derived agonists for γδ T cells are described. These plant-derived agonists impart innate effector functions upon distinct γδ T cell subsets. Plant tannins represent one class of γδ T cell agonist and preferentially activate the mucosal population. Mucosal γδ T cells function to modulate tissue immune responses and induce epithelium repair. Select tannins, isolated from apple peel, rapidly induce immune gene transcription in γδ T cells, leading to cytokine production and increased responsiveness to secondary signals. Activity of these tannin preparations tracks to the procyanidin fraction, with the procyanidin trimer (C1) having the most robust activity defined to date. The response to the procyanidins is evolutionarily conserved in that responses are seen with human, bovine, and murine γδ T cells. Procyanidin-induced responses described in this review likely account for the expansion of mucosal γδ T cells seen in mice and rats fed soluble extracts of tannins. Procyanidins may represent a novel approach for treatment of tissue damage, chronic infection, and autoimmune therpies. PMID:19166386

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

PubMed Central

Wheeler, Richard; Turner, Robert D.; Bailey, Richard G.; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A. S.; Hayhurst, Emma J.; Horsburgh, Malcolm; Hobbs, Jamie K.

2015-01-01

ABSTRACT Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. PMID:26220963

A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

NASA Astrophysics Data System (ADS)

Lei, Yuguo; Schaffer, David V.

2013-12-01

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

Expansion of brain T cells in homeostatic conditions in lymphopenic Rag2(-/-) mice.

PubMed

Song, Chang; Nicholson, James D; Clark, Sarah M; Li, Xin; Keegan, Achsah D; Tonelli, Leonardo H

2016-10-01

The concept of the brain as an immune privileged organ is rapidly evolving in light of new findings outlining the sophisticated relationship between the central nervous and the immune systems. The role of T cells in brain development and function, as well as modulation of behavior has been demonstrated by an increasing number of studies. Moreover, recent studies have redefined the existence of a brain lymphatic system and the presence of T cells in specific brain structures, such as the meninges and choroid plexus. Nevertheless, much information is needed to further the understanding of brain T cells and their relationship with the central nervous system under non-inflammatory conditions. In the present study we employed the Rag2(-/-) mouse model of lymphocyte deficiency and reconstitution by adoptive transfer to study the temporal and anatomical expansion of T cells in the brain under homeostatic conditions. Lymphopenic Rag2(-/-) mice were reconstituted with 10 million lymphoid cells and studied at one, two and four weeks after transfer. Moreover, lymphoid cells and purified CD4(+) and CD8(+) T cells from transgenic GFP expressing mice were used to define the neuroanatomical localization of transferred cells. T cell numbers were very low in the brain of reconstituted mice up to one week after transfer and significantly increased by 2weeks, reaching wild type values at 4weeks after transfer. CD4(+) T cells were the most abundant lymphocyte subtype found in the brain followed by CD8(+) T cells and lastly B cells. Furthermore, proliferation studies showed that CD4(+) T cells expand more rapidly than CD8(+) T cells. Lymphoid cells localize abundantly in meningeal structures, choroid plexus, and circumventricular organs. Lymphocytes were also found in vascular and perivascular spaces and in the brain parenchyma across several regions of the brain, in particular in structures rich in white matter content. These results provide proof of concept that the brain meningeal system, as well as vascular and perivascular spaces, are homing sites of lymphocytes and suggest the possibility of a brain specific T cell subtype. Published by Elsevier Inc.

Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand.

PubMed

Proulx, Chantal; Boyer, Lucie; Hurnanen, Darin R; Lemieux, Réal

2003-04-01

The high proliferative potential of cord blood (CB) stem cells and the identification of the key factor of megakaryopoiesis, thrombopoietin (TPO), permit the ex vivo expansion of megakaryocytes (MKs) for possible use in early post-transplant support of patients and the production of functional platelets for transfusion. However, culture conditions for the generation of adequate MKs for this purpose are not yet optimized. Therefore, we sought to define the mixture of early-acting cytokines and TPO that would promote the expansion of MK progenitors over other lineages and result in overall better MK expansion and platelet yields. CB CD34(+)-enriched cells were cultured in serum-free medium for 17 days in presence of TPO alone or in various combinations with early-acting cytokines used at different concentrations and addition times. MK expansion and polyploidy and platelet production were monitored by flow cytometry analysis using specific surface markers (CD41 and CD42b) and propidium iodide labeling. Our results showed that the use of high concentrations of stem cell factor (SCF) and Flt-3 ligand (FL) in early CB TPO-supplemented cultures was more favorable to monocytic and granulocytic cell expansion. However, we observed that their presence in limiting amounts was required for the preferential expansion of MK progenitors. The addition of SCF, FL, TPO, and interleukin-6 (IL-6) at high concentrations in secondary cultures of these expanded MKs resulted in optimal MK proportion (approximately 25% of MKs) and expansion (>300 MK per seeded cell), highest proportions of polyploid MKs (22% of mature MKs > or = 8N), and best platelet yields. Our results indicate that TPO-induced MK progenitors are more sensitive to early-acting cytokines than non-MK cells. We propose that MKs generated in the optimized conditions, in combination with immature stem/progenitor cells, could prove useful for the short-term platelet recovery following CB transplantation.

Can microcarrier-expanded chondrocytes synthesize cartilaginous tissue in vitro?

PubMed

Surrao, Denver C; Khan, Aasma A; McGregor, Aaron J; Amsden, Brian G; Waldman, Stephen D

2011-08-01

Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on Millicell™ filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue.

Multiple Inflammatory Cytokines Converge to Regulate CD8+ T cell Expansion and Function During Tuberculosis

PubMed Central

Booty, Matthew G.; Nunes-Alves, Cláudio; Carpenter, Stephen M.; Jayaraman, Pushpa; Behar, Samuel M.

2015-01-01

The differentiation of effector CD8+ T cells is a dynamically regulated process that varies during different infections and is influenced by the inflammatory milieu of the host. Here, we define three signals regulating CD8+ T cell responses during tuberculosis by focusing on cytokines known to affect disease outcome: IL-12, type I IFN, and IL-27. Using mixed bone marrow chimeras, we compared wild type and cytokine receptor knockout CD8+ T cells within the same mouse following aerosol infection with Mycobacterium tuberculosis. Four weeks post-infection, IL-12, type 1 IFN, and IL-27 were all required for efficient CD8+ T cell expansion in the lungs. We next determined if these cytokines directly promote CD8+ T cell priming or are required only for expansion in the lungs. Utilizing retrogenic CD8+ T cells specific for the Mtb antigen TB10.4 (EsxH), we observed that IL-12 is the dominant cytokine driving both CD8+ T cell priming in the lymph node and expansion in the lungs; however, type I IFN and IL-27 have non-redundant roles supporting pulmonary CD8+ T cell expansion. Thus, IL-12 is a major signal promoting priming in the lymph node, but a multitude of inflammatory signals converge in the lung to promote continued expansion. Furthermore, these cytokines regulate the differentiation and function of CD8+ T cells during tuberculosis. These data demonstrate distinct and overlapping roles for each of the cytokines examined and underscore the complexity of CD8+ T cell regulation during tuberculosis. PMID:26755819

Human mesenchymal stem cells promote CD34+ hematopoietic stem cell proliferation with preserved red blood cell differentiation capacity.

PubMed

Lau, Show Xuan; Leong, Yin Yee; Ng, Wai Hoe; Ng, Albert Wee Po; Ismail, Ida Shazrina; Yusoff, Narazah Mohd; Ramasamy, Rajesh; Tan, Jun Jie

2017-06-01

Studies showed that co-transplantation of mesenchymal stem cells (MSCs) and cord blood-derived CD34 + hematopoietic stem cells (HSCs) offered greater therapeutic effects but little is known regarding the effects of human Wharton's jelly derived MSCs on HSC expansion and red blood cell (RBC) generation in vitro. This study aimed to investigate the effects of MSCs on HSC expansion and differentiation. HSCs were co-cultured with MSCs or with 10% MSCs-derived conditioned medium, with HSCs cultured under standard medium served as a control. Cell expansion rates, number of mononuclear cell post-expansion and number of enucleated cells post-differentiation were evaluated. HSCs showed superior proliferation in the presence of MSC with mean expansion rate of 3.5 × 10 8  ± 1.8 × 10 7 after day 7 compared to the conditioned medium and the control group (8.9 × 10 7  ± 1.1 × 10 8 and 7.0 × 10 7  ± 3.3 × 10 6 respectively, P < 0.001). Although no significant differences in RBC differentiation were observed between groups at passage IV, the number of enucleated cell was greater compared to earlier passages, indicating successful RBC differentiation. Cord blood-derived CD34 + HSCs can be greatly expanded by co-culturing with MSCs without affecting the RBC differentiation capability, suggesting the importance of direct MSC-HSCs contact in HSC expansion and RBC differentiation. © 2017 International Federation for Cell Biology.

Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in CD34+CD133+CXCR4low fraction.

PubMed

Lapostolle, Véronique; Chevaleyre, Jean; Duchez, Pascale; Rodriguez, Laura; Vlaski-Lafarge, Marija; Sandvig, Ioanna; Brunet de la Grange, Philippe; Ivanovic, Zoran

2018-06-01

Feasibility of ex vivo expansion allows us to consider the steady-state peripheral blood as an alternative source of hematopoietic stem progenitor cells for transplantation when growth factor-induced cell mobilization is contraindicated or inapplicable. Ex vivo expansion dramatically enhances the in vivo reconstituting cell population from steady-state blood. In order to investigate phenotype and the expression of homing molecules, CD34, CD133, CD90, CD45RA, CD26 and CD9 expression was determined on sorted CD34+ cells according to CXCR4 (neg, low, bright) and CD133 expression before and after ex vivo expansion. Hematopoietic stem cell activity was determined in vivo on the basis of hematopoietic repopulation of primary and secondary recipients - NSG immuno-deficient mice. In vivo reconstituting cells in steady-state blood CD34+ cell fraction before expansion belong to the CD133+ population and are CXCR4low or, to a lesser extent, CXCR4neg, while after ex vivo expansion they are contained in only the CD133+CXCR4low cells. The failure of CXCR4bright population to engraft is probably due to the exclusive expression of CD26 by these cells. The limiting-dilution analysis showed that both repopulating cell number and individual proliferative capacity were enhanced by ex vivo expansion. Thus, steady-state peripheral blood cells exhibit a different phenotype compared to mobilized and cord blood ones, as well as to those issued from the bone marrow. This data represent the first phenotypic characterization of steady-state blood cells exhibiting short and long term hematopoietic reconstituting potential, which can be expanded ex vivo, a sine qua non for their subsequent use for transplantation. Copyright © 2018, Ferrata Storti Foundation.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

PubMed

Schindelman, G; Morikami, A; Jung, J; Baskin, T I; Carpita, N C; Derbyshire, P; McCann, M C; Benfey, P N

2001-05-01

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis

PubMed Central

Schindelman, Gary; Morikami, Atsushi; Jung, Jee; Baskin, Tobias I.; Carpita, Nicholas C.; Derbyshire, Paul; McCann, Maureen C.; Benfey, Philip N.

2001-01-01

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion. PMID:11331607

A human dendritic cell-based in vitro model to assess Mycobacterium tuberculosis SO2 vaccine immunogenicity.

PubMed

Etna, Marilena P; Giacomini, Elena; Severa, Martina; Pardini, Manuela; Aguilo, Nacho; Martin, Carlos; Coccia, Eliana M

2014-01-01

Among the tuberculosis (TB) vaccine candidates, SO2 is the prototype of the first live-attenuated vaccine that recently entered into clinical trials. To investigate the capacity of SO2 to stimulate an appropriate immune response in vitro within a human immunological context, a comparative analysis of the effects promoted by SO2, the current Bacille Calmette-Guerin (BCG) vaccine and Mycobacterium tuberculosis (Mtb) was conducted in human primary dendritic cells (DC), which are critical modulators of vaccine-induced immunity. In particular, we found that SO2 promotes the expression of maturation markers similarly to BCG but at a lower extent than Mtb. Moreover, SO2-infected DC released higher levels of interleukin (IL)-23 than BCG-infected cells, which account for the expansion of interferon (IFN)-γ-producing T cells in an IL-12-independent manner. In the autologous mixed leukocyte reaction setting, the expansion of IL-17-producing T cells was also observed in response to SO2 infection. Interestingly, apoptosis and autophagic flux, events required for the antigen presentation within MHC class II complex, were not affected in DC infected with SO2, conversely to what observed upon Mtb stimulation. Collectively, our results indicate that SO2 represents a promising TB vaccine candidate, which displays an attenuated phenotype and promotes in DC a stronger capacity to stimulate the Th response than BCG vaccine. Interestingly, the data obtained by using the human DC-based experimental setting mirrored the results derived from studies in animal models, suggesting that this system could be used for an efficient and rapid down-selection of new TB vaccine candidates, contributing to achieve the "3Rs" objective.

Expansion and hepatic differentiation of rat multipotent adult progenitor cells in microcarrier suspension culture.

PubMed

Park, Y; Subramanian, K; Verfaillie, C M; Hu, W S

2010-10-01

Many potential applications of stem cells require large quantities of cells, especially those involving large organs such as the liver. For such applications, a scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiation competent or differentiated cells. We employed a microcarrier culture system for the expansion of undifferentiated rat multipotent adult progenitor cells (rMAPC) as well as for directed differentiation of these cells to hepatocyte-like cells. During the 4-day expansion culture, cell concentration increased by 85-fold while expression level of pluripotency markers were maintained, as well as the MAPC differentiation potential. Directed differentiation into hepatocyte-like cells on the microcarriers themselves gave comparable results as observed with cells cultured in static cultures. The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Microcarrier culture thus offers the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment. Copyright © 2010 Elsevier B.V. All rights reserved.

Moving with the flow: what transport laws reveal about cell division and expansion.

PubMed

Silk, Wendy Kuhn

2006-01-01

This material was presented as a keynote talk for the symposium, "Crosstalk between cell division and expansion," organized by G.T.S. Beemster and H. Tsukaya at the International Botanical Congress, Vienna in July, 2005. The review focuses on the utility of continuity equations to understand relationships among cell size, division and expansion; insights from Lagrangian or cell-specific descriptions of developmental variables; and a growth-diffusion equation to show effects of root growth zones on the surrounding soil.

The many ways tissue phagocytes respond to dying cells

PubMed Central

Blander, J. Magarian

2017-01-01

Summary Apoptosis is an important component of normal tissue physiology, and the prompt removal of apoptotic cells is equally essential to avoid the undesirable consequences of their accumulation and disintegration. Professional phagocytes are highly specialized for engulfing apoptotic cells. The recent ability to track cells that have undergone apoptosis in situ has revealed a division of labor among the tissue resident phagocytes that sample them. Macrophages are uniquely programmed to process internalized apoptotic cell-derived fatty acids, cholesterol and nucleotides, as a reflection of their dominant role in clearing the bulk of apoptotic cells. Dendritic cells carry apoptotic cells to lymph nodes where they signal the emergence and expansion of highly suppressive regulatory CD4 T cells. A broad suppression of inflammation is executed through distinct phagocyte-specific mechanisms. A clever induction of negative regulatory nodes is notable in dendritic cells serving to simultaneously shut down multiple pathways of inflammation. Several of the genes and pathways modulated in phagocytes in response to apoptotic cells have been linked to chronic inflammatory and autoimmune diseases such as atherosclerosis, inflammatory bowel disease and systemic lupus erythematosus. Our collective understanding of old and new phagocyte functions after apoptotic cell phagocytosis demonstrates the enormity of ways to mediate immune suppression and enforce tissue homeostasis. PMID:28462530

The many ways tissue phagocytes respond to dying cells.

PubMed

Blander, J Magarian

2017-05-01

Apoptosis is an important component of normal tissue physiology, and the prompt removal of apoptotic cells is equally essential to avoid the undesirable consequences of their accumulation and disintegration. Professional phagocytes are highly specialized for engulfing apoptotic cells. The recent ability to track cells that have undergone apoptosis in situ has revealed a division of labor among the tissue resident phagocytes that sample them. Macrophages are uniquely programmed to process internalized apoptotic cell-derived fatty acids, cholesterol and nucleotides, as a reflection of their dominant role in clearing the bulk of apoptotic cells. Dendritic cells carry apoptotic cells to lymph nodes where they signal the emergence and expansion of highly suppressive regulatory CD4 T cells. A broad suppression of inflammation is executed through distinct phagocyte-specific mechanisms. A clever induction of negative regulatory nodes is notable in dendritic cells serving to simultaneously shut down multiple pathways of inflammation. Several of the genes and pathways modulated in phagocytes in response to apoptotic cells have been linked to chronic inflammatory and autoimmune diseases such as atherosclerosis, inflammatory bowel disease and systemic lupus erythematosus. Our collective understanding of old and new phagocyte functions after apoptotic cell phagocytosis demonstrates the enormity of ways to mediate immune suppression and enforce tissue homeostasis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Pre- and Post-Evaluation of Integrating Sustainability Curriculum by Inserting Okala Modules into an Interior Design Materials and Methods Course

ERIC Educational Resources Information Center

Schneiderman, Deborah; Freihoefer, Kara

2012-01-01

Purpose: The purpose of this paper is to examine the integration of Okala curriculum into Interior Design coursework. Okala, as a teaching package, is utilized extensively in industrial design education. However, this study examines the expansion and insertion of Okala modules in an existing interior design curriculum. The Okala modules included…

Enhancing Intergenerational Contact.

ERIC Educational Resources Information Center

Ralston, Penny A.; And Others

This document is a curriculum module designed to assist secondary school home economic teachers in teaching students about aging. The three major purposes of this module are to provide data-based information on aging related to needs of students to enhance intergenerational contact; to facilitate the expansion of intergenerational contacts by…

Vγ1+γδT, early cardiac infiltrated innate population dominantly producing IL-4, protect mice against CVB3 myocarditis by modulating IFNγ+ T response.

PubMed

Wan, Fangfang; Yan, Kepeng; Xu, Dan; Qian, Qian; Liu, Hui; Li, Min; Xu, Wei

2017-01-01

Viral myocarditis (VMC) is an inflammation of the myocardium closely associated with Coxsackievirus B3 (CVB3) infection. Vγ1 + γδT cells, one of early cardiac infiltrated innate population, were reported to protect CVB3 myocarditis while the precise mechanism not fully addressed. To explore cytokine profiles and kinetics of Vγ1 + γδT and mechanism of protection against VMC, flow cytometry was conducted on cardiac Vγ1 cells in C57BL/6 mice following CVB3 infection. The level of cardiac inflammation, transthoracic echocardiography and viral replication were evaluated after monoclonal antibody depletion of Vγ1γδT. We found that Vγ1 + γδT cells infiltration peaked in the heart at day3 post CVB3 infection and constituted a minor source of IFN-γ but major producers for early IL-4. Vγ1γδT cells were activated earlier holding a higher IL-4-producing efficiency than CD4 + Th cells in the heart. Depletion of Vγ1 + γδT resulted in a significantly exacerbated cardiac infiltration, increased T, macrophage and neutrophil population in heart homogenates and worse cardiomyopathy; which was accompanied by a significant expansion of peripheral IFNγ + CD4+ and CD8+T cells. Neutralization of IL-4 in mice resulted in an exacerbated acute myocarditis confirming the IL-4-mediated protective mechanism of Vγ1. Our findings identify a unique property of Vγ1 + γδT cells as one dominant early producers of IL-4 upon CVB3 acute infection which is a key mediator to protect mice against acute myocarditis by modulating IFNγ-secreting T response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pleural mesothelial cells promote expansion of IL-17-producing CD8+ T cells in tuberculous pleural effusion.

PubMed

Li, X; Zhou, Q; Yang, W B; Xiong, X Z; Du, R H; Zhang, J C

2013-05-01

IL-17-producing CD8(+) T lymphocytes (Tc17 cells) have recently been detected in many cancers and autoimmune diseases. However, the possible implication of Tc17 cells in tuberculous pleural effusion remains unclarified. In this study, distribution and phenotypic features of Tc17 cells in both tuberculous pleural effusion (TPE) and peripheral blood from patients with tuberculosis were determined. The effects of proinflammatory cytokines and local accessory cells (pleural mesothelial cells) on Tc17 cell expansion were also explored. We found that TPE contained more Tc17 cells than the blood. Compared with IFN-γ-producing CD8(+) T cells, Tc17 cells displayed higher expression of chemokine receptors (CCRs) and lower expression of cytotoxic molecules. In particularly, Tc17 cells in TPE exhibited high expression levels of CCR6, which could migrate in response to CCL20. Furthermore, IL-1β, IL-6, IL-23, or their various combinations could promote Tc17 cell expansion from CD8(+) T cells, whereas the proliferative response of Tc17 cells to above cytokines was lower than that of Th17 cells. Pleural mesothelial cells (PMCs) were able to stimulate Tc17 cell expansion via cell contact in an IL-1β/IL-6/IL-23 independent fashion. Thus this study demonstrates that Tc17 cells marks a subset of non-cytotoxic, CCR6(+) CD8(+) T lymphocytes with low proliferative capacity. The overrepresentation of Tc17 cells in TPE may be due to Tc17 cell expansion stimulated by pleural proinflammatory cytokines and to recruitment of Tc17 cells from peripheral blood. Additionally, PMCs may promote the production of IL-17 by CD8(+) T cells at sites of TPE via cell-cell interactions.

Dectin-1 Positive Dendritic Cells Expand after Infection with Leishmania major Parasites and Represent Promising Targets for Vaccine Development

PubMed Central

Zimara, Nicole; Chanyalew, Menberework; Aseffa, Abraham; van Zandbergen, Ger; Lepenies, Bernd; Schmid, Maximilian; Weiss, Richard; Rascle, Anne; Wege, Anja Kathrin; Jantsch, Jonathan; Schatz, Valentin; Brown, Gordon D.; Ritter, Uwe

2018-01-01

Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L.) parasites. Healing correlates with a T helper (Th) cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR)-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c+ DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major-infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL). Both mouse strains responded with an expansion of Dectin-1+ DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1+ DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1+ DCs in adaptive immunity against L. major, we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs) in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania-specific CD4+ T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1−/− BALB/c mice still developed the fatal course of disease after Curdlan treatment. Furthermore, the vaccination of BALB/c mice with a combination of soluble L. major antigens and Curdlan was able to provide a partial protection from severe leishmaniasis. These findings indicate that the ligation of Dectin-1 on DCs acts as an important checkpoint in adaptive immunity against L. major and should therefore be considered in future whole-organism vaccination strategies. PMID:29535708

Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

PubMed Central

Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul

2014-01-01

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment. PMID:24641164

Dendritic Cells Limit Fibro-Inflammatory Injury in NASH

PubMed Central

Henning, Justin R.; Graffeo, Christopher S.; Rehman, Adeel; Fallon, Nina C.; Zambirinis, Constantinos P.; Ochi, Atsuo; Barilla, Rocky; Jamal, Mohsin; Deutsch, Michael; Greco, Stephanie; Ego-Osuala, Melvin; Saeed, Usama Bin; Rao, Raghavendra S.; Badar, Sana; Quesada, Juan P.; Acehan, Devrim; Miller, George

2013-01-01

Non-alcoholic steatohepatitis (NASH) is the most common etiology of chronic liver dysfunction in the United States and can progress to cirrhosis and liver failure. Inflammatory insult resulting from fatty infiltration of the liver is central to disease pathogenesis. Dendritic cells (DC) are antigen presenting cells with an emerging role in hepatic inflammation. We postulated that DC are important in the progression of NASH. We found that intrahepatic DC expand and mature in NASH liver and assume an activated immune-phenotype. However, rather than mitigating the severity of NASH, DC depletion markedly exacerbated intrahepatic fibro-inflammation. Our mechanistic studies support a regulatory role for DC in NASH by limiting sterile inflammation via their role in clearance of apoptotic cells and necrotic debris. We found that DC limit CD8+ T cell expansion and restrict Toll-like receptor expression and cytokine production in innate immune effector cells in NASH, including Kupffer cells, neutrophils, and inflammatory monocytes. Consistent with their regulatory role in NASH, during the recovery phase of disease, ablation of DC populations results in delayed resolution of intrahepatic inflammation and fibroplasia. Conclusion Our findings support a role for DC in modulating NASH. Targeting DC functional properties may hold promise for therapeutic intervention in NASH. PMID:23322710

The Peripheral Myeloid Expansion Driven by Murine Cancer Progression Is Reversed by Radiation Therapy of the Tumor

PubMed Central

Crittenden, Marka R.; Savage, Talicia; Cottam, Benjamin; Bahjat, Keith S.; Redmond, William L.; Bambina, Shelly; Kasiewicz, Melissa; Newell, Pippa; Jackson, Andrew M.; Gough, Michael J.

2013-01-01

Expansion of myeloid-lineage leukocytes in tumor-bearing mice has been proposed as a cause of systemic immunosuppression. We demonstrate that radiation therapy of tumors leads to a decline in myeloid cell numbers in the blood and a decrease in spleen size. The frequency of myeloid cells does not decline to the level seen in tumor-free mice: we demonstrate that metastatic disease can prevent myeloid cell numbers from returning to baseline, and that tumor recurrence from residual disease correlates with re-expansion of myeloid lineage cells. Radiation therapy results in increased proliferation of T cells in the spleen and while T cell responses to foreign antigens are not altered by tumor burden or myeloid cell expansion, responses to tumor-associated antigens are increased after radiation therapy. These data demonstrate that myeloid cell numbers are directly linked to primary tumor burden, that this population contracts following radiation therapy, and that radiation therapy may open a therapeutic window for immunotherapy of residual disease. PMID:23936036

Model-based analysis of Arabidopsis leaf epidermal cells reveals distinct division and expansion patterns for pavement and guard cells.

PubMed

Asl, Leila Kheibarshekan; Dhondt, Stijn; Boudolf, Véronique; Beemster, Gerrit T S; Beeckman, Tom; Inzé, Dirk; Govaerts, Willy; De Veylder, Lieven

2011-08-01

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

PubMed Central

LAPTEVA, NATALIA; DURETT, APRIL G.; SUN, JIALI; ROLLINS, LISA A.; HUYE, LESLIE L.; FANG, JIAN; DANDEKAR, VARADA; MEI, ZHUYONG; JACKSON, KIMBERLEY; VERA, JUAN; ANDO, JUN; NGO, MINHTRAN C.; COUSTAN-SMITH, ELAINE; CAMPANA, DARIO; SZMANIA, SUSANN; GARG, TARUN; MORENO-BOST, AMBERLY; VANRHEE, FRITS; GEE, ADRIAN P.; ROONEY, CLIONA M.

2016-01-01

Background aims Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results Using this system we produced up to 19 × 109 functional NK cells from unseparated apheresis products, starting with 15 × 107 CD3− CD56+ NK cells, within 8–10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8+ T cells within the NK cultures. However, these CD3+ T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy. PMID:22900959

Model-Based Analysis of Arabidopsis Leaf Epidermal Cells Reveals Distinct Division and Expansion Patterns for Pavement and Guard Cells1[W][OA

PubMed Central

Asl, Leila Kheibarshekan; Dhondt, Stijn; Boudolf, Véronique; Beemster, Gerrit T.S.; Beeckman, Tom; Inzé, Dirk; Govaerts, Willy; De Veylder, Lieven

2011-01-01

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery. PMID:21693673

Genetic Interaction of Centrosomin and Bazooka in Apical Domain Regulation in Drosophila Photoreceptor

PubMed Central

Chen, Geng; Rogers, Alicia K.; League, Garrett P.; Nam, Sang-Chul

2011-01-01

Background Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes' role in photoreceptor morphogenesis. Methodology/Principal Findings Here, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn's gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs). Conclusions/Significance These results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor. PMID:21253601

Inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules

PubMed Central

Chen, Xu; Grandont, Laurie; Li, Hongjiang; Hauschild, Robert; Paque, Sébastien; Abuzeineh, Anas; Rakusová, Hana; Benkova, Eva; Perrot-Rechenmann, Catherine; Friml, Jiří

2014-01-01

The prominent and evolutionary ancient effect of the plant hormone auxin is the regulation of cell expansion1. Cell expansion requires ordered cytoskeleton arrangement2 but molecular mechanisms underlying its regulation by signaling molecules including auxin are unknown. Here we show in the model plant Arabidopsis thaliana that in elongating cells exogenous application of auxin or redistribution of endogenous auxin induces very rapid microtubule reorientation from transversal to longitudinal, coherent with the inhibition of cell expansion. This fast auxin effect requires Auxin Binding Protein1 (ABP1) and involves a contribution of downstream signaling components such as ROP6 GTPase, ROP-interactive protein RIC1 and microtubule severing protein Katanin. These components are required for rapid auxin and ABP1-mediated reorientation of microtubules to regulate cell elongation in roots and dark grown hypocotyls as well as asymmetric growth during gravitropic responses. PMID:25409144

Nanoparticle curcumin ameliorates experimental colitis via modulation of gut microbiota and induction of regulatory T cells

PubMed Central

Ohno, Masashi; Sugitani, Yoshihiko; Nishino, Kyohei; Inatomi, Osamu; Sugimoto, Mitsushige; Kawahara, Masahiro; Andoh, Akira

2017-01-01

Background and Aims Curcumin is a hydrophobic polyphenol derived from turmeric, a traditional Indian spice. Curcumin exhibits various biological functions, but its clinical application is limited due to its poor absorbability after oral administration. A newly developed nanoparticle curcumin shows improved absorbability in vivo. In this study, we examined the effects of nanoparticle curcumin (named Theracurmin) on experimental colitis in mice. Methods BALB/c mice were fed with 3% dextran sulfate sodium (DSS) in water. Mucosal cytokine expression and lymphocyte subpopulation were analyzed by real-time PCR and flow cytometry, respectively. The profile of the gut microbiota was analyzed by real-time PCR. Results Treatment with nanoparticle curcumin significantly attenuated body weight loss, disease activity index, histological colitis score and significantly improved mucosal permeability. Immunoblot analysis showed that NF-κB activation in colonic epithelial cells was significantly suppressed by treatment with nanoparticle curcumin. Mucosal mRNA expression of inflammatory mediators was significantly suppressed by treatment with nanoparticle curcumin. Treatment with nanoparticle curcumin increased the abundance of butyrate-producing bacteria and fecal butyrate level. This was accompanied by increased expansion of CD4+ Foxp3+ regulatory T cells and CD103+ CD8α− regulatory dendritic cells in the colonic mucosa. Conclusions Treatment with nanoparticle curcumin suppressed the development of DSS-induced colitis potentially via modulation of gut microbial structure. These responses were associated with induction of mucosal immune cells with regulatory properties. Nanoparticle curcumin is one of the promising candidates as a therapeutic option for the treatment of IBD. PMID:28985227

The role of microbial signals in plant growth and development

PubMed Central

Ortíz-Castro, Randy; Contreras-Cornejo, Hexon Angel; Macías-Rodríguez, Lourdes

2009-01-01

Plant growth and development involves a tight coordination of the spatial and temporal organization of cell division, cell expansion and cell differentiation. Orchestration of these events requires the exchange of signaling molecules between the root and shoot, which can be affected by both biotic and abiotic factors. The interactions that occur between plants and their associated microorganisms have long been of interest, as knowledge of these processes could lead to the development of novel agricultural applications. Plants produce a wide range of organic compounds including sugars, organic acids and vitamins, which can be used as nutrients or signals by microbial populations. On the other hand, microorganisms release phytohormones, small molecules or volatile compounds, which may act directly or indirectly to activate plant immunity or regulate plant growth and morphogenesis. In this review, we focus on recent developments in the identification of signals from free-living bacteria and fungi that interact with plants in a beneficial way. Evidence has accumulated indicating that classic plant signals such as auxins and cytokinins can be produced by microorganisms to efficiently colonize the root and modulate root system architecture. Other classes of signals, including N-acyl-L-homoserine lactones, which are used by bacteria for cell-to-cell communication, can be perceived by plants to modulate gene expression, metabolism and growth. Finally, we discuss the role played by volatile organic compounds released by certain plant growth-promoting rhizobacteria in plant immunity and developmental processes. The picture that emerges is one in which plants and microbes communicate themselves through transkingdom signaling systems involving classic and novel signals. PMID:19820333

Epstein-Barr virus-driven B Cell Proliferation with CD4+ T Cell Expansion: A Lymphomatoid Granulomatosis-like Disease Related to Hyperinterleukin-10 Secretion of Remarkably Favourable Outcome with Rituximab.

PubMed

Cervera, P; Guihot, A; Gorochov, G; Lassoued, K; Coppo, P

2015-12-01

Granulomatous lymphomatosis is an Epstein-Barr virus (EBV)-driven B cell proliferation associated with an exuberant CD4(+) T cell reaction with usually histopathological pictures of angiocentrism. So far, the characteristics of CD4(+) T cells in granulomatous lymphomatosis and the mechanism leading to their expansion remain poorly explored. We report a 56-year-old female with a past history of cold agglutinin disease, which was successfully treated with 4 weekly infusions of rituximab. She presented one year later with features of granulomatous lymphomatosis that resulted in severe lung and bone marrow infiltration. We provide evidence that CD4(+) T cell expansion was oligoclonal, involved anergic cells and did not result from an EBV-driven stimulation. Rather, it resulted possibly from a high production of interleukin-10 by immunoblastic EBV-positive B cells. The outcome was remarkably favourable with rituximab and steroids. Our results suggest that an EBV-driven B cell proliferation should be investigated in patients presenting with a CD4(+) T cells alveolitis or other systemic manifestations resulting from a CD4(+) T cell expansion. These features should prompt to introduce an immunosuppressive therapy including steroids and rituximab. Our results deserve further investigations to confirm our pathophysiological hypotheses in CD4(+) T cell expansions associated with EBV-driven B cell proliferations and to assess whether granulomatous lymphomatosis could result from comparable mechanisms. © 2015 The Foundation for the Scandinavian Journal of Immunology.

Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype

PubMed Central

Gołąb, Karolina; Grose, Randall; Placencia, Veronica; Wickrema, Amittha; Solomina, Julia; Tibudan, Martin; Konsur, Evelyn; Ciepły, Kamil; Marek-Trzonkowska, Natalia; Trzonkowski, Piotr; Millis, J. Michael; Fung, John; Witkowski, Piotr

2018-01-01

The first clinical trials with adoptive Treg therapy have shown safety and potential efficacy. Feasibility of such therapy could be improved if cells are cryopreserved and stored until optimal timing for infusion. Herein, we report the evaluation of two cell-banking strategies for Treg therapy: 1) cryopreservation of CD4+ cells for subsequent Treg isolation/expansion and 2) cryopreservation of ex-vivo expanded Tregs (CD4+CD25hiCD127lo/- cells). First, we checked how cryopreservation affects cell viability and Treg markers expression. Then, we performed Treg isolation/expansion with the final products release testing. We observed substantial decrease in cell number recovery after thawing and overnight culture. This observation might be explained by the high percentage of necrotic and apoptotic cells found just after thawing. Furthermore, we noticed fluctuations in percentage of CD4+CD25hiCD127- and CD4+FoxP3+ cells obtained from cryopreserved CD4+ as well as Treg cells. However, after re-stimulation Tregs expanded well, presented a stable phenotype and fulfilled the release criteria at the end of expansions. Cryopreservation of CD4+ cells for subsequent Treg isolation/expansion and cryopreservation of expanded Tregs with re-stimulation and expansion after thawing, are promising solutions to overcome detrimental effects of cryopreservation. Both of these cell-banking strategies for Treg therapy can be applied when designing new clinical trials. PMID:29515766

Cell banking for regulatory T cell-based therapy: strategies to overcome the impact of cryopreservation on the Treg viability and phenotype.

PubMed

Gołąb, Karolina; Grose, Randall; Placencia, Veronica; Wickrema, Amittha; Solomina, Julia; Tibudan, Martin; Konsur, Evelyn; Ciepły, Kamil; Marek-Trzonkowska, Natalia; Trzonkowski, Piotr; Millis, J Michael; Fung, John; Witkowski, Piotr

2018-02-09

The first clinical trials with adoptive Treg therapy have shown safety and potential efficacy. Feasibility of such therapy could be improved if cells are cryopreserved and stored until optimal timing for infusion. Herein, we report the evaluation of two cell-banking strategies for Treg therapy: 1) cryopreservation of CD4 + cells for subsequent Treg isolation/expansion and 2) cryopreservation of ex-vivo expanded Tregs (CD4 + CD25 hi CD127 lo/- cells). First, we checked how cryopreservation affects cell viability and Treg markers expression. Then, we performed Treg isolation/expansion with the final products release testing. We observed substantial decrease in cell number recovery after thawing and overnight culture. This observation might be explained by the high percentage of necrotic and apoptotic cells found just after thawing. Furthermore, we noticed fluctuations in percentage of CD4 + CD25 hi CD127 - and CD4 + FoxP3 + cells obtained from cryopreserved CD4 + as well as Treg cells. However, after re-stimulation Tregs expanded well, presented a stable phenotype and fulfilled the release criteria at the end of expansions. Cryopreservation of CD4 + cells for subsequent Treg isolation/expansion and cryopreservation of expanded Tregs with re-stimulation and expansion after thawing, are promising solutions to overcome detrimental effects of cryopreservation. Both of these cell-banking strategies for Treg therapy can be applied when designing new clinical trials.

Physalis floridana Cell Number Regulator1 encodes a cell membrane-anchored modulator of cell cycle and negatively controls fruit size

PubMed Central

Li, Zhichao; He, Chaoying

2015-01-01

Physalis species show a significant variation in berry size; however, the underlying molecular basis is unknown. In this work, we showed that cell division difference in the ovaries might contribute to the ultimate berry size variation within Physalis species, and that mRNA abundance of Physalis floridana Cell Number Regulator1 (PfCNR1), the putative orthologue of the tomato fruit weight 2.2 (FW2.2), was negatively correlated with cell division in the ovaries. Moreover, heterochronic expression variation of the PfCNR1 genes in the ovaries concomitantly correlated with berry weight variation within Physalis species. In transgenic Physalis, multiple organ sizes could be negatively controlled by altering PfCNR1 levels, and cell division instead of cell expansion was primarily affected. PfCNR1 was shown to be anchored in the plasma membrane and to interact with PfAG2 (an AGAMOUS-like protein determining ovary identity). The expression of PfCYCD2;1, a putative orthologue of the mitosis-specific gene CyclinD2;1 in the cell cycle was negatively correlated with the PfCNR1 mRNA levels. PfAG2 was found to selectively bind to the CArG-box in the PfCYCD2;1 promoter and to repress PfCYCD2;1 expression, thus suggesting a PfAG2-mediated pathway for PfCNR1 to regulate cell division. The interaction of PfCNR1 with PfAG2 enhanced the repression of PfCYCD2;1 expression. The nuclear import of PfAG2 was essential in the proposed pathway. Our data provide new insights into the developmental pathways of a cell membrane-anchored protein that modulates cell division and governs organ size determination. This study also sheds light on the link between organ identity and organ growth in plants. PMID:25305759

Multiple Inflammatory Cytokines Converge To Regulate CD8+ T Cell Expansion and Function during Tuberculosis.

PubMed

Booty, Matthew G; Nunes-Alves, Cláudio; Carpenter, Stephen M; Jayaraman, Pushpa; Behar, Samuel M

2016-02-15

The differentiation of effector CD8(+) T cells is a dynamically regulated process that varies during different infections and is influenced by the inflammatory milieu of the host. In this study, we define three signals regulating CD8(+) T cell responses during tuberculosis by focusing on cytokines known to affect disease outcome: IL-12, type I IFN, and IL-27. Using mixed bone marrow chimeras, we compared wild-type and cytokine receptor knockout CD8(+) T cells within the same mouse following aerosol infection with Mycobacterium tuberculosis. Four weeks postinfection, IL-12, type 1 IFN, and IL-27 were all required for efficient CD8(+) T cell expansion in the lungs. We next determined if these cytokines directly promote CD8(+) T cell priming or are required only for expansion in the lungs. Using retrogenic CD8(+) T cells specific for the M. tuberculosis Ag TB10.4 (EsxH), we observed that IL-12 is the dominant cytokine driving both CD8(+) T cell priming in the lymph node and expansion in the lungs; however, type I IFN and IL-27 have nonredundant roles supporting pulmonary CD8(+) T cell expansion. Thus, IL-12 is a major signal promoting priming in the lymph node, but a multitude of inflammatory signals converge in the lung to promote continued expansion. Furthermore, these cytokines regulate the differentiation and function of CD8(+) T cells during tuberculosis. These data demonstrate distinct and overlapping roles for each of the cytokines examined and underscore the complexity of CD8(+) T cell regulation during tuberculosis. Copyright © 2016 by The American Association of Immunologists, Inc.

Defective fluid shear stress mechanotransduction mediates hereditary hemorrhagic telangiectasia

PubMed Central

Baeyens, Nicolas; Larrivée, Bruno; Ola, Roxana; Hayward-Piatkowskyi, Brielle; Dubrac, Alexandre; Huang, Billy; Ross, Tyler D.; Coon, Brian G.; Min, Elizabeth; Tsarfati, Maya; Tong, Haibin; Eichmann, Anne

2016-01-01

Morphogenesis of the vascular system is strongly modulated by mechanical forces from blood flow. Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal-dominant disease in which arteriovenous malformations and telangiectasias accumulate with age. Most cases are linked to heterozygous mutations in Alk1 or Endoglin, receptors for bone morphogenetic proteins (BMPs) 9 and 10. Evidence suggests that a second hit results in clonal expansion of endothelial cells to form lesions with poor mural cell coverage that spontaneously rupture and bleed. We now report that fluid shear stress potentiates BMPs to activate Alk1 signaling, which correlates with enhanced association of Alk1 and endoglin. Alk1 is required for BMP9 and flow responses, whereas endoglin is only required for enhancement by flow. This pathway mediates both inhibition of endothelial proliferation and recruitment of mural cells; thus, its loss blocks flow-induced vascular stabilization. Identification of Alk1 signaling as a convergence point for flow and soluble ligands provides a molecular mechanism for development of HHT lesions. PMID:27646277

Multi-tissue analysis of co-expression networks by higher-order generalized singular value decomposition identifies functionally coherent transcriptional modules.

PubMed

Xiao, Xiaolin; Moreno-Moral, Aida; Rotival, Maxime; Bottolo, Leonardo; Petretto, Enrico

2014-01-01

Recent high-throughput efforts such as ENCODE have generated a large body of genome-scale transcriptional data in multiple conditions (e.g., cell-types and disease states). Leveraging these data is especially important for network-based approaches to human disease, for instance to identify coherent transcriptional modules (subnetworks) that can inform functional disease mechanisms and pathological pathways. Yet, genome-scale network analysis across conditions is significantly hampered by the paucity of robust and computationally-efficient methods. Building on the Higher-Order Generalized Singular Value Decomposition, we introduce a new algorithmic approach for efficient, parameter-free and reproducible identification of network-modules simultaneously across multiple conditions. Our method can accommodate weighted (and unweighted) networks of any size and can similarly use co-expression or raw gene expression input data, without hinging upon the definition and stability of the correlation used to assess gene co-expression. In simulation studies, we demonstrated distinctive advantages of our method over existing methods, which was able to recover accurately both common and condition-specific network-modules without entailing ad-hoc input parameters as required by other approaches. We applied our method to genome-scale and multi-tissue transcriptomic datasets from rats (microarray-based) and humans (mRNA-sequencing-based) and identified several common and tissue-specific subnetworks with functional significance, which were not detected by other methods. In humans we recapitulated the crosstalk between cell-cycle progression and cell-extracellular matrix interactions processes in ventricular zones during neocortex expansion and further, we uncovered pathways related to development of later cognitive functions in the cortical plate of the developing brain which were previously unappreciated. Analyses of seven rat tissues identified a multi-tissue subnetwork of co-expressed heat shock protein (Hsp) and cardiomyopathy genes (Bag3, Cryab, Kras, Emd, Plec), which was significantly replicated using separate failing heart and liver gene expression datasets in humans, thus revealing a conserved functional role for Hsp genes in cardiovascular disease.

Reciprocal interactions between endothelial cells and macrophages in angiogenic vascular niches

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

Baer, Caroline; Squadrito, Mario Leonardo; Iruela-Arispe, M. Luisa, E-mail: arispe@mcdb.ucla.edu

The ability of macrophages to promote vascular growth has been associated with the secretion and local delivery of classic proangiogenic factors (e.g., VEGF-A and proteases). More recently, a series of studies have also revealed that physical contact of macrophages with growing blood vessels coordinates vascular fusion of emerging sprouts. Interestingly, the interactions between macrophages and vascular endothelial cells (ECs) appear to be bidirectional, such that activated ECs also support the expansion and differentiation of proangiogenic macrophages from myeloid progenitors. Here, we discuss recent findings suggesting that dynamic angiogenic vascular niches might also exist in vivo, e.g. in tumors, where sproutingmore » blood vessels and immature myeloid cells like monocytes engage in heterotypic interactions that are required for angiogenesis. Finally, we provide an account of emerging mechanisms of cell-to-cell communication that rely on secreted microvesicles, such as exosomes, which can offer a vehicle for the rapid exchange of molecules and genetic information between macrophages and ECs engaged in angiogenesis. -- Highlights: • Macrophages promote angiogenesis by secreting proangiogenic factors. • Macrophages modulate angiogenesis via cell-to-cell contacts with endothelial cells. • Endothelial cells promote the differentiation of proangiogenic macrophages. • Macrophages and endothelial cells may cooperate to form angiogenic vascular niches.« less

Di-Ethylhexylphthalate (DEHP) Modulates Cell Invasion, Migration and Anchorage Independent Growth through Targeting S100P in LN-229 Glioblastoma Cells

PubMed Central

Sims, Jennifer Nicole; Graham, Barbara; Pacurari, Maricica; Leggett, Sophia S.; Tchounwou, Paul B.; Ndebele, Kenneth

2014-01-01

Glioblastoma multiforme (GBM) is the most aggressive brain cancer with a median survival of 1–2 years. The treatment of GBM includes surgical resection, radiation and chemotherapy, which minimally extends survival. This poor prognosis necessitates the identification of novel molecular targets associated with glioblastoma. S100P is associated with drug resistance, metastasis, and poor clinical outcomes in many malignancies. The functional role of S100P in glioblastoma has not been fully investigated. In this study, we examined the role of S100P mediating the effects of the environmental contaminant, DEHP, in glioblastoma cells (LN-229) by assessing cell proliferation, apoptosis, anchorage independent growth, cell migration and invasion following DEHP exposure. Silencing S100P and DEHP treatment inhibited LN-229 glioblastoma cell proliferation and induced apoptosis. Anchorage independent growth study revealed significantly decreased colony formation in shS100P cells. We also observed reduced cell migration in cells treated with DEHP following S100P knockdown. Similar results were observed in spheroid formation and expansion. This study is the first to demonstrate the effects of DEHP on glioblastoma cells, and implicates S100P as a potential therapeutic target that may be useful as a drug response biomarker. PMID:24821384

Enhanced Ex Vivo Expansion of Human Hematopoietic Progenitors on Native and Spin Coated Acellular Matrices Prepared from Bone Marrow Stromal Cells

PubMed Central

Wasnik, Samiksha; Kantipudi, Suma; Kirkland, Mark A.; Pande, Gopal

2016-01-01

The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion. PMID:26981135

Rab7a modulates ER stress and ER morphology.

PubMed

Mateus, Duarte; Marini, Elettra Sara; Progida, Cinzia; Bakke, Oddmund

2018-05-01

The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called "ER matrices" together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22 N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells. Copyright © 2018 Elsevier B.V. All rights reserved.

In vivo Expansion of Naïve CD4+CD25high FOXP3+ Regulatory T Cells in Patients with Colorectal Carcinoma after IL-2 Administration

PubMed Central

Beyer, Marc; Schumak, Beatrix; Weihrauch, Martin R.; Andres, Bettina; Giese, Thomas; Endl, Elmar; Knolle, Percy A.; Classen, Sabine; Limmer, Andreas; Schultze, Joachim L.

2012-01-01

Regulatory T cells (Treg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of Treg cells was established. In IL-2 treated cancer patients a further Treg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional Treg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve Treg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve Treg cells indicate IL-2 dependent thymic generation of naïve Treg cells as a mechanism leading to increased frequencies of Treg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine Treg cells after IL-2 administration. These results point to a more complex regulation of Treg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve Treg cells. PMID:22276195

Adenovirus-Based Vaccines against Rhesus Lymphocryptovirus EBNA-1 Induce Expansion of Specific CD8+ and CD4+ T Cells in Persistently Infected Rhesus Macaques

PubMed Central

Leskowitz, R.; Fogg, M. H.; Zhou, X. Y.; Kaur, A.; Silveira, E. L. V.; Villinger, F.; Lieberman, P. M.; Wang, F.

2014-01-01

ABSTRACT The impact of Epstein-Barr virus (EBV) on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA-1) is an important target for vaccination because it is the only protein expressed in all EBV-associated malignancies. We have designed and tested two therapeutic EBV vaccines that target the rhesus (rh) lymphocryptovirus (LCV) EBNA-1 to determine if ongoing T cell responses during persistent rhLCV infection in rhesus macaques can be expanded upon vaccination. Vaccines were based on two serotypes of E1-deleted simian adenovirus and were administered in a prime-boost regimen. To further modulate the response, rhEBNA-1 was fused to herpes simplex virus glycoprotein D (HSV-gD), which acts to block an inhibitory signaling pathway during T cell activation. We found that vaccines expressing rhEBNA-1 with or without functional HSV-gD led to expansion of rhEBNA-1-specific CD8+ and CD4+ T cells in 33% and 83% of the vaccinated animals, respectively. Additional animals developed significant changes within T cell subsets without changes in total numbers. Vaccination did not increase T cell responses to rhBZLF-1, an immediate early lytic phase antigen of rhLCV, thus indicating that increases of rhEBNA-1-specific responses were a direct result of vaccination. Vaccine-induced rhEBNA-1-specific T cells were highly functional and produced various combinations of cytokines as well as the cytolytic molecule granzyme B. These results serve as an important proof of principle that functional EBNA-1-specific T cells can be expanded by vaccination. IMPORTANCE EBV is a common human pathogen that establishes a persistent infection through latency in B cells, where it occasionally reactivates. EBV infection is typically benign and is well controlled by the host adaptive immune system; however, it is considered carcinogenic due to its strong association with lymphoid and epithelial cell malignancies. Latent EBNA-1 is a promising target for a therapeutic vaccine, as it is the only antigen expressed in all EBV-associated malignancies. The goal was to determine if rhEBNA-1-specific T cells could be expanded upon vaccination of infected animals. Results were obtained with vaccines that target EBNA-1 of rhLCV, a virus closely related to EBV. We found that vaccination led to expansion of rhEBNA-1 immune cells that exhibited functions fit for controlling viral infection. This confirms that rhEBNA-1 is a suitable target for therapeutic vaccines. Future work should aim to generate more-robust T cell responses through modified vaccines. PMID:24522914

Increasing Stem Cell Dose Promotes Posttransplant Immune Reconstitution.

PubMed

Xu, Ning; Shen, Sylvie; Dolnikov, Alla

2017-04-01

Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.

Mechanical feedback coordinates cell wall expansion and assembly in yeast mating morphogenesis

PubMed Central

2018-01-01

The shaping of individual cells requires a tight coordination of cell mechanics and growth. However, it is unclear how information about the mechanical state of the wall is relayed to the molecular processes building it, thereby enabling the coordination of cell wall expansion and assembly during morphogenesis. Combining theoretical and experimental approaches, we show that a mechanical feedback coordinating cell wall assembly and expansion is essential to sustain mating projection growth in budding yeast (Saccharomyces cerevisiae). Our theoretical results indicate that the mechanical feedback provided by the Cell Wall Integrity pathway, with cell wall stress sensors Wsc1 and Mid2 increasingly activating membrane-localized cell wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating projection growth without affecting cell shape. Experimental perturbation of the osmotic pressure and cell wall mechanics, as well as compromising the mechanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes that support the theoretical predictions. Our results indicate that while the existence of mechanical feedback is essential to stabilize mating projection growth, the shape and size of the cell are insensitive to the feedback. PMID:29346368

Clinical proof-of-concept study with MSDC-0160, a prototype mTOT-modulating insulin sensitizer.

PubMed

Colca, J R; VanderLugt, J T; Adams, W J; Shashlo, A; McDonald, W G; Liang, J; Zhou, R; Orloff, D G

2013-04-01

It may be possible to achieve insulin sensitivity through the recently identified mitochondrial target of thiazolidinediones (mTOT), thereby avoiding peroxisome proliferator-activated receptor-γ (PPAR-γ)-dependent side effects. In this phase IIb clinical trial, 258 patients with type 2 diabetes completed a 12-week protocol with 50, 100, or 150 mg of MSDC-0160 (an mTOT modulator), 45 mg pioglitazone HCl (a PPAR-γ agonist), or a placebo. The two active treatments lowered fasting glucose levels to the same extent. The decreases in glycated hemoglobin (HbA1c) observed with the two higher doses of MSDC-0160 were not different from those associated with pioglitazone. By contrast, fluid retention as evidenced by reduction in hematocrit, red blood cells, and total hemoglobin was 50% less in the MSDC-0160-treated groups. There was also a smaller increase in high-molecular-weight (HMW) adiponectin with MSDC-0160 than with pioglitazone (P < 0.0001), suggesting that MSDC-0160 produces less expansion of white adipose tissue. Thus, mTOT modulators may have glucose-lowering effects similar to those of pioglitazone but without the adverse effects associated with PPAR-γ agonists.

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

PubMed

Fujita, Miki; Lechner, Bettina; Barton, Deborah A; Overall, Robyn L; Wasteneys, Geoffrey O

2012-02-01

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule-cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.

MutSβ abundance and Msh3 ATP hydrolysis activity are important drivers of CTG•CAG repeat expansions

PubMed Central

Keogh, Norma; Chan, Kara Y.; Li, Guo-Min

2017-01-01

Abstract CTG•CAG repeat expansions cause at least twelve inherited neurological diseases. Expansions require the presence, not the absence, of the mismatch repair protein MutSβ (Msh2-Msh3 heterodimer). To evaluate properties of MutSβ that drive expansions, previous studies have tested under-expression, ATPase function or polymorphic variants of Msh2 and Msh3, but in disparate experimental systems. Additionally, some variants destabilize MutSβ, potentially masking the effects of biochemical alterations of the variations. Here, human Msh3 was mutated to selectively inactivate MutSβ. Msh3−/− cells are severely defective for CTG•CAG repeat expansions but show full activity on contractions. Msh3−/− cells provide a single, isogenic system to add back Msh3 and test key biochemical features of MutSβ on expansions. Msh3 overexpression led to high expansion activity and elevated levels of MutSβ complex, indicating that MutSβ abundance drives expansions. An ATPase-defective Msh3 expressed at normal levels was as defective in expansions as Msh3−/− cells, indicating that Msh3 ATPase function is critical for expansions. Expression of two Msh3 polymorphic variants at normal levels showed no detectable change in expansions, suggesting these polymorphisms primarily affect Msh3 protein stability, not activity. In summary, CTG•CAG expansions are limited by the abundance of MutSβ and rely heavily on Msh3 ATPase function. PMID:28973443

MutSβ abundance and Msh3 ATP hydrolysis activity are important drivers of CTG•CAG repeat expansions.

PubMed

Keogh, Norma; Chan, Kara Y; Li, Guo-Min; Lahue, Robert S

2017-09-29

CTG•CAG repeat expansions cause at least twelve inherited neurological diseases. Expansions require the presence, not the absence, of the mismatch repair protein MutSβ (Msh2-Msh3 heterodimer). To evaluate properties of MutSβ that drive expansions, previous studies have tested under-expression, ATPase function or polymorphic variants of Msh2 and Msh3, but in disparate experimental systems. Additionally, some variants destabilize MutSβ, potentially masking the effects of biochemical alterations of the variations. Here, human Msh3 was mutated to selectively inactivate MutSβ. Msh3-/- cells are severely defective for CTG•CAG repeat expansions but show full activity on contractions. Msh3-/- cells provide a single, isogenic system to add back Msh3 and test key biochemical features of MutSβ on expansions. Msh3 overexpression led to high expansion activity and elevated levels of MutSβ complex, indicating that MutSβ abundance drives expansions. An ATPase-defective Msh3 expressed at normal levels was as defective in expansions as Msh3-/- cells, indicating that Msh3 ATPase function is critical for expansions. Expression of two Msh3 polymorphic variants at normal levels showed no detectable change in expansions, suggesting these polymorphisms primarily affect Msh3 protein stability, not activity. In summary, CTG•CAG expansions are limited by the abundance of MutSβ and rely heavily on Msh3 ATPase function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Histone methylation mediates plasticity of human FOXP3+ regulatory T cells by modulating signature gene expressions

PubMed Central

He, Haiqi; Ni, Bing; Tian, Yi; Tian, Zhiqiang; Chen, Yanke; Liu, Zhengwen; Yang, Xiaomei; Lv, Yi; Zhang, Yong

2014-01-01

CD4+ FOXP3+ regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4+ CD25high CD127low/− Treg cells convert to two subpopulations with distinctive FOXP3+ and FOXP3− phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events. PMID:24152290

Intestinal Helminths Regulate Lethal Acute Graft Versus Host Disease and Preserve Graft Versus Tumor Effect in Mice

PubMed Central

Li, Yue; Chen, Hung-lin; Bannick, Nadine; Henry, Michael; Holm, Adrian N.; Metwali, Ahmed; Urban, Joseph F.; Rothman, Paul B.; Weiner, George J.; Blazar, Bruce R.; Elliott, David E.; Ince, M. Nedim

2014-01-01

Donor T lymphocyte transfer with hematopoietic stem cells suppresses residual tumor growth (graft-versus-tumor; GVT) in cancer patients undergoing bone marrow transplantation (BMT). However, donor T cell reactivity to host organs causes severe and potentially lethal inflammation, called graft-versus-host disease (GVHD). High dose steroids or other immune suppressives are used to treat GVHD that have limited ability to control the inflammation while incurring long-term toxicity. Novel strategies are needed to modulate GVHD, preserve GVT and improve the outcome of BMT. Regulatory T cells (Tregs) control alloantigen-sensitized inflammation of GVHD, sustain GVT and prevent mortality in bone marrow transplantation. Helminths colonizing the alimentary tract dramatically increase the Treg activity, thereby modulating intestinal or systemic inflammatory responses. These observations led us to hypothesize that helminths can regulate GVHD and maintain GVT in mice. Acute GVHD was induced in helminth (Heligmosomoides polygyrus)-infected or uninfected Balb/C recipients of C57BL/6 donor grafts. Helminth infection suppressed donor T cell inflammatory cytokine generation along with reduction in GVHD lethality and maintenance of GVT. H. polygyrus colonization promoted the survival of TGFβ generating recipient Tregs after a conditioning regimen with total body irradiation and led to a TGFβ-dependent in vivo expansion/maturation of donor Tregs after BMT. Helminths did not control GVHD, when T cells unresponsive to TGFβ-mediated immune regulation were used as donor T lymphocytes. These results suggest that helminths suppress acute GVHD, employing regulatory T cells and TGFβ-dependent pathways in mice. Helminthic regulation of GVHD and GVT through intestinal immune conditioning may improve the outcome of BMT. PMID:25527786

Elements of the niche for adult stem cell expansion

PubMed Central

Redondo, Patricia A; Pavlou, Marina; Loizidou, Marilena; Cheema, Umber

2017-01-01

Adult stem cells are crucial for tissue homeostasis. These cells reside within exclusive locations in tissues, termed niches, which protect adult stem cell fidelity and regulate their many functions through biophysical-, biochemical- and cellular-mediated mechanisms. There is a growing understanding of how these mechanisms and their components contribute towards maintaining stem cell quiescence, self-renewal, expansion and differentiation patterns. In vitro expansion of adult stem cells is a powerful tool for understanding stem cell biology, and for tissue engineering and regenerative medicine applications. However, it is technically challenging, since adult stem cell removal from their native microenvironment has negative repercussions on their sustainability. In this review, we overview specific elements of the biomimetic niche and how recreating such elements can help in vitro propagation of adult stem cells. PMID:28890779

Elements of the niche for adult stem cell expansion.

PubMed

Redondo, Patricia A; Pavlou, Marina; Loizidou, Marilena; Cheema, Umber

2017-01-01

Adult stem cells are crucial for tissue homeostasis. These cells reside within exclusive locations in tissues, termed niches, which protect adult stem cell fidelity and regulate their many functions through biophysical-, biochemical- and cellular-mediated mechanisms. There is a growing understanding of how these mechanisms and their components contribute towards maintaining stem cell quiescence, self-renewal, expansion and differentiation patterns. In vitro expansion of adult stem cells is a powerful tool for understanding stem cell biology, and for tissue engineering and regenerative medicine applications. However, it is technically challenging, since adult stem cell removal from their native microenvironment has negative repercussions on their sustainability. In this review, we overview specific elements of the biomimetic niche and how recreating such elements can help in vitro propagation of adult stem cells.

Natural killer cell activity, lymphocyte proliferation, and cytokine profile in tumor-bearing mice treated with MAPA, a magnesium aggregated polymer from Aspergillus oryzae.

PubMed

Justo, G Z; Durán, N; Queiroz, M L S

2003-08-01

The present study examined the effects of MAPA, an antitumor aggregated polymer of protein magnesium ammonium phospholinoleate-palmitoleate anhydride, isolated from Aspergillus oryzae, on concanavalin A (Con A)-induced spleen cell proliferation, cytokine production and on natural killer (NK) cell activity in Ehrlich ascites tumor-bearing mice. The Ehrlich ascites tumor (EAT) growth led to diminished mitogen-induced expansion of spleen cell populations and total NK activity. This was accompanied by striking spleen enlargement, with a marked increase in total cell counts. Moreover, a substantial enhancement in IL-10 levels, paralleled by a significant decrease in IL-2 was observed, while production of IL-4 and interferon-gamma (IFN-gamma) was not altered. Treatment of mice with 5 mg/kg MAPA for 7 days promoted spleen cell proliferation, IL-2 production and NK cell activity regardless of tumor outgrowth. In addition, MAPA treatment markedly enhanced IFN-gamma levels and reduced IL-10 production relative to EAT mice. A 35% reduction in splenomegaly with normal number of nucleated cells was also found. Altogether, our results suggest that MAPA directly and/or indirectly modulates immune cell activity, and probably disengages tumor-induced suppression of these responses. Clearly, MAPA has an impact and may delay tumor outgrowth through immunotherapeutic mechanisms.

Multidisciplinary insight into clonal expansion of HTLV-1-infected cells in adult T-cell leukemia via modeling by deterministic finite automata coupled with high-throughput sequencing.

PubMed

Farmanbar, Amir; Firouzi, Sanaz; Park, Sung-Joon; Nakai, Kenta; Uchimaru, Kaoru; Watanabe, Toshiki

2017-01-31

Clonal expansion of leukemic cells leads to onset of adult T-cell leukemia (ATL), an aggressive lymphoid malignancy with a very poor prognosis. Infection with human T-cell leukemia virus type-1 (HTLV-1) is the direct cause of ATL onset, and integration of HTLV-1 into the human genome is essential for clonal expansion of leukemic cells. Therefore, monitoring clonal expansion of HTLV-1-infected cells via isolation of integration sites assists in analyzing infected individuals from early infection to the final stage of ATL development. However, because of the complex nature of clonal expansion, the underlying mechanisms have yet to be clarified. Combining computational/mathematical modeling with experimental and clinical data of integration site-based clonality analysis derived from next generation sequencing technologies provides an appropriate strategy to achieve a better understanding of ATL development. As a comprehensively interdisciplinary project, this study combined three main aspects: wet laboratory experiments, in silico analysis and empirical modeling. We analyzed clinical samples from HTLV-1-infected individuals with a broad range of proviral loads using a high-throughput methodology that enables isolation of HTLV-1 integration sites and accurate measurement of the size of infected clones. We categorized clones into four size groups, "very small", "small", "big", and "very big", based on the patterns of clonal growth and observed clone sizes. We propose an empirical formal model based on deterministic finite state automata (DFA) analysis of real clinical samples to illustrate patterns of clonal expansion. Through the developed model, we have translated biological data of clonal expansion into the formal language of mathematics and represented the observed clonality data with DFA. Our data suggest that combining experimental data (absolute size of clones) with DFA can describe the clonality status of patients. This kind of modeling provides a basic understanding as well as a unique perspective for clarifying the mechanisms of clonal expansion in ATL.

High-scale expansion of melanoma-reactive TIL by a polyclonal stimulus: predictability and relation with disease advancement.

PubMed

Pandolfino, M C; Labarrière, N; Tessier, M H; Cassidanius, A; Bercegeay, S; Lemarre, P; Dehaut, F; Dréno, B; Jotereau, F

2001-05-01

The rationale of treating melanoma patients by infusion with tumor-infiltrating leukocytes (TIL) is to perform an adoptive therapy through injection of tumor-specific T cells. Nonetheless, methods currently used for ex vivo TIL expansion have not been evaluated for their efficacy to expand TAA-specific T cells. We have addressed this question here, using a culture method in which high TIL growth was induced by a polyclonal T cell stimulus. Intracellular cytokine assays were performed to measure the proportion of T cells responding to autologous tumor cells among the lymphocytes from lymph node biopsies (TIL) of 26 patients with stage III melanoma. The data show that TIL from 18 of these patients contained detectable amounts of tumor-specific T cells before expansion. Although they decreased somewhat in percent abundance during expansion, they were still present afterwards, ranging from 0.3 to 13.8%. Since a median number of 1.7 x 10(10) TIL was obtained from these patients (starting from 3.6 x 10(6) TIL), a total amount of tumor-reactive cytokine-secreting TIL of between 2.8 x 10(6) and 1.12 x 10(9) was obtained in each case from 18 patients. The TIL populations from 8 patients did not contain tumor-reactive T cells: neither before expansion, nor after expansion. Lack of tumor-reactive TIL only occurs for patients bearing several tumor-invaded lymph nodes (40%), but not for those having a single invaded lymph node. Therefore, high numbers of tumor-reactive T cells can be produced, through a polyclonal TIL stimulation, from most early stage III melanoma patients but from only about half of the patients with a more disseminated disease. For this last group, the possibility of getting tumor-reactive TIL can be predicted by checking the presence of these cells before expansion.

Spontaneous mutation of Dock7 results in lower trabecular bone mass and impaired periosteal expansion in aged female Misty mice.

PubMed

Le, Phuong T; Bishop, Kathleen A; Maridas, David E; Motyl, Katherine J; Brooks, Daniel J; Nagano, Kenichi; Baron, Roland; Bouxsein, Mary L; Rosen, Clifford J

2017-12-01

Misty mice (m/m) have a loss of function mutation in Dock7 gene, a guanine nucleotide exchange factor, resulting in low bone mineral density, uncoupled bone remodeling and reduced bone formation. Dock7 has been identified as a modulator of osteoblast number and in vitro osteogenic differentiation in calvarial osteoblast culture. In addition, m/m exhibit reduced preformed brown adipose tissue innervation and temperature as well as compensatory increase in beige adipocyte markers. While the low bone mineral density phenotype is in part due to higher sympathetic nervous system (SNS) drive in young mice, it is unclear what effect aging would have in mice homozygous for the mutation in the Dock7 gene. We hypothesized that age-related trabecular bone loss and periosteal envelope expansion would be altered in m/m. To test this hypothesis, we comprehensively characterized the skeletal phenotype of m/m at 16, 32, 52, and 78wks of age. When compared to age-matched wild-type control mice (+/+), m/m had lower areal bone mineral density (aBMD) and areal bone mineral content (aBMC). Similarly, both femoral and vertebral BV/TV, Tb.N, and Conn.D were decreased in m/m while there was also an increase in Tb.Sp. As low bone mineral density and decreased trabecular bone were already present at 16wks of age in m/m and persisted throughout life, changes in age-related trabecular bone loss were not observed highlighting the role of Dock7 in controlling trabecular bone acquisition or bone loss prior to 16wks of age. Cortical thickness was also lower in the m/m across all ages. Periosteal and endosteal circumferences were higher in m/m compared to +/+ at 16wks. However, endosteal and periosteal expansion were attenuated in m/m, resulting in m/m having lower periosteal and endosteal circumferences by 78wks of age compared to +/+, highlighting the critical role of Dock7 in appositional bone expansion. Histomorphometry revealed that osteoblasts were nearly undetectable in m/m and marrow adipocytes were elevated 3.5 fold over +/+ (p=0.014). Consistent with reduced bone formation, osteoblast gene expression of Alp, Col1a1, Runx-2, Sp7, and Bglap was significantly decreased in m/m whole bone. Furthermore, markers of osteoclasts were either unchanged or suppressed. Bone marrow stromal cell migration and motility were inhibited in culture and changes in senescence markers suggest that osteoblast function may also be inhibited with loss of Dock7 expression in m/m. Finally, increased Oil Red O staining in m/m ear mesenchymal stem cells during adipogenesis highlights a potential shift of cells from the osteogenic to adipogenic lineages. In summary, loss of Dock7 in the aging m/m resulted in an impairment of periosteal and endocortical envelope expansion, but did not alter age-related trabecular bone loss. These studies establish Dock7 as a critical regulator of both cortical and trabecular bone mass, and demonstrate for the first time a novel role of Dock7 in modulating compensatory changes in the periosteum with aging. Copyright © 2017 Elsevier Inc. All rights reserved.

Artifacts Introduced in the Point Evaluation of Functions Expanded into a Degree 360 Spherical Harmonic Series

NASA Technical Reports Server (NTRS)

Rapp, R.

1999-01-01

An expansion of a function initially given in 1deg cells was carried out to degree 360 by using 30'cells whose value was initially assigned to be the value of the 1deg cell in which it fell. The evaluation of point values of the function from the degree 360 expansion revealed spurious patterns attributed to the coefficients from degree 181 to 360. Expansion of the original function in 1deg cells to degree 180 showed no problems in the point evaluation. Mean 1deg values computed from both degree 180 to 360 expansions showed close agreement with the original function. The artifacts could be removed if the 30' values were interpolated by spline procedures from adjacent I' cells. These results led to an examination of the gravity anomalies and geoid undulations from EGM96 in areas where I' values were "split up" to form 30'cells. The area considered was 75degS to 85degS, 100degE to 120degE where the split up cells were basically south of 81 degS. A small, latitude related, and possibly spurious effect might be detectable in anomaly variations in the region. These results suggest that point values of a function computed from a high degree expansion may have spurious signals unless the cell size is compatible with the maximum degree of expansion. The spurious signals could be eliminated by using a spline interpolation procedure to obtain the 30'values from the 1deg values.

Single-Pass, Closed-System Rapid Expansion of Lymphocyte Cultures for Adoptive Cell Therapy

PubMed Central

Klapper, Jacob A.; Thomasian, Armen A.; Smith, Douglas M.; Gorgas, Gayle C.; Wunderlich, John R.; Smith, Franz O.; Hampson, Brian S.; Rosenberg, Steven A.; Dudley, Mark E.

2009-01-01

Adoptive cell therapy (ACT) for metastatic melanoma involves the ex vivo expansion and re-infusion of tumor infiltrating lymphocytes (TIL) obtained from resected specimens. With an overall objective response rate of fifty-six percent, this T-cell immunotherapy provides an appealing alternative to other therapies, including conventional therapies with lower response rates. However, there are significant regulatory and logistical concerns associated with the ex vivo activation and large scale expansion of these cells. The best current practice uses a rapid expansion protocol (REP) consisting of an ex vivo process that occurs in tissue culture flasks (T-flasks) and gas-permeable bags, utilizes OKT3 (anti-CD3 monoclonal antibody), recombinant human interleukin-2, and irradiated peripheral blood mononuclear cells to initiate rapid lymphocyte growth. A major limitation to the widespread delivery of therapy to large numbers of melanoma patients is the open system in which a REP is initiated. To address this problem, we have investigated the initiation, expansion and harvest at clinical scale of TIL in a closed-system continuous perfusion bioreactor. Each cell product met all safety criteria for patient treatment and by head-to-head comparison had a similar potency and phenotype as cells grown in control T-flasks and gas-permeable bags. However, the currently available bioreactor cassettes were limited in the total cell numbers that could be generated. This bioreactor may simplify the process of the rapid expansion of TIL under stringent regulatory conditions thereby enabling other institutions to pursue this form of ACT. PMID:19389403

COBRA, an Arabidopsis Extracellular Glycosyl-Phosphatidyl Inositol-Anchored Protein, Specifically Controls Highly Anisotropic Expansion through Its Involvement in Cellulose Microfibril OrientationW⃞

PubMed Central

Roudier, François; Fernandez, Anita G.; Fujita, Miki; Himmelspach, Regina; Borner, Georg H.H.; Schindelman, Gary; Song, Shuang; Baskin, Tobias I.; Dupree, Paul; Wasteneys, Geoffrey O.; Benfey, Philip N.

2005-01-01

The orientation of cell expansion is a process at the heart of plant morphogenesis. Cellulose microfibrils are the primary anisotropic material in the cell wall and thus are likely to be the main determinant of the orientation of cell expansion. COBRA (COB) has been identified previously as a potential regulator of cellulose biogenesis. In this study, characterization of a null allele, cob-4, establishes the key role of COB in controlling anisotropic expansion in most developing organs. Quantitative polarized-light and field-emission scanning electron microscopy reveal that loss of anisotropic expansion in cob mutants is accompanied by disorganization of the orientation of cellulose microfibrils and subsequent reduction of crystalline cellulose. Analyses of the conditional cob-1 allele suggested that COB is primarily implicated in microfibril deposition during rapid elongation. Immunodetection analysis in elongating root cells revealed that, in agreement with its substitution by a glycosylphosphatidylinositol anchor, COB was polarly targeted to both the plasma membrane and the longitudinal cell walls and was distributed in a banding pattern perpendicular to the longitudinal axis via a microtubule-dependent mechanism. Our observations suggest that COB, through its involvement in cellulose microfibril orientation, is an essential factor in highly anisotropic expansion during plant morphogenesis. PMID:15849274

COBRA, an Arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation.

PubMed

Roudier, François; Fernandez, Anita G; Fujita, Miki; Himmelspach, Regina; Borner, Georg H H; Schindelman, Gary; Song, Shuang; Baskin, Tobias I; Dupree, Paul; Wasteneys, Geoffrey O; Benfey, Philip N

2005-06-01

The orientation of cell expansion is a process at the heart of plant morphogenesis. Cellulose microfibrils are the primary anisotropic material in the cell wall and thus are likely to be the main determinant of the orientation of cell expansion. COBRA (COB) has been identified previously as a potential regulator of cellulose biogenesis. In this study, characterization of a null allele, cob-4, establishes the key role of COB in controlling anisotropic expansion in most developing organs. Quantitative polarized-light and field-emission scanning electron microscopy reveal that loss of anisotropic expansion in cob mutants is accompanied by disorganization of the orientation of cellulose microfibrils and subsequent reduction of crystalline cellulose. Analyses of the conditional cob-1 allele suggested that COB is primarily implicated in microfibril deposition during rapid elongation. Immunodetection analysis in elongating root cells revealed that, in agreement with its substitution by a glycosylphosphatidylinositol anchor, COB was polarly targeted to both the plasma membrane and the longitudinal cell walls and was distributed in a banding pattern perpendicular to the longitudinal axis via a microtubule-dependent mechanism. Our observations suggest that COB, through its involvement in cellulose microfibril orientation, is an essential factor in highly anisotropic expansion during plant morphogenesis.

Architectured Materials to Improve the Reliability of Power Electronics Modules: Substrate and Lead-Free Solder

NASA Astrophysics Data System (ADS)

Kaabi, Abderrahmen; Bienvenu, Yves; Ryckelynck, David; Pierre, Bertrand

2014-03-01

Power electronics modules (>100 A, >500 V) are essential components for the development of electrical and hybrid vehicles. These modules are formed from silicon chips (transistors and diodes) assembled on copper substrates by soldering. Owing to the fact that the assembly is heterogeneous, and because of thermal gradients, shear stresses are generated in the solders and cause premature damage to such electronics modules. This work focuses on architectured materials for the substrate and on lead-free solders to reduce the mechanical effects of differential expansion, improve the reliability of the assembly, and achieve a suitable operating temperature (<175°C). These materials are composites whose thermomechanical properties have been optimized by numerical simulation and validated experimentally. The substrates have good thermal conductivity (>280 W m-1 K-1) and a macroscopic coefficient of thermal expansion intermediate between those of Cu and Si, as well as limited structural evolution in service conditions. An approach combining design, optimization, and manufacturing of new materials has been followed in this study, leading to improved thermal cycling behavior of the component.

Crystalline phases by an improved gradient expansion technique

NASA Astrophysics Data System (ADS)

Carignano, S.; Mannarelli, M.; Anzuini, F.; Benhar, O.

2018-02-01

We develop an innovative technique for studying inhomogeneous phases with a spontaneous broken symmetry. The method relies on the knowledge of the exact form of the free energy in the homogeneous phase and on a specific gradient expansion of the order parameter. We apply this method to quark matter at vanishing temperature and large chemical potential, which is expected to be relevant for astrophysical considerations. The method is remarkably reliable and fast as compared to performing the full numerical diagonalization of the quark Hamiltonian in momentum space and is designed to improve the standard Ginzburg-Landau expansion close to the phase transition points. For definiteness, we focus on inhomogeneous chiral symmetry breaking, accurately reproducing known results for one-dimensional and two-dimensional modulations and examining novel crystalline structures, as well. Consistently with previous results, we find that the energetically favored modulation is the so-called one-dimensional real-kink crystal. We propose a qualitative description of the pairing mechanism to motivate this result.

Melanoma induced immunosuppression is mediated by hematopoietic dysregulation.

PubMed

Kamran, Neha; Li, Youping; Sierra, Maria; Alghamri, Mahmoud S; Kadiyala, Padma; Appelman, Henry D; Edwards, Marta; Lowenstein, Pedro R; Castro, Maria G

2018-01-01

Tumors are associated with expansion of immunosuppressive cells such as tumor associated macrophages (TAMs), regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs). These cells promote tumor growth, angiogenesis, metastasis and immune escape. Cancer patients frequently present symptoms such as anemia, leukocytosis and/or cytopenia; associated with poor prognosis. To uncover tumor-mediated hematopoietic abnormalities and identify novel targets that can be harnessed to improve tumor-specific immune responses, we investigated the hematopoietic stem and progenitor cell compartment in melanoma bearing mice. We show that melanoma growth results in expansion of myeloid lineages such as MDSCs, macrophages and DCs along with a reduction in mature RBCs and platelets. Mature B lymphocytes in the blood and BM of melanoma mice were also reduced. Mice bearing melanoma showed extramedullary hematopoiesis in the spleen. Increased expansion of myeloid lineages occurred directly at the level of stem and progenitor cells. The reduction in mature B lymphocytes resulted from a block at the Pro-B cell stage in the bone marrow. Addition of recombinant IL-3 to bone marrow cells resulted in the expansion of committed myeloid progenitors including common myeloid precursors, granulocyte-monocyte precursors and megakaryocyte-erythrocyte precursors. In vivo , IL-3 receptor stimulation in melanoma bearing mice using an IL-3 antibody also resulted in a robust expansion of committed myeloid progenitors and hematopoietic stem cells. Collectively our findings demonstrate that tumor growth plays a pivotal role in reprogramming the host immune system by impacting hematopoiesis directly at the level of stem cell compartment.

Association of monoclonal expansion of Epstein-Barr virus-negative CD158a+ NK cells secreting large amounts of gamma interferon with hemophagocytic lymphohistiocytosis.

PubMed

López-Alvarez, María R; Martínez-Sánchez, María V; Salgado-Cecilia, María G; Campillo, José A; Heine-Suñer, Damian; Villar-Permuy, Florentina; Fuster, José L; Bas, Agueda; Gil-Herrera, Juana; Muro, Manuel; García-Alonso, Ana M; Alvarez-López, María R; Minguela, Alfredo

2009-01-01

We report the first case of hemophagocytic lymphohistiocytosis (HLH) induced by the monoclonal expansion of Epstein-Barr virus (EBV)-negative NK cells. Consanguinity of the patient's parents made it necessary to discard familial HLH in the patient and her sister with identical HLA markers and demonstrate that no cause other than the expansion of NK cells, which secrete high levels of gamma interferon, was inducing HLH in this patient.

Expansions of CD8+CD28- and CD8+TcRVbeta5.2+ T cells in peripheral blood of heavy alcohol drinkers.

PubMed

Arosa, F A; Porto, G; Cabeda, J M; Lacerda, R; Resende, D; Cruz, E; Cardoso, C; Fonseca, M; Simões, C; Rodrigues, P; Bravo, F; Oliveira, J C; Alves, H; Fraga, J; Justiça, B; de Sousa, M

2000-04-01

Despite heavy alcohol consumption, only a low percentage of heavy drinkers develop liver disease. Imbalances in T-cell subsets and iron metabolism parameters are common findings in heavy drinkers, yet the possible role played by discrete T-lymphocyte subsets under heavy alcohol consumption remains unclear. To gain new insights into the possible role played by T lymphocytes during alcohol consumption, characterization of CD28 expression and TcR repertoire in peripheral blood CD4+ and CD8+ T cells by two and three-color flow cytometry was performed. A group of heavy alcohol drinkers (AHD, n = 71) and a group of age-matched controls (n = 81), both HLA-phenotyped and HFE-genotyped, constituted the groups under study. Marked expansions of CD28- T cells within the CD8+ but not the CD4+ T-cell pool were observed in AHD compared with controls. These CD8+CD28- expansions were paralleled by expansions of CD8+ T cells bearing specific TcR Valpha/beta chains, namely VP5.2. Moreover, AHD, but not controls, carrying the H63D mutation in the HFE gene showed significantly higher percentages of CD28- T cells within the CD8+ T-cell pool than AHD carrying the normal HFE gene. Finally, high numbers of CD8+CD28- T cells in AHD were associated with lower levels of the liver-related enzymes ALT and GGT. This study showed that under active ethanol consumption, expansions of discrete CD8+ T-cell subsets occur within the CD8+ T-cell pool, that molecules of the MHC-class I locus seem to influence the extent of the expansions, and that high numbers of CD8+CD28- T cells are associated with low levels of liver enzymes in AHD.

Advances in cell culture: anchorage dependence

PubMed Central

Merten, Otto-Wilhelm

2015-01-01

Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000–6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems—microcarrier/microcarrier-clump cultures using stirred-tank reactors—for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes. PMID:25533097

Rapid expansion of T cells: Effects of culture and cryopreservation and importance of short-term cell recovery.

PubMed

Sadeghi, Arian; Ullenhag, Gustav; Wagenius, Gunnar; Tötterman, Thomas H; Eriksson, Fredrik

2013-06-01

Successful cell therapy relies on the identification and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP = 16 ± 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP = 356 ± 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF-β and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artificial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specificity, as determined by IFNγ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artificial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specificity.

Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor.

PubMed

Tsai, Ang-Chen; Ma, Teng

2016-01-01

Human mesenchymal stem cells (hMSCs) are considered as a primary candidate in cell therapy owing to their self-renewability, high differentiation capabilities, and secretions of trophic factors. In clinical application, a large quantity of therapeutically competent hMSCs is required that cannot be produced in conventional petri dish culture. Bioreactors are scalable and have the capacity to meet the production demand. Microcarrier suspension culture in stirred-tank bioreactors is the most widely used method to expand anchorage dependent cells in a large scale. Stirred-tank bioreactors have the potential to scale up and microcarriers provide the high surface-volume ratio. As a result, a spinner flask bioreactor with microcarriers has been commonly used in large scale expansion of adherent cells. This chapter describes a detailed culture protocol for hMSC expansion in a 125 mL spinner flask using microcarriers, Cytodex I, and a procedure for cell seeding, expansion, metabolic sampling, and quantification and visualization using microculture tetrazolium (MTT) reagent.

Species-specific chitin-binding module 18 expansion in the amphibian pathogen Batrachochytrium dendrobatidis.

PubMed

Abramyan, John; Stajich, Jason E

2012-01-01

Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity. Amphibian populations are declining worldwide at an unprecedented rate. Although various factors are thought to contribute to this phenomenon, chytridiomycosis has been identified as one of the leading causes. This deadly fungal disease is cause by Batrachochytrium dendrobatidis, a chytrid fungus species unique in its pathogenicity and, furthermore, its specificity to amphibians. Despite more than two decades of research, the biology of this fungus species and its deadly interaction with amphibians had been notoriously difficult to unravel. Due to the alarming rate of worldwide spread and associated decline in amphibian populations, it is imperative to incorporate novel genomic and genetic techniques into the study of this species. In this study, we present the first reported potential pathogenicity factors in B. dendrobatidis. In silico studies such as this allow us to identify putative targets for more specific molecular analyses, furthering our hope for the control of this pathogen.

Degree of bioresorbable vascular scaffold expansion modulates loss of essential function.

PubMed

Ferdous, Jahid; Kolachalama, Vijaya B; Kolandaivelu, Kumaran; Shazly, Tarek

2015-10-01

Drug-eluting bioresorbable vascular scaffolds (BVSs) have the potential to restore lumen patency, enable recovery of the native vascular environment, and circumvent late complications associated with permanent endovascular devices. To ensure therapeutic effects persist for sufficient times prior to scaffold resorption and resultant functional loss, many factors dictating BVS performance must be identified, characterized and optimized. While some factors relate to BVS design and manufacturing, others depend on device deployment and intrinsic vascular properties. Importantly, these factors interact and cannot be considered in isolation. The objective of this study is to quantify the extent to which degree of radial expansion modulates BVS performance, specifically in the context of modifying device erosion kinetics and evolution of structural mechanics and local drug elution. We systematically varied degree of radial expansion in model BVS constructs composed of poly dl-lactide-glycolide and generated in vitro metrics of device microstructure, degradation, erosion, mechanics and drug release. Experimental data permitted development of computational models that predicted transient concentrations of scaffold-derived soluble species and drug in the arterial wall, thus enabling speculation on the short- and long-term effects of differential expansion. We demonstrate that degree of expansion significantly affects scaffold properties critical to functionality, underscoring its relevance in BVS design and optimization. Bioresorbable vascular scaffold (BVS) therapy is beginning to transform the treatment of obstructive artery disease, owing to effective treatment of short term vessel closure while avoiding long term consequences such as in situ, late stent thrombosis - a fatal event associated with permanent implants such as drug-eluting stents. As device scaffolding and drug elution are temporary for BVS, the notion of using this therapy in lieu of existing, clinically approved devices seems attractive. However, there is still a limited understanding regarding the optimal lifetime and performance characteristics of erodible endovascular implants. Several engineering criteria must be met and clinical endpoints confirmed to ensure these devices are both safe and effective. In this manuscript, we sought to establish general principles for the design and deployment of erodible, drug-eluting endovascular scaffolds, with focus on how differential expansion can modulate device performance. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Athermal design for the potassium titanyl phosphate electro-optical modulator

NASA Astrophysics Data System (ADS)

Zheng, Guoliang; Xu, Jie; Chen, Lixiang; Wang, Hongcheng; She, Weilong

2007-09-01

An athermal design for the KTP electro-optical modulator is presented. By using the wave coupling theory of linear electro-optic effect and taking account of thermal expansion, the more accurate athermal static phase retardation (ASPR) directions in potassium titanyl phosphate (KTP) are found, and the optimized design for a transverse amplitude modulator at ASPR orientation is obtained. The numerical results show that the modulator with an athermal Soleil-Babinet compensator is of excellent thermal stability, and the acceptable error of the ASPR direction is less than 0.1°.

In vitro expansion of the mammary stem/progenitor cell population by xanthosinetreatment

USDA-ARS?s Scientific Manuscript database

Background: Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine (Xs) treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo ...

Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification.

PubMed

Eguiara, Arrate; Holgado, Olaia; Beloqui, Izaskun; Abalde, Leire; Sanchez, Yolanda; Callol, Carles; Martin, Angel G

2011-11-01

The cancer stem cell is defined by its capacity to self-renew, the potential to differentiate into all cells of the tumor and the ability to proliferate and drive the expansion of the tumor. Thus, targeting these cells may provide novel anti-cancer treatment strategies. Breast cancer stem cells have been isolated according to surface marker expression, ability to efflux fluorescent dyes, increased activity of aldehyde dehydrogenase or the capacity to form spheres in non-adherent culture conditions. In order to test novel drugs directed towards modulating self-renewal of cancer stem cells, rapid, easy and inexpensive assays must be developed. Using 2 days-post-fertilization (dpf) zebrafish embryos as transplant recipients, we show that cells grown in mammospheres from breast carcinoma cell lines migrate to the tail of the embryo and form masses with a significantly higher frequency than parental monolayer populations. When stem-like self-renewal was targeted in the parental population by the use of the dietary supplement curcumin, cell migration and mass formation were reduced, indicating that these effects were associated with stem-like cell content. This is a proof of principle report that proposes a rapid and inexpensive assay to target in vivo cancer stem-like cells, which may be used to unravel basic cancer stem cell biology and for drug screening.

Tunable Volumetric Density and Porous Structure of Spherical Poly-ε-caprolactone Microcarriers, as Applied in Human Mesenchymal Stem Cell Expansion.

PubMed

Li, Jian; Lam, Alan Tin-Lun; Toh, Jessica Pei Wen; Reuveny, Shaul; Oh, Steve Kah-Weng; Birch, William R

2017-03-28

Polymeric microspheres may serve as microcarrier (MC) matrices, for the expansion of anchorage-dependent stem cells. They require surface properties that promote both initial cell adhesion and the subsequent spreading of cells, which is a prerequisite for successful expansion. When implemented in a three-dimensional culture environment, under agitation, their suspension under low shear rates depends on the MCs having a modest negative buoyancy, with a density of 1.02-1.05 g/cm 3 . Bioresorbable poly-ε-caprolactone (PCL), with a density of 1.14 g/cm 3 , requires a reduction in volumetric density, for the microspheres to achieve high cell viability and yields. Uniform-sized droplets, from solutions of PCL dissolved in dichloromethane (DCM), were generated by coaxial microfluidic geometry. Subsequent exposure to ethanol rapidly extracted the DCM solvent, solidifying the droplets and yielding monodisperse microspheres with a porous structure, which was demonstrated to have tunable porosity and a hollow inner core. The variation in process parameters, including the molecular weight of PCL, its concentration in DCM, and the ethanol concentration, served to effectively alter the diffusion flux between ethanol and DCM, resulting in a broad spectrum of volumetric densities of 1.04-1.11 g/cm 3 . The solidified microspheres are generally covered by a smooth thin skin, which provides a uniform cell culture surface and masks their internal porous structure. When coated with a cationic polyelectrolyte and extracellular matrix protein, monodisperse microspheres with a diameter of approximately 150 μm and densities ranging from 1.05-1.11 g/cm 3 are capable of supporting the expansion of human mesenchymal stem cells (hMSCs). Validation of hMSC expansion was carried out with a positive control of commercial Cytodex 3 MCs and a negative control of uncoated low-density PCL MCs. Static culture conditions generated more than 70% cell attachment and similar yields of sixfold cell expansion on all coated MCs, with poor cell attachment and growth on the negative control. Under agitation, coated porous microspheres, with a low density of 1.05 g/cm 3 , achieved robust cell attachment and resulted in high cell yields of ninefold cell expansion, comparable with those generated by commercial Cytodex 3 MCs.

Late Graft Rejection in Association With T-Large Granular Lymphocyte Expansion of Recipient Origin After Human Leukocyte Antigen-Haploidentical Stem Cell Transplantation: A Case Report.

PubMed

Nakagawa, N; Yamazaki, H; Aoki, G; Kondo, Y; Nakao, S

2016-11-01

Large granular lymphocyte (LGL) expansion occasionally occurs after allogeneic stem cell transplantation (allo-SCT), and is thought to be a good prognostic sign that is associated with a lower relapse rate. However, there have been no reports of late graft failure (LGF) due to graft rejection in association with oligoclonal LGL expansion. We herein report a case of LGF associated with the transient expansion of recipient-derived T-LGL after allo-SCT. A 65-year-old man underwent peripheral blood stem cell transplantation (PBSCT) from his human leukocyte antigen (HLA)-haploidentical son for the treatment of acute myeloid leukemia, which had evolved from a myelodysplastic syndrome (MDS). Neutrophil engraftment occurred on day 20. A chimerism analysis on day 29 showed both granulocytes and mononuclear cells in the peripheral blood to be completely of donor origin. However, his neutrophil count gradually decreased and a chimerism analysis on day 61 showed that 84% of the patient's T cells were of recipient origin while the granulocytes were 100% donor-derived. His LGLs rapidly increased to 4.01 × 10 9 /L on day 113 and decreased thereafter. The percentage of donor cells in his granulocytes gradually decreased, and the patient's leukocytes were completely replaced by recipient cells on day 177. The clinical course suggests that the expansion of recipient-derived T-LGLs after allo-SCT can be a sign of graft rejection. Early intervention may be needed if the LGL expansion is recipient-derived. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

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

Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan

2010-11-26

Research highlights: {yields} Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. {yields} Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. {yields} LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In thismore » study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133{sup +} cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and decreased cellular sensitivity to anticancer treatments. These findings may provide pivotal insights in the context of fractionated radiation-based therapeutic interventions in brain cancer.« less

Cell growth and water relations of the halophyte, Atriplex nummularia L., in response to NaCl.

PubMed

Casas, A M; Bressan, R A; Hasegawa, P M

1991-06-01

Growth reduction or cessation is an initial response of Atriplex nummularia L. cells to NaCl. However, A. nummularia L. cells that are adapted to 342 and 428 mM NaCl are capable of sustained growth in the presence of salt. Cells that are adapted to NaCl exhibit a reduced rate of division compared to unadapted cells. Unlike salt adapted cells of the glycophyte Nicotiana tabacum L., A. nummularia L. cells do not exhibit reduced rate of cell expansion after adaptation. However, the cell expansion rate of unadapted A. nummularia L. cells is considerably slower than that of unadapted glycophyte cells and this normally low rate of cell expansion may contribute to the enhanced capacity of the halophyte to tolerate salt. Turgor of NaCl adapted cells was equivalent to unadapted cells indicating that the cells of the halophyte do not respond to salt by osmotic "over adjustment" as reported for the glycophyte tobacco (Binzel et al. 1985, Plant Physiol. 79:118-125).

Long-term Blue Light Effects on the Histology of Lettuce and Soybean Leaves and Stems

NASA Technical Reports Server (NTRS)

Dougher, Tracy A. O.; Bugbee, Bruce

2004-01-01

Blue light (320 to 496 nm) alters hypocotyl and stem elongation and leaf expansion in short-term, cell-level experiments, but histological effects of blue light in long-term studies of whole plants have not been described. We measured cell size and number in stems of soybean (Glycine max L.) and leaves of soybean and lettuce (Lactuca sativa L.), at two blue light fractions. Short-term studies have shown that cell expansion in stems is rapidly inhibited when etiolated tissue is exposed to blue light. However, under long-term light exposure, an increase in the blue light fraction from less than 0.1% to 26% decreased internode length, specifically by inhibiting soybean cell division in stems. In contrast, an increase in blue light fraction from 6% to 26% reduced soybean leaf area by decreasing cell expansion. Surprisingly, lettuce leaf area increased with increasing blue light fraction (0% to 6%), which was attributed to a 3.1-fold increase in cell expansion and a 1.6-fold increase in cell division.

A BTLA-mediated bait-and-switch strategy permits Listeria expansion in CD8α+ DCs to promote long term T cell responses

PubMed Central

Yang, Xuanming; Zhang, Xunmin; Sun, Yonglian; Tu, Tony; Fu, May Lynne; Miller, Mendy; Fu, Yang-Xin

2014-01-01

SUMMARY Listeria monocytogenes infected CD8α+ DCs in the spleen are essential for CD8+ T cell generation. CD8α+ DCs are also necessary for Listeria expansion and dissemination within the host. The mechanisms that regulate CD8α+ DCs to allow Listeria expansion are unclear. We find that activating the B and T lymphocyte attenuator (BTLA), a co-inhibitory receptor on CD8α+ DCs, suppresses, while blocking BTLA enhances both the primary and memory CD8 T cell responses against Listeria. Btla−/− mice have lower effector and memory CD8+ T cells while paradoxically also being more resistant to Listeria. Although bacterial entry into Btla−/− CD8α+ DCs is unaffected, Listeria fails to expand within these cells. BTLA signaling limits Fas/FasL-mediated suppression of Listeria expansion within CD8α+ DCs to more effectively alert adaptive immune cells. This study uncovers a BTLA-mediated strategy used by the host that permits Listeria proliferation to enable increasing T cell responses for long-term protection. PMID:25011109

Regulatory T cells as suppressors of anti-tumor immunity: Role of metabolism.

PubMed

De Rosa, Veronica; Di Rella, Francesca; Di Giacomo, Antonio; Matarese, Giuseppe

2017-06-01

Novel concepts in immunometabolism support the hypothesis that glucose consumption is also used to modulate anti-tumor immune responses, favoring growth and expansion of specific cellular subsets defined in the past as suppressor T cells and currently reborn as regulatory T (Treg) cells. During the 1920s, Otto Warburg and colleagues observed that tumors consumed high amounts of glucose compared to normal tissues, even in the presence of oxygen and completely functioning mitochondria. However, the role of the Warburg Effect is still not completely understood, particularly in the context of an ongoing anti-tumor immune response. Current experimental evidence suggests that tumor-derived metabolic restrictions can drive T cell hyporesponsiveness and immune tolerance. For example, several glycolytic enzymes, deregulated in cancer, contribute to tumor progression independently from their canonical metabolic activity. Indeed, they can control apoptosis, gene expression and activation of specific intracellular pathways, thus suggesting a direct link between metabolic switches and pro-tumorigenic transcriptional programs. Focus of this review is to define the specific metabolic pathways controlling Treg cell immunobiology in the context of anti-tumor immunity and tumor progression. Copyright © 2017 Elsevier Ltd. All rights reserved.

OGR1/GPR68 Modulates the Severity of Experimental Autoimmune Encephalomyelitis and Regulates Nitric Oxide Production by Macrophages

PubMed Central

D’Souza, Cheryl A.; Zhao, Fei Linda; Li, Xujian; Xu, Yan; Dunn, Shannon E.; Zhang, Li

2016-01-01

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a proton-sensing molecule that can detect decreases in extracellular pH that occur during inflammation. Although OGR1 has been shown to have pro-inflammatory functions in various diseases, its role in autoimmunity has not been examined. We therefore sought to determine whether OGR1 has a role in the development of T cell autoimmunity by contrasting the development of experimental autoimmune encephalomyelitis between wild type and OGR1-knockout mice. OGR1-knockout mice showed a drastically attenuated clinical course of disease that was associated with a profound reduction in the expansion of myelin oligodendrocyte glycoprotein 35-55-reactive T helper 1 (Th1) and Th17 cells in the periphery and a reduced accumulation of Th1 and Th17 effectors in the central nervous system. We determined that these impaired T cell responses in OGR1-knockout mice associated with a reduced frequency and number of dendritic cells in draining lymph nodes during EAE and a higher production of nitric oxide by macrophages. Our studies suggest that OGR1 plays a key role in regulating T cell responses during autoimmunity. PMID:26828924

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.

Adipose-Derived Stromal Vascular Fraction Differentially Expands Breast Progenitors in Tissue Adjacent to Tumors Compared to Healthy Breast Tissue

PubMed Central

Chatterjee, Sumanta; Laliberte, Mike; Blelloch, Sarah; Ratanshi, Imran; Safneck, Janice; Buchel, Ed

2015-01-01

Background: Autologous fat grafts supplemented with adipose-derived stromal vascular fraction are used in reconstructive and cosmetic breast procedures. Stromal vascular fraction contains adipose-derived stem cells that are thought to encourage wound healing, tissue regeneration, and graft retention. Although use of stromal vascular fraction has provided exciting perspectives for aesthetic procedures, no studies have yet been conducted to determine whether its cells contribute to breast tissue regeneration. The authors examined the effect of these cells on the expansion of human breast epithelial progenitors. Methods: From patients undergoing reconstructive breast surgery following mastectomies, abdominal fat, matching tissue adjacent to breast tumors, and the contralateral non–tumor-containing breast tissue were obtained. Ex vivo co-cultures using breast epithelial cells and the stromal vascular fraction cells were used to study the expansion potential of breast progenitors. Breast reduction samples were collected as a source of healthy breast cells. Results: The authors observed that progenitors present in healthy breast tissue or contralateral non–tumor-containing breast tissue showed significant and robust expansion in the presence of stromal vascular fraction (5.2- and 4.8-fold, respectively). Whereas the healthy progenitors expanded up to 3-fold without the stromal vascular fraction cells, the expansion of tissue adjacent to breast tumor progenitors required the presence of stromal vascular fraction cells, leading to a 7-fold expansion, which was significantly higher than the expansion of healthy progenitors with stromal vascular fraction. Conclusions: The use of stromal vascular fraction might be more beneficial to reconstructive operations following mastectomies compared with cosmetic corrections of the healthy breast. Future studies are required to examine the potential risk factors associated with its use. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V. PMID:26090768

Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

PubMed

Wuchter, Patrick; Vetter, Marcel; Saffrich, Rainer; Diehlmann, Anke; Bieback, Karen; Ho, Anthony D; Horn, Patrick

2016-06-01

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Slow Cholinergic Modulation of Spike Probability in Ultra-Fast Time-Coding Sensory Neurons

PubMed Central

Goyer, David; Kurth, Stefanie; Rübsamen, Rudolf

2016-01-01

Abstract Sensory processing in the lower auditory pathway is generally considered to be rigid and thus less subject to modulation than central processing. However, in addition to the powerful bottom-up excitation by auditory nerve fibers, the ventral cochlear nucleus also receives efferent cholinergic innervation from both auditory and nonauditory top–down sources. We thus tested the influence of cholinergic modulation on highly precise time-coding neurons in the cochlear nucleus of the Mongolian gerbil. By combining electrophysiological recordings with pharmacological application in vitro and in vivo, we found 55–72% of spherical bushy cells (SBCs) to be depolarized by carbachol on two time scales, ranging from hundreds of milliseconds to minutes. These effects were mediated by nicotinic and muscarinic acetylcholine receptors, respectively. Pharmacological block of muscarinic receptors hyperpolarized the resting membrane potential, suggesting a novel mechanism of setting the resting membrane potential for SBC. The cholinergic depolarization led to an increase of spike probability in SBCs without compromising the temporal precision of the SBC output in vitro. In vivo, iontophoretic application of carbachol resulted in an increase in spontaneous SBC activity. The inclusion of cholinergic modulation in an SBC model predicted an expansion of the dynamic range of sound responses and increased temporal acuity. Our results thus suggest of a top–down modulatory system mediated by acetylcholine which influences temporally precise information processing in the lower auditory pathway. PMID:27699207

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.

Ultraviolet B irradiation induces expansion of intraepithelial tumor cells in a tissue model of early cancer progression.

PubMed

Mudgil, Adarsh V; Segal, Nadav; Andriani, Frank; Wang, Youai; Fusenig, Norbert E; Garlick, Jonathan A

2003-07-01

Ultraviolet B irradiation is thought to enable skin cancer progression as clones of genetically damaged keratinocytes escape apoptosis and expand at the expense of adjacent normal cells. Mechanisms through which potentially malignant cells in human skin undergo clonal expansion, however, are not well understood. The goal of this study was to characterize the role of ultraviolet B irradiation on the intraepithelial expansion of early stage human tumor cells in organotypic skin cultures. To accomplish this, we have studied the effect of ultraviolet B irradiation on organotypic cultures that were fabricated by mixing normal human keratinocytes with beta-galactosidase-marked, intraepithelial tumor cells (HaCaT-ras, clone II-4), which bear mutations in both p53 alleles and harbor an activated H-ras oncogene. We found that when organotypic mixtures were exposed to an ultraviolet B dose of 50 mJ per cm2, intraepithelial tumor cells underwent a significant degree of proliferative expansion compared to nonirradiated cultures. To understand this response, organotypic cultures of nor-mal keratinocytes were exposed to ultraviolet B and showed a dose-dependent increase in numbers of sunburn cells and TUNEL-positive cells although their proliferation was suppressed. In contrast, neither the apoptotic nor the proliferative response of II-4 cells was altered by ultraviolet B in organotypic cultures. The differential response of these cell types suggested that II-4 cells were resistant to ultraviolet-B-induced alterations, which allowed these intraepithelial tumor cells to gain a selective growth and survival advantage relative to neighboring normal cells. These findings demonstrate that ultraviolet B exposure can induce the intraepithelial expansion of apoptosis-resistant, p53-mutant, and ras-activated keratinocytes, suggesting that this agent can act to promote the early stages of epithelial carcinogenesis.

Promoting effects of serotonin on hematopoiesis: ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis.

PubMed

Yang, Mo; Li, Karen; Ng, Pak Cheung; Chuen, Carmen Ka Yee; Lau, Tze Kin; Cheng, Yuan Shan; Liu, Yuan Sheng; Li, Chi Kong; Yuen, Patrick Man Pan; James, Anthony Edward; Lee, Shuk Man; Fok, Tai Fai

2007-07-01

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5-hydroxytryptamine (5-HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony-forming unit-fibroblast (CFU-F) formation, and antiapoptosis of megakaryoblastic M-07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony-forming unit-mixed [CFU-GEMM]) and multilineage committed progenitors (burst-forming unit/colony-forming unit-erythroid [BFU/CFU-E], colony-forming unit-granulocyte macrophage, colony-forming unit-megakaryocyte, CD61+ CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU-E, and CFU-GEMM engraftment in BM of animals 6 weeks post-transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor stimulated BM CFU-F formation. In M-07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase-3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5-HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation. Disclosure of potential conflicts of interest is found at the end of this article.

Oven wall panel construction

DOEpatents

Ellison, Kenneth; Whike, Alan S.

1980-04-22

An oven roof or wall is formed from modular panels, each of which comprises an inner fabric and an outer fabric. Each such fabric is formed with an angle iron framework and somewhat resilient tie-bars or welded at their ends to flanges of the angle irons to maintain the inner and outer frameworks in spaced disposition while minimizing heat transfer by conduction and permitting some degree of relative movement on expansion and contraction of the module components. Suitable thermal insulation is provided within the module. Panels or skins are secured to the fabric frameworks and each such skin is secured to a framework and projects laterally so as slidingly to overlie the adjacent frame member of an adjacent panel in turn to permit relative movement during expansion and contraction.

The SMS4 cryptographic system design based on dynamic partial self-reconfiguration technology

NASA Astrophysics Data System (ADS)

Wang, Jianxin; Gao, Xianwei; Li, Xiuying; Sui, Meili

2013-03-01

This paper describes SMS4 algorithm by using dynamic partial self-reconfiguration. The design is implemented on Xilinx VirtexII-Pro XC2VP30 FPGA devices. The partial self-reconfiguration encryption/decryption module data throughput is up to 50Mb/s, key expansion and encryption/decryption modules use 1606 and 1570 slices respectively, and the resource utilization ratio of the key expansion by using partial self-reconfiguration technology is less 32.03% and slices are less 757 than the non-reconfiguration technology. SMS4 implementation gets a good balance between high performance and low complexity in area. The theoretical and practical research of dynamic partial self-reconfiguration has a broad space for development and application prospect.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment.

PubMed

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition "modules" to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo . Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion.

PubMed

Baxter, Melissa A; Wynn, Robert F; Jowitt, Simon N; Wraith, J Ed; Fairbairn, Leslie J; Bellantuono, Ilaria

2004-01-01

Human marrow stromal cells (MSCs) can be isolated from bone marrow and differentiate into multiple tissues in vitro and in vivo. These properties make them promising tools in cell and gene therapy. The lack of a specific MSC marker and the low frequency of MSCs in bone marrow necessitate their isolation by in vitro expansion prior to clinical use. This may severely reduce MSC proliferative capacity to the point that the residual proliferative potential is insufficient to maintain long-term tissue regeneration upon reinfusion. In this study we determined the effect of in vitro expansion on the replicative capacity of MSCs by correlating their rate of telomere loss during in vitro expansion with their behavior in vivo. We report that even protocols that involve minimal expansion induce a rapid aging of MSCs, with losses equivalent to about half their total replicative lifespan.

Intestinal helminths regulate lethal acute graft-versus-host disease and preserve the graft-versus-tumor effect in mice.

PubMed

Li, Yue; Chen, Hung-Lin; Bannick, Nadine; Henry, Michael; Holm, Adrian N; Metwali, Ahmed; Urban, Joseph F; Rothman, Paul B; Weiner, George J; Blazar, Bruce R; Elliott, David E; Ince, M Nedim

2015-02-01

Donor T lymphocyte transfer with hematopoietic stem cells suppresses residual tumor growth (graft-versus-tumor [GVT]) in cancer patients undergoing bone marrow transplantation (BMT). However, donor T cell reactivity to host organs causes severe and potentially lethal inflammation called graft-versus-host disease (GVHD). High-dose steroids or other immunosuppressive drugs are used to treat GVHD that have limited ability to control the inflammation while incurring long-term toxicity. Novel strategies are needed to modulate GVHD, preserve GVT, and improve the outcome of BMT. Regulatory T cells (Tregs) control alloantigen-sensitized inflammation of GVHD, sustain GVT, and prevent mortality in BMT. Helminths colonizing the alimentary tract dramatically increase the Treg activity, thereby modulating intestinal or systemic inflammatory responses. These observations led us to hypothesize that helminths can regulate GVHD and maintain GVT in mice. Acute GVHD was induced in helminth (Heligmosomoides polygyrus)-infected or uninfected BALB/c recipients of C57BL/6 donor grafts. Helminth infection suppressed donor T cell inflammatory cytokine generation and reduced GVHD-related mortality, but maintained GVT. H. polygyrus colonization promoted the survival of TGF-β-generating recipient Tregs after a conditioning regimen with total body irradiation and led to a TGF-β-dependent in vivo expansion/maturation of donor Tregs after BMT. Helminths did not control GVHD when T cells unresponsive to TGF-β-mediated immune regulation were used as donor T lymphocytes. These results suggest that helminths suppress acute GVHD using Tregs and TGF-β-dependent pathways in mice. Helminthic regulation of GVHD and GVT through intestinal immune conditioning may improve the outcome of BMT. Copyright © 2015 by The American Association of Immunologists, Inc.

Dendritic cells limit fibroinflammatory injury in nonalcoholic steatohepatitis in mice.

PubMed

Henning, Justin R; Graffeo, Christopher S; Rehman, Adeel; Fallon, Nina C; Zambirinis, Constantinos P; Ochi, Atsuo; Barilla, Rocky; Jamal, Mohsin; Deutsch, Michael; Greco, Stephanie; Ego-Osuala, Melvin; Bin-Saeed, Usama; Rao, Raghavendra S; Badar, Sana; Quesada, Juan P; Acehan, Devrim; Miller, George

2013-08-01

Nonalcoholic steatohepatitis (NASH) is the most common etiology of chronic liver dysfunction in the United States and can progress to cirrhosis and liver failure. Inflammatory insult resulting from fatty infiltration of the liver is central to disease pathogenesis. Dendritic cells (DCs) are antigen-presenting cells with an emerging role in hepatic inflammation. We postulated that DCs are important in the progression of NASH. We found that intrahepatic DCs expand and mature in NASH liver and assume an activated immune phenotype. However, rather than mitigating the severity of NASH, DC depletion markedly exacerbated intrahepatic fibroinflammation. Our mechanistic studies support a regulatory role for DCs in NASH by limiting sterile inflammation through their role in the clearance of apoptotic cells and necrotic debris. We found that DCs limit CD8(+) T-cell expansion and restrict Toll-like receptor expression and cytokine production in innate immune effector cells in NASH, including Kupffer cells, neutrophils, and inflammatory monocytes. Consistent with their regulatory role in NASH, during the recovery phase of disease, ablation of DC populations results in delayed resolution of intrahepatic inflammation and fibroplasia. Our findings support a role for DCs in modulating NASH. Targeting DC functional properties may hold promise for therapeutic intervention in NASH. Copyright © 2013 American Association for the Study of Liver Diseases.

Expansion and redifferentiation of chondrocytes from osteoarthritic cartilage: cells for human cartilage tissue engineering.

PubMed

Hsieh-Bonassera, Nancy D; Wu, Iwen; Lin, Jonathan K; Schumacher, Barbara L; Chen, Albert C; Masuda, Koichi; Bugbee, William D; Sah, Robert L

2009-11-01

To determine if selected culture conditions enhance the expansion and redifferentiation of chondrocytes isolated from human osteoarthritic cartilage with yields appropriate for creation of constructs for treatment of joint-scale cartilage defects, damage, or osteoarthritis. Chondrocytes isolated from osteoarthritic cartilage were analyzed to determine the effects of medium supplement on cell expansion in monolayer and then cell redifferentiation in alginate beads. Expansion was assessed as cell number estimated from DNA, growth rate, and day of maximal growth. Redifferentiation was evaluated quantitatively from proteoglycan and collagen type II content, and qualitatively by histology and immunohistochemistry. Using either serum or a growth factor cocktail (TFP: transforming growth factor beta1, fibroblast growth factor 2, and platelet-derived growth factor type bb), cell growth rate in monolayer was increased to 5.5x that of corresponding conditions without TFP, and cell number increased 100-fold within 17 days. In subsequent alginate bead culture with human serum or transforming growth factor beta1 and insulin-transferrin-selenium-linoleic acid-bovine serum albumin, redifferentiation was enhanced with increased proteoglycan and collagen type II production. Effects of human serum were dose dependent, and 5% or higher induced formation of chondron-like structures with abundant proteoglycan-rich matrix. Chondrocytes from osteoarthritic cartilage can be stimulated to undergo 100-fold expansion and then redifferentiation, suggesting that they may be useful as a cell source for joint-scale cartilage tissue engineering.

Clonal expansion under the microscope: studying lymphocyte activation and differentiation using live-cell imaging.

PubMed

Polonsky, Michal; Chain, Benjamin; Friedman, Nir

2016-03-01

Clonal expansion of lymphocytes is a hallmark of vertebrate adaptive immunity. A small number of precursor cells that recognize a specific antigen proliferate into expanded clones, differentiate and acquire various effector and memory phenotypes, which promote effective immune responses. Recent studies establish a large degree of heterogeneity in the level of expansion and in cell state between and within expanding clones. Studying these processes in vivo, while providing insightful information on the level of heterogeneity, is challenging due to the complex microenvironment and the inability to continuously track individual cells over extended periods of time. Live cell imaging of ex vivo cultures within micro fabricated arrays provides an attractive methodology for studying clonal expansion. These experiments facilitate continuous acquisition of a large number of parameters on cell number, proliferation, death and differentiation state, with single-cell resolution on thousands of expanding clones that grow within controlled environments. Such data can reveal stochastic and instructive mechanisms that contribute to observed heterogeneity and elucidate the sequential order of differentiation events. Intercellular interactions can also be studied within these arrays by following responses of a controlled number of interacting cells, all trapped within the same microwell. Here we describe implementations of live-cell imaging within microwell arrays for studies of lymphocyte clonal expansion, portray insights already gained from these experiments and outline directions for future research. These tools, together with in vivo experiments tracking single-cell responses, will expand our understanding of adaptive immunity and the ways by which it can be manipulated.

Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

NASA Astrophysics Data System (ADS)

Weber, C.; Pohl, S.; Poertner, R.; Pino-Grace, Pablo; Freimark, D.; Wallrapp, C.; Geigle, P.; Czermak, P.

Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

Human red blood cells have an enhancing effect on the relative expansion of CD8+ T lymphocytes in vitro.

PubMed

Porto, B; Fonseca, A M; Godinho, I; Arosa, F A; Porto, G

2001-12-01

The present study was designed to analyse the effect of red blood cells on T-cell proliferation and expansion. A comparative study was done in peripheral blood cell cultures stimulated with phytohemagglutinin, with or without red blood cells. The presence of red blood cells had a consistent enhancing effect on T lymphocyte proliferation, as determined by an increase in both the mitotic index and thymidine uptake. Phenotypic characterization of T cell blasts by flow cytometry revealed that, in the presence of red blood cells, expanding cells were preferentially CD8+ cells. Accordingly, proliferation of CD8+ lymphocytes from two patients with CD8+ hyperlymphocytosis was dependent on the presence of red blood cells. In contrast, proliferation of CD4+ lymphocytes from two patients with CD4+ hyperlymphocytosis was strongly inhibited by the presence of red blood cells. This is the first reported evidence that human red blood cells have an enhancing effect on the expansion of CD8+ lymphocytes in vitro.

Thrombopoietin to replace megakaryocyte-derived growth factor: impact on stem and progenitor cells during ex vivo expansion of CD34+ cells mobilized in peripheral blood.

PubMed

Duchez, Pascale; Chevaleyre, Jean; Vlaski, Marija; Dazey, Bernard; Bijou, Fontanet; Lafarge, Xavier; Milpied, Noël; Boiron, Jean-Michel; Ivanovic, Zoran

2011-02-01

The first protocol of ex vivo expansion that enabled almost total abrogation of postmyeloablative chemotherapy neutropenia was based on a three-cytokine cocktail (stem cell factor [SCF], granulocyte-colony-stimulating factor [G-CSF], pegylated-megakaryocyte growth and development factor [PEG-MGDF]) in a serum-free medium. Since the clinical-grade molecule MGDF is no longer available on the market, we evaluated its substitution by thrombopoietin (TPO). CD34+ cells of myeloma patients were expanded for 10 days in serum-free cultures with SCF, G-CSF, or MGDF (100 ng/mL) or with TPO (2.5, 10, 20, 50, and 100 ng/mL) instead of MGDF. Day 10 amplifications of total nucleated cells, CD34+ cells, committed progenitors (CFCs), the capacity of engraftment of NOD/SCID mice (SCID repopulating cells [SRCs]), and the immunophenotype of cells in expansion product (CD13, CD14, CD33, CD41, CD61) were analyzed. TPO in doses of 2.5 and 10 ng/mL exhibits an effect comparable to that of MGDF (100 ng/mL) on total, CD34+, and CFCs amplification. Compared to MGDF, TPO (starting at 10 ng/mL) enhances two- to threefold the percentage of megakaryocyte lineage cells (CD41+ and CD61+). Finally, TPO maintains or even enhances (depending on dose) SRC activity. The use of TPO instead of MGDF in our protocol is feasible without any negative effect on progenitor cell expansion. Furthermore, applied in dose of 10 or 100 ng/mL it could enhance both the stem cell activity and the percentage of megakaryocyte lineage cells in expansion product. © 2010 American Association of Blood Banks.

The Important Role of FLT3-L in Ex Vivo Expansion of Hematopoietic Stem Cells following Co-Culture with Mesenchymal Stem Cells.

PubMed

Oubari, Farhad; Amirizade, Naser; Mohammadpour, Hemn; Nakhlestani, Mozhdeh; Zarif, Mahin Nikougoftar

2015-01-01

Hematopoietic stem cells (HSCs) transplantation using umbilical cord blood (UCB) has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand (FLT3-L) as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell (MSC) feeder. In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count (TNC), cluster of differentiation 34+(CD34(+)) cell count, colony forming unit assay (CFU), long-term culture initiating cell (LTC-IC), homeobox protein B4 (HoxB4) mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines (P<0.05). FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased (P<0.05). FLT3-L co-culture with MSCs can induce high yield expansion of HSCs and be a substitute for the universal cocktail of SCF, TPO and FLT3-L in feeder-free culture.

Expansion of mesenchymal stem cells under atmospheric carbon dioxide.

PubMed

Brodsky, Arthur Nathan; Zhang, Jing; Visconti, Richard P; Harcum, Sarah W

2013-01-01

Stem cells are needed for an increasing number of scientific applications, including both fundamental research and clinical disease treatment. To meet this rising demand, improved expansion methods to generate high quantities of high quality stem cells must be developed. Unfortunately, the bicarbonate buffering system - which relies upon an elevated CO2 environment - typically used to maintain pH in stem cell cultures introduces several unnecessary limitations in bioreactor systems. In addition to artificially high dissolved CO2 levels negatively affecting cell growth, but more importantly, the need to sparge CO2 into the system complicates the ability to control culture parameters. This control is especially important for stem cells, whose behavior and phenotype is highly sensitive to changes in culture conditions such as dissolved oxygen and pH. As a first step, this study developed a buffer to support expansion of mesenchymal stem cells (MSC) under an atmospheric CO2 environment in static cultures. MSC expanded under atmospheric CO2 with this buffer achieved equivalent growth rates without adaptation compared to those grown in standard conditions and also maintained a stem cell phenotype, self-renewal properties, and the ability to differentiate into multiple lineages after expansion. © 2013 American Institute of Chemical Engineers.

Modeling future U.S. forest sector market and trade impacts of expansion in wood energy consumption

Treesearch

Peter J. Ince; Andrew D. Kramp; Kenneth E. Skog; Do-il Yoo; V. Alaric Sample

2011-01-01

This paper describes an approach to modeling U.S. forest sector market and trade impacts of expansion in domestic wood energy consumption under hypothetical future U.S. wood biomass energy policy scenarios. The U.S. Forest Products Module (USFPM) was created to enhance the modeling of the U.S. forest sector within the Global Forest Products Model (GFPM), providing a...

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

PubMed

Abecasis, Bernardo; Aguiar, Tiago; Arnault, Émilie; Costa, Rita; Gomes-Alves, Patricia; Aspegren, Anders; Serra, Margarida; Alves, Paula M

2017-03-20

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day -1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×10 6 cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors. Copyright © 2017 Elsevier B.V. All rights reserved.

Src Homology 2–containing 5-Inositol Phosphatase (SHIP) Suppresses an Early Stage of Lymphoid Cell Development through Elevated Interleukin-6 Production by Myeloid Cells in Bone Marrow

PubMed Central

Nakamura, Koji; Kouro, Taku; Kincade, Paul W.; Malykhin, Alexander; Maeda, Kazuhiko; Coggeshall, K. Mark

2004-01-01

The Src homology (SH)2–containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP−/− animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP−/− mice. Lineage-negative marrow (Lin−) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP−/− mice. Furthermore, culture supernatants of SHIP−/− Lin− cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP−/− Lin− cells contained more IL-6 transcripts than wild-type Lin− cells, and neutralizing anti–IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP−/− Lin− cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin− bone marrow cells reproduced the phenotype of SHIP−/− bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow. PMID:14718513

Expansion and Harvesting of hMSC-TERT

PubMed Central

Weber, Christian; Pohl, Sebastian; Pörtner, Ralf; Wallrapp, Christine; Kassem, Moustapha; Geigle, Peter; Czermak, Peter

2007-01-01

The expansion of human mesenchymal stem cells as suspension culture by means of spinner flasks and microcarriers, compared to the cultivation in tissue culture flasks, offers the advantage of reducing the requirements of large incubator capacities as well as reducing the handling effort during cultivation and harvesting. Nonporous microcarriers are preferable when the cells need to be kept in viable condition for further applications like tissue engineering or cell therapy. In this study, the qualification of Biosilon, Cytodex 1, Cytodex 3, RapidCell and P102-L for expansion of hMSC-TERT with an associated harvesting process using either trypsin, accutase, collagenase or a trypsin-accutase mixture was investigated. A subsequent adipogenic differentiation of harvested hMSC-TERT was performed in order to observe possible negative effects on their (adipogenic) differentiation potential as a result of the cultivation and harvesting method. The cultivated cells showed an average growth rate of 0.52 d-1. The cells cultivated on Biosilon, RapidCell and P102-L were harvested succesfully achieving high cell yield and vitalities near 100%. This was not the case for cells on Cytodex 1 and Cytodex 3. The trypsin-accutase mix was most effective. After spinner expansion and harvesting the cells were successfully differentiated to adipocytes. PMID:19662126

Islet transplantation in patients with autoimmune diabetes induces homeostatic cytokines that expand autoreactive memory T cells

PubMed Central

Monti, Paolo; Scirpoli, Miriam; Maffi, Paola; Ghidoli, Nadia; De Taddeo, Francesca; Bertuzzi, Federico; Piemonti, Lorenzo; Falcone, Marika; Secchi, Antonio; Bonifacio, Ezio

2008-01-01

Successful transplantation requires the prevention of allograft rejection and, in the case of transplantation to treat autoimmune disease, the suppression of autoimmune responses. The standard immunosuppressive treatment regimen given to patients with autoimmune type 1 diabetes who have received an islet transplant results in the loss of T cells. In many other situations, the immune system responds to T cell loss through cytokine-dependant homeostatic proliferation of any remaining T cells. Here we show that T cell loss after islet transplantation in patients with autoimmune type 1 diabetes was associated with both increased serum concentrations of IL-7 and IL-15 and in vivo proliferation of memory CD45RO+ T cells, highly enriched in autoreactive glutamic acid decarboxylase 65–specific T cell clones. Immunosuppression with FK506 and rapamycin after transplantation resulted in a chronic homeostatic expansion of T cells, which acquired effector function after immunosuppression was removed. In contrast, the cytostatic drug mycophenolate mofetil efficiently blocked homeostatic T cell expansion. We propose that the increased production of cytokines that induce homeostatic expansion could contribute to recurrent autoimmunity in transplanted patients with autoimmune disease and that therapy that prevents the expansion of autoreactive T cells will improve the outcome of islet transplantation. PMID:18431516

Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality

PubMed Central

Schneidawind, Dominik; Baker, Jeanette; Pierini, Antonio; Buechele, Corina; Luong, Richard H.; Meyer, Everett H.

2015-01-01

Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4+ iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4+ iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4+ iNKT cells from third-party mice were as protective as CD4+ iNKT cells from donor mice although third-party CD4+ iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4+ iNKT cells resulted in a robust expansion of donor CD4+CD25+FoxP3+ regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation. PMID:25795920

Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures.

PubMed

Roy, A; Krzykwa, E; Lemieux, R; Néron, S

2001-12-01

Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.

MERTK as negative regulator of human T cell activation

PubMed Central

Cabezón, Raquel; Carrera-Silva, E. Antonio; Flórez-Grau, Georgina; Errasti, Andrea E.; Calderón-Gómez, Elisabeth; Lozano, Juan José; España, Carolina; Ricart, Elena; Panés, Julián; Rothlin, Carla Vanina; Benítez-Ribas, Daniel

2015-01-01

The aim of this study was to test the hypothesis whether MERTK, which is up-regulated in human DCs treated with immunosuppressive agents, is directly involved in modulating T cell activation. MERTK is a member of the TAM family and contributes to regulating innate immune response to ACs by inhibiting DC activation in animal models. However, whether MERTK interacts directly with T cells has not been addressed. Here, we show that MERTK is highly expressed on dex-induced human tol-DCs and participates in their tolerogenic effect. Neutralization of MERTK in allogenic MLR, as well as autologous DC–T cell cultures, leads to increased T cell proliferation and IFN-γ production. Additionally, we identify a previously unrecognized noncell-autonomous regulatory function of MERTK expressed on DCs. Mer-Fc protein, used to mimic MERTK on DCs, suppresses naïve and antigen-specific memory T cell activation. This mechanism is mediated by the neutralization of the MERTK ligand PROS1. We find that MERTK and PROS1 are expressed in human T cells upon TCR activation and drive an autocrine proproliferative mechanism. Collectively, these results suggest that MERTK on DCs controls T cell activation and expansion through the competition for PROS1 interaction with MERTK in the T cells. In conclusion, this report identified MERTK as a potent suppressor of T cell response. PMID:25624460

Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma gondii.

PubMed

John, Beena; Harris, Tajie H; Tait, Elia D; Wilson, Emma H; Gregg, Beth; Ng, Lai Guan; Mrass, Paulus; Roos, David S; Dzierszinski, Florence; Weninger, Wolfgang; Hunter, Christopher A

2009-07-01

To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

PubMed Central

John, Beena; Harris, Tajie H.; Tait, Elia D.; Wilson, Emma H.; Gregg, Beth; Ng, Lai Guan; Mrass, Paulus; Roos, David S.; Dzierszinski, Florence; Weninger, Wolfgang; Hunter, Christopher A.

2009-01-01

To better understand the initiation of CD8+ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8+ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8+ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8+ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8+ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis. PMID:19578440

Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor

PubMed Central

2012-01-01

Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols. PMID:22475724

Clonal Expansion (CE) Models in Cancer Risk Assessment

EPA Science Inventory

Cancer arises when cells accumulate sufficient critical mutations. Carcinogens increase the probability of mutation during cell division or promote clonal expansion within stages. Multistage CE models recapitulate this process and provide a framework for incorporating relevant da...

Scaled-down particle-in-cell simulation of cathode plasma expansion in magnetically insulated coaxial diode

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

Zhu, Danni; Zhang, Jun, E-mail: zhangjun@nudt.edu.cn; Zhong, Huihuang

2016-03-15

The expansion of cathode plasma in magnetically insulated coaxial diode (MICD) is investigated in theory and particle-in-cell (PIC) simulation. The temperature and density of the cathode plasma are about several eV and 10{sup 13}–10{sup 16 }cm{sup −3}, respectively, and its expansion velocity is of the level of few cm/μs. Through hydrodynamic theory analysis, expressions of expansion velocities in axial and radial directions are obtained. The characteristics of cathode plasma expansion have been simulated through scaled-down PIC models. Simulation results indicate that the expansion velocity is dominated by the ratio of plasma density other than the static electric field. The electric fieldmore » counteracts the plasma expansion reverse of it. The axial guiding magnetic field only reduces the radial transport coefficients by a correction factor, but not the axial ones. Both the outward and inward radial expansions of a MICD are suppressed by the much stronger guiding magnetic field and even cease.« less

Vascular remodeling: A redox-modulated mechanism of vessel caliber regulation.

PubMed

Tanaka, Leonardo Y; Laurindo, Francisco R M

2017-08-01

Vascular remodeling, i.e. whole-vessel structural reshaping, determines lumen caliber in (patho)physiology. Here we review mechanisms underlying vessel remodeling, with emphasis in redox regulation. First, we discuss confusing terminology and focus on strictu sensu remodeling. Second, we propose a mechanobiological remodeling paradigm based on the concept of tensional homeostasis as a setpoint regulator. We first focus on shear-mediated models as prototypes of remodeling closely dominated by highly redox-sensitive endothelial function. More detailed discussions focus on mechanosensors, integrins, extracellular matrix, cytoskeleton and inflammatory pathways as potential of mechanisms potentially coupling tensional homeostasis to redox regulation. Further discussion of remodeling associated with atherosclerosis and injury repair highlights important aspects of redox vascular responses. While neointima formation has not shown consistent responsiveness to antioxidants, vessel remodeling has been more clearly responsive, indicating that despite the multilevel redox signaling pathways, there is a coordinated response of the whole vessel. Among mechanisms that may orchestrate redox pathways, we discuss roles of superoxide dismutase activity and extracellular protein disulfide isomerase. We then discuss redox modulation of aneurysms, a special case of expansive remodeling. We propose that the redox modulation of vascular remodeling may reflect (1) remodeling pathophysiology is dominated by a particularly redox-sensitive cell type, e.g., endothelial cells (2) redox pathways are temporospatially coordinated at an organ level across distinct cellular and acellular structures or (3) the tensional homeostasis setpoint is closely connected to redox signaling. The mechanobiological/redox model discussed here can be a basis for improved understanding of remodeling and helps clarifying mechanisms underlying prevalent hard-to-treat diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Tumor-secreted miR-214 induces regulatory T cells: a major link between immune evasion and tumor growth

PubMed Central

Yin, Yuan; Cai, Xing; Chen, Xi; Liang, Hongwei; Zhang, Yujing; Li, Jing; Wang, Zuoyun; Chen, Xiulan; Zhang, Wen; Yokoyama, Seiji; Wang, Cheng; Li, Liang; Li, Limin; Hou, Dongxia; Dong, Lei; Xu, Tao; Hiroi, Takachika; Yang, Fuquan; Ji, Hongbin; Zhang, Junfeng; Zen, Ke; Zhang, Chen-Yu

2014-01-01

An increased population of CD4+CD25highFoxp3+ regulatory T cells (Tregs) in the tumor-associated microenvironment plays an important role in cancer immune evasion. However, the underlying mechanism remains unclear. Here we observed an increased secretion of miR-214 in various types of human cancers and mouse tumor models. Tumor-secreted miR-214 was sufficiently delivered into recipient T cells by microvesicles (MVs). In targeted mouse peripheral CD4+ T cells, tumor-derived miR-214 efficiently downregulated phosphatase and tensin homolog (PTEN) and promoted Treg expansion. The miR-214-induced Tregs secreted higher levels of IL-10 and promoted tumor growth in nude mice. Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-214 resulted in enhanced immune suppression and tumor implantation/growth in mice. The MV delivery of anti-miR-214 antisense oligonucleotides (ASOs) into mice implanted with tumors blocked Treg expansion and tumor growth. Our study reveals a novel mechanism through which cancer cell actively manipulates immune response via promoting Treg expansion. PMID:25223704

An insulin signaling feedback loop regulates pancreas progenitor cell differentiation during islet development and regeneration

PubMed Central

Ye, Lihua; Robertson, Morgan A.; Mastracci, Teresa L.; Anderson, Ryan M.

2016-01-01

As one of the key nutrient sensors, insulin signaling plays an important role in integrating environmental energy cues with organism growth. In adult organisms, relative insufficiency of insulin signaling induces compensatory expansion of insulin-secreting pancreatic beta (β) cells. However, little is known about how insulin signaling feedback might influence neogenesis of β cells during embryonic development. Using genetic approaches and a unique cell transplantation system in developing zebrafish, we have uncovered a novel role for insulin signaling in the negative regulation of pancreatic progenitor cell differentiation. Blocking insulin signaling in the pancreatic progenitors hastened the expression of the essential β cell genes insulin and pdx1, and promoted β cell fate at the expense of alpha cell fate. In addition, loss of insulin signaling promoted β cell regeneration and destabilization of alpha cell character. These data indicate that insulin signaling constitutes a tunable mechanism for β cell compensatory plasticity during early development. Moreover, using a novel blastomere-to-larva transplantation strategy, we found that loss of insulin signaling in endoderm-committed blastomeres drove their differentiation into β cells. Furthermore, the extent of this differentiation was dependent on the function of the β cell mass in the host. Altogether, our results indicate that modulation of insulin signaling will be crucial for the development of β cell restoration therapies for diabetics; further clarification of the mechanisms of insulin signaling in β cell progenitors will reveal therapeutic targets for both in vivo and in vitro β cell generation. PMID:26658317

Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During β-Cell Mass Expansion In Vivo.

PubMed

Horn, Signe; Kirkegaard, Jeannette S; Hoelper, Soraya; Seymour, Philip A; Rescan, Claude; Nielsen, Jens H; Madsen, Ole D; Jensen, Jan N; Krüger, Marcus; Grønborg, Mads; Ahnfelt-Rønne, Jonas

2016-01-01

Diabetes is characterized by insulin insufficiency due to a relative paucity of functional β-cell mass. Thus, strategies for increasing β-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust β-cell mass expansion, however, the mechanisms driving this expansion are not fully understood. Thus, the aim of this study was to characterize pregnancy-induced changes in the islet proteome at the peak of β-cell proliferation in mice. Islets from pregnant and nonpregnant littermates were compared via 2 proteomic strategies. In vivo pulsed stable isotope labeling of amino acids in cell culture was used to monitor de novo protein synthesis during the first 14.5 days of pregnancy. In parallel, protein abundance was determined using ex vivo dimethyl labelling at gestational day 14.5. Comparison of the 2 datasets revealed 170 islet proteins to be up regulated as a response to pregnancy. These included several proteins, not previously associated with pregnancy-induced islet expansion, such as CLIC1, STMN1, MCM6, PPIB, NEDD4, and HLTF. Confirming the validity of our approach, we also identified proteins encoded by genes known to be associated with pregnancy-induced islet expansion, such as CHGB, IGFBP5, MATN2, EHHADH, IVD, and BMP1. Bioinformatic analyses demonstrated enrichment and activation of the biological functions: "protein synthesis" and "proliferation," and predicted the transcription factors HNF4α, MYC, MYCN, E2F1, NFE2L2, and HNF1α as upstream regulators of the observed expressional changes. As the first characterization of the islet-proteome during pregnancy, this study provides novel insight into the mechanisms involved in promoting pregnancy-induced β-cell mass expansion and function.

Fibronectin regulates Wnt7a signaling and satellite cell expansion

PubMed Central

Bentzinger, C. Florian; Wang, Yu Xin; von Maltzahn, Julia; Soleimani, Vahab D.; Yin, Hang; Rudnicki, Michael A.

2012-01-01

SUMMARY The influence of the extracellular matrix (ECM) within the stem cell niche remains poorly understood. We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a co-receptor complex in satellite cells and that binding of the ECM glycoprotein Fibronectin (FN) to Sdc4 stimulates the ability of Wnt7a to induce the symmetric expansion of satellite stem cells. Newly activated satellite cells dynamically remodel their niche by transient high-level expression of FN. Knockdown of FN in prospectively isolated satellite cells severely impaired their ability to repopulate the satellite cell niche. Conversely, in vivo over-expression of FN with Wnt7a dramatically stimulated the expansion of satellite stem cells in regenerating muscle. Therefore, activating satellite cells remodel their niche through autologous expression of FN that provides feedback to stimulate Wnt7a signaling through the Fzd7/Sdc4 co-receptor complex. Thus, FN and Wnt7a together regulate the homeostatic levels of satellite stem cells and satellite myogenic cells during regenerative myogenesis. PMID:23290138

Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy.

PubMed

Urak, Ryan; Walter, Miriam; Lim, Laura; Wong, ChingLam W; Budde, Lihua E; Thomas, Sandra; Forman, Stephen J; Wang, Xiuli

2017-01-01

Insufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model. Various T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 μM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments. We found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once adoptively transferred into CD19+ tumor-bearing mice, Akti treated CD19CAR T cells exhibited more antitumor activity than did untreated CD19CAR T cells. Inhibition of Akt signaling during ex vivo priming and expansion gives rise to CD19CAR T cell populations that display comparatively higher antitumor activity.

Unstart coupling mechanism analysis of multiple-modules hypersonic inlet.

PubMed

Hu, Jichao; Chang, Juntao; Wang, Lei; Cao, Shibin; Bao, Wen

2013-01-01

The combination of multiplemodules in parallel manner is an important way to achieve the much higher thrust of scramjet engine. For the multiple-modules scramjet engine, when inlet unstarted oscillatory flow appears in a single-module engine due to high backpressure, how to interact with each module by massflow spillage, and whether inlet unstart occurs in other modules are important issues. The unstarted flowfield and coupling characteristic for a three-module hypersonic inlet caused by center module II and side module III were, conducted respectively. The results indicate that the other two hypersonic inlets are forced into unstarted flow when unstarted phenomenon appears on a single-module hypersonic inlet due to high backpressure, and the reversed flow in the isolator dominates the formation, expansion, shrinkage, and disappearance of the vortexes, and thus, it is the major factor of unstart coupling of multiple-modules hypersonic inlet. The coupling effect among multiple modules makes hypersonic inlet be more likely unstarted.

Umbilical cord blood regulatory T-cell expansion and functional effects of tumor necrosis factor receptor family members OX40 and 4-1BB expressed on artificial antigen-presenting cells

PubMed Central

Harker-Murray, Paul; Porter, Stephen B.; Merkel, Sarah C.; Londer, Aryel; Taylor, Dawn K.; Bina, Megan; Panoskaltsis-Mortari, Angela; Rubinstein, Pablo; Van Rooijen, Nico; Golovina, Tatiana N.; Suhoski, Megan M.; Miller, Jeffrey S.; Wagner, John E.; June, Carl H.; Riley, James L.

2008-01-01

Previously, we showed that human umbilical cord blood (UCB) regulatory T cells (Tregs) could be expanded approximately 100-fold using anti-CD3/28 monoclonal antibody (mAb)–coated beads to provide T-cell receptor and costimulatory signals. Because Treg numbers from a single UCB unit are limited, we explored the use of cell-based artificial antigen-presenting cells (aAPCs) preloaded with anti-CD3/28 mAbs to achieve higher levels of Treg expansion. Compared with beads, aAPCs had similar expansion properties while significantly increasing transforming growth factor β (TGF-β) secretion and the potency of Treg suppressor function. aAPCs modified to coexpress OX40L or 4-1BBL expanded UCB Tregs to a significantly greater extent than bead- or nonmodified aAPC cultures, reaching mean expansion levels exceeding 1250-fold. Despite the high expansion and in contrast to studies using other Treg sources, neither OX40 nor 4-1BB signaling of UCB Tregs reduced in vitro suppression. UCB Tregs expanded with 4-1BBL expressing aAPCs had decreased levels of proapoptotic bim. UCB Tregs expanded with nonmodified or modified aAPCs versus beads resulted in higher survival associated with increased Treg persistence in a xeno-geneic graft-versus-host disease lethality model. These data offer a novel approach for UCB Treg expansion using aAPCs, including those coexpressing OX40L or 4-1BBL. PMID:18645038

Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.

PubMed

Shah, Nina; Martin-Antonio, Beatriz; Yang, Hong; Ku, Stephanie; Lee, Dean A; Cooper, Laurence J N; Decker, William K; Li, Sufang; Robinson, Simon N; Sekine, Takuya; Parmar, Simrit; Gribben, John; Wang, Michael; Rezvani, Katy; Yvon, Eric; Najjar, Amer; Burks, Jared; Kaur, Indreshpal; Champlin, Richard E; Bollard, Catherine M; Shpall, Elizabeth J

2013-01-01

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56(+)/CD3(-)) and less than 1% CD3(+) cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Characterization of Postinfusion Phenotypic Differences in Fresh Versus Cryopreserved TCR Engineered Adoptive Cell Therapy Products.

PubMed

Nowicki, Theodore S; Escuin-Ordinas, Helena; Avramis, Earl; Chmielowski, Bartosz; Chodon, Thinle; Berent-Maoz, Beata; Wang, Xiaoyan; Kaplan-Lefko, Paula; Yang, Lili; Baltimore, David; Economou, James S; Ribas, Antoni; Comin-Anduix, Begoña

2018-06-01

Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort's superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols.

Characterization of Postinfusion Phenotypic Differences in Fresh Versus Cryopreserved TCR Engineered Adoptive Cell Therapy Products

PubMed Central

Nowicki, Theodore S.; Escuin-Ordinas, Helena; Avramis, Earl; Chmielowski, Bartosz; Chodon, Thinle; Berent-Maoz, Beata; Wang, Xiaoyan; Kaplan-Lefko, Paula; Yang, Lili; Baltimore, David; Economou, James S.; Ribas, Antoni

2018-01-01

Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort’s superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols. PMID:29470191

Scalable Expansion of Human Pluripotent Stem Cell-Derived Neural Progenitors in Stirred Suspension Bioreactor Under Xeno-free Condition.

PubMed

Nemati, Shiva; Abbasalizadeh, Saeed; Baharvand, Hossein

2016-01-01

Recent advances in neural differentiation technology have paved the way to generate clinical grade neural progenitor populations from human pluripotent stem cells. These cells are an excellent source for the production of neural cell-based therapeutic products to treat incurable central nervous system disorders such as Parkinson's disease and spinal cord injuries. This progress can be complemented by the development of robust bioprocessing technologies for large scale expansion of clinical grade neural progenitors under GMP conditions for promising clinical use and drug discovery applications. Here, we describe a protocol for a robust, scalable expansion of human neural progenitor cells from pluripotent stem cells as 3D aggregates in a stirred suspension bioreactor. The use of this platform has resulted in easily expansion of neural progenitor cells for several passages with a fold increase of up to 4.2 over a period of 5 days compared to a maximum 1.5-2-fold increase in the adherent static culture over a 1 week period. In the bioreactor culture, these cells maintained self-renewal, karyotype stability, and cloning efficiency capabilities. This approach can be also used for human neural progenitor cells derived from other sources such as the human fetal brain.

Endothelial Cells Promote Expansion of Long‐Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

PubMed Central

Gori, Jennifer L.; Butler, Jason M.; Kunar, Balvir; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Norgaard, Zachary K.; Adair, Jennifer E.; Rafii, Shahin

2016-01-01

Abstract Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self‐renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self‐renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+C38− HSPCs cocultured with ECs expanded up to 17‐fold, with a significant increase in hematopoietic colony‐forming activity compared with cells cultured with cytokines alone (colony‐forming unit‐granulocyte‐erythroid‐macrophage‐monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long‐term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864–876 PMID:28297579

Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells.

PubMed

de Soure, António M; Fernandes-Platzgummer, Ana; da Silva, Cláudia L; Cabral, Joaquim M S

2016-10-20

Due to their unique features, mesenchymal stem/stromal cells (MSC) have been exploited in clinical settings as therapeutic candidates for the treatment of a variety of diseases. However, the success in obtaining clinically-relevant MSC numbers for cell-based therapies is dependent on efficient isolation and ex vivo expansion protocols, able to comply with good manufacturing practices (GMP). In this context, the 2-dimensional static culture systems typically used for the expansion of these cells present several limitations that may lead to reduced cell numbers and compromise cell functions. Furthermore, many studies in the literature report the expansion of MSC using fetal bovine serum (FBS)-supplemented medium, which has been critically rated by regulatory agencies. Alternative platforms for the scalable manufacturing of MSC have been developed, namely using microcarriers in bioreactors, with also a considerable number of studies now reporting the production of MSC using xenogeneic/serum-free medium formulations. In this review we provide a comprehensive overview on the scalable manufacturing of human mesenchymal stem/stromal cells, depicting the various steps involved in the process from cell isolation to ex vivo expansion, using different cell tissue sources and culture medium formulations and exploiting bioprocess engineering tools namely microcarrier technology and bioreactors. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolism of murine TH 17 cells: Impact on cell fate and function.

PubMed

Wang, Ran; Solt, Laura A

2016-04-01

An effective adaptive immune response relies on the ability of lymphocytes to rapidly act upon a variety of insults. In T lymphocytes, this response includes cell growth, clonal expansion, differentiation, and cytokine production, all of which place a significant energy burden on the cell. Recent evidence shows that T-cell metabolic reprogramming is an essential component of the adaptive immune response and specific metabolic pathways dictate T-cell fate decisions, including the development of TH 17 versus T regulatory (Treg) cells. TH 17 cells have garnered significant attention due to their roles in the pathology of immune-mediated inflammatory diseases. Attempts to characterize TH 17 cells have demonstrated that they are highly dynamic, adjusting their function to environmental cues, which dictate their metabolic program. In this review, we highlight recent data demonstrating the impact of cellular metabolism on the TH 17/Treg balance and present factors that mediate TH 17-cell metabolism. Some examples of these include the differential impact of the mTOR signaling complexes on T-helper-cell differentiation, hypoxia inducible factor 1 alpha (HIF1α) promotion of glycolysis to favor TH 17-cell development, and ACC1-dependent de novo fatty acid synthesis favoring TH 17-cell development over Treg cells. Finally, we discuss the potential therapeutic options and the implications of modulating TH 17-cell metabolism for the treatment of TH 17-mediated diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of a French medical multi-terminology indexer for the manual annotation of natural language medical reports of healthcare-associated infections.

PubMed

Sakji, Saoussen; Gicquel, Quentin; Pereira, Suzanne; Kergourlay, Ivan; Proux, Denys; Darmoni, Stéfan; Metzger, Marie-Hélène

2010-01-01

Surveillance of healthcare-associated infections is essential to prevention. A new collaborative project, namely ALADIN, was launched in January 2009 and aims to develop an automated detection tool based on natural language processing of medical documents. The objective of this study was to evaluate the annotation of natural language medical reports of healthcare-associated infections. A software MS Access application (NosIndex) has been developed to interface ECMT XML answer and manual annotation work. ECMT performances were evaluated by an infection control practitioner (ICP). Precision was evaluated for the 2 modules and recall only for the default module. Exclusion rate was defined as ratio between medical terms not found by ECMT and total number of terms evaluated. The medical discharge summaries were randomly selected in 4 medical wards. From the 247 medical terms evaluated, ECMT proposed 428 and 3,721 codes, respectively for the default and expansion modules. The precision was higher with the default module (P1=0.62) than with the expansion (P2=0.47). Performances of ECMT as support tool for the medical annotation were satisfactory.

A novel nonlinear adaptive filter using a pipelined second-order Volterra recurrent neural network.

PubMed

Zhao, Haiquan; Zhang, Jiashu

2009-12-01

To enhance the performance and overcome the heavy computational complexity of recurrent neural networks (RNN), a novel nonlinear adaptive filter based on a pipelined second-order Volterra recurrent neural network (PSOVRNN) is proposed in this paper. A modified real-time recurrent learning (RTRL) algorithm of the proposed filter is derived in much more detail. The PSOVRNN comprises of a number of simple small-scale second-order Volterra recurrent neural network (SOVRNN) modules. In contrast to the standard RNN, these modules of a PSOVRNN can be performed simultaneously in a pipelined parallelism fashion, which can lead to a significant improvement in its total computational efficiency. Moreover, since each module of the PSOVRNN is a SOVRNN in which nonlinearity is introduced by the recursive second-order Volterra (RSOV) expansion, its performance can be further improved. Computer simulations have demonstrated that the PSOVRNN performs better than the pipelined recurrent neural network (PRNN) and RNN for nonlinear colored signals prediction and nonlinear channel equalization. However, the superiority of the PSOVRNN over the PRNN is at the cost of increasing computational complexity due to the introduced nonlinear expansion of each module.

HOT CELL BUILDING, TRA632. FLOOR PLAN OF EXPANSION SHOWS LOCATION ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

HOT CELL BUILDING, TRA-632. FLOOR PLAN OF EXPANSION SHOWS LOCATION OF NEW CELLS, "HEAVY" CELL AT WEST END, "LIGHT" CELLS AT EAST. MOCK-UP AND STORAGE AREAS IN SOUTH HALF OF FLOOR. H.K. FERGUSON 895-MTR-ETR-632-A1, 12/1958. INL INDEX NO. 531-0632-00-279-101924, REV. 4. - Idaho National Engineering Laboratory, Test Reactor Area, Materials & Engineering Test Reactors, Scoville, Butte County, ID

Photovoltaic cell module and method of forming

DOEpatents

Howell, Malinda; Juen, Donnie; Ketola, Barry; Tomalia, Mary Kay

2017-12-12

A photovoltaic cell module, a photovoltaic array including at least two modules, and a method of forming the module are provided. The module includes a first outermost layer and a photovoltaic cell disposed on the first outermost layer. The module also includes a second outermost layer disposed on the photovoltaic cell and sandwiching the photovoltaic cell between the second outermost layer and the first outermost layer. The method of forming the module includes the steps of disposing the photovoltaic cell on the first outermost layer, disposing a silicone composition on the photovoltaic cell, and compressing the first outermost layer, the photovoltaic cell, and the second layer to form the photovoltaic cell module.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

PubMed

Klöß, Stephan; Oberschmidt, Olaf; Morgan, Michael; Dahlke, Julia; Arseniev, Lubomir; Huppert, Volker; Granzin, Markus; Gardlowski, Tanja; Matthies, Nadine; Soltenborn, Stephanie; Schambach, Axel; Koehl, Ulrike

2017-10-01

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56 + CD3 - ) was carried out with the CliniMACS Prodigy ® in a single process, starting with approximately 1.2 × 10 9 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 6 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO ™ 10, CellGro ® , TexMACS ™ , and NK MACS ® ). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56 + CD3 - target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56 dim CD16 pos and CD56 bright CD16 dim&neg NK subsets on day 14 with cells cultivated in NK MACS ® media. Moreover, effector cell expansion in manually performed experiments with NK MACS ® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123 pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia.

PubMed

Giudice, Valentina; Feng, Xingmin; Lin, Zenghua; Hu, Wei; Zhang, Fanmao; Qiao, Wangmin; Ibanez, Maria Del Pilar Fernandez; Rios, Olga; Young, Neal S

2018-05-01

Oligoclonal expansion of CD8 + CD28 - lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8 + CD28 - cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8 + CD28 - CD57 + cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8 + CD57 + cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8 + cells from aplastic anemia patients with CD8 + CD57 + cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8 + CD57 + cells, which also showed decreased diversity compared to total CD4 + and CD8 + cell pools. From analysis of complementarity-determining region 3 sequences in the CD8 + cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8 + T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064 ). Copyright © 2018 Ferrata Storti Foundation.

MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation

PubMed Central

Huang, Y; Thoms, JAI; Tursky, ML; Knezevic, K; Beck, D; Chandrakanthan, V; Suryani, S; Olivier, J; Boulton, A; Glaros, EN; Thomas, SR; Lock, RB; MacKenzie, KL; Bushweller, JH; Wong, JWH; Pimanda, JE

2018-01-01

Aberrant ERG (v-ets avian erythroblastosis virus E26 oncogene homolog) expression drives leukemic transformation in mice and high expression is associated with poor patient outcomes in acute myeloid leukemia (AML) and T-acute lymphoblastic leukemia (T-ALL). Protein phosphorylation regulates the activity of many ETS factors but little is known about ERG in leukemic cells. To characterize ERG phosphorylation in leukemic cells, we applied liquid chromatography coupled tandem mass spectrometry and identified five phosphorylated serines on endogenous ERG in T-ALL and AML cells. S283 was distinct as it was abundantly phosphorylated in leukemic cells but not in healthy hematopoietic stem and progenitor cells (HSPCs). Overexpression of a phosphoactive mutant (S283D) increased expansion and clonogenicity of primary HSPCs over and above wild-type ERG. Using a custom antibody, we screened a panel of primary leukemic xenografts and showed that ERG S283 phosphorylation was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and in turn regulated expression of components of this pathway. S283 phosphorylation facilitates ERG enrichment and transactivation at the ERG +85 HSPC enhancer that is active in AML and T-ALL with poor prognosis. Taken together, we have identified a specific post-translational modification in leukemic cells that promotes progenitor proliferation and is a potential target to modulate ERG-driven transcriptional programs in leukemia. PMID:27055868

Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system.

PubMed

Mizukami, Amanda; Orellana, Maristela D; Caruso, Sâmia R; de Lima Prata, Karen; Covas, Dimas T; Swiech, Kamilla

2013-01-01

The need for efficient and reliable technologies for clinical-scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood-derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10(7) cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10(8) cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra-Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier-based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical-scale production system. Copyright © 2013 American Institute of Chemical Engineers.

Intravenous apoptotic spleen cell infusion induces a TGF-beta-dependent regulatory T-cell expansion

PubMed Central

Kleinclauss, François M.; Perruche, Sylvain; Masson, Emeline; De Carvalho Bittencourt, Marcelo; Biichle, Sabeha; Remy-Martin, Jean-Paul; Ferrand, Christophe; Martin, Mael; Bittard, Hugues; Chalopin, Jean-Marc; Seilles, Estelle; Tiberghien, Pierre; Saas, Philippe

2006-01-01

Apoptotic leukocytes are endowed with immunomodulatory properties that can be used to enhance hematopoietic engraftment and prevent graft-versus-host disease. This apoptotic cell-induced tolerogenic effect is mediated by host macrophages and not recipient dendritic cells or donor phagocytes present in the bone marrow graft as evidenced by selective cell depletion and trafficking experiments. Furthermore, apoptotic cell infusion is associated with TGF-β-dependent donor CD4+CD25+ T cell expansion. Such cells have a regulatory phenotype (CD62Lhigh and intracellular CTLA-4+), express high levels of Foxp3 mRNA and exert ex vivo suppressive activity through a cell-to-cell contact mechanism. In vivo CD25 depletion after apoptotic cell infusion prevents the apoptotic spleen cell-induced beneficial effects on engraftment and graft-versus-host disease occurrence. This highlights the role of regulatory T cells in the tolerogenic effect of apoptotic spleen cell infusion. This novel association between apoptosis and regulatory T cell expansion may also contribute to preventing deleterious auto-immune responses during normal turnover. PMID:15962005

Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

PubMed Central

Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J. T.; Alves, Paula M.

2011-01-01

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications. PMID:21850261

Engineering a fibrocartilage spectrum through modulation of aggregate redifferentiation.

PubMed

Murphy, Meghan K; Masters, Taylor E; Hu, Jerry C; Athanasiou, Kyriacos A

2015-01-01

Expanded costochondral cells provide a clinically relevant cell source for engineering both fibrous and hyaline articular cartilage. Expanding chondrocytes in a monolayer results in a shift toward a proliferative, fibroblastic phenotype. Three-dimensional aggregate culture may, however, be used to recover chondrogenic matrix production. This study sought to engineer a spectrum of fibrous to hyaline neocartilage from a single cell source by varying the duration of three-dimensional culture following expansion. In third passage porcine costochondral cells, the effects of aggregate culture duration were assessed after 0, 8, 11, 14, and 21 days of aggregate culture and after 4 subsequent weeks of neocartilage formation. Varying the duration of aggregate redifferentiation generated a spectrum of fibrous to hyaline neocartilage. Within 8 days of aggregation, proliferation ceased, and collagen and glycosaminoglycan production increased, compared with monolayer cells. In self-assembled neocartilage, type II-to-I collagen ratio increased with increasing aggregate duration, yet glycosaminoglycan content varied minimally. Notably, 14 days of aggregate redifferentiation increased collagen content by 25%, tensile modulus by over 110%, and compressive moduli by over 50%, compared with tissue formed in the absence of redifferentiation. A spectrum of fibrous to hyaline cartilage was generated using a single, clinically relevant cell source, improving the translational potential of engineered cartilage.

IL-2 immunotherapy in chronically SIV-infected Rhesus Macaques

PubMed Central

2012-01-01

Background Despite inducing a sustained increase in CD4+ T cell counts, intermittent recombinant IL-2 (rIL-2) therapy did not confer a better clinical outcome in HIV-infected patients enrolled in large phase III clinical trials ESPRIT and SILCAAT. Several hypotheses were evoked to explain these discrepancies. Here, we investigated the impact of low and high doses of IL-2 in Rhesus macaques of Chinese origin infected with SIVmac251 in the absence of antiretroviral therapy (ART). Results We demonstrated that rIL-2 induced a dose dependent expansion of CD4+ and CD8+ T cells without affecting viral load. rIL-2 increased CD4 and CD8 Treg cells as defined by the expression of CD25highFoxP3+CD127low. We also showed that rIL-2 modulated spontaneous and Fas-mediated CD4+ and CD8+ T cell apoptosis. The higher dose exhibited a dramatic pro-apoptotic effect on both CD4+ and CD8+ T cell populations. Finally, all the animals treated with rIL-2 developed a wasting syndrome in the month following treatment simultaneously to a dramatic decrease of circulating effector T cells. Conclusion These data contribute to the understanding of the homeostatic and dosage effects of IL-2 in the context of SIV/HIV infection. PMID:23021024

Engineering a Fibrocartilage Spectrum Through Modulation of Aggregate Redifferentiation

PubMed Central

Murphy, Meghan K.; Masters, Taylor E.; Hu, Jerry C.; Athanasiou, Kyriacos A.

2015-01-01

Expanded costochondral cells provide a clinically relevant cell source for engineering both fibrous and hyaline articular cartilage. Expanding chondrocytes in monolayer results in a shift toward a proliferative, fibroblastic phenotype. Three-dimensional aggregate culture may, however, be used to recover chondrogenic matrix production. This study sought to engineer a spectrum of fibrous to hyaline neocartilage from a single cell source by varying the duration of three-dimensional culture following expansion. In third passage porcine costochondral cells, the effects of aggregate culture duration were assessed after 0, 8, 11, 14, and 21 days of aggregate culture and after 4 subsequent weeks of neocartilage formation. Varying the duration of aggregate redifferentiation generated a spectrum of fibrous to hyaline neocartilage. Within 8 days of aggregation, proliferation ceased, and collagen and glycosaminoglycan production increased, compared with monolayer cells. In self-assembled neocartilage, type II to I collagen ratio increased with increasing aggregate duration, yet glycosaminoglycan content varied minimally. Notably, 14 days of aggregate redifferentiation increased collagen content by 25%, tensile modulus by over 110%, and compressive moduli by over 50%, compared with tissue formed in the absence of redifferentiation. A spectrum of fibrous to hyaline cartilage was generated using a single, clinically relevant cell source, improving the translational potential of engineered cartilage. PMID:24380383

Oncogenic Viruses and Tumor Glucose Metabolism: Like Kids in a Candy Store

PubMed Central

Noch, Evan; Khalili, Kamel

2011-01-01

Oncogenic viruses represent a significant public health burden in light of the multitude of malignancies resulting from chronic or spontaneous viral infection and transformation. Though many of the molecular signaling pathways underlying virus-mediated cellular transformation are known, the impact of these viruses on metabolic signaling and phenotype within proliferating tumor cells is less well understood. Whether the interaction of oncogenic viruses with metabolic signaling pathways involves enhanced glucose uptake and glycolysis, both hallmark features of transformed cells, or dysregulation of molecular pathways regulating oxidative stress, viruses are adept at facilitating tumor expansion. Through their effects on cell proliferation pathways, such as the PI3K and MAPK pathways, the cell cycle regulatory proteins, p53 and ATM, and the cell stress response proteins, HIF-1α and AMPK, viruses exert control over critical metabolic signaling cascades. Additionally, oncogenic viruses modulate the tumor metabolomic profile through direct and indirect interaction with glucose transporters, such as GLUT1, and specific glycolytic enzymes, including pyruvate kinase, glucose 6-phosphate dehydrogenase, and hexokinase. Through these pathways, oncogenic viruses alter the phenotypic characteristics of transformed cells and their methods of energy utilization, and it may be possible to develop novel anti-glycolytic therapies to target these dysregulated pathways in virus-derived malignancies. PMID:22234809

Inflatable habitation for the lunar base

NASA Technical Reports Server (NTRS)

Roberts, M.

1992-01-01

Inflatable structures have a number of advantages over rigid modules in providing habitation at a lunar base. Some of these advantages are packaging efficiency, convenience of expansion, flexibility, and psychological benefit to the inhabitants. The relatively small, rigid cylinders fitted to the payload compartment of a launch vehicle are not as efficient volumetrically as a collapsible structure that fits into the same space when packaged, but when deployed is much larger. Pressurized volume is a valuable resource. By providing that resource efficiently, in large units, labor intensive external expansion (such as adding additional modules to the existing base) can be minimized. The expansive interior in an inflatable would facilitate rearrangement of the interior to suite the evolving needs of the base. This large, continuous volume would also relieve claustrophobia, enhancing habitability and improving morale. The purpose of this paper is to explore some of the aspects of inflatable habitat design, including structural, architectural, and environmental considerations. As a specific case, the conceptual design of an inflatable lunar habitat, developed for the Lunar Base Systems Study at the Johnson Space Center, is described.

HOXB7 overexpression in lung cancer is a hallmark of acquired stem-like phenotype.

PubMed

Monterisi, Simona; Lo Riso, Pietro; Russo, Karin; Bertalot, Giovanni; Vecchi, Manuela; Testa, Giuseppe; Di Fiore, Pier Paolo; Bianchi, Fabrizio

2018-03-26

HOXB7 is a homeodomain (HOX) transcription factor involved in regional body patterning of invertebrates and vertebrates. We previously identified HOXB7 within a ten-gene prognostic signature for lung adenocarcinoma, where increased expression of HOXB7 was associated with poor prognosis. This raises the question of how HOXB7 overexpression can influence the metastatic behavior of lung adenocarcinoma. Here, we analyzed publicly available microarray and RNA-seq lung cancer expression datasets and found that HOXB7-overexpressing tumors are enriched in gene signatures characterizing adult and embryonic stem cells (SC), and induced pluripotent stem cells (iPSC). Experimentally, we found that HOXB7 upregulates several canonical SC/iPSC markers and sustains the expansion of a subpopulation of cells with SC characteristics, through modulation of LIN28B, an emerging cancer gene and pluripotency factor, which we discovered to be a direct target of HOXB7. We validated this new circuit by showing that HOXB7 enhances reprogramming to iPSC with comparable efficiency to LIN28B or its target c-MYC, which is a canonical reprogramming factor.

Experimental murine fascioliasis derives early immune suppression with increased levels of TGF-β and IL-4.

PubMed

Chung, Joon-Yong; Bae, Young-An; Yun, Doo-Hee; Yang, Hyun-Jong; Kong, Yoon

2012-12-01

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19(+) B cells was observed as early as week 1 post-infection while CD4(+)/CD8(+) T cells were down-regulated. Accumulation of Mac1(+) cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1β expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-β were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-β and IL-4 during the early stages of infection.

Complementarity-Determining Region 3 Size Spectratypes of T Cell Receptor β Chains in CD8+ T Cells following Antiviral Treatment of Chronic Hepatitis B▿

PubMed Central

Ma, Shi-Wu; Li, Yong-Yin; Zhang, Guang-Wen; Huang, Xuan; Sun, Jian; Li, Chris; Abbott, William G. H.; Hou, Jin-Lin

2011-01-01

An increased CD8+ T cell response to hepatitis B virus (HBV) peptides occurs between 12 and 24 weeks after starting antiviral therapy for chronic hepatitis B. It is not known whether these cells have antiviral function. The aim of this study was to determine whether clonal expansions of CD8+ T cells at these time points predict the virological response to therapy. Peripheral blood CD8+ T cells were obtained from 20 patients treated with lamivudine or telbivudine for chronic hepatitis B at baseline, 12 weeks, and 24 weeks. The CDR3 spectratype of each T cell receptor (TCR) β chain variable region (Vβ) gene family was analyzed, and the changes in the numbers of Vβ families with clonal expansions were compared in subjects with (n = 12) and without (n = 8) a virological response (52 week HBV DNA < 300 copies/ml). The number of CD8+ TCR Vβ families with clonal expansions at 12 weeks relative to baseline (median [10th to 90th percentile], +2.5 [0 to +7] versus +1 [0 to +2], P = 0.03) and at 24 weeks relative to 12 weeks (+1 [0 to +2] versus −1 [−3 to +4], P = 0.006) was higher in subjects with a virological response versus subjects without a virological response, as were interleukin-2 (IL-2) but not IL-21 mRNA levels in peripheral blood mononuclear cells. The duration of new expansions at 12 weeks was higher (P < 0.0001) in responders. Increased numbers of CD8+ T cell expansions after antiviral therapy are associated with a virological response to treatment. These CD8+ T cells are a potential target for a therapeutic vaccine for chronic hepatitis B. PMID:21098256

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

PubMed Central

Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

PubMed

Kusuma, Gina D; Brennecke, Shaun P; O'Connor, Andrea J; Kalionis, Bill; Heath, Daniel E

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

G-protein signaling modulator 1 deficiency accelerates cystic disease in an orthologous mouse model of autosomal dominant polycystic kidney disease

PubMed Central

Kwon, Michelle; Pavlov, Tengis S.; Nozu, Kandai; Rasmussen, Shauna A.; Ilatovskaya, Daria V.; Lerch-Gaggl, Alexandra; North, Lauren M.; Kim, Hyunho; Qian, Feng; Sweeney, William E.; Avner, Ellis D.; Blumer, Joe B.; Staruschenko, Alexander; Park, Frank

2012-01-01

Polycystic kidney diseases are the most common genetic diseases that affect the kidney. There remains a paucity of information regarding mechanisms by which G proteins are regulated in the context of polycystic kidney disease to promote abnormal epithelial cell expansion and cystogenesis. In this study, we describe a functional role for the accessory protein, G-protein signaling modulator 1 (GPSM1), also known as activator of G-protein signaling 3, to act as a modulator of cyst progression in an orthologous mouse model of autosomal dominant polycystic kidney disease (ADPKD). A complete loss of Gpsm1 in the Pkd1V/V mouse model of ADPKD, which displays a hypomorphic phenotype of polycystin-1, demonstrated increased cyst progression and reduced renal function compared with age-matched cystic Gpsm1+/+ and Gpsm1+/− mice. Electrophysiological studies identified a role by which GPSM1 increased heteromeric polycystin-1/polycystin-2 ion channel activity via Gβγ subunits. In summary, the present study demonstrates an important role for GPSM1 in controlling the dynamics of cyst progression in an orthologous mouse model of ADPKD and presents a therapeutic target for drug development in the treatment of this costly disease. PMID:23236168

AXISYMMETRIC, THROTTLEABLE NON-GIMBALLED ROCKET ENGINE

NASA Technical Reports Server (NTRS)

Sackheim, Robert L. (Inventor); Hutt, John J. (Inventor); Anderson, William E. (Inventor); Dressler, Gordon A. (Inventor)

2005-01-01

A rocket engine assembly is provided for a vertically launched rocket vehicle. A rocket engine housing of the assembly includes two or more combustion chambers each including an outlet end defining a sonic throat area. A propellant supply for the combustion chambers includes a throttling injector, associated with each of the combustion chambers and located opposite to sonic throat area, which injects the propellant into the associated combustion chamber. A modulator, which may form part of the injector, and which is controlled by a controller, modulates the flow rate of the propellant to the combustion chambers so that the chambers provide a vectorable net thrust. An expansion nozzle or body located downstream of the throat area provides expansion of the combustion gases produced by the combustion chambers so as to increase the net thrust.

Continuous 4-1BB co-stimulatory signals for the optimal expansion of tumor-infiltrating lymphocytes for adoptive T-cell therapy.

PubMed

Chacon, Jessica Ann; Pilon-Thomas, Shari; Sarnaik, Amod A; Radvanyi, Laszlo G

2013-09-01

Co-stimulation through members of the tumor necrosis factor receptor (TNFR) family appears to be critical for the generation of T cells with optimal effector-memory properties for adoptive cell therapy. Our work suggests that continuous 4-1BB/CD137 co-stimulation is required for the expansion of T cells with an optimal therapeutic profile and that the administration of 4-1BB agonists upon adoptive cell transfer further improves antitumor T-cell functions.

Selective Expansion of Skeletal Muscle Stem Cells from Bulk Muscle Cells in Soft Three‐Dimensional Fibrin Gel

PubMed Central

Zhu, Pei; Zhou, Yalu; Wu, Furen; Hong, Yuanfan; Wang, Xin; Shekhawat, Gajendra; Mosenson, Jeffrey

2017-01-01

Abstract Muscle stem cells (MuSCs) exhibit robust myogenic potential in vivo, thus providing a promising curative treatment for muscle disorders. Ex vivo expansion of adult MuSCs is highly desired to achieve a therapeutic cell dose because of their scarcity in limited muscle biopsies. Sorting of pure MuSCs is generally required for all the current culture systems. Here we developed a soft three‐dimensional (3D) salmon fibrin gel culture system that can selectively expand mouse MuSCs from bulk skeletal muscle preparations without cell sorting and faithfully maintain their regenerative capacity in culture. Our study established a novel platform for convenient ex vivo expansion of MuSCs, thus greatly advancing stem cell‐based therapies for various muscle disorders. Stem Cells Translational Medicine 2017;6:1412–1423 PMID:28244269

Elucidation of conditions allowing conversion of penicillin G and other penicillins to deacetoxycephalosporins by resting cells and extracts of Streptomyces clavuligerus NP1

PubMed Central

Cho, Hiroshi; Adrio, José L.; Luengo, José M.; Wolfe, Saul; Ocran, Simeon; Hintermann, Gilberto; Piret, Jacqueline M.; Demain, Arnold L.

1998-01-01

Using resting cells and extracts of Streptomyces clavuligerus NP1, we have been able to convert penicillin G (benzylpenicillin) to deacetoxycephalosporin G. Conversion was achieved by increasing by 45× the concentration of FeSO4 (1.8 mM) and doubling the concentration of α-ketoglutarate (1.28 mM) as compared with standard conditions used for the normal cell-free conversion of penicillin N to deacetoxycephalosporin C. ATP, MgSO4, KCl, and DTT, important in cell-free expansion of penicillin N, did not play a significant role in the ring expansion of penicillin G by resting cells or cell-free extracts. When these conditions were used with 14 other penicillins, ring expansion was achieved in all cases. PMID:9751702

Metal-air cells comprising collapsible foam members and means for minimizing internal pressure buildup

NASA Technical Reports Server (NTRS)

Putt, Ronald A. (Inventor); Woodruff, Glenn (Inventor)

1994-01-01

This invention provides a prismatic zinc-air cell including, in general, a prismatic container having therein an air cathode, a separator and a zinc anode. The container has one or more oxygen access openings, and the air cathode is disposed in the container in gaseous communication with the oxygen access openings so as to allow access of oxygen to the cathode. The separator has a first side in electrolytic communication with the air cathode and a second side in electrolytic communication with the zinc anode. The separator isolates the cathode and the zinc anode from direct electrical contact and allows passage of electrolyte therebetween. An expansion chamber adjacent to the zinc anode is provided which accommodates expansion of the zinc anode during discharge of the cell. A suitable collapsible foam member generally occupies the expansion space, providing sufficient resistance tending to oppose movement of the zinc anode away from the separator while collapsing upon expansion of the zinc anode during discharge of the cell. One or more vent openings disposed in the container are in gaseous communication with the expansion space, functioning to satisfactorily minimize the pressure buildup within the container by venting gasses expelled as the foam collapses during cell discharge.

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells

PubMed Central

Himburg, Heather A; Muramoto, Garrett G; Daher, Pamela; Meadows, Sarah K; Russell, J Lauren; Doan, Phuong; Chi, Jen-Tsan; Salter, Alice B; Lento, William E; Reya, Tannishtha; Chao, Nelson; Chute, John P

2013-01-01

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC counts in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34+CDCD38−Lin− cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration PMID:20305662

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells.

PubMed

Himburg, Heather A; Muramoto, Garrett G; Daher, Pamela; Meadows, Sarah K; Russell, J Lauren; Doan, Phuong; Chi, Jen-Tsan; Salter, Alice B; Lento, William E; Reya, Tannishtha; Chao, Nelson J; Chute, John P

2010-04-01

Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC numbers in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34(+)CDCD38(-)Lin(-) cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration.

A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue.

PubMed

Shelley, Brandon C; Gowing, Geneviève; Svendsen, Clive N

2014-06-15

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as "chopping" that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

Summary of Expansions, Updates, and Results in GREET 2017 Suite of Models

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

Wang, Michael; Elgowainy, Amgad; Han, Jeongwoo

This report provides a technical summary of the expansions and updates to the 2017 release of Argonne National Laboratory’s Greenhouse Gases, Regulated Emissions, and Energy Use in Transportation (GREET®) model, including references and links to key technical documents related to these expansions and updates. The GREET 2017 release includes an updated version of the GREET1 (the fuel-cycle GREET model) and GREET2 (the vehicle-cycle GREET model), both in the Microsoft Excel platform and in the GREET.net modeling platform. Figure 1 shows the structure of the GREET Excel modeling platform. The .net platform integrates all GREET modules together seamlessly.

Enhancing the Migration Ability of Mesenchymal Stromal Cells by Targeting the SDF-1/CXCR4 Axis

PubMed Central

Marquez-Curtis, Leah A.

2013-01-01

Mesenchymal stromal cells (MSCs) are currently being investigated in numerous clinical trials of tissue repair and various immunological disorders based on their ability to secrete trophic factors and to modulate inflammatory responses. MSCs have been shown to migrate to sites of injury and inflammation in response to soluble mediators including the chemokine stromal cell-derived factor-(SDF-)1, but during in vitro culture expansion MSCs lose surface expression of key homing receptors particularly of the SDF-1 receptor, CXCR4. Here we review studies on enhancement of SDF-1-directed migration of MSCs with the premise that their improved recruitment could translate to therapeutic benefits. We describe our studies on approaches to increase the CXCR4 expression in in vitro-expanded cord blood-derived MSCs, namely, transfection, using the commercial liposomal reagent IBAfect, chemical treatment with the histone deacetylase inhibitor valproic acid, and exposure to recombinant complement component C1q. These methodologies will be presented in the context of other cell targeting and delivery strategies that exploit pathways involved in MSC migration. Taken together, these findings indicate that MSCs can be manipulated in vitro to enhance their in vivo recruitment and efficacy for tissue repair. PMID:24381939

Reishi Protein LZ-8 Induces FOXP3+ Treg Expansion via a CD45-Dependent Signaling Pathway and Alleviates Acute Intestinal Inflammation in Mice

PubMed Central

Hsu, Hsien-Yeh; Kuan, Yen-Chou; Lin, Tung-Yi; Tsao, Shu-Ming; Hsu, Jason; Ma, Li-Juan; Sheu, Fuu

2013-01-01

LZ-8, an immunomodulatory protein isolated from Ganoderma lucidum (also known as Ling-Zhi or Reishi), has been shown to promote cell proliferation and IL-2 production in T cells. In this study, we show that LZ-8 induces the expansion of both murine and human CD4+ T cells into FOXP3+ regulatory T (Treg) cells. LZ-8 treatment was found to stimulate a 4-fold and a 10-fold expansion in the Treg populations of murine and human primary CD4+ T cells, respectively. In addition, the expression of CTLA-4 and IL-10 was induced in LZ-8-treated CD4+ T cells. Using neutralizing antibodies and gene-deficient T-cell lines, we also found that LZ-8 promotes Treg expansion through a CD45-mediated signaling pathway and that the CD18-dependent induction of IL-2 was involved in Treg formation and IL-10 production. The suppressive activity of LZ-8 was confirmed using a murine model of DSS-induced colitis; the disease was alleviated by the adoptive transfer of LZ-8-treated CD4+ T cells. In conclusion, a new regulatory function for LZ-8 was identified, and the molecular mechanisms underlying this function were elucidated. PMID:23864893

Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

PubMed

Gori, Jennifer L; Butler, Jason M; Kunar, Balvir; Poulos, Michael G; Ginsberg, Michael; Nolan, Daniel J; Norgaard, Zachary K; Adair, Jennifer E; Rafii, Shahin; Kiem, Hans-Peter

2017-03-01

Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34 + cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34 + C38 - HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34 + cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34 + cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864-876. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Statistical Enrichment of Epigenetic States Around Triplet Repeats that Can Undergo Expansions

PubMed Central

Essebier, Alexandra; Vera Wolf, Patricia; Cao, Minh Duc; Carroll, Bernard J.; Balasubramanian, Sureshkumar; Bodén, Mikael

2016-01-01

More than 30 human genetic diseases are linked to tri-nucleotide repeat expansions. There is no known mechanism that explains repeat expansions in full, but changes in the epigenetic state of the associated locus has been implicated in the disease pathology for a growing number of examples. A comprehensive comparative analysis of the genomic features associated with diverse repeat expansions has been lacking. Here, in an effort to decipher the propensity of repeats to undergo expansion and result in a disease state, we determine the genomic coordinates of tri-nucleotide repeat tracts at base pair resolution and computationally establish epigenetic profiles around them. Using three complementary statistical tests, we reveal that several epigenetic states are enriched around repeats that are associated with disease, even in cells that do not harbor expansion, relative to a carefully stratified background. Analysis of over one hundred cell types reveals that epigenetic states generally tend to vary widely between genic regions and cell types. However, there is qualified consistency in the epigenetic signatures of repeats associated with disease suggesting that changes to the chromatin and the DNA around an expanding repeat locus are likely to be similar. These epigenetic signatures may be exploited further to develop models that could explain the propensity of repeats to undergo expansions. PMID:27013954

Analysis of calibration-free wavelength-scanned wavelength modulation spectroscopy for practical gas sensing using tunable diode lasers

NASA Astrophysics Data System (ADS)

Sun, K.; Chao, X.; Sur, R.; Goldenstein, C. S.; Jeffries, J. B.; Hanson, R. K.

2013-12-01

A novel strategy has been developed for analysis of wavelength-scanned, wavelength modulation spectroscopy (WMS) with tunable diode lasers (TDLs). The method simulates WMS signals to compare with measurements to determine gas properties (e.g., temperature, pressure and concentration of the absorbing species). Injection-current-tuned TDLs have simultaneous wavelength and intensity variation, which severely complicates the Fourier expansion of the simulated WMS signal into harmonics of the modulation frequency (fm). The new method differs from previous WMS analysis strategies in two significant ways: (1) the measured laser intensity is used to simulate the transmitted laser intensity and (2) digital lock-in and low-pass filter software is used to expand both simulated and measured transmitted laser intensities into harmonics of the modulation frequency, WMS-nfm (n = 1, 2, 3,…), avoiding the need for an analytic model of intensity modulation or Fourier expansion of the simulated WMS harmonics. This analysis scheme is valid at any optical depth, modulation index, and at all values of scanned-laser wavelength. The method is demonstrated and validated with WMS of H2O dilute in air (1 atm, 296 K, near 1392 nm). WMS-nfm harmonics for n = 1 to 6 are extracted and the simulation and measurements are found in good agreement for the entire WMS lineshape. The use of 1f-normalization strategies to realize calibration-free wavelength-scanned WMS is also discussed.

Optimal ex vivo expansion of neutrophils from PBSC CD34+ cells by a combination of SCF, Flt3-L and G-CSF and its inhibition by further addition of TPO.

PubMed

Tura, Olga; Barclay, G Robin; Roddie, Huw; Davies, John; Turner, Marc L

2007-10-30

Autologous mobilised peripheral blood stem cell (PBSC) transplantation is now a standard approach in the treatment of haematological diseases to reconstitute haematopoiesis following myeloablative chemotherapy. However, there remains a period of severe neutropenia and thrombocytopenia before haematopoietic reconstitution is achieved. Ex vivo expanded PBSC have been employed as an adjunct to unmanipulated HSC transplantation, but have tended to be produced using complex cytokine mixtures aimed at multilineage (neutrophil and megakaryocyte) progenitor expansion. These have been reported to reduce or abrogate neutropenia but have little major effect on thrombocytopenia. Selective megakaryocyte expansion has been to date ineffective in reducing thrombocytopenia. This study was implemented to evaluate neutrophil specific rather than multilineage ex vivo expansion of PBSC for specifically focusing on reduction or abrogation of neutropenia. CD34+ cells (PBSC) were enriched from peripheral blood mononuclear cells following G-CSF-mobilisation and cultured with different permutations of cytokines to determine optimal cytokine combinations and doses for expansion and functional differentiation and maturation of neutrophils and their progenitors. Results were assessed by cell number, morphology, phenotype and function. A simple cytokine combination, SCF + Flt3-L + G-CSF, synergised to optimally expand and mature neutrophil progenitors assessed by cell number, phenotype, morphology and function (superoxide respiratory burst measured by chemiluminescence). G-CSF appears mandatory for functional maturation. Addition of other commonly employed cytokines, IL-3 and IL-6, had no demonstrable additive effect on numbers or function compared to this optimal combination. Addition of TPO, commonly included in multilineage progenitor expansion for development of megakaryocytes, reduced the maturation of neutrophil progenitors as assessed by number, morphology and function (respiratory burst activity). Given that platelet transfusion support is available for autologous PBSC transplantation but granulocyte transfusion is generally lacking, and that multilineage expanded PBSC do not reduce thrombocytopenia, we suggest that instead of multilineage expansion selective neutrophil expansion based on this relatively simple cytokine combination might be prioritized for development for clinical use as an adjunct to unmanipulated PBSC transplantation to reduce or abrogate post-transplant neutropenia.

Modulation of human multipotent and pluripotent stem cells using surface nanotopographies and surface-immobilised bioactive signals: A review.

PubMed

Wang, Peng-Yuan; Thissen, Helmut; Kingshott, Peter

2016-11-01

The ability to control the interactions of stem cells with synthetic surfaces is proving to be effective and essential for the quality of passaged stem cells and ultimately the success of regenerative medicine. The stem cell niche is crucial for stem cell self-renewal and differentiation. Thus, mimicking the stem cell niche, and here in particular the extracellular matrix (ECM), in vitro is an important goal for the expansion of stem cells and their applications. Here, surface nanotopographies and surface-immobilised biosignals have been identified as major factors that control stem cell responses. The development of tailored surfaces having an optimum nanotopography and displaying suitable biosignals is proposed to be essential for future stem cell culture, cell therapy and regenerative medicine applications. While early research in the field has been restricted by the limited availability of micro- and nanofabrication techniques, new approaches involving the use of advanced fabrication and surface immobilisation methods are starting to emerge. In addition, new cell types such as induced pluripotent stem cells (iPSCs) have become available in the last decade, but have not been fully understood. This review summarises significant advances in the area and focuses on the approaches that are aimed at controlling the behavior of human stem cells including maintenance of their self-renewal ability and improvement of their lineage commitment using nanotopographies and biosignals. More specifically, we discuss developments in biointerface science that are an important driving force for new biomedical materials and advances in bioengineering aiming at improving stem cell culture protocols and 3D scaffolds for clinical applications. Cellular responses revolve around the interplay between the surface properties of the cell culture substrate and the biomolecular composition of the cell culture medium. Determination of the precise role played by each factor, as well as the synergistic effects amongst the factors, all of which influence stem cell responses is essential for future developments. This review provides an overview of the current state-of-the-art in the design of complex material surfaces aimed at being the next generation of tools tailored for applications in cell culture and regenerative medicine. This review focuses on the effect of surface nanotopographies and surface-bound biosignals on human stem cells. Recently, stem cell research attracts much attention especially the induced pluripotent stem cells (iPSCs) and direct lineage reprogramming. The fast advance of stem cell research benefits disease treatment and cell therapy. On the other hand, surface property of cell adhered materials has been demonstrated very important for in vitro cell culture and regenerative medicine. Modulation of cell behavior using surfaces is costeffective and more defined. Thus, we summarise the recent progress of modulation of human stem cells using surface science. We believe that this review will capture a broad audience interested in topographical and chemical patterning aimed at understanding complex cellular responses to biomaterials. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Immune Regulatory Properties of CD117pos Amniotic Fluid Stem Cells Vary According to Gestational Age

PubMed Central

Di Trapani, Mariano; Bassi, Giulio; Fontana, Emanuela; Giacomello, Luca; Pozzobon, Michela; Guillot, Pascale V.; De Coppi, Paolo

2015-01-01

Amniotic Fluid Stem (AFS) cells are broadly multipotent fetal stem cells derived from the positive selection and ex vivo expansion of amniotic fluid CD117/c-kitpos cells. Considering the differentiation potential in vitro toward cell lineages belonging to the three germ layers, AFS cells have raised great interest as a new therapeutic tool, but their immune properties still need to be assessed. We analyzed the in vitro immunological properties of AFS cells from different gestational age in coculture with T, B, and natural killer (NK) cells. Nonactivated (resting) first trimester-AFS cells showed lower expression of HLA class-I molecules and NK-activating ligands than second and third trimester-AFS cells, whose features were associated with lower sensitivity to NK cell-mediated lysis. Nevertheless, inflammatory priming with interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) enhanced resistance of all AFS cell types to NK cytotoxicity. AFS cells modulated lymphocyte proliferation in a different manner according to gestational age: first trimester-AFS cells significantly inhibited T and NK cell proliferation, while second and third trimester-AFS cells were less efficient. In addition, only inflammatory-primed second trimester-AFS cells could suppress B cell proliferation, which was not affected by the first and third trimester-AFS cells. Indolamine 2,3 dioxygenase pathway was significantly involved only in T cell suppression mediated by second and third trimester-AFS cells. Overall, this study shows a number of significant quantitative differences among AFS cells of different gestational age that have to be considered in view of their clinical application. PMID:25072397

Development of large-scale manufacturing of adipose-derived stromal cells for clinical applications using bioreactors and human platelet lysate.

PubMed

Haack-Sørensen, Mandana; Juhl, Morten; Follin, Bjarke; Harary Søndergaard, Rebekka; Kirchhoff, Maria; Kastrup, Jens; Ekblond, Annette

2018-04-17

In vitro expanded adipose-derived stromal cells (ASCs) are a useful resource for tissue regeneration. Translation of small-scale autologous cell production into a large-scale, allogeneic production process for clinical applications necessitates well-chosen raw materials and cell culture platform. We compare the use of clinical-grade human platelet lysate (hPL) and fetal bovine serum (FBS) as growth supplements for ASC expansion in the automated, closed hollow fibre quantum cell expansion system (bioreactor). Stromal vascular fractions were isolated from human subcutaneous abdominal fat. In average, 95 × 10 6 cells were suspended in 10% FBS or 5% hPL medium, and loaded into a bioreactor coated with cryoprecipitate. ASCs (P0) were harvested, and 30 × 10 6 ASCs were reloaded for continued expansion (P1). Feeding rate and time of harvest was guided by metabolic monitoring. Viability, sterility, purity, differentiation capacity, and genomic stability of ASCs P1 were determined. Cultivation of SVF in hPL medium for in average nine days, yielded 546 × 10 6 ASCs compared to 111 × 10 6 ASCs, after 17 days in FBS medium. ASCs P1 yields were in average 605 × 10 6 ASCs (PD [population doublings]: 4.65) after six days in hPL medium, compared to 119 × 10 6 ASCs (PD: 2.45) in FBS medium, after 21 days. ASCs fulfilled ISCT criteria and demonstrated genomic stability and sterility. The use of hPL as a growth supplement for ASCs expansion in the quantum cell expansion system provides an efficient expansion process compared to the use of FBS, while maintaining cell quality appropriate for clinical use. The described process is an obvious choice for manufacturing of large-scale allogeneic ASC products.

Behavior of the potential-induced degradation of photovoltaic modules fabricated using flat mono-crystalline silicon cells with different surface orientations

NASA Astrophysics Data System (ADS)

Yamaguchi, Seira; Masuda, Atsushi; Ohdaira, Keisuke

2016-04-01

This paper deals with the dependence of the potential-induced degradation (PID) of flat, p-type mono-crystalline silicon solar cell modules on the surface orientation of solar cells. The investigated modules were fabricated from p-type mono-crystalline silicon cells with a (100) or (111) surface orientation using a module laminator. PID tests were performed by applying a voltage of -1000 V to shorted module interconnector ribbons with respect to an Al plate placed on the cover glass of the modules at 85 °C. A decrease in the parallel resistance of the (100)-oriented cell modules is more significant than that of the (111)-oriented cell modules. Hence, the performance of the (100)-oriented-cell modules drastically deteriorates, compared with that of the (111)-oriented-cell modules. This implies that (111)-oriented cells offer a higher PID resistance.

mTOR and MEK1/2 inhibition differentially modulate tumor growth and the immune microenvironment in syngeneic models of oral cavity cancer

PubMed Central

Cash, Harrison; Shah, Sujay; Moore, Ellen; Caruso, Andria; Uppaluri, Ravindra; Van Waes, Carter; Allen, Clint

2015-01-01

We investigated the effects of mTOR and MEK1/2 inhibition on tumor growth and the tumor microenvironment in immunogenic and poorly immunogenic models of murine oral cancer. In vitro, rapamycin and PD901 inhibited signaling through expected downstream targets, but only PD901 reduced viability and altered function of MOC cells. Following transplantation of MOC cells into immune-competent mice, effects on both cancer and infiltrating immune cells were characterized following rapamycin and/or PD901 treatment for 21 days. In vivo, both rapamycin and PD901 inhibition reduced primary growth of established MOC tumors on treatment. Following withdrawal of PD901, rapid rebound of tumor growth limited survival, whereas durable tumor control was observed following rapamycin treatment in immunogenic MOC1 tumors despite more robust inhibition of oncogenic signaling by PD901. Characterization of the immune microenvironment revealed diminished infiltration and activation of antigen-specific CD8+ T-cells and other immune cells following PD901 but not rapamycin in immunogenic tumors. Subsequent in vitro T-cell assays validated robust inhibition of T-cell expansion and activation following MEK inhibition compared to mTOR inhibition. CD8 cell depletion abrogated rapamycin-induced primary tumor growth inhibition in MOC1 mice. These data have critical implications in the design of combination targeted and immune therapies in oral cancer. PMID:26506415

Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System

PubMed Central

Hanley, Patrick J.; Mei, Zhuyong; Durett, April G.; Cabreira-Harrison, Marie da Graca; Klis, Mariola; Li, Wei; Zhao, Yali; Yang, Bing; Parsha, Kaushik; Mir, Osman; Vahidy, Farhaan; Bloom, Debra; Rice, R. Brent; Hematti, Peiman; Savitz, Sean I; Gee, Adrian P.

2014-01-01

Background The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host-disease, diabetes, ischemic cardiomyopathy, and Crohn's disease has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells needs to be cost effective, safe, and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures, and require prolonged culture times. Methods We evaluated the Quantum Cell Expansion system for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results to a flask-based method currently in clinical trials. Results After only two passages, we were able to expand a mean of 6.6×108 MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts, and adipocytes. The Quantum was able to generate the target cell number of 2.0×108 cells in an average of 9-fewer days and in half the number of passages required during flask-based expansion. We estimated the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. Discussion The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared to flasks. In the Quantum, the risk of contamination is substantially reduced due to the substantial decrease in open procedures. PMID:24726657

The hollow fiber bioreactor as a stroma-supported, serum-free ex vivo expansion platform for human umbilical cord blood cells.

PubMed

Xue, Cao; Kwek, Kenneth Y C; Chan, Jerry K Y; Chen, Qingfeng; Lim, Mayasari

2014-07-01

The bone marrow microenvironment plays an integral role in the regulation of hematopoiesis. Residing stromal cells and the extracellular matrix in the bone marrow microenvironment provide biological signals that control hematopoietic stem cell (HSC) function. In this study, we developed a bio-mimetic co-culture platform using the hollow fiber bioreactor (HFBR) for ex vivo expansion of HSCs. We evaluated the efficacy of such a platform in comparison to standard cultures performed on tissue culture polystyrene (TCP), using a human stromal cell line (HS-5) as stromal support, co-cultured with lineage-depleted human cord blood cells in serum-free medium supplemented with a cytokine cocktail. Our results showed that the performance of the HFBR in supporting total cell and CD34(+) progenitor cell expansion was comparable to that of cultures on TCP. Cells harvested from the HFBR had a higher clonogenic ability. The performance of ex vivo-expanded cells from the HFBR in hematopoietic reconstitution in humanized mice was comparable to that of the TCP control. Scanning electron microscopy revealed that stroma cell growth inside the HFBR created a three-dimensional cell matrix architecture. These findings demonstrate the feasibility of utilizing the HFBR for creating a complex cell matrix architecture, which may provide good in vitro mimicry of the bone marrow, supporting large-scale expansion of HSCs. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell Expansion and Endoreduplication Show a Large Genetic Variability in Pericarp and Contribute Strongly to Tomato Fruit Growth1

PubMed Central

Cheniclet, Catherine; Rong, Wen Ying; Causse, Mathilde; Frangne, Nathalie; Bolling, Laurence; Carde, Jean-Pierre; Renaudin, Jean-Pierre

2005-01-01

Postanthesis growth of tomato (Solanum lycopersicon) as of many types of fruit relies on cell division and cell expansion, so that some of the largest cells to be found in plants occur in fleshy fruit. Endoreduplication is known to occur in such materials, which suggests its involvement in cell expansion, although no data have demonstrated this hypothesis as yet. We have analyzed pattern formation, cell size, and ploidy in tomato fruit pericarp. A first set of data was collected in one cherry tomato line throughout fruit development. A second set of data was obtained from 20 tomato lines displaying a large weight range in fruit, which were compared as ovaries at anthesis and as fully grown fruit at breaker stage. A remarkable conservation of pericarp pattern, including cell layer number and cell size, is observed in all of the 20 tomato lines at anthesis, whereas large variations of growth occur afterward. A strong, positive correlation, combining development and genetic diversity, is demonstrated between mean cell size and ploidy, which holds for mean cell diameters from 10 to 350 μm (i.e. a 32,000-times volume variation) and for mean ploidy levels from 3 to 80 C. Fruit weight appears also significantly correlated with cell size and ploidy. These data provide a framework of pericarp patterning and growth. They strongly suggest the quantitative importance of polyploidy-associated cell expansion as a determinant of fruit weight in tomato. PMID:16306145

TACI is required for efficient plasma cell differentiation in response to T-independent type 2 antigens.

PubMed

Mantchev, George T; Cortesão, Catarina S; Rebrovich, Michelle; Cascalho, Marilia; Bram, Richard J

2007-08-15

The control of systemic infection by encapsulated microorganisms requires T-independent type II (TI-2) Ab responses to bacterial polysaccharides. To understand how such responses evolve, we explored the function of transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI), a member of the TNFR family, required for TI-2 Ab production. Quasimonoclonal (QM) mice produce robust TI-2 responses to 4-hydroxy-3-nitrophenylacetate (NP)-Ficoll, owing to the high precursor frequency of NP-specific B cells in the marginal zone of the spleen. QM mice that lack TACI produce decreased numbers of IgM (2-fold) and IgG (1.6-fold) NP-specific ASCs, compared with TACI-positive QM mice in response to immunization with NP-Ficoll. Our studies indicate that TACI acts at a remote time from activation because TACI is not necessary for activation and proliferation of B cells both in vitro and in vivo. Instead, TACI-deficient QM B cells remained in the cell cycle longer than TACI-proficient QM cells and had impaired plasma cell differentiation in response to NP-Ficoll. We conclude that TACI has dual B cell-autonomous functions, inhibiting prolonged B cell proliferation and stimulating plasma cell differentiation, thus resolving the longstanding paradox that TACI may have both B cell-inhibitory and -stimulatory functions. By promoting plasma cell differentiation earlier during clonal expansion, TACI may decrease the chances of autoantibody production by somatic hypermutation of Ig genes in response to T-independent Ags.

Human platelet lysate in mesenchymal stromal cell expansion according to a GMP grade protocol: a cell factory experience.

PubMed

Becherucci, Valentina; Piccini, Luisa; Casamassima, Serena; Bisin, Silvia; Gori, Valentina; Gentile, Francesca; Ceccantini, Riccardo; De Rienzo, Elena; Bindi, Barbara; Pavan, Paola; Cunial, Vanessa; Allegro, Elisa; Ermini, Stefano; Brugnolo, Francesca; Astori, Giuseppe; Bambi, Franco

2018-05-02

The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production.

Effect of IL-18 on the Expansion and Phenotype of Human Natural Killer Cells: Application to Cancer Immunotherapy.

PubMed

Senju, Hiroaki; Kumagai, Asuka; Nakamura, Yoichi; Yamaguchi, Hiroyuki; Nakatomi, Katsumi; Fukami, Shota; Shiraishi, Kengo; Harada, Yuka; Nakamura, Mitsuhiro; Okamura, Haruki; Tanaka, Yoshimasa; Mukae, Hiroshi

2018-01-01

When pathogenic stresses are recognized by innate immune cells, inflammasomes are assembled and caspase-1 is activated, resulting in the conversion of pro-IL-18 into mature IL-18. Because natural killer (NK) cells express IL-18 receptors, IL-18 may play roles in immune functions of NK cells. In the present study, we examined the effect of IL-18 on NK cells derived from lung cancer patients and healthy adult volunteers. When peripheral blood NK cells were stimulated with IL-2, the cells formed clusters beginning on day 5-6 and proliferated thereafter, in which the number of NK cells increased by 10-fold in 10 days. When IL-18 was added, cell clusters were observed as early as on day 4 and NK cells proliferated vigorously. On day 10, the expansion rate was 56-fold on average, showing that IL-18 promoted the expansion of NK cells. It was also notable that IL-18 enhanced the expression of CD80, CD86, HLA-DR and HLA-DQ on NK cells, suggesting that IL-18 conferred NK cells an APC-like phenotype. When cellular cytotoxicity was determined, APC-like NK cells efficiently killed tumor cells and anti-tumor activity was augmented by the addition of tumor antigen-specific mAbs. In addition, IFN-γ was produced by APC-like NK cells in response to tumor cells, and the cytokine production was further enhanced by mAbs. Taken together, IL-18 not only promoted the expansion of NK cells, but also changed the phenotype of NK cells. IL-2/IL-18-induced NK cells might, therefore, serve as a bridge between innate immunity and adaptive immunity and be useful for cancer immunotherapy.

Analyzing Pulse-Code Modulation On A Small Computer

NASA Technical Reports Server (NTRS)

Massey, David E.

1988-01-01

System for analysis pulse-code modulation (PCM) comprises personal computer, computer program, and peripheral interface adapter on circuit board that plugs into expansion bus of computer. Functions essentially as "snapshot" PCM decommutator, which accepts and stores thousands of frames of PCM data, sifts through them repeatedly to process according to routines specified by operator. Enables faster testing and involves less equipment than older testing systems.

Multiple types of data are required to identify the mechanisms influencing the spatial expansion of melanoma cell colonies.

PubMed

Treloar, Katrina K; Simpson, Matthew J; Haridas, Parvathi; Manton, Kerry J; Leavesley, David I; McElwain, D L Sean; Baker, Ruth E

2013-12-12

The expansion of cell colonies is driven by a delicate balance of several mechanisms including cell motility, cell-to-cell adhesion and cell proliferation. New approaches that can be used to independently identify and quantify the role of each mechanism will help us understand how each mechanism contributes to the expansion process. Standard mathematical modelling approaches to describe such cell colony expansion typically neglect cell-to-cell adhesion, despite the fact that cell-to-cell adhesion is thought to play an important role. We use a combined experimental and mathematical modelling approach to determine the cell diffusivity, D, cell-to-cell adhesion strength, q, and cell proliferation rate, λ, in an expanding colony of MM127 melanoma cells. Using a circular barrier assay, we extract several types of experimental data and use a mathematical model to independently estimate D, q and λ. In our first set of experiments, we suppress cell proliferation and analyse three different types of data to estimate D and q. We find that standard types of data, such as the area enclosed by the leading edge of the expanding colony and more detailed cell density profiles throughout the expanding colony, does not provide sufficient information to uniquely identify D and q. We find that additional data relating to the degree of cell-to-cell clustering is required to provide independent estimates of q, and in turn D. In our second set of experiments, where proliferation is not suppressed, we use data describing temporal changes in cell density to determine the cell proliferation rate. In summary, we find that our experiments are best described using the range D=161-243μm2 hour-1, q=0.3-0.5 (low to moderate strength) and λ=0.0305-0.0398 hour-1, and with these parameters we can accurately predict the temporal variations in the spatial extent and cell density profile throughout the expanding melanoma cell colony. Our systematic approach to identify the cell diffusivity, cell-to-cell adhesion strength and cell proliferation rate highlights the importance of integrating multiple types of data to accurately quantify the factors influencing the spatial expansion of melanoma cell colonies.

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. II. Quantity and quality of light required

NASA Technical Reports Server (NTRS)

Van Volkenburgh, E.; Cleland, R. E.; Watanabe, M.

1990-01-01

The quantity and quality of light required for light-stimulated cell expansion in leaves of Phaseolus vulgaris L. have been determined. Seedlings were grown in dim red light (RL; 4 micromoles photons m-2 s-1) until cell division in the primary leaves was completed, then excised discs were incubated in 10 mM sucrose plus 10 mM KCl in a variety of light treatments. The growth response of discs exposed to continuous white light (WL) for 16 h was saturated at 100 micromoles m-2 s-1, and did not show reciprocity. Extensive, but not continuous, illumination was needed for maximal growth. The wavelength dependence of disc expansion was determined from fluence-response curves obtained from 380 to 730 nm provided by the Okazaki Large Spectrograph. Blue (BL; 460 nm) and red light (RL; 660 nm) were most effective in promoting leaf cell growth, both in photosynthetically active and inhibited leaf discs. Far-red light (FR; 730 nm) reduced the effectiveness of RL, but not BL, indicating that phytochrome and a separate blue-light receptor mediate expansion of leaf cells.

Abnormal degradation of the neuronal stress-protective transcription factor HSF1 in Huntington's disease

PubMed Central

Gomez-Pastor, Rocio; Burchfiel, Eileen T.; Neef, Daniel W.; Jaeger, Alex M.; Cabiscol, Elisa; McKinstry, Spencer U.; Doss, Argenia; Aballay, Alejandro; Lo, Donald C.; Akimov, Sergey S.; Ross, Christopher A.; Eroglu, Cagla; Thiele, Dennis J.

2017-01-01

Huntington's Disease (HD) is a neurodegenerative disease caused by poly-glutamine expansion in the Htt protein, resulting in Htt misfolding and cell death. Expression of the cellular protein folding and pro-survival machinery by heat shock transcription factor 1 (HSF1) ameliorates biochemical and neurobiological defects caused by protein misfolding. We report that HSF1 is degraded in cells and mice expressing mutant Htt, in medium spiny neurons derived from human HD iPSCs and in brain samples from patients with HD. Mutant Htt increases CK2α′ kinase and Fbxw7 E3 ligase levels, phosphorylating HSF1 and promoting its proteasomal degradation. An HD mouse model heterozygous for CK2α′ shows increased HSF1 and chaperone levels, maintenance of striatal excitatory synapses, clearance of Htt aggregates and preserves body mass compared with HD mice homozygous for CK2α′. These results reveal a pathway that could be modulated to prevent neuronal dysfunction and muscle wasting caused by protein misfolding in HD. PMID:28194040

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients.

PubMed

Siegler, Uwe; Meyer-Monard, Sandrine; Jörger, Simon; Stern, Martin; Tichelli, André; Gratwohl, Alois; Wodnar-Filipowicz, Aleksandra; Kalberer, Christian P

2010-10-01

Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use. We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells. NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML. The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

Moclobemide upregulated Bcl-2 expression and induced neural stem cell differentiation into serotoninergic neuron via extracellular-regulated kinase pathway

PubMed Central

Chiou, Shih-Hwa; Ku, Hung-Hai; Tsai, Tung-Hu; Lin, Heng-Liang; Chen, Li-Hsin; Chien, Chan-Shiu; Ho, Larry L -T; Lee, Chen-Hsen; Chang, Yuh-Lih

2006-01-01

Moclobemide (MB) is an antidepressant drug that selectively and reversibly inhibits monoamine oxidase-A. Recent studies have revealed that antidepressant drugs possess the characters of potent growth-promoting factors for the development of neurogenesis and improve the survival rate of serotonin (5-hydroxytrytamine; 5-HT) neurons. However, whether MB comprises neuroprotection effects or modulates the proliferation of neural stem cells (NSCs) needs to be elucidated. In this study, firstly, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to demonstrate that 50 μM MB can increase the cell viability of NSCs. The result of real-time reverse transcription–polymerase chain reaction (RT–PCR) showed that the induction of MB can upregulate the gene expressions of Bcl-2 and Bcl-xL. By using caspases 8 and 3, ELISA and terminal dUTP nick-end labeling (TUNEL) assay, our data further confirmed that 50 μM MB-treated NSCs can prevent FasL-induced apoptosis. The morphological findings also supported the evidence that MB can facilitate the dendritic development and increase the neurite expansion of NSCs. Moreover, we found that MB treatment increased the expression of Bcl-2 in NSCs through activating the extracellular-regulated kinase (ERK) phosphorylation. By using the triple-staining immunofluorescent study, the percentages of serotonin- and MAP-2-positive cells in the day 7 culture of MB-treated NSCs were significantly increased (P<0.01). Furthermore, our data supported that MB treatment increased functional production of serotonin in NSCs via the modulation of ERK1/2. In sum, the study results support that MB can upregulate Bcl-2 expression and induce the differentiation of NSCs into serotoninergic neuron via ERK pathway. PMID:16702990

Moclobemide upregulated Bcl-2 expression and induced neural stem cell differentiation into serotoninergic neuron via extracellular-regulated kinase pathway.

PubMed

Chiou, Shih-Hwa; Ku, Hung-Hai; Tsai, Tung-Hu; Lin, Heng-Liang; Chen, Li-Hsin; Chien, Chan-Shiu; Ho, Larry L-T; Lee, Chen-Hsen; Chang, Yuh-Lih

2006-07-01

1. Moclobemide (MB) is an antidepressant drug that selectively and reversibly inhibits monoamine oxidase-A. Recent studies have revealed that antidepressant drugs possess the characters of potent growth-promoting factors for the development of neurogenesis and improve the survival rate of serotonin (5-hydroxytrytamine; 5-HT) neurons. However, whether MB comprises neuroprotection effects or modulates the proliferation of neural stem cells (NSCs) needs to be elucidated. 2. In this study, firstly, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to demonstrate that 50 microM MB can increase the cell viability of NSCs. The result of real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the induction of MB can upregulate the gene expressions of Bcl-2 and Bcl-xL. By using caspases 8 and 3, ELISA and terminal dUTP nick-end labeling (TUNEL) assay, our data further confirmed that 50 microM MB-treated NSCs can prevent FasL-induced apoptosis. 3. The morphological findings also supported the evidence that MB can facilitate the dendritic development and increase the neurite expansion of NSCs. Moreover, we found that MB treatment increased the expression of Bcl-2 in NSCs through activating the extracellular-regulated kinase (ERK) phosphorylation. 4. By using the triple-staining immunofluorescent study, the percentages of serotonin- and MAP-2-positive cells in the day 7 culture of MB-treated NSCs were significantly increased (P<0.01). Furthermore, our data supported that MB treatment increased functional production of serotonin in NSCs via the modulation of ERK1/2. In sum, the study results support that MB can upregulate Bcl-2 expression and induce the differentiation of NSCs into serotoninergic neuron via ERK pathway.

Oleanolic acid acetate inhibits rheumatoid arthritis by modulating T cell immune responses and matrix-degrading enzymes

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

Choi, Jin Kyeong; Molecular Immunology Section, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892; Kim, Sung-Wan

ABSTRACT: Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with a combination of synovium joint inflammation, synovium hyperplasia, and destruction of cartilage and bone. Oleanolic acid acetate (OAA), a compound isolated from Vigna angularis, has been known to possess pharmacological activities, including anti-inflammation and anti-bone destruction. In this study, we investigated the effects of OAA on RA and the underlying mechanisms of action by using a type-II collagen-induced arthritis (CIA) mouse model and tumor necrosis factor (TNF)-α-stimulated RA synovial fibroblasts. Oral administration of OAA decreased the clinical arthritis symptoms, paw thickness, histologic and radiologic changes, and serum total andmore » anti-type II collagen IgG, IgG1, and IgG2a levels. OAA administration reduced Th1/Th17 phenotype CD4{sup +} T lymphocyte expansions and inflammatory cytokine productions in T cell activated draining lymph nodes and spleen. OAA reduced the expression and production of inflammatory mediators, such as cytokines and matrix metalloproteinase (MMP)-1/3, in the ankle joint tissue and RA synovial fibroblasts by down-regulating Akt, mitogen-activated protein kinases, and nuclear factor-κB. Our results clearly support that OAA plays a therapeutic role in RA pathogenesis by modulating helper T cell immune responses and matrix-degrading enzymes. The immunosuppressive effects of OAA were comparable to dexamethasone and ketoprofen. We provide evidences that OAA could be a potential therapeutic candidate for RA. - Highlights: • OAA attenuated chronic CIA symptoms. • OAA had a regulating effect on the T helper cell immune reaction for CIA. • The effect of OAA on the RA was comparable to the dexamethasone or ketoprofen. • OAA might be a candidate for the treatment of arthritic diseases.« less

Enhancing Post-Expansion Chondrogenic Potential of Costochondral Cells in Self-Assembled Neocartilage

PubMed Central

Murphy, Meghan K.; Huey, Daniel J.; Reimer, Andrew J.; Hu, Jerry C.; Athanasiou, Kyriacos A.

2013-01-01

The insufficient healing capacity of articular cartilage necessitates mechanically functional biologic tissue replacements. Using cells to form biomimetic cartilage implants is met with the challenges of cell scarcity and donor site morbidity, requiring expanded cells that possess the ability to generate robust neocartilage. To address this, this study assesses the effects of expansion medium supplementation (bFGF, TFP, FBS) and self-assembled construct seeding density (2, 3, 4 million cells/5 mm dia. construct) on the ability of costochondral cells to generate biochemically and biomechanically robust neocartilage. Results show TFP (1 ng/mL TGF-β1, 5 ng/mL bFGF, 10 ng/mL PDGF) supplementation of serum-free chondrogenic expansion medium enhances the post-expansion chondrogenic potential of costochondral cells, evidenced by increased glycosaminoglycan content, decreased type I/II collagen ratio, and enhanced compressive properties. Low density (2 million cells/construct) enhances matrix synthesis and tensile and compressive mechanical properties. Combined, TFP and Low density interact to further enhance construct properties. That is, with TFP, Low density increases type II collagen content by over 100%, tensile stiffness by over 300%, and compressive moduli by over 140%, compared with High density. In conclusion, the interaction of TFP and Low density seeding enhances construct material properties, allowing for a mechanically functional, biomimetic cartilage to be formed using clinically relevant costochondral cells. PMID:23437288

Pharmacological preconditioning for short-term ex vivo expansion of human umbilical cord blood hematopoietic stem cells by filgrastim

PubMed Central

Grigoriadis, Nikolaos G; Grigoriadis, Ioannis G; Markoula, Sofia; Paschopoulos, Minas; Zikopoulos, Konstantinos; Apostolakopoulos, Panagiotis Gr; Vizirianakis, Ioannis S; Georgiou, Ioannis

2016-01-01

Although umbilical cord blood (UCB) hematopoietic stem cell transplantation (UCBT) has emerged as a promising haematological reconstitution therapy for leukemias and other related disorders, the insufficient UCB stem cell dosage still hinders better clinical outcomes. Previous research efforts, by focusing on ex vivo UCB expansion capabilities have sought to benefit from well-known mechanisms of self-renewal characteristics of UCB stem cells. However, the long-term (> 21 days) in vitro culture period and the low neutrophil recovery significantly reduce the transplantability of such ex vivo expanded UCB stem cells. To overcome the latter hurdles in this study, a post-thaw, short-term ex vivo expansion methodology of UCB mononuclear (UCB-MN) and CD34+ cells has been established. Notably, such effort was achieved through pharmacological preconditioned of UCB cultures by filgrastim agent already used in the clinical setting. In crucial cell populations implicated in the promotion of functional engraftment, the progression of free survival rates (PFS), a marked increase of 6.65 to 9.34 fold for UCB-MN and 35 to 49 fold for CD34+ cells has been noticed. Overall, these results indicate that transplantation of pharmacologically-preconditioned ex vivo expansion of UCB stem and progenitor cells keep high promise upon transplantation to enhance therapeutic potential in everyday clinical practice. PMID:27335700

Forever Young: The Role of Ubiquitin Receptor DA1 and E3 Ligase BIG BROTHER in Controlling Leaf Growth and Development.

PubMed

Vanhaeren, Hannes; Nam, Youn-Jeong; De Milde, Liesbeth; Chae, Eunyoung; Storme, Veronique; Weigel, Detlef; Gonzalez, Nathalie; Inzé, Dirk

2017-02-01

The final size of plant organs is determined by a combination of cell proliferation and cell expansion. Leaves account for a large part of above-ground biomass and provide energy to complete the plant's life cycle. Although the final size of leaves is remarkably constant under fixed environmental conditions, several genes have been described to enhance leaf growth when their expression is modulated. In Arabidopsis (Arabidopsis thaliana), mutations in DA1 and BB increase leaf size, an effect that is synergistically enhanced in the double mutant. Here, we show that overexpression of a dominant-negative version of DA1 enhances leaf size in a broad range of natural accessions of this species, indicating a highly conserved role of this protein in controlling organ size. We also found that during early stages of development, leaves of da1-1 and bb/eod1-2 mutants were already larger than the isogenic Col-0 wild type, but this phenotype was triggered by different cellular mechanisms. Later during development, da1-1 and bb/eod1-2 leaves showed a prolonged longevity, which was enhanced in the double mutant. Conversely, ectopic expression of DA1 or BB restricted growth and promoted leaf senescence. In concert, shortly upon induction of DA1 and BB expression, several marker genes for the transition from proliferation to expansion were highly up-regulated. Additionally, multiple genes involved in maintaining the mitotic cell cycle were rapidly down-regulated and senescence genes were strongly up-regulated, particularly upon BB induction. With these results, we demonstrate that DA1 and BB restrict leaf size and promote senescence through converging and different mechanisms. © 2017 American Society of Plant Biologists. All Rights Reserved.

Characterization of the procera Tomato Mutant Shows Novel Functions of the SlDELLA Protein in the Control of Flower Morphology, Cell Division and Expansion, and the Auxin-Signaling Pathway during Fruit-Set and Development1[C][W

PubMed Central

Carrera, Esther; Ruiz-Rivero, Omar; Peres, Lazaro Eustaquio Pereira; Atares, Alejandro; Garcia-Martinez, Jose Luis

2012-01-01

procera (pro) is a tall tomato (Solanum lycopersicum) mutant carrying a point mutation in the GRAS region of the gene encoding SlDELLA, a repressor in the gibberellin (GA) signaling pathway. Consistent with the SlDELLA loss of function, pro plants display a GA-constitutive response phenotype, mimicking wild-type plants treated with GA3. The ovaries from both nonemasculated and emasculated pro flowers had very strong parthenocarpic capacity, associated with enhanced growth of preanthesis ovaries due to more and larger cells. pro parthenocarpy is facultative because seeded fruits were obtained by manual pollination. Most pro pistils had exserted stigmas, thus preventing self-pollination, similar to wild-type pistils treated with GA3 or auxins. However, Style2.1, a gene responsible for long styles in noncultivated tomato, may not control the enhanced style elongation of pro pistils, because its expression was not higher in pro styles and did not increase upon GA3 application. Interestingly, a high percentage of pro flowers had meristic alterations, with one additional petal, sepal, stamen, and carpel at each of the four whorls, respectively, thus unveiling a role of SlDELLA in flower organ development. Microarray analysis showed significant changes in the transcriptome of preanthesis pro ovaries compared with the wild type, indicating that the molecular mechanism underlying the parthenocarpic capacity of pro is complex and that it is mainly associated with changes in the expression of genes involved in GA and auxin pathways. Interestingly, it was found that GA activity modulates the expression of cell division and expansion genes and an auxin signaling gene (tomato AUXIN RESPONSE FACTOR7) during fruit-set. PMID:22942390

Forever Young: The Role of Ubiquitin Receptor DA1 and E3 Ligase BIG BROTHER in Controlling Leaf Growth and Development1[OPEN

PubMed Central

Vanhaeren, Hannes; De Milde, Liesbeth

2017-01-01

The final size of plant organs is determined by a combination of cell proliferation and cell expansion. Leaves account for a large part of above-ground biomass and provide energy to complete the plant’s life cycle. Although the final size of leaves is remarkably constant under fixed environmental conditions, several genes have been described to enhance leaf growth when their expression is modulated. In Arabidopsis (Arabidopsis thaliana), mutations in DA1 and BB increase leaf size, an effect that is synergistically enhanced in the double mutant. Here, we show that overexpression of a dominant-negative version of DA1 enhances leaf size in a broad range of natural accessions of this species, indicating a highly conserved role of this protein in controlling organ size. We also found that during early stages of development, leaves of da1-1 and bb/eod1-2 mutants were already larger than the isogenic Col-0 wild type, but this phenotype was triggered by different cellular mechanisms. Later during development, da1-1 and bb/eod1-2 leaves showed a prolonged longevity, which was enhanced in the double mutant. Conversely, ectopic expression of DA1 or BB restricted growth and promoted leaf senescence. In concert, shortly upon induction of DA1 and BB expression, several marker genes for the transition from proliferation to expansion were highly up-regulated. Additionally, multiple genes involved in maintaining the mitotic cell cycle were rapidly down-regulated and senescence genes were strongly up-regulated, particularly upon BB induction. With these results, we demonstrate that DA1 and BB restrict leaf size and promote senescence through converging and different mechanisms. PMID:28003326

Wall relaxation and the driving forces for cell expansive growth

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.

1987-01-01

When water uptake by growing cells is prevented, the turgor pressure and the tensile stress in the cell wall are reduced by continued wall loosening. This process, termed in vivo stress relaxation, provides a new way to study the dynamics of wall loosening and to measure the wall yield threshold and the physiological wall extensibility. Stress relaxation experiments indicate that wall stress supplies the mechanical driving force for wall yielding. Cell expansion also requires water absorption. The driving force for water uptake during growth is created by wall relaxation, which lowers the water potential of the expanding cells. New techniques for measuring this driving force show that it is smaller than believed previously; in elongating stems it is only 0.3 to 0.5 bar. This means that the hydraulic resistance of the water transport pathway is small and that rate of cell expansion is controlled primarily by wall loosening and yielding.

[Cytomegalovirus-associated infectious mononucleosis-like syndrome accompanied by transient monoclonal expansion of CD8+ T-cells].

PubMed

Yonezawa, Akihito; Onaka, Takashi; Imada, Kazunori

2009-08-01

Most cases of infectious mononucleosis (IM) are caused by Epstein-Barr virus (EBV). Other pathogens have been reported to cause heterophile-negative mononucleosis-like syndrome, including cytomegalovirus (CMV) and human immunodeficiency virus type-1 (HIV-1). Primary CMV infection is often asymptomatic in immunocompetent individuals. In this article, we describe a patient with prolonged fever and fatigue, who developed transient monoclonal CD8+ T-cell lymphocytosis after primary CMV infection. Monoclonal gene rearrangement of T-cell receptor (TCR) beta locus was transiently detected in DNA from peripheral lymphocytes. Monoclonal rearrangement and atypical lymphocytosis disappeared after treatment with anti-viral agents. These observations imply that monoclonal expansion of T-cells could be a reactive phenomenon of primary CMV infection and TCR gene rearrangement is not specific for malignancy. Physicians should carefully follow patients with monoclonal expansion of CD8+ T-cells after CMV-IM in order to rule out T cell malignancy.

Optimizing T Cell Expansion in a Hollow-Fiber Bioreactor.

PubMed

Nankervis, Brian; Jones, Mark; Vang, Boah; Brent Rice, R; Coeshott, Claire; Beltzer, Jim

2018-01-01

Recent developments in regenerative medicine have precipitated the need to expand gene-modified human T cells to numbers that exceed the capacity of well-plate-based, and flask-based processes. This review discusses the changes in process development that are needed to meet the cell expansion requirements by utilizing hollow-fiber bioreactors . Maintenance of cell proliferation over long periods can become limited by unfilled demands for nutrients and oxygen and by the accumulation of waste products in the local environment. Perfusion feeding, improved gas exchange, and the efficient removal of lactate can increase the yield of T cells from an average of 10.8E +09 to more than 28E +09 in only 10 days. Aggressively feeding cells and actively keeping cells in the bioreactor improves gas exchange and metabolite management over semi-static methods. The ability to remove the environmental constraints that can limit cell expansion by using a two-chamber hollow-fiber bioreactor will be discussed.

Specification of Drosophila Corpora Cardiaca Neuroendocrine Cells from Mesoderm Is Regulated by Notch Signaling

PubMed Central

Park, Sangbin; Bustamante, Erika L.; Antonova, Julie; McLean, Graeme W.; Kim, Seung K.

2011-01-01

Drosophila neuroendocrine cells comprising the corpora cardiaca (CC) are essential for systemic glucose regulation and represent functional orthologues of vertebrate pancreatic α-cells. Although Drosophila CC cells have been regarded as developmental orthologues of pituitary gland, the genetic regulation of CC development is poorly understood. From a genetic screen, we identified multiple novel regulators of CC development, including Notch signaling factors. Our studies demonstrate that the disruption of Notch signaling can lead to the expansion of CC cells. Live imaging demonstrates localized emergence of extra precursor cells as the basis of CC expansion in Notch mutants. Contrary to a recent report, we unexpectedly found that CC cells originate from head mesoderm. We show that Tinman expression in head mesoderm is regulated by Notch signaling and that the combination of Daughterless and Tinman is sufficient for ectopic CC specification in mesoderm. Understanding the cellular, genetic, signaling, and transcriptional basis of CC cell specification and expansion should accelerate discovery of molecular mechanisms regulating ontogeny of organs that control metabolism. PMID:21901108

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis.

PubMed

Carpenter, Stephen M; Nunes-Alves, Cláudio; Booty, Matthew G; Way, Sing Sing; Behar, Samuel M

2016-01-01

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

PubMed Central

Carpenter, Stephen M.; Nunes-Alves, Cláudio; Booty, Matthew G.; Way, Sing Sing; Behar, Samuel M.

2016-01-01

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8+ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8+ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8+ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8+ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8+ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8+ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection. PMID:26745507

Competition for Space Is Controlled by Apoptosis-Induced Change of Local Epithelial Topology.

PubMed

Tsuboi, Alice; Ohsawa, Shizue; Umetsu, Daiki; Sando, Yukari; Kuranaga, Erina; Igaki, Tatsushi; Fujimoto, Koichi

2018-06-11

During the initial stage of tumor progression, oncogenic cells spread despite spatial confinement imposed by surrounding normal tissue. This spread of oncogenic cells (winners) is thought to be governed by selective killing of surrounding normal cells (losers) through a phenomenon called "cell competition" (i.e., supercompetition). Although the mechanisms underlying loser elimination are increasingly apparent, it is not clear how winner cells selectively occupy the space made available following loser apoptosis. Here, we combined live imaging analyses of two different oncogenic clones (Yki/YAP activation and Ras activation) in the Drosophila epithelium with computer simulation of tissue mechanics to elucidate such a mechanism. Contrary to the previous expectation that cell volume loss after apoptosis of loser cells was simply compensated for by the faster proliferation of winner cells, we found that the lost volume was compensated for by rapid cell expansion of winners. Mechanistically, the rapid winner-dominated cell expansion was driven by apoptosis-induced epithelial junction remodeling, which causes re-connection of local cellular connectivity (cell topology) in a manner that selectively increases winner apical surface area. In silico experiments further confirmed that repetition of loser elimination accelerates tissue-scale winner expansion through topological changes over time. Our proposed mechanism for linking loser death and winner expansion provides a new perspective on how tissue homeostasis disruption can initiate from an oncogenic mutation. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Development of Low Cost, High Energy-Per-Unit-Area Solar Cell Modules

NASA Technical Reports Server (NTRS)

Jones, G. T.; Chitre, S.

1977-01-01

Work on the development of low cost, high energy per unit area solar cell modules was conducted. Hexagonal solar cell and module efficiencies, module packing ratio, and solar cell design calculations were made. The cell grid structure and interconnection pattern was designed and the module substrates were fabricated for the three modules to be used. It was demonstrated that surface macrostructures significantly improve cell power output and photovoltaic energy conversion efficiency.

Recovery from Cyclophosphamide-Induced Lymphopenia Results in Expansion of Immature Dendritic Cells Which Can Mediate Enhanced Prime-Boost Vaccination Antitumor Responses In Vivo When Stimulated with the TLR3 Agonist Poly(I:C)1

PubMed Central

Salem, Mohamed L.; Díaz-Montero, C. Marcela; Al-Khami, Amir A.; El-Naggar, Sabry A.; Naga, Osama; Montero, Alberto J.; Khafagy, Ahmed; Cole, David J.

2011-01-01

Recent preclinical studies suggest that vaccination following adoptive transfer of CD8+ T cells into a lymphopenic host can augment the therapeutic antitumor responses of the transferred cells. However, the mechanism by which the lymphopenic microenvironment benefits Ag-specific CD8+ T cell responses remains elusive. We show herein that induction of lymphodepletion by a single 4 mg cyclophosphamide (CTX) treatment induces a marked expansion of immature dendritic cells (DCs) in the peripheral blood on days 8–16 post-CTX (termed restoration phase). In vitro, these DCs were functional, because they showed normal phagocytosis and effective Ag presentation capability upon activation. In vivo, administration of the TLR3 agonist poly(I:C) at the peak of DC expansion (day 12 postlymphopenia) induced inflammatory cytokine production and increases in the number of activated DCs in lymph nodes. Importantly, boosting with gp10025–33 melanoma peptide combined with poly(I:C) 12 days after an initial priming with the same regimen significantly increased the expansion and the antitumor efficacy of adoptively transferred pmel-1 CD8+ T cells. These responses were abrogated after depletion of activated DCs during Ag boosting. In conclusion, our data show that CTX treatment induces, during the restoration phase, expansion of immature DCs, which are functional and can be exploited in vivo to foster more effective antitumor adoptive immunotherapy strategies. PMID:19201856

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

PubMed

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A S; Zhou, Pengbo; Mesuraca, Maria; Bond, Heather Mandy; Morrone, Giovanni

2017-07-04

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

Defined Serum-Free Medium for Bioreactor Culture of an Immortalized Human Erythroblast Cell Line.

PubMed

Lee, Esmond; Lim, Zhong Ri; Chen, Hong-Yu; Yang, Bin Xia; Lam, Alan Tin-Lun; Chen, Allen Kuan-Liang; Sivalingam, Jaichandran; Reuveny, Shaul; Loh, Yuin-Han; Oh, Steve Kah-Weng

2018-04-01

Anticipated shortages in donated blood supply have prompted investigation of alternative approaches for in vitro production of red blood cells (RBCs), such as expansion of conditional immortalization erythroid progenitors. However, there is a bioprocessing challenge wherein factors promoting maximal cell expansion and growth-limiting inhibitory factors are yet to be investigated. The authors use an erythroblast cell line (ImEry) derived from immortalizing CD71+CD235a+ erythroblast from adult peripheral blood for optimization of expansion culture conditions. Design of experiments (DOE) is used in media formulation to explore relationships and interactive effects between factors which affect cell expansion. Our in-house optimized medium formulation produced significantly higher cell densities (3.62 ± 0.055) × 10 6  cells mL -1 , n = 3) compared to commercial formulations (2.07 ± 0.055) × 10 6  cells mL -1 , n = 3; at 209 h culture). Culture media costs per unit of blood is shown to have a 2.96-3.09 times cost reduction. As a proof of principle for scale up, ImEry are expanded in a half-liter stirred-bioreactor under controlled settings. Growth characteristics, metabolic, and molecular profile of the cells are evaluated. ImEry has identical O 2 binding capacity to adult erythroblasts. Amino acid supplementation results in further yield improvements. The study serves as a first step for scaling up erythroblast expansion in controlled bioreactors. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells

PubMed Central

Codispoti, Bruna; Rinaldo, Nicola; Chiarella, Emanuela; Lupia, Michela; Spoleti, Cristina Barbara; Marafioti, Maria Grazia; Aloisio, Annamaria; Scicchitano, Stefania; Giordano, Marco; Nappo, Giovanna; Lucchino, Valeria; Moore, Malcolm A.S.; Zhou, Pengbo; Mesuraca, Maria

2017-01-01

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery. Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes. PMID:28187462

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

PubMed Central

Redkar, Amey; Hoser, Rafal; Schilling, Lena; Zechmann, Bernd; Krzymowska, Magdalena; Walbot, Virginia; Doehlemann, Gunther

2015-01-01

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize. PMID:25888589

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

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

Wang, Wei; Li, Yonggang; Zhou, Liya

2011-07-15

Highlights: {yields} JAK/STAT activity is graded in the Drosophila optic lobe neuroepithelium. {yields} Inactivation of JAK signaling causes disintegration of the optic lobe neuroepithelium and depletion of the neuroepithelial stem cells. {yields} JAK pathway overactivation promotes neuroepithelial overgrowth. {yields} Notch signaling acts downstream of JAK/STAT to promote neuroepithelial growth and expansion. -- Abstract: During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. In this study, we findmore » that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. We further show that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth.« less

IL-10 Enhances IgE-Mediated Mast Cell Responses and Is Essential for the Development of Experimental Food Allergy in IL-10-Deficient Mice.

PubMed

Polukort, Stephanie H; Rovatti, Jeffrey; Carlson, Logan; Thompson, Chelsea; Ser-Dolansky, Jennifer; Kinney, Shannon R M; Schneider, Sallie S; Mathias, Clinton B

2016-06-15

IL-10 is a key pleiotropic cytokine that can both promote and curb Th2-dependent allergic responses. In this study, we demonstrate a novel role for IL-10 in promoting mast cell expansion and the development of IgE-mediated food allergy. Oral OVA challenge in sensitized BALB/c mice resulted in a robust intestinal mast cell response accompanied by allergic diarrhea, mast cell activation, and a predominance of Th2 cytokines, including enhanced IL-10 expression. In contrast, the development of intestinal anaphylaxis, including diarrhea, mast cell activation, and Th2 cytokine production, was significantly attenuated in IL-10(-/-) mice compared with wild-type (WT) controls. IL-10 also directly promoted the expansion, survival, and activation of mast cells; increased FcεRI expression on mast cells; and enhanced the production of mast cell cytokines. IL-10(-/-) mast cells had reduced functional capacity, which could be restored by exogenous IL-10. Similarly, attenuated passive anaphylaxis in IL-10(-/-) mice could be restored by IL-10 administration. The adoptive transfer of WT mast cells restored allergic symptoms in IL-10(-/-) mice, suggesting that the attenuated phenotype observed in these animals is due to a deficiency in IL-10-responding mast cells. Lastly, transfer of WT CD4 T cells also restored allergic diarrhea and intestinal mast cell numbers in IL-10(-/-) mice, suggesting that the regulation of IL-10-mediated intestinal mast cell expansion is T cell dependent. Our observations demonstrate a critical role for IL-10 in driving mucosal mast cell expansion and activation, suggesting that, in its absence, mast cell function is impaired, leading to attenuated food allergy symptoms. Copyright © 2016 by The American Association of Immunologists, Inc.

Histone Deacetylase Inhibitors Stimulate Dedifferentiation of Human Breast Cancer Cells through WNT/β-catenin Signaling

PubMed Central

Debeb, Bisrat G; Lacerda, Lara; Xu, Wei; Larson, Richard; Solley, Travis; Atkinson, Rachel; Sulman, Erik P.; Ueno, Naoto T; Krishnamurthy, Savitri; Reuben, James M; Buchholz, Thomas A; Woodward, Wendy A

2015-01-01

Recent studies have shown that differentiated cancer cells can de-differentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti-cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self renewal and expansion of hematopietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells towards the more resistant stem cell-like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA-231, were FACS-sorted based on ALDH activity and subsequently ALDH-negative and ALDH-positive cells were treated with one of two known HDAC inhibitors, VA or SAHA (suberoylanilide hydroxamic acid). In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single cell sorted ALDH- negative cells spontaneously generated ALDH-positive cells in vitro. Treatment of ALDH-negative cells with HDAC inhibitors promoted the expansion of ALDH-positive cells and increased mammosphere forming efficiency. Most importantly, it significantly increased the tumor-initiating capacity of ALDH- negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β-catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC-inhibitor induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer. PMID:22961641

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

PubMed Central

Fraietta, Joseph A.; Beckwith, Kyle A.; Patel, Prachi R.; Ruella, Marco; Zheng, Zhaohui; Barrett, David M.; Lacey, Simon F.; Melenhorst, Jan Joseph; McGettigan, Shannon E.; Cook, Danielle R.; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B.; Cogdill, Alexandria P.; Gill, Saar; Porter, David L.; Woyach, Jennifer A.; Long, Meixiao; Johnson, Amy J.; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L.; June, Carl H.; Byrd, John C.

2016-01-01

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. PMID:26813675

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

PubMed

Fraietta, Joseph A; Beckwith, Kyle A; Patel, Prachi R; Ruella, Marco; Zheng, Zhaohui; Barrett, David M; Lacey, Simon F; Melenhorst, Jan Joseph; McGettigan, Shannon E; Cook, Danielle R; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B; Cogdill, Alexandria P; Gill, Saar; Porter, David L; Woyach, Jennifer A; Long, Meixiao; Johnson, Amy J; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L; June, Carl H; Byrd, John C; Maus, Marcela V

2016-03-03

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. © 2016 by The American Society of Hematology.

De Novo Chromosome Copy Number Variation in Fanconi Anemia-Associated Hematopoietic Defects

DTIC Science & Technology

2012-04-01

Appendix 1. Expansion of monoclonal populations of FA-A hTERT and FA-A + FANCA hTERT cells Appendix 2. Expansion of monoclonal populations of FA...marrow failure (BMF) and pronounced cancer susceptibility. The FA proteins and the major breast cancer susceptibility gene products BRCA1 and BRCA2...Correction of FA-A, FA-C, and FA-D2 hTERT cells with pLenti6.2/V5- FANCA , -FANCC, and FANCD2, respectively. Sub-task 1. Selection and expansion of clonal

Hybrid proline-rich proteins: novel players in plant cell elongation?

PubMed Central

Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas

2012-01-01

Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464

Endogenous T-Cell Therapy: Clinical Experience.

PubMed

Yee, Cassian; Lizee, Greg; Schueneman, Aaron J

2015-01-01

Adoptive cellular therapy represents a robust means of augmenting the tumor-reactive effector population in patients with cancer by adoptive transfer of ex vivo expanded T cells. Three approaches have been developed to achieve this goal: the use of tumor-infiltrating lymphocytes or tumor-infiltrating lymphocytess extracted from patient biopsy material; the redirected engineering of lymphocytes using vectors expressing a chimeric antigen receptor and T-cell receptor; and third, the isolation and expansion of often low-frequency endogenous T cells (ETCs) reactive to tumor antigens from the peripheral blood of patients. This last form of adoptive transfer of T cells, known as ETC therapy, requires specialized methods to isolate and expand from peripheral blood the very low-frequency tumor-reactive T cells, methods that have been developed over the last 2 decades, to the point where such an approach may be broadly applicable not only for the treatment of melanoma but also for that of other solid tumor malignancies. One compelling feature of ETC is the ability to rapidly deploy clinical trials following identification of a tumor-associated target epitope, a feature that may be exploited to develop personalized antigen-specific T-cell therapy for patients with almost any solid tumor. With a well-validated antigen discovery pipeline in place, clinical studies combining ETC with agents that modulate the immune microenvironment can be developed that will transform ETC into a feasible treatment modality.

Expansion of Multipotent Stem Cells from the Adult Human Brain

PubMed Central

Murrell, Wayne; Palmero, Emily; Bianco, John; Stangeland, Biljana; Joel, Mrinal; Paulson, Linda; Thiede, Bernd; Grieg, Zanina; Ramsnes, Ingunn; Skjellegrind, Håvard K.; Nygård, Ståle; Brandal, Petter; Sandberg, Cecilie; Vik-Mo, Einar; Palmero, Sheryl; Langmoen, Iver A.

2013-01-01

The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells. PMID:23967194

High-Dose Nicotinamide Suppresses ROS Generation and Augments Population Expansion during CD8(+) T Cell Activation.

PubMed

Choi, Ho Jin; Jang, So-Young; Hwang, Eun Seong

2015-10-01

During T cell activation, mitochondrial content increases to meet the high energy demand of rapid cell proliferation. With this increase, the level of reactive oxygen species (ROS) also increases and causes the rapid apoptotic death of activated cells, thereby facilitating T cell homeostasis. Nicotinamide (NAM) has previously been shown to enhance mitochondria quality and extend the replicative life span of human fibroblasts. In this study, we examined the effect of NAM on CD8(+) T cell activation. NAM treatment attenuated the increase of mitochondrial content and ROS in T cells activated by CD3/CD28 antibodies. This was accompanied by an accelerated and higher-level clonal expansion resulting from attenuated apoptotic death but not increased division of the activated cells. Attenuation of ROS-triggered pro-apoptotic events and upregulation of Bcl-2 expression appeared to be involved. Although cells activated in the presence of NAM exhibited compromised cytokine gene expression, our results suggest a means to augment the size of T cell expansion during activation without consuming their limited replicative potential.

Differences in Normal Tissue Response in the Esophagus Between Proton and Photon Radiation Therapy for Non-Small Cell Lung Cancer Using In Vivo Imaging Biomarkers.

PubMed

Niedzielski, Joshua S; Yang, Jinzhong; Mohan, Radhe; Titt, Uwe; Mirkovic, Dragan; Stingo, Francesco; Liao, Zhongxing; Gomez, Daniel R; Martel, Mary K; Briere, Tina M; Court, Laurence E

2017-11-15

To determine whether there exists any significant difference in normal tissue toxicity between intensity modulated radiation therapy (IMRT) or proton therapy for the treatment of non-small cell lung cancer. A total of 134 study patients (n=49 treated with proton therapy, n=85 with IMRT) treated in a randomized trial had a previously validated esophageal toxicity imaging biomarker, esophageal expansion, quantified during radiation therapy, as well as esophagitis grade (Common Terminology Criteria for Adverse Events version 3.0), on a weekly basis during treatment. Differences between the 2 modalities were statically analyzed using the imaging biomarker metric value (Kruskal-Wallis analysis of variance), as well as the incidence and severity of esophagitis grade (χ 2 and Fisher exact tests, respectively). The dose-response of the imaging biomarker was also compared between modalities using esophageal equivalent uniform dose, as well as delivered dose to an isotropic esophageal subvolume. No statistically significant difference in the distribution of esophagitis grade, the incidence of grade ≥3 esophagitis (15 and 11 patients treated with IMRT and proton therapy, respectively), or the esophageal expansion imaging biomarker between cohorts (P>.05) was found. The distribution of imaging biomarker metric values had similar distributions between treatment arms, despite a slightly higher dose volume in the proton arm (P>.05). Imaging biomarker dose-response was similar between modalities for dose quantified as esophageal equivalent uniform dose and delivered esophageal subvolume dose. Regardless of treatment modality, there was high variability in imaging biomarker response, as well as esophagitis grade, for similar esophageal doses between patients. There was no significant difference in esophageal toxicity from either proton- or photon-based radiation therapy as quantified by esophagitis grade or the esophageal expansion imaging biomarker. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiparameter comparative analysis reveals differential impacts of various cytokines on CART cell phenotype and function ex vivo and in vivo

PubMed Central

Xu, Xiao-Jun; Song, De-Gang; Poussin, Mathilde; Ye, Qunrui; Sharma, Prannda; Rodríguez-García, Alba; Tang, Yong-Min; Powell, Daniel J.

2016-01-01

Exogenous cytokines are widely applied to enhance the anti-tumor ability of immune cells. However, systematic comparative studies of their effects on chimeric antigen receptor (CAR)-engineered T (CART) cells are lacking. In this study, CART cells targeting folate receptor-alpha were generated and expanded ex vivo in the presence of different cytokines (IL-2, IL-7, IL-15, IL-18, and IL-21), and their expansion, phenotype and cytotoxic capacity were evaluated, in vitro and in vivo. Moreover, the effect of the administration of these cytokines along with CART cells in vivo was also studied. IL-2, IL-7, and IL-15 favored the ex vivo expansion of CART cells compared to other cytokines or no cytokine treatment. IL-7 induced the highest proportion of memory stem cell-like CART cells in the final product, and IL-21 supported the expansion of CART cells with a younger phenotype, while IL-2 induced more differentiated CART cells. IL-2 and IL-15-exposed CART cells secreted more proinflammatory cytokines and presented stronger tumor-lysis ability in vitro. However, when tested in vivo, CART cells exposed to IL-2 ex vivo showed the least anti-tumor effect. In contrast, the administration of IL-15 and IL-21 in combination with CART cells in vivo increased their tumor killing capacity. According to our results, IL-7 and IL-15 show promise to promote ex vivo expansion of CART cells, while IL-15 and IL-21 seem better suited for in vivo administration after CART cell infusion. Collectively, these results may have a profound impact on the efficacy of CART cells in both hematologic and solid cancers. PMID:27409425

Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

PubMed

Fukuda, Takayuki; Takayama, Kazuo; Hirata, Mitsuhi; Liu, Yu-Jung; Yanagihara, Kana; Suga, Mika; Mizuguchi, Hiroyuki; Furue, Miho K

2017-03-15

Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A chemically defined substrate for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells.

PubMed

Tsai, Yihuan; Cutts, Josh; Kimura, Azuma; Varun, Divya; Brafman, David A

2015-07-01

Due to the limitation of current pharmacological therapeutic strategies, stem cell therapies have emerged as a viable option for treating many incurable neurological disorders. Specifically, human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs), a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS), could provide an unlimited source of cells for such cell-based therapies. However the clinical application of these cells will require (i) defined, xeno-free conditions for their expansion and neuronal differentiation and (ii) scalable culture systems that enable their expansion and neuronal differentiation in numbers sufficient for regenerative medicine and drug screening purposes. Current extracellular matrix protein (ECMP)-based substrates for the culture of hNPCs are expensive, difficult to isolate, subject to batch-to-batch variations, and, therefore, unsuitable for clinical application of hNPCs. Using a high-throughput array-based screening approach, we identified a synthetic polymer, poly(4-vinyl phenol) (P4VP), that supported the long-term proliferation and self-renewal of hNPCs. The hNPCs cultured on P4VP maintained their characteristic morphology, expressed high levels of markers of multipotency, and retained their ability to differentiate into neurons. Such chemically defined substrates will eliminate critical roadblocks for the utilization of hNPCs for human neural regenerative repair, disease modeling, and drug discovery. Copyright © 2015. Published by Elsevier B.V.

Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension Bioreactors.

PubMed

Almutawaa, Walaa; Rohani, Leili; Rancourt, Derrick E

2016-01-01

Human induced pluripotent stem cells (hiPSCs) hold great promise as a cell source for therapeutic applications and regenerative medicine. Traditionally, hiPSCs are expanded in two-dimensional static culture as colonies in the presence or absence of feeder cells. However, this expansion procedure is associated with lack of reproducibility and low cell yields. To fulfill the large cell number demand for clinical use, robust large-scale production of these cells under defined conditions is needed. Herein, we describe a scalable, low-cost protocol for expanding hiPSCs as aggregates in a lab-scale bioreactor.

Unstart Coupling Mechanism Analysis of Multiple-Modules Hypersonic Inlet

PubMed Central

Wang, Lei; Cao, Shibin

2013-01-01

The combination of multiplemodules in parallel manner is an important way to achieve the much higher thrust of scramjet engine. For the multiple-modules scramjet engine, when inlet unstarted oscillatory flow appears in a single-module engine due to high backpressure, how to interact with each module by massflow spillage, and whether inlet unstart occurs in other modules are important issues. The unstarted flowfield and coupling characteristic for a three-module hypersonic inlet caused by center module II and side module III were, conducted respectively. The results indicate that the other two hypersonic inlets are forced into unstarted flow when unstarted phenomenon appears on a single-module hypersonic inlet due to high backpressure, and the reversed flow in the isolator dominates the formation, expansion, shrinkage, and disappearance of the vortexes, and thus, it is the major factor of unstart coupling of multiple-modules hypersonic inlet. The coupling effect among multiple modules makes hypersonic inlet be more likely unstarted. PMID:24348146

Early and Definitive Diagnosis of Toxic Shock Syndrome by Detection of Marked Expansion of T-Cell-Receptor Vβ2-Positive T Cells

PubMed Central

Kato, Hidehito; Yamada, Ritsuko; Okano, Hiroya; Ohta, Hiroaki; Imanishi, Ken’ichi; Kikuchi, Ken; Totsuka, Kyouichi; Uchiyama, Takehiko

2003-01-01

We describe two cases of early toxic shock syndrome, caused by the superantigen produced from methicillin-resistant Staphylococcus aureus and diagnosed on the basis of an expansion of T-cell-receptor Vβ2-positive T cells. One case-patient showed atypical symptoms. Our results indicate that diagnostic systems incorporating laboratory techniques are essential for rapid, definitive diagnosis of toxic shock syndrome. PMID:12643839

Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.

PubMed Central

Kirby, S L; Cook, D N; Walton, W; Smithies, O

1996-01-01

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells. Images Fig. 3 PMID:8790342

On nonlinear evolution of low-frequency Alfvén waves in weakly-expanding solar wind plasmas

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

Nariyuki, Y.

A multi-dimensional nonlinear evolution equation for Alfvén waves in weakly-expanding solar wind plasmas is derived by using the reductive perturbation method. The expansion of solar wind plasma parcels is modeled by an expanding box model, which includes the accelerating expansion. It is shown that the resultant equation agrees with the Wentzel-Kramers-Brillouin prediction of the low-frequency Alfvén waves in the linear limit. In the cold and one-dimensional limit, a modified derivative nonlinear Schrodinger equation is obtained. Direct numerical simulations are carried out to discuss the effect of the expansion on the modulational instability of monochromatic Alfvén waves and the propagation ofmore » Alfvén solitons. By using the instantaneous frequency, it is quantitatively shown that as far as the expansion rate is much smaller than wave frequencies, effects of the expansion are almost adiabatic. It is also confirmed that while shapes of Alfvén solitons temporally change due to the expansion, some of them can stably propagate after their collision in weakly-expanding plasmas.« less

Vaccine-elicited memory CD4+ T cell expansion is impaired in the lungs during tuberculosis.

PubMed

Carpenter, Stephen M; Yang, Jason D; Lee, Jinhee; Barreira-Silva, Palmira; Behar, Samuel M

2017-11-01

Immunological memory is the key biological process that makes vaccines possible. Although tuberculosis vaccines elicit protective immunity in animals, few provide durable protection. To understand why protection is transient, we evaluated the ability of memory CD4+ T cells to expand, differentiate, and control Mycobacterium tuberculosis. Both naïve and memory CD4+ T cells initially proliferated exponentially, and the accumulation of memory T cells in the lung correlated with early bacterial control. However, later during infection, memory CD4+ T cell proliferation was curtailed and no protection was observed. We show that memory CD4+ T cells are first activated in the LN and their recruitment to the lung attenuates bacterial growth. However, their interaction with Mtb-infected macrophages does not promote continued proliferation. We conclude that a lack of sustained expansion by memory-derived T cells in the lung limits the durability of their protection, linking their slower expansion with transient protection in vaccinated mice.

Lhx4 and Prop1 are required for cell survival and expansion of the pituitary primordia.

PubMed

Raetzman, Lori T; Ward, Robert; Camper, Sally A

2002-09-01

Deficiencies in the homeobox transcription factors LHX4 and PROP1 cause pituitary hormone deficiency in both humans and mice. Lhx4 and Prop1 mutants exhibit severe anterior pituitary hypoplasia resulting from limited differentiation and expansion of most specialized cell types. Little is known about the mechanism through which these genes promote pituitary development. In this study we determined that the hypoplasia in Lhx4 mutants results from increased cell death and that the reduced differentiation is attributable to a temporal shift in Lhx3 activation. In contrast, Prop1 mutants exhibit normal cell proliferation and cell survival but show evidence of defective dorsal-ventral patterning. Molecular genetic analyses reveal that Lhx4 and Prop1 have overlapping functions in early pituitary development. Double mutants exhibit delayed corticotrope specification and complete failure of all other anterior pituitary cell types to differentiate. Thus, Lhx4 and Prop1 have critical, but mechanistically different roles in specification and expansion of specialized anterior pituitary cells.

A new subtype of progenitor cell in the mouse embryonic neocortex

PubMed Central

Wang, Xiaoqun; Tsai, Jin-Wu; LaMonica, Bridget; Kriegstein, Arnold R.

2011-01-01

A hallmark of mammalian brain evolution is cortical expansion, which reflects an increase in the number of cortical neurons established by the progenitor cell subtypes present and the number of their neurogenic divisions. Recent studies have revealed a new class of radial glia-like (oRG) progenitor cells in the human brain, which reside in the outer subventricular zone. Expansion of the subventricular zone and appearance of oRG cells may have been essential evolutionary steps leading from lissencephalic to gyrencephalic neocortex. Here we show that oRG-like progenitor cells are present in the mouse embryonic neocortex. They arise from asymmetric divisions of radial glia and undergo self-renewing asymmetric divisions to generate neurons. Moreover, mouse oRG cells undergo mitotic somal translocation whereby centrosome movement into the basal process during interphase preceeds nuclear translocation. Our finding of oRG cells in the developing rodent brain fills a gap in our understanding of neocortical expansion. PMID:21478886

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective

PubMed Central

Sonnaert, Maarten; Luyten, Frank P.; Papantoniou, Ioannis

2015-01-01

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion. PMID:26313143

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective.

PubMed

Sonnaert, Maarten; Luyten, Frank P; Schrooten, Jan; Papantoniou, Ioannis

2015-01-01

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion.

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

Trypanosomatid Infections: How Do Parasites and Their Excreted–Secreted Factors Modulate the Inducible Metabolism of l-Arginine in Macrophages?

PubMed Central

Holzmuller, Philippe; Geiger, Anne; Nzoumbou-Boko, Romaric; Pissarra, Joana; Hamrouni, Sarra; Rodrigues, Valérie; Dauchy, Frédéric-Antoine; Lemesre, Jean-Loup; Vincendeau, Philippe; Bras-Gonçalves, Rachel

2018-01-01

Mononuclear phagocytes (monocytes, dendritic cells, and macrophages) are among the first host cells to face intra- and extracellular protozoan parasites such as trypanosomatids, and significant expansion of macrophages has been observed in infected hosts. They play essential roles in the outcome of infections caused by trypanosomatids, as they can not only exert a powerful antimicrobial activity but also promote parasite proliferation. These varied functions, linked to their phenotypic and metabolic plasticity, are exerted via distinct activation states, in which l-arginine metabolism plays a pivotal role. Depending on the environmental factors and immune response elements, l-arginine metabolites contribute to parasite elimination, mainly through nitric oxide (NO) synthesis, or to parasite proliferation, through l-ornithine and polyamine production. To survive and adapt to their hosts, parasites such as trypanosomatids developed mechanisms of interaction to modulate macrophage activation in their favor, by manipulating several cellular metabolic pathways. Recent reports emphasize that some excreted–secreted (ES) molecules from parasites and sugar-binding host receptors play a major role in this dialog, particularly in the modulation of the macrophage’s inducible l-arginine metabolism. Preventing l-arginine dysregulation by drugs or by immunization against trypanosomatid ES molecules or by blocking partner host molecules may control early infection and is a promising way to tackle neglected diseases including Chagas disease, leishmaniases, and African trypanosomiases. The present review summarizes recent knowledge on trypanosomatids and their ES factors with regard to their influence on macrophage activation pathways, mainly the NO synthase/arginase balance. The review ends with prospects for the use of biological knowledge to develop new strategies of interference in the infectious processes used by trypanosomatids, in particular for the development of vaccines or immunotherapeutic approaches. PMID:29731753

β2-Microglobulin as a potential factor for the expansion of mesenchymal stem cells

PubMed Central

Zhu, Ying; Su, Yongping; Cheng, Tianmin; Chung, Leland W. K.

2010-01-01

Multipotent mesenchymal stem cells (MSCs) hold great promise in regenerative medicine, but one of the biggest challenges facing for their application is the ex vivo expansion to obtain enough undifferentiated cells. Fetal bovine serum (FBS), which can elicit possible contaminations of prion, virus, zoonosis or immunological reaction against xenogenic serum antigens, still remains essential to the culture formulations. There is an urgent need to identify potential factors for the undifferentiated expansion of MSCs to reduce the use of FBS or eventually replace it. A previously recognized housekeeping gene, β2-microglobulin (β2M), is demonstrated to act as a novel growth factor to stimulate the undifferentiated ex vivo expansion and preserve the pluripotency of adult MSCs from various sources. The use of β2M might have promising implications for future clinical application of MSCs. PMID:19466557

Transient Proliferation of Proanthocyanidin-Accumulating Cells on the Epidermal Apex Contributes to Highly Aluminum-Resistant Root Elongation in Camphor Tree1[W

PubMed Central

Osawa, Hiroki; Endo, Izuki; Hara, Yukari; Matsushima, Yuki; Tange, Takeshi

2011-01-01

Aluminum (Al) is a harmful element that rapidly inhibits the elongation of plant roots in acidic soils. The release of organic anions explains Al resistance in annual crops, but the mechanisms that are responsible for superior Al resistance in some woody plants remain unclear. We examined cell properties at the surface layer of the root apex in the camphor tree (Cinnamomum camphora) to understand its high Al resistance mechanism. Exposure to 500 μm Al for 8 d, more than 20-fold higher concentration and longer duration than what soybean (Glycine max) can tolerate, only reduced root elongation in the camphor tree to 64% of the control despite the slight induction of citrate release. In addition, Al content in the root apices was maintained at low levels. Histochemical profiling revealed that proanthocyanidin (PA)-accumulating cells were present at the adjacent outer layer of epidermis cells at the root apex, having distinctive zones for cell division and the early phase of cell expansion. Then the PA cells were gradually detached off the root, leaving thin debris behind, and the root surface was replaced with the elongating epidermis cells at the 3- to 4-mm region behind the tip. Al did not affect the proliferation of PA cells or epidermis cells, except for the delay in the start of expansion and the accelerated detachment of the former. In soybean roots, the innermost lateral root cap cells were absent in both PA accumulation and active cell division and failed to protect the epidermal cell expansion at 25 μm Al. These results suggest that transient proliferation and detachment of PA cells may facilitate the expansion of epidermis cells away from Al during root elongation in camphor tree. PMID:21045123

Transient proliferation of proanthocyanidin-accumulating cells on the epidermal apex contributes to highly aluminum-resistant root elongation in camphor tree.

PubMed

Osawa, Hiroki; Endo, Izuki; Hara, Yukari; Matsushima, Yuki; Tange, Takeshi

2011-01-01

Aluminum (Al) is a harmful element that rapidly inhibits the elongation of plant roots in acidic soils. The release of organic anions explains Al resistance in annual crops, but the mechanisms that are responsible for superior Al resistance in some woody plants remain unclear. We examined cell properties at the surface layer of the root apex in the camphor tree (Cinnamomum camphora) to understand its high Al resistance mechanism. Exposure to 500 μm Al for 8 d, more than 20-fold higher concentration and longer duration than what soybean (Glycine max) can tolerate, only reduced root elongation in the camphor tree to 64% of the control despite the slight induction of citrate release. In addition, Al content in the root apices was maintained at low levels. Histochemical profiling revealed that proanthocyanidin (PA)-accumulating cells were present at the adjacent outer layer of epidermis cells at the root apex, having distinctive zones for cell division and the early phase of cell expansion. Then the PA cells were gradually detached off the root, leaving thin debris behind, and the root surface was replaced with the elongating epidermis cells at the 3- to 4-mm region behind the tip. Al did not affect the proliferation of PA cells or epidermis cells, except for the delay in the start of expansion and the accelerated detachment of the former. In soybean roots, the innermost lateral root cap cells were absent in both PA accumulation and active cell division and failed to protect the epidermal cell expansion at 25 μm Al. These results suggest that transient proliferation and detachment of PA cells may facilitate the expansion of epidermis cells away from Al during root elongation in camphor tree.

The THO Complex Non-Cell-Autonomously Represses Female Germline Specification through the TAS3-ARF3 Module.

PubMed

Su, Zhenxia; Zhao, Lihua; Zhao, Yuanyuan; Li, Shaofang; Won, SoYoun; Cai, Hanyang; Wang, Lulu; Li, Zhenfang; Chen, Piaojuan; Qin, Yuan; Chen, Xuemei

2017-06-05

In most sexually reproducing plants, a single somatic, sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell, which is committed to giving rise to the female germline. However, it remains unclear how intercellular signaling among somatic cells results in only one cell in the sub-epidermal layer differentiating into the megaspore mother cell. Here we uncovered a role of the THO complex in restricting the megaspore mother cell fate to a single cell. Mutations in TEX1, HPR1, and THO6, components of the THO/TREX complex, led to the formation of multiple megaspore mother cells, which were able to initiate gametogenesis. We demonstrated that TEX1 repressed the megaspore mother cell fate by promoting the biogenesis of TAS3-derived trans-acting small interfering RNA (ta-siRNA), which represses ARF3 expression. The TEX1 protein was present in epidermal cells, but not in the germline, and, through TAS3-derived ta-siRNA, restricted ARF3 expression to the medio domain of ovule primordia. Expansion of ARF3 expression into lateral epidermal cells in a TAS3 ta-siRNA-insensitive mutant led to the formation of supernumerary megaspore mother cells, suggesting that TEX1- and TAS3-mediated restriction of ARF3 expression limits excessive megaspore mother cell formation non-cell-autonomously. Our findings reveal the role of a small-RNA pathway in the regulation of female germline specification in Arabidopsis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Association of Regulatory T-Cell Expansion With Progression of Amyotrophic Lateral Sclerosis: A Study of Humans and a Transgenic Mouse Model.

PubMed

Sheean, Rebecca K; McKay, Fiona C; Cretney, Erika; Bye, Christopher R; Perera, Nirma D; Tomas, Doris; Weston, Richard A; Scheller, Karlene J; Djouma, Elvan; Menon, Parvathi; Schibeci, Stephen D; Marmash, Najwa; Yerbury, Justin J; Nutt, Stephen L; Booth, David R; Stewart, Graeme J; Kiernan, Mathew C; Vucic, Steve; Turner, Bradley J

2018-06-01

Neuroinflammation appears to be a key modulator of disease progression in amyotrophic lateral sclerosis (ALS) and thereby a promising therapeutic target. The CD4+Foxp3+ regulatory T-cells (Tregs) infiltrating into the central nervous system suppress neuroinflammation and promote the activation of neuroprotective microglia in mouse models of ALS. To our knowledge, the therapeutic association of host Treg expansion with ALS progression has not been studied in vivo. To assess the role of Tregs in regulating the pathophysiology of ALS in humans and the therapeutic outcome of increasing Treg activity in a mouse model of the disease. This prospective multicenter human and animal study was performed in hospitals, outpatient clinics, and research institutes. Clinical and function assessment, as well as immunological studies, were undertaken in 33 patients with sporadic ALS, and results were compared with 38 healthy control participants who were consecutively recruited from the multidisciplinary ALS clinic at Westmead Hospital between February 1, 2013, and December 31, 2014. All data analysis on patients with ALS was undertaken between January 2015 and December 2016. Subsequently, we implemented a novel approach to amplify the endogenous Treg population using peripheral injections of interleukin 2/interleukin 2 monoclonal antibody complexes (IL-2c) in transgenic mice that expressed mutant superoxide dismutase 1 (SOD1), a gene associated with motor neuron degeneration. In patients with ALS, Treg levels were determined and then correlated with disease progression. Circulating T-cell populations, motor neuron size, glial cell activation, and T-cell and microglial gene expression in spinal cords were determined in SOD1G93A mice, as well as the association of Treg amplification with disease onset and survival time in mice. The cohort of patients with ALS included 24 male patients and 9 female patients (mean [SD] age at assessment, 58.9 [10.9] years). There was an inverse correlation between total Treg levels (including the effector CD45RO+ subset) and rate of disease progression (R = -0.40, P = .002). Expansion of the effector Treg population in the SOD1G93A mice was associated with a significant slowing of disease progression, which was accompanied by an increase in survival time (IL-2c-treated mice: mean [SD], 160.6 [10.8] days; control mice: mean [SD], 144.9 [10.6] days; P = .003). Importantly, Treg expansion was associated with preserved motor neuron soma size and marked suppression of astrocytic and microglial immunoreactivity in the spinal cords of SOD1G93A mice, as well as elevated neurotrophic factor gene expression in spinal cord and peripheral nerves. These findings establish a neuroprotective effect of Tregs, possibly mediated by suppression of toxic neuroinflammation in the central nervous system. Strategies aimed at enhancing the Treg population and neuroprotective activity from the periphery may prove therapeutically useful for patients with ALS.

Comparison of photovoltaic cell temperatures in modules operating with exposed and enclosed back surfaces

NASA Technical Reports Server (NTRS)

Namkoong, D.; Simon, F. F.

1981-01-01

Four different photovoltaic module designs were tested to determine the cell temperature of each design. The cell temperatures were compared to those obtained on identical design, using the same nominal operating cell temperature (NOCT) concept. The results showed that the NOCT procedure does not apply to the enclosed configurations due to continuous transient conditions. The enclosed modules had higher cell temperatures than the open modules, and insulated modules higher than the uninsulated. The severest performance loss - when translated from cell temperatures - 17.5 % for one enclosed, insulated module as a compared to that module mounted openly.

Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.

PubMed

Capmany, G; Querol, S; Cancelas, J A; García, J

1999-08-01

The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.

Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem and Progenitor Cells from Non‐Enriched Umbilical Cord Blood with Azole Compounds

PubMed Central

Bari, Sudipto; Zhong, Qixing; Fan, Xiubo; Poon, Zhiyong; Lim, Alvin Soon Tiong; Lim, Tse Hui; Dighe, Niraja; Li, Shang; Chiu, Gigi Ngar Chee; Chai, Christina Li Lin

2018-01-01

Abstract Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non‐enriched mononucleated cells (MNC) using novel azole‐based small molecules. Freshly‐thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure‐activity‐relationship studies of SB203580, a known p38‐MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8‐fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7‐fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600‐fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+CD34+ cells in the PB and BM by day 21 compared to non‐expanded and cytokine expanded grafts. The C7 expanded grafts maintained long‐term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre‐culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376–393 PMID:29392885

NK cell-derived IL-10 is critical for DC-NK cell dialogue at the maternal-fetal interface.

PubMed

Blois, Sandra M; Freitag, Nancy; Tirado-González, Irene; Cheng, Shi-Bin; Heimesaat, Markus M; Bereswill, Stefan; Rose, Matthias; Conrad, Melanie L; Barrientos, Gabriela; Sharma, Surendra

2017-05-19

DC-NK cell interactions are thought to influence the development of maternal tolerance and de novo angiogenesis during early gestation. However, it is unclear which mechanism ensures the cooperative dialogue between DC and NK cells at the feto-maternal interface. In this article, we show that uterine NK cells are the key source of IL-10 that is required to regulate DC phenotype and pregnancy success. Upon in vivo expansion of DC during early gestation, NK cells expressed increased levels of IL-10. Exogenous administration of IL-10 was sufficient to overcome early pregnancy failure in dams treated to achieve simultaneous DC expansion and NK cell depletion. Remarkably, DC expansion in IL-10 -/- dams provoked pregnancy loss, which could be abrogated by the adoptive transfer of IL-10 +/+ NK cells and not by IL-10 -/- NK cells. Furthermore, the IL-10 expressing NK cells markedly enhanced angiogenic responses and placental development in DC expanded IL-10 -/- dams. Thus, the capacity of NK cells to secrete IL-10 plays a unique role facilitating the DC-NK cell dialogue during the establishment of a healthy gestation.

Programmed Death 1 Regulates Memory Phenotype CD4 T Cell Accumulation, Inhibits Expansion of the Effector Memory Phenotype Subset and Modulates Production of Effector Cytokines

PubMed Central

Charlton, Joanna J.; Tsoukatou, Debbie; Mamalaki, Clio; Chatzidakis, Ioannis

2015-01-01

Memory phenotype CD4 T cells are found in normal mice and arise through response to environmental antigens or homeostatic mechanisms. The factors that regulate the homeostasis of memory phenotype CD4 cells are not clear. In the present study we demonstrate that there is a marked accumulation of memory phenotype CD4 cells, specifically of the effector memory (TEM) phenotype, in lymphoid organs and tissues of mice deficient for the negative co-stimulatory receptor programmed death 1 (PD-1). This can be correlated with decreased apoptosis but not with enhanced homeostatic turnover potential of these cells. PD-1 ablation increased the frequency of memory phenotype CD4 IFN-γ producers but decreased the respective frequency of IL-17A-producing cells. In particular, IFN-γ producers were more abundant but IL-17A producing cells were more scarce among PD-1 KO TEM-phenotype cells relative to WT. Transfer of peripheral naïve CD4 T cells suggested that accumulated PD-1 KO TEM-phenotype cells are of peripheral and not of thymic origin. This accumulation effect was mediated by CD4 cell-intrinsic mechanisms as shown by mixed bone marrow chimera experiments. Naïve PD-1 KO CD4 T cells gave rise to higher numbers of TEM-phenotype lymphopenia-induced proliferation memory cells. In conclusion, we provide evidence that PD-1 has an important role in determining the composition and functional aspects of memory phenotype CD4 T cell pool. PMID:25803808

Polymeric nanofibrous substrates stimulate pluripotent stem cells to form three-dimensional multilayered patty-like spheroids in feeder-free culture and maintain their pluripotency.

PubMed

Alamein, Mohammad A; Wolvetang, Ernst J; Ovchinnikov, Dmitry A; Stephens, Sebastien; Sanders, Katherine; Warnke, Patrick H

2015-09-01

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. Copyright © 2014 John Wiley & Sons, Ltd.

PRC2 inhibition counteracts the culture-associated loss of engraftment potential of human cord blood-derived hematopoietic stem and progenitor cells.

PubMed

Varagnolo, Linda; Lin, Qiong; Obier, Nadine; Plass, Christoph; Dietl, Johannes; Zenke, Martin; Claus, Rainer; Müller, Albrecht M

2015-07-22

Cord blood hematopoietic stem cells (CB-HSCs) are an outstanding source for transplantation approaches. However, the amount of cells per donor is limited and culture expansion of CB-HSCs is accompanied by a loss of engraftment potential. In order to analyze the molecular mechanisms leading to this impaired potential we profiled global and local epigenotypes during the expansion of human CB hematopoietic stem and progenitor cells (HPSCs). Human CB-derived CD34+ cells were cultured in serum-free medium together with SCF, TPO, FGF, with or without Igfbp2 and Angptl5 (STF/STFIA cocktails). As compared to the STF cocktail, the STFIA cocktail maintains in vivo repopulation capacity of cultured CD34+ cells. Upon expansion, CD34+ cells genome-wide remodel their epigenotype and depending on the cytokine cocktail, cells show different H3K4me3 and H3K27me3 levels. Expanding cells without Igfbp2 and Angptl5 leads to higher global H3K27me3 levels. ChIPseq analyses reveal a cytokine cocktail-dependent redistribution of H3K27me3 profiles. Inhibition of the PRC2 component EZH2 counteracts the culture-associated loss of NOD scid gamma (NSG) engraftment potential. Collectively, our data reveal chromatin dynamics that underlie the culture-associated loss of engraftment potential. We identify PRC2 component EZH2 as being involved in the loss of engraftment potential during the in vitro expansion of HPSCs.

Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy.

PubMed

Seay, Howard R; Putnam, Amy L; Cserny, Judit; Posgai, Amanda L; Rosenau, Emma H; Wingard, John R; Girard, Kate F; Kraus, Morey; Lares, Angela P; Brown, Heather L; Brown, Katherine S; Balavage, Kristi T; Peters, Leeana D; Bushdorf, Ashley N; Atkinson, Mark A; Bluestone, Jeffrey A; Haller, Michael J; Brusko, Todd M

2017-03-17

Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs) are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA)-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP)-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 10 9 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR). Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.

Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

PubMed Central

2013-01-01

Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

Severity of Acute Infectious Mononucleosis Correlates with Cross-Reactive Influenza CD8 T-Cell Receptor Repertoires.

PubMed

Aslan, Nuray; Watkin, Levi B; Gil, Anna; Mishra, Rabinarayan; Clark, Fransenio G; Welsh, Raymond M; Ghersi, Dario; Luzuriaga, Katherine; Selin, Liisa K

2017-12-05

Fifty years after the discovery of Epstein-Barr virus (EBV), it remains unclear how primary infection with this virus leads to massive CD8 T-cell expansion and acute infectious mononucleosis (AIM) in young adults. AIM can vary greatly in severity, from a mild transient influenza-like illness to a prolonged severe syndrome. We questioned whether expansion of a unique HLA-A2.01-restricted, cross-reactive CD8 T-cell response between influenza virus A-M1 58 (IAV-M1) and EBV BMLF1 280 (EBV-BM) could modulate the immune response to EBV and play a role in determining the severity of AIM in 32 college students. Only ex vivo total IAV-M1 and IAV-M1+EBV-BM cross-reactive tetramer + frequencies directly correlated with AIM severity and were predictive of severe disease. Expansion of specific cross-reactive memory IAV-M1 T-cell receptor (TCR) Vβ repertoires correlated with levels of disease severity. There were unique profiles of qualitatively different functional responses in the cross-reactive and EBV-specific CD8 T-cell responses in each of the three groups studied, severe-AIM patients, mild-AIM patients, and seropositive persistently EBV-infected healthy donors, that may result from differences in TCR repertoire use. IAV-M1 tetramer + cells were functionally cross-reactive in short-term cultures, were associated with the highest disease severity in AIM, and displayed enhanced production of gamma interferon, a cytokine that greatly amplifies immune responses, thus frequently contributing to induction of immunopathology. Altogether, these data link heterologous immunity via CD8 T-cell cross-reactivity to CD8 T-cell repertoire selection, function, and resultant disease severity in a common and important human infection. In particular, it highlights for the first time a direct link between the TCR repertoire with pathogenesis and the diversity of outcomes upon pathogen encounter. IMPORTANCE The pathogenic impact of immune responses that by chance cross-react to unrelated viruses has not been established in human infections. Here, we demonstrate that the severity of acute infectious mononucleosis (AIM), an Epstein-Barr virus (EBV)-induced disease prevalent in young adults but not children, is associated with increased frequencies of T cells cross-reactive to EBV and the commonly acquired influenza A virus (IAV). The T-cell receptor (TCR) repertoire and functions of these cross-reactive T cells differed between mild- and severe-AIM patients, most likely because these two groups of patients had selected different memory TCR repertoires in response to IAV infections encountered earlier. This heterologous immunity may explain variability in disease outcome and why young adults with more-developed IAV-specific memory T-cell pools have more-severe disease than children, who have less-developed memory pools. This study provides a new framework for understanding the role of heterologous immunity in human health and disease and highlights an important developing field examining the role of T-cell repertoires in the mediation of immunopathology. Copyright © 2017 Aslan et al.

Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance.

PubMed

Ito, Kyoko; Turcotte, Raphaël; Cui, Jinhua; Zimmerman, Samuel E; Pinho, Sandra; Mizoguchi, Toshihide; Arai, Fumio; Runnels, Judith M; Alt, Clemens; Teruya-Feldstein, Julie; Mar, Jessica C; Singh, Rajat; Suda, Toshio; Lin, Charles P; Frenette, Paul S; Ito, Keisuke

2016-12-02

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2 + HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2 + HSCs. Activation of the PPAR (peroxisome proliferator-activated receptor)-fatty acid oxidation pathway promotes expansion of Tie2 + HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2 + HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways. Copyright © 2016, American Association for the Advancement of Science.

Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance

PubMed Central

Ito, Kyoko; Turcotte, Raphaël; Cui, Jinhua; Zimmerman, Samuel E.; Pinho, Sandra; Mizoguchi, Toshihide; Arai, Fumio; Runnels, Judith M.; Alt, Clemens; Teruya-Feldstein, Julie; Mar, Jessica C.; Singh, Rajat; Suda, Toshio; Lin, Charles P.; Frenette, Paul S.; Ito, Keisuke

2016-01-01

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2+ HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2+ HSCs. Activation of the PPAR (peroxisome proliferator–activated receptor)–fatty acid oxidation pathway promotes expansion of Tie2+ HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2+ HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways. PMID:27738012

Following the 'tracks': Tramtrack69 regulates epithelial tube expansion in the Drosophila ovary through Paxillin, Dynamin, and the homeobox protein Mirror.

PubMed

Peters, Nathaniel C; Thayer, Nathaniel H; Kerr, Scott A; Tompa, Martin; Berg, Celeste A

2013-06-15

Epithelial tubes are the infrastructure for organs and tissues, and tube morphogenesis requires precise orchestration of cell signaling, shape, migration, and adhesion. Follicle cells in the Drosophila ovary form a pair of epithelial tubes whose lumens act as molds for the eggshell respiratory filaments, or dorsal appendages (DAs). DA formation is a robust and accessible model for studying the patterning, formation, and expansion of epithelial tubes. Tramtrack69 (TTK69), a transcription factor that exhibits a variable embryonic DNA-binding preference, controls DA lumen volume and shape by promoting tube expansion; the tramtrack mutation twin peaks (ttk(twk)) reduces TTK69 levels late in oogenesis, inhibiting this expansion. Microarray analysis of wild-type and ttk(twk) ovaries, followed by in situ hybridization and RNAi of candidate genes, identified the Phospholipase B-like protein Lamina ancestor (LAMA), the scaffold protein Paxillin, the endocytotic regulator Shibire (Dynamin), and the homeodomain transcription factor Mirror, as TTK69 effectors of DA-tube expansion. These genes displayed enriched expression in DA-tube cells, except lama, which was expressed in all follicle cells. All four genes showed reduced expression in ttk(twk) mutants and exhibited RNAi phenotypes that were enhanced in a ttk(twk)/+ background, indicating ttk(twk) genetic interactions. Although previous studies show that Mirror patterns the follicular epithelium prior to DA tubulogenesis, we show that Mirror has an independent, novel role in tube expansion, involving positive regulation of Paxillin. Thus, characterization of ttk(twk)-differentially expressed genes expands the network of TTK69 effectors, identifies novel epithelial tube-expansion regulators, and significantly advances our understanding of this vital developmental process. Copyright © 2013 Elsevier Inc. All rights reserved.

Nucleoid halo expansion indirectly measures DNA damage in single cells.

PubMed

Thomas, E A; Thomas, C A

1989-07-01

A simple test has been developed that measures how much DNA damage has occurred in a single mammalian cell. The procedure is based on the microscopic examination of "halos" of nucleoids that adhere to coverslips. Nucleoids are produced by flowing salt solutions containing detergents over the attached cells. The nucleoid halos are thought to be a tangle of loops of free DNA that emanate from the remnants of the nucleus. When visualized by staining with ethidium bromide the nucleoid halos first expand, and then contract as the concentration of ethidium increases. Exposure of nucleoids to very low levels of DNA chain-breaking treatments results in the incremental expansion of the halos to a maximum of 15 microns or more. Our assay is based upon quantitating the degree of halo expansion. If intact cells are exposed to DNA-damaging treatments, then allowed increasing periods of post-treatment growth before forming nucleoids, the DNA repair processes result first in expanded and then in contracted halos. By admixing a supercoiled plasma DNA of known length (38 kb) to nucleoids with contracted halos, the fractional halo expansion and the fraction of surviving plasmid supercoils can be measured from the same solution. Use of photodynamic DNA damage showed that the halo expansion was 11.6 times more sensitive than plasmid relaxation. Use of gamma-irradiation showed that the halo expansion was 3.6 times more sensitive than plasmid relaxation. The latter value demonstrates that one break per 137,000 bp results in the expansion of the halos to 63% of their maximal value. We estimate that this method will detect about 5000 breaks per nucleus containing 5 x 10(9) bp.

Promotion of initiated cells by radiation-induced cell inactivation.

PubMed

Heidenreich, W F; Paretzke, H G

2008-11-01

Cells on the way to carcinogenesis can have a growth advantage relative to normal cells. It has been hypothesized that a radiation-induced growth advantage of these initiated cells might be induced by an increased cell replacement probability of initiated cells after inactivation of neighboring cells by radiation. Here Monte Carlo simulations extend this hypothesis for larger clones: The effective clonal expansion rate decreases with clone size. This effect is stronger for the two-dimensional than for the three-dimensional situation. The clones are irregular, far from a circular shape. An exposure-rate dependence of the effective clonal expansion rate could come in part from a minimal recovery time of the initiated cells for symmetric cell division.

Ex-vivo expansion of CFU-GM and BFU-E in unselected PBMC cultures with Flt3L is enhanced by autologous plasma.

PubMed

Guo, M; Miller, W M; Papoutsakis, E T; Patel, S; James, C; Goolsby, C; Winter, J N

1999-01-01

Previous ex-vivo expansion studies in our laboratory, comparing unselected and CD34(+)-selected PBMC, have shown no advantage for CD34(+) cell selection, in terms of the expansion achieved. Our goal was to develop procedures for consistent generation of large numbers of hematopoietic progenitor and post-progenitor cells from unselected PBMC. Unselected PBMC, collected from cancer patients undergoing apheresis prior to high-dose chemotherapy and autologous stem cell rescue, were expanded ex vivo in static cultures, without a stromal layer, in the presence of Flt3 ligand (Flt3L), a recombinant GM-CSF/IL-3 fusion protein (PIXY321), G-CSF and GM-CSF for 10 days. The addition of 2% autologous plasma to this cytokine combination enhanced expansion of total cell numbers (3.2 fold versus 1.9 fold; p < 0.01), colony-forming units granulocyte-macrophage (CFU-GM) (22.0 fold versus 8.1 fold, p < 0.01) and burst-forming units erythroid (BFU-E) (17.6 fold versus 7.0 fold, 0.01 < p < 0.02). The optimal seeding density for a given specimen was inversely related to the frequency of CD34(+) cells in the sample. CFU-GM expansion with the Flt3L-containing cytokine cocktail was equivalent to that obtained with IL-3, IL-6, G-CSF and SCF, whether or not the cultures were supplemented with autologous plasma. In plasma-free cultures, BFU-E expansion was significantly higher with IL-3, IL-6, G-CSF and SCF than with Flt3L, PIXY321, G-CSF and GM-CSF. In the presence of autologous plasma, however BFU-E expansion was higher in the Flt3L-containing media. In comparison studies, autologous plasma suppressed BFU-E expansion in SCF-containing cultures. Consistent with our colony assay results, dual-parameter flow cytometric analysis of the expanded cell population revealed that supplementation with autologous plasma yielded a significant increase in the numbers of myeloid progenitors in Flt3L-containing cultures. Unselected PBMC from cancer patients can be effectively expanded ex vivo in Flt3L, PIXY321, G-CSF and GM-CSF, supplemented with autologous plasma, yielding high numbers of myeloid and erythroid progenitors.

Retinal pigment epithelium expansion around the neural retina occurs in two separate phases with distinct mechanisms.

PubMed

Cechmanek, Paula Bernice; McFarlane, Sarah

2017-08-01

The retinal pigment epithelium (RPE) is a specialized monolayer of epithelial cells that forms a tight barrier surrounding the neural retina. RPE cells are indispensable for mature photoreceptor renewal and survival, yet how the initial RPE cell population expands around the neural retina during eye development is poorly understood. Here we characterize the differentiation, proliferation, and movements of RPE progenitors in the Zebrafish embryo over the period of optic cup morphogenesis. RPE progenitors are present in the dorsomedial eye vesicle shortly after eye vesicle evagination. We define two separate phases that allow for full RPE expansion. The first phase involves a previously uncharacterized antero-wards expansion of the RPE progenitor domain in the inner eye vesicle leaflet, driven largely by an increase in cell number. During this phase, RPE progenitors start to express differentiation markers. In the second phase, the progenitor domain stretches in the dorsoventral and posterior axes, involving cell movements and shape changes, and coinciding with optic cup morphogenesis. Significantly, cell division is not required for RPE expansion. RPE development to produce the monolayer epithelium that covers the back of the neural retina occurs in two distinct phases driven by distinct mechanisms. Developmental Dynamics 246:598-609, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Skin cell isolation and expansion for cell transplantation is limited in patients using tobacco, alcohol, or are exhibiting diabetes mellitus.

PubMed

Johnen, Christa; Hartmann, Bernd; Steffen, Ingo; Bräutigam, Kirsten; Witascheck, Tom; Toman, Nidal; Küntscher, Markus V; Gerlach, Jörg C

2006-03-01

The aim of this exploratory study was to investigate the isolation and expansion of keratinocytes and fibroblasts from donors with certain medical histories. Biopsies were taken from donors (N=32) falling into one or more of the following categories: a history of heavy smoking and/or alcohol abuse, drug abuse, diabetes mellitus or steroid treatment. Cells from donors who did not fall into any of the above-mentioned categories were used as controls. Proliferation and growth behaviour of cells were analyzed by measurement of passage duration, absorbance (MTT-assay) and light microscopy. Donors with a specific medical history required larger biopsy areas than the control group for isolating a sufficient number of fibroblasts and keratinocytes. Times to confluence were significantly prolonged and absorbances (MTT) were significantly reduced in several donor groups when compared to control cultures. Biopsies from donors with steroid treatment, drug abuse and combined nicotine and alcohol abuse could not be established beyond passage 0 degrees or 1 degree, respectively. We conclude that isolation and expansion of skin cells from donors with certain medical histories may require larger biopsies, prolonged expansion times or may even result in failure. These findings may therefore be of clinical importance in the field of autologous skin cell transplantation.

An organ-specific role for ethylene in rose petal expansion during dehydration and rehydration

PubMed Central

Liu, Daofeng; Liu, Xiaojing; Meng, Yonglu; Sun, Cuihui; Tang, Hongshu; Jiang, Yudong; Khan, Muhammad Ali; Xue, Jingqi; Ma, Nan; Gao, Junping

2013-01-01

Dehydration is a major factor resulting in huge loss from cut flowers during transportation. In the present study, dehydration inhibited petal cell expansion and resulted in irregular flowers in cut roses, mimicking ethylene-treated flowers. Among the five floral organs, dehydration substantially elevated ethylene production in the sepals, whilst rehydration caused rapid and elevated ethylene levels in the gynoecia and sepals. Among the five ethylene biosynthetic enzyme genes (RhACS1–5), expression of RhACS1 and RhACS2 was induced by dehydration and rehydration in the two floral organs. Silencing both RhACS1 and RhACS2 significantly suppressed dehydration- and rehydration-induced ethylene in the sepals and gynoecia. This weakened the inhibitory effect of dehydration on petal cell expansion. β-glucuronidase activity driven by both the RhACS1 and RhACS2 promoters was dramatically induced in the sepals, pistil, and stamens, but not in the petals of transgenic Arabidopsis. This further supports the organ-specific induction of these two genes. Among the five rose ethylene receptor genes (RhETR1–5), expression of RhETR3 was predominantly induced by dehydration and rehydration in the petals. RhETR3 silencing clearly aggravated the inhibitory effect of dehydration on petal cell expansion. However, no significant difference in the effect between RhETR3-silenced flowers and RhETR-genes-silenced flowers was observed. Furthermore, RhETR-genes silencing extensively altered the expression of 21 cell expansion-related downstream genes in response to ethylene. These results suggest that induction of ethylene biosynthesis by dehydration proceeds in an organ-specific manner, indicating that ethylene can function as a mediator in dehydration-caused inhibition of cell expansion in rose petals. PMID:23599274

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

PubMed Central

Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

TGFβ Pathway Inhibition Redifferentiates Human Pancreatic Islet β Cells Expanded In Vitro

PubMed Central

Toren-Haritan, Ginat; Efrat, Shimon

2015-01-01

In-vitro expansion of insulin-producing cells from adult human pancreatic islets could provide an abundant cell source for diabetes therapy. However, proliferation of β-cell-derived (BCD) cells is associated with loss of phenotype and epithelial-mesenchymal transition (EMT). Nevertheless, BCD cells maintain open chromatin structure at β-cell genes, suggesting that they could be readily redifferentiated. The transforming growth factor β (TGFβ) pathway has been implicated in EMT in a range of cell types. Here we show that human islet cell expansion in vitro involves upregulation of the TGFβ pathway. Blocking TGFβ pathway activation using short hairpin RNA (shRNA) against TGFβ Receptor 1 (TGFBR1, ALK5) transcripts inhibits BCD cell proliferation and dedifferentiation. Treatment of expanded BCD cells with ALK5 shRNA results in their redifferentiation, as judged by expression of β-cell genes and decreased cell proliferation. These effects, which are reproducible in cells from multiple human donors, are mediated, at least in part, by AKT-FOXO1 signaling. ALK5 inhibition synergizes with a soluble factor cocktail to promote BCD cell redifferentiation. The combined treatment may offer a therapeutically applicable way for generating an abundant source of functional insulin-producing cells following ex-vivo expansion. PMID:26418361

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

PubMed

Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS. ePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS.