Progesterone, Inflammatory Cytokine (TNF-α), and Oxidative Stress (H2O2) Regulate Progesterone Receptor Membrane Component 1 Expression in Fetal Membrane Cells.

PubMed

Meng, Yan; Murtha, Amy P; Feng, Liping

2016-09-01

Progesterone receptor membrane component 1 (PGRMC1) is an important novel mediator of progesterone (P4) function in fetal membrane cells. We demonstrated previously that PGRMC1 is differentially expressed in fetal membranes among pregnancy subjects and diminished in preterm premature rupture of membrane subjects. In the current study, we aim to elucidate whether PGRMC1 expression is regulated by P4, tumor necrosis factor α (TNF-α), and H2O2 in fetal membrane cells. Primary cultured membrane cells were serum starved for 24 hours followed by treatments of P4, 17 hydroxyprogesterone caproate, and medroxyprogesterone 17 acetate (MPA) at 10(-7) mol/L with ethanol as vehicle control; TNF-α at 10, 20, and 50 ng/mL with phosphate-buffered saline (PBS) as control; and H2O2 at 10 and 100 μmol/L with culture media as control for 24, 48, and 72 hours. The messenger RNA (mRNA) and protein expression of PGRMC1 was quantified using polymerase chain reaction and Western blotting, respectively. We found that PGRMC1 protein expression was regulated by MPA, TNF-α, and H2O2 in a dose-dependent manner. This regulation is also specific to the type of cell (amnion, chorion, or decidua). The upregulation of PGRMC1 by MPA might be mediated through glucocorticoid receptor (GR) demonstrated using amnion and chorion cells model with GR knockdown by specific small interfering RNA transfection. The mRNA expression of PGRMC1 was decreased by H2O2 (100 μmol/L) treatment in amnion cells, which might ultimately result in downregulation of PGRMC1 protein as our data demonstrated. None of other treatments changed PGRMC1 mRNA level in these cells. We conclude that these stimuli act as regulatory factors of PGRMC1 in a cell-specific manner. © The Author(s) 2016.

Amnion allografts prepared in the Central Tissue Bank in Warsaw.

PubMed

Tyszkiewicz, J T; Uhrynowska-Tyszkiewicz, I A; Kaminski, A; Dziedzic-Goclawska, A

1999-01-01

Applications of allogenic amnion grafts range from wound dressing of severe burns, dermabrasions and lower extremity ulcer treatments to plastic surgery, laryngology and ophthalmology. The aim of the present study was to elaborate the method of processing, preservation and sterilization of human amnion allografts prepared as wound dressing used mainly for burned patients. During the amniotic sac processing (after separation of chorion) special attention was paid to ensure that the epithelial side of amnion is placed directly on polyester net used as a support. After application on the wound, the epithelial side with the basement membrane is facing outwards; this will promote migration, attachment and spreading of the host cells encouraging epithelialization. Human amnion allografts were preserved by lyophilization or deep-freezing and subsequently radiation-sterilized with a dose of 35 kGy. It has been observed, however, that lyophilized irradiated allografts are resorbed within a few days, while frozen irradiated ones better adhere to wound and persist even 3 weeks after grafting, therefore, it has been decided to preserve amnion by deep-freezing. Since the beginning of 1998 over 400 preserved radiation-sterilized amnion allografts (with a total surface area over 40,000 cm2) have been prepared at the Central Tissue Bank in Warsaw and distributed to clinics and hospitals throughout the country.

Increased oxidative stress in human fetal membranes overlying the cervix from term non-labouring and post labour deliveries.

PubMed

Chai, M; Barker, G; Menon, R; Lappas, M

2012-08-01

Enzymatic breakdown of the collagen-rich extracellular matrix (ECM) that connects the amnion and chorion layers of the fetal membranes is one of the key events leading to rupture of membranes. Oxidant stress caused by increased formation of reactive oxygen species and/or reduced antioxidant capacity may predispose to membrane rupture, a major cause of preterm birth. The aim of this study was to determine the effect of human labour and supracervical (SC) apposition on antioxidant enzymes and 8-isoprostane (a marker of lipid peroxidation). To determine the effect of human labour on oxidative stress status, fetal membranes from the SC site (SCS) were collected from women at term Caesarean section (no labour), and from the site of membrane rupture (SOR) after spontaneous labour onset and delivery (post labour). To determine the effect of SC apposition on oxidative stress status, amnion was collected from the SCS and a distal site (DS) in women at term Caesarean section in the absence of labour. The release of 8-isoprostane was significantly higher in amnion from the SCS compared to DS, and in fetal membranes from the SOR compared to the SCS. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were lower in amnion from the SC compared to DS. SOD gene expression and enzyme activity were lower in fetal membranes after labour. There was no difference in expression or activity in catalase, GPx and glutathione reductase (GSR) between no labour and post labour fetal membranes. In primary amnion cells, SOD supplementation significantly augmented IL-1β induced MMP-9 expression and activity. In summary, non-labouring SC fetal membranes are characterised by reduced antioxidant enzyme activity when compared to distal membranes, and, as such, may be more susceptible to oxidative damage and thus membrane rupture. Copyright © 2012 Elsevier Ltd. All rights reserved.

Evaluation of the cranial base in amnion rupture sequence involving the anterior neural tube: implications regarding recurrence risk.

PubMed

Jones, Kenneth Lyons; Robinson, Luther K; Benirschke, Kurt

2006-09-01

Amniotic bands can cause disruption of the cranial end of the developing fetus, leading in some cases to a neural tube closure defect. Although recurrence for unaffected parents of an affected child with a defect in which the neural tube closed normally but was subsequently disrupted by amniotic bands is negligible; for a primary defect in closure of the neural tube to which amnion has subsequently adhered, recurrence risk is 1.7%. In that primary defects of neural tube closure are characterized by typical abnormalities of the base of the skull, evaluation of the cranial base in such fetuses provides an approach for making a distinction between these 2 mechanisms. This distinction has implications regarding recurrence risk. The skull base of 2 fetuses with amnion rupture sequence involving the cranial end of the neural tube were compared to that of 1 fetus with anencephaly as well as that of a structurally normal fetus. The skulls were cleaned, fixed in 10% formalin, recleaned, and then exposed to 10% KOH solution. After washing and recleaning, the skulls were exposed to hydrogen peroxide for bleaching and photography. Despite involvement of the anterior neural tube in both fetuses with amnion rupture sequence, in Case 3 the cranial base was normal while in Case 4 the cranial base was similar to that seen in anencephaly. This technique provides a method for determining the developmental pathogenesis of anterior neural tube defects in cases of amnion rupture sequence. As such, it provides information that can be used to counsel parents of affected children with respect to recurrence risk.

Modulation of Cytokine-Induced Cyclooxygenase 2 Expression by PPARG Ligands Through NFκB Signal Disruption in Human WISH and Amnion Cells1

PubMed Central

Ackerman, William E.; Zhang, Xiaolan L.; Rovin, Brad H.; Kniss, Douglas A.

2006-01-01

Cyclooxygenase (COX) activity increases in the human amnion in the settings of term and idiopathic preterm labor, contributing to the generation of uterotonic prostaglandins (PGs) known to participate in mammalian parturition. Augmented COX activity is highly correlated with increased COX2 (also known as prostaglandin-endoperoxide synthase 2, PTGS2) gene expression. We and others have demonstrated an essential role for nuclear factor κB (NFκB) in cytokine-driven COX2 expression. Peroxisome proliferator-activated receptor gamma (PPARG), a member of the nuclear hormone receptor superfamily, has been shown to antagonize NFκB activation and inflammatory gene expression, including COX2. We hypothesized that PPARG activation might suppress COX2 expression during pregnancy. Using primary amnion and WISH cells, we evaluated the effects of pharmacological (thiazolidinediones) and putative endogenous (15-deoxy-Δ12,14-prostaglandin J2, 15d-PGJ2) PPARG ligands on cytokine-induced NFκB activation, COX2 expression, and PGE2 production. We observed that COX2 expression and PGE2 production induced by tumor necrosis factor alpha (TNF) were significantly abrogated by 15d-PGJ2. The thiazolidinediones rosiglitazone (ROSI) and troglitazone (TRO) had relatively little effect on cytokine-induced COX2 expression except at high concentrations, at which these agents tended to increase COX2 abundance relative to cells treated with TNF alone. Interestingly, treatment with ROSI, but not TRO, led to augmentation of TNF-stimulated PGE2 production. Mechanistically, we observed that 15d-PGJ2 markedly diminished cytokine-induced activity of the NFκB transcription factor, whereas thiazolidinediones had no discernable effect on this system. Our data suggest that pharmacological and endogenous PPARG ligands use both receptor-dependent and -independent mechanisms to influence COX2 expression. PMID:15843495

Standardized limbal epithelial stem cell graft generation and transplantation.

PubMed

Zakaria, Nadia; Koppen, Carina; Van Tendeloo, Viggo; Berneman, Zwi; Hopkinson, Andrew; Tassignon, Marie-José

2010-10-01

To describe a standardized, xenogenic-free protocol for the manufacture of limbal epithelial stem cell grafts and a "no touch" surgical technique for its standardized transplantation. Antwerp University Hospital, Antwerp, Belgium. The limbo-amnion composite graft is generated by cultivating limbal epithelial stem cells on a standardized (thermolysin treated and spongy layer removed) amniotic membrane, stretched within an interlockable amnion ring. The cells are cultured in CnT-20 medium with the addition of 1% human AB serum for a period of 2 weeks. Fibrin glue is applied to the surgically prepared recipient's cornea and in one fluid motion, the composite graft within the amnion ring construct is transferred from culture and positioned onto the graft bed. The required size is cut out at the level of the limbus by means of a trephine and/or microsurgical scissors. The lightweight, plastic interlockable ring offered stability to the graft during culture, transport, and transplantation. The use of the standardized amniotic membrane, within the amnion ring construct, improves reproducibility of the results and therefore heralds elective surgery. Rapid transplantation of a wrinkle-free graft, using a sutureless, “no touch" technique was achieved and this allowed precise tailoring of the graft to the recipient bed. This is the first time a standardized, clinical grade protocol has been described for manufacturing limbal epithelial grafts with an efficient surgical technique that prevents postsurgical graft shrinkage and improves corneal integration. The quick, sutureless, and manipulation-free technique ensured transplantation of viable, proliferating limbal epithelial stem cells.

Large Extremity Peripheral Nerve Repair

DTIC Science & Technology

2014-10-01

has also been shown to produce human-beta-3-defensin. These antimicrobial peptides are implicated in the resistance of epithelial surfaces to...gonadotrophin receptors that regulate prostaglandin production and activity. Epithelial cells manufacture multiple vasoactive peptides , growth factors...200734 P-RCT (n Z 102) PT burns Processed Amnion vs topical antimicrobials . Significantly less dressing changes with amnion. Time to healing, length

Fetal Fibronectin Signaling Induces Matrix Metalloproteases and Cyclooxygenase-2 (COX-2) in Amnion Cells and Preterm Birth in Mice*

PubMed Central

Mogami, Haruta; Kishore, Annavarapu Hari; Shi, Haolin; Keller, Patrick W.; Akgul, Yucel; Word, R. Ann

2013-01-01

Fetal fibronectin (fFN) in cervical and vaginal secretions has been used as a predictor of preterm delivery. Here, we clarified the pathological function of fFN on cell type-specific matrix metalloproteinases (MMPs) and prostaglandin synthesis in fetal membranes. Treatment of amnion mesenchymal cells with fFN resulted in dramatic increases in MMP-1 and MMP-9 mRNA and enzymatic activity as well as COX-2 mRNA and prostaglandin E2 synthesis, activating both NFκB and ERK1/2 signaling. Fetal FN-induced increases in MMPs and COX-2 were mediated through its extra domain A and Toll-like receptor 4 expressed in mesenchymal cells. Lipopolysaccharide and TNF-α increased the release of free FN in medium of amnion epithelial cells in culture. Finally, injection of fFN in pregnant mice resulted in preterm birth. Collectively, these results indicate that fFN is not only a marker of preterm delivery but also plays a significant role in the pathogenesis of preterm labor and premature rupture of fetal membranes. PMID:23184961

Hepatitis A virus cellular receptor 2 (HAVCR2) is decreased with viral infection and regulates pro-labour mediators OA.

PubMed

Liong, Stella; Lim, Ratana; Barker, Gillian; Lappas, Martha

2017-07-01

Intrauterine infection caused by viral infection has been implicated to contribute to preterm birth. Hepatitis A virus cellular receptor 2 (HAVCR2) regulates inflammation in non-gestational tissues in response to viral infection. The aims of this study were to determine the effect of: (i) viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) on HAVCR2 expression; and (ii) HAVCR2 silencing by siRNA (siHAVCR2) in primary amnion and myometrial cells on poly(I:C)-induced inflammation. In human foetal membranes and myometrium, HAVCR2 mRNA and protein expression was decreased when exposed to poly(I:C). Treatment of primary amnion and myometrial cells with poly(I:C) significantly increased the expression and release of pro-inflammatory cytokines TNF, IL1A, IL1B and IL6; the expression of chemokines CXCL8 and CCL2; the expression and secretion of adhesion molecules ICAM1 and VCAM1; and PTGS2 and PTGFR mRNA expression and the release of prostaglandin PGF 2α . This increase was significantly augmented in cells transfected with siHAVCR2. Furthermore, mRNA expression of anti-inflammatory cytokines IL4 and IL10 was significantly decreased. Collectively, our data suggest that HAVCR2 regulates cytokines, chemokines, prostaglandins and cell adhesion molecules in the presence of viral infection. This suggests a potential for HAVCR2 activators as therapeutics for the management of preterm birth associated with viral infections. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaluation of Amnion-derived Multipotent Progenitor (AMP) Cells and Amnion-derived Cellular Cytokine Solution (ST266) in Promoting Craniomaxillofacial Regenerative Bone Healing in Critical Size Calvarial Defects

DTIC Science & Technology

2017-10-10

action. MATERIALS AND METHODS Subjects Subjects used in the study include male Fischer 344 (CDF®) rats weighing 280-300 grams obtained from...PBS before being transferred to a flat bottom 96-well plate. Absorbance was then read at 450nm on a Biotek microplate reader. Live/Dead Staining of...incubation, live/dead staining of the scaffolds was imaged on a confocal microscope (Nikon). AMP Cell Differentiation To determine whether AMP

Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing.

PubMed

Koob, Thomas J; Lim, Jeremy J; Zabek, Nicole; Massee, Michelle

2015-07-01

Human amniotic membrane allografts have proven effective at improving healing of cutaneous wounds. The mechanism of action for these therapeutic effects is poorly understood but is thought to involve the resident growth factors present in near term amniotic tissue. To determine the relative cytokine contribution of the amnion and chorion in amniotic allografts, the content of 18 cytokines involved in wound healing were measured in samples of PURION® Processed dehydrated amnion, chorion, and amnion/chorion membrane (dHACM) grafts by multiplex enzyme-linked immunosorbent assay array. Both amnion and chorion contained similar amounts of each factor when normalized per dry weight; however, when calculated per surface area of tissue applied to a wound, amnion contained on average only 25% as much of each factor as the chorion. Therefore, an allograft containing both amnion and chorion would contain four to five times more cytokine than a single layer amnion allograft alone. Both single layer amnion and multilayer allografts containing amnion and chorion are currently marketed for wound repair. To examine the role of tissue processing technique in cytokine retention, cytokine contents in representative dehydrated single layer wound care products were measured. The results demonstrated that cytokine content varied significantly among the allografts tested, and that PURION® Processed single layer amnion grafts contained more cytokines than other single layer products. These results suggest that PURION® Processed dHACM contains substantially more cytokines than single layer amnion products, and therefore dHACM may be more effective at delivering growth factors to a healing wound than amnion alone. © 2014 Wiley Periodicals, Inc.

Generation of Two Biological Wound Dressings as a Potential Delivery System of Human Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Brena-Molina, Ana; Martínez-López, Valentín; Melgarejo-Ramírez, Yaaziel; Tamay de Dios, Lenin; Gómez-García, Ricardo; Reyes-Frías, Ma. de Lourdes; Rodríguez-Rodríguez, Lourdes; Garciadiego-Cázares, David; Lugo-Martínez, Haydée; Ibarra, Clemente

2015-01-01

Human adipose-derived mesenchymal stem cells (hADMSCs) are believed to be potential key factors for starting the regenerative process after tissue injury. However, an efficient method of delivering these regenerative cells to an external wound site is still lacking. Human amnion and pig skin have long been used as skin wound dressings for the treatment of burns and other skin lesions. Herein, we present the generation of two constructs using these two biomaterials as effective scaffolds for the culture of hADMSCs. It was found that hADMSCs seeded onto radiosterilized human amnion and pig skin are viable and proliferate. These cells are able to migrate over these scaffolds as demonstrated by using time-lapse microscopy. In addition, the scaffolds induce hADMSCs to secrete interleukin-10, an important negative regulator of inflammation, and interleukin-1β, a proinflammatory protein. The interplay between these two proteins has been proven to be vital for a balanced restoration of all necessary tissues. Thus, radiosterilized human amnion and pig skin are likely suitable scaffolds for delivery of hADMSCs transplants that could promote tissue regeneration in skin injuries like patients with burn injuries. PMID:26418201

Amnion-derived stem cells: in quest of clinical applications

PubMed Central

2011-01-01

In the promising field of regenerative medicine, human perinatal stem cells are of great interest as potential stem cells with clinical applications. Perinatal stem cells could be isolated from normally discarded human placentae, which are an ideal cell source in terms of availability, the fewer number of ethical concerns, less DNA damage, and so on. Numerous studies have demonstrated that some of the placenta-derived cells possess stem cell characteristics like pluripotent differentiation ability, particularly in amniotic epithelial (AE) cells. Term human amniotic epithelium contains a relatively large number of stem cell marker-positive cells as an adult stem cell source. In this review, we introduce a model theory of why so many AE cells possess stem cell characteristics. We also describe previous work concerning the therapeutic applications and discuss the pluripotency of the AE cells and potential pitfalls for amnion-derived stem cell research. PMID:21596003

Human amnion epithelial cells expressing HLA-G as novel cell-based treatment for liver disease.

PubMed

Strom, Stephen C; Gramignoli, Roberto

2016-09-01

Despite routine liver transplantation and supporting medical therapies, thousands of patients currently wait for an organ and there is an unmet need for more refined and widely available regenerative strategies to treat liver diseases. Cell transplants attempt to maximize the potential for repair and/or regeneration in liver and other organs. Over 40years of laboratory pre-clinical research and 25years of clinical procedures have shown that certain liver diseases can be treated by the infusion of isolated cells (hepatocyte transplant). However, like organ transplants, hepatocyte transplant suffers from a paucity of tissues useful for cell production. Alternative sources have been investigated, yet with limited success. The tumorigenic potential of pluripotent stem cells together with their primitive level of hepatic differentiation, have limited the use of stem cell populations. Stem cell sources from human placenta, and the amnion tissue in particular are receiving renewed interest in the field of regenerative medicine. Unlike pluripotent stem cells, human amnion epithelial (AE) cells are easily available without ethical or religious concerns; they do not express telomerase and are not immortal or tumorigenic when transplanted. In addition, AE cells have been reported to express genes normally expressed in mature liver, when transplanted into the liver. Moreover, because of the possibility of an immune-privileged status related to their expression of HLA-G, it might be possible to transplant human AE cells without immunosuppression of the recipient. Copyright © 2016. Published by Elsevier Inc.

Comparative Sensitivity of Tissue Cultures to Rubella Virus: Use of Guinea Pig Cells for Virus Titration

PubMed Central

Horta-Barbosa, L.; Warren, Joel

1969-01-01

A series of 19 different primary and serial tissue cultures were investigated for their sensitivity to virulent or attenuated rubella virus (RV). Primary guinea pig tissues, a serial passage of baby hamster kidney, and primary human amnion were comparable to African green monkey kidney tissue cultures in their sensitivity. In general, primary human tissues were relatively insusceptible to the Gilchrist strain of RV. RV interfered with the growth of vesicular stomatitis virus. Based on this finding, it was possible to develop an assay method in guinea pig tissue cultures by using VSV as the challenge virus. This system appeared to be comparable in sensitivity to the use of primary monkey kidney tissue cultures for the detection of small amounts of RV and offers the advantages of economy, rapidity, and safety. PMID:4979943

Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care.

PubMed

Magnusson, Skuli; Baldursson, Baldur Tumi; Kjartansson, Hilmar; Rolfsson, Ottar; Sigurjonsson, Gudmundur Fertram

2017-03-01

Improvised explosive devices and new directed energy weapons are changing warfare injuries from penetrating wounds to large surface area thermal and blast injuries. Acellular fish skin is used for tissue repair and during manufacturing subjected to gentle processing compared to biologic materials derived from mammals. This is due to the absence of viral and prion disease transmission risk, preserving natural structure and composition of the fish skin graft. The aim of this study was to assess properties of acellular fish skin relevant for severe battlefield injuries and to compare those properties with those of dehydrated human amnion/chorion membrane. We evaluated cell ingrowth capabilities of the biological materials with microscopy techniques. Bacterial barrier properties were tested with a 2-chamber model. The microstructure of the acellular fish skin is highly porous, whereas the microstructure of dehydrated human amnion/chorion membrane is mostly nonporous. The fish skin grafts show superior ability to support 3-dimensional ingrowth of cells compared to dehydrated human amnion/chorion membrane (p < 0.0001) and the fish skin is a bacterial barrier for 24 to 48 hours. The unique biomechanical properties of the acellular fish skin graft make it ideal to be used as a conformal cover for severe trauma and burn wounds in the battlefield. Reprint & Copyright © 2017 Association of Military Surgeons of the U.S.

Mesenchymal Stem or Stromal Cells from Amnion and Umbilical Cord Tissue and Their Potential for Clinical Applications

PubMed Central

Lindenmair, Andrea; Hatlapatka, Tim; Kollwig, Gregor; Hennerbichler, Simone; Gabriel, Christian; Wolbank, Susanne; Redl, Heinz; Kasper, Cornelia

2012-01-01

Mesenchymal stem or stromal cells (MSC) have proven to offer great promise for cell-based therapies and tissue engineering applications, as these cells are capable of extensive self-renewal and display a multilineage differentiation potential. Furthermore, MSC were shown to exhibit immunomodulatory properties and display supportive functions through parakrine effects. Besides bone marrow (BM), still today the most common source of MSC, these cells were found to be present in a variety of postnatal and extraembryonic tissues and organs as well as in a large variety of fetal tissues. Over the last decade, the human umbilical cord and human amnion have been found to be a rich and valuable source of MSC that is bio-equivalent to BM-MSC. Since these tissues are discarded after birth, the cells are easily accessible without ethical concerns. PMID:24710543

Amnion as a surrogate tissue reporter of the effects of maternal preeclampsia on the fetus.

PubMed

Suzuki, Masako; Maekawa, Ryo; Patterson, Nicole E; Reynolds, David M; Calder, Brent R; Reznik, Sandra E; Heo, Hye J; Einstein, Francine Hughes; Greally, John M

2016-01-01

Preeclampsia, traditionally characterized by high blood pressure and proteinuria, is a common pregnancy complication, which affects 2-8 % of all pregnancies. Although children born to women with preeclampsia have a higher risk of hypertension in later life, the mechanism of this increased risk is unknown. DNA methylation is an epigenetic modification that has been studied as a mediator of cellular memory of adverse exposures in utero. Since each cell type in the body has a unique DNA profile, cell subtype composition is a major confounding factor in studies of tissues with heterogeneous cell types. The best way to avoid this confounding effect is by using purified cell types. However, using purified cell types in large cohort translational studies is difficult. The amnion, the inner layer of the fetal membranes of the placenta, is derived from the epiblast and consists of two cell types, which are easy to isolate from the delivered placenta. In this study, we demonstrate the value of using amnion samples for DNA methylation studies, revealing distinctive patterns between fetuses exposed to proteinuria or hypertension and fetuses from normal pregnancies. We performed a genome-wide DNA methylation analysis, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP)-tagging, on 62 amnion samples from the placentas of uncomplicated, normal pregnancies and from those with complications of preeclampsia or hypertension. Using a regression model approach, we found 123, 85, and 99 loci with high-confidence hypertension-associated, proteinuria-associated, and hypertension- and proteinuria-associated DNA methylation changes, respectively. A gene ontology analysis showed DNA methylation changes to be selecting genes with different biological processes in exposure status. We also found that these differentially methylated regions overlap loci previously reported as differentially methylated regions in preeclampsia. Our findings support prior observations that preeclampsia is associated with changes of DNA methylation near genes that have previously been found to be dysregulated in preeclampsia. We propose that amniotic membranes represent a valuable surrogate fetal tissue on which to perform epigenome-wide association studies of adverse intrauterine conditions.

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators.

PubMed

Lim, R; Barker, G; Lappas, M

2015-04-01

In non-gestational tissues, the activation of adenosine monophosphate (AMP)-activated kinase (AMPK) is associated with potent anti-inflammatory actions. Infection and/or inflammation, by stimulating pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9, play a central role in the rupture of fetal membranes. However, no studies have examined the role of AMPK in human labour. Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and after preterm pre-labour rupture of membranes (PPROM). AMPK activity was assessed by Western blotting of phosphorylated AMPK expression. To determine the effect of AMPK activators on pro-inflammatory cytokines, fetal membranes were pre-treated with AMPK activators then stimulated with bacterial products LPS and flagellin or viral dsDNA analogue poly(I:C). Primary amnion cells were used to determine the effect of AMPK activators on IL-1β-stimulated MMP-9 expression. AMPK activity was decreased with term labour. There was no effect of preterm labour. AMPK activity was also decreased in preterm fetal membranes, in the absence of labour, with PROM compared to intact membranes. AMPK activators AICAR, phenformin and A769662 significantly decreased IL-6 and IL-8 stimulated by LPS, flagellin and poly(I:C). Primary amnion cells treated with AMPK activators significantly decreased IL-1β-induced MMP-9 expression. The decrease in AMPK activity in fetal membranes after spontaneous term labour and PPROM indicates an anti-inflammatory role for AMPK in human labour and delivery. The use of AMPK activators as possible therapeutics for threatened preterm labour would be an exciting future avenue of research. Copyright © 2015 Elsevier Ltd. All rights reserved.

Improved amino acid, bioenergetic metabolite and neurotransmitter profiles following human amnion epithelial cell transplant in intermediate maple syrup urine disease mice.

PubMed

Skvorak, Kristen J; Dorko, Kenneth; Marongiu, Fabio; Tahan, Veysel; Hansel, Marc C; Gramignoli, Roberto; Arning, Erland; Bottiglieri, Teodoro; Gibson, K Michael; Strom, Stephen C

2013-06-01

Orthotopic liver transplant (OLT) significantly improves patient outcomes in maple syrup urine disease (MSUD; OMIM: 248600), yet organ shortages point to the need for alternative therapies. Hepatocyte transplantation has shown both clinical and preclinical efficacy as an intervention for metabolic liver diseases, yet the availability of suitable livers for hepatocyte isolation is also limited. Conversely, human amnion epithelial cells (hAEC) may have utility as a hepatocyte substitute, and they share many of the characteristics of pluripotent embryonic stem cells while lacking their safety and ethical concerns. We reported that like hepatocytes, transplantation of hAEC significantly improved survival and lifespan, normalized body weight, and significantly improved branched-chain amino acid (BCAA) levels in sera and brain in a transgenic murine model of intermediate maple syrup urine disease (imsud). In the current report, we detail the neural and peripheral metabolic improvements associated with hAEC transplant in imsud mice, including amino acids associated with bioenergetics, the urea cycle, as well as the neurotransmitter systems for serotonin, dopamine, and gamma-aminobutyric acid (GABA). This stem cell therapy results in significant global correction of the metabolic profile that characterizes the disease, both in the periphery and the central nervous system, the target organ for toxicity in iMSUD. The significant correction of the disease phenotype, coupled with the theoretical benefits of hAEC, particularly their lack of immunogenicity and tumorigenicity, suggests that human amnion epithelial cells deserve serious consideration for clinical application to treat metabolic liver diseases. Copyright © 2013. Published by Elsevier Inc.

First steps to define murine amniotic fluid stem cell microenvironment.

PubMed

Bertin, E; Piccoli, M; Franzin, C; Spiro, G; Donà, S; Dedja, A; Schiavi, F; Taschin, E; Bonaldo, P; Braghetta, P; De Coppi, P; Pozzobon, M

2016-11-15

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit + cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit + cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP + embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP + sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells.

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

PubMed Central

Kim, Eun Young; Lee, Kyung-Bon; Kim, Min Kyu

2014-01-01

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140] PMID:24499672

Amnion and Chorion Allografts in Combination with Coronally Advanced Flap in the Treatment of Gingival Recession: A Clinical Study

PubMed Central

Chakraborthy, Sonali; Sambashivaiah, Savita; Bilchodmath, Shivaprasad

2015-01-01

Background Guided tissue regeneration (GTR) based root coverage using different allograft membranes has been utilized to correct gingival recession defects with promising results. Amnion and chorion allograft membranes of alternative origin derived from human placental tissue has been advocated in the treatment of gingival recession. However, chorion membrane has been used in combination with amnion membrane no study has compared these allograft membranes in the treatment of gingival recession. Therefore, the purpose of this study was to clinically evaluate and compare the efficacy of amnion membrane and chorion membrane in combination with coronally advanced flap in the treatment of gingival recessions. Materials and Methods Twelve systemically healthy patients having at least 2 bilateral Miller’s Class I or Class II gingival recession were recruited and coronally advanced flap was performed with amnion membrane or chorion membrane. Clinical parameters such as gingival Index, plaque index, length of the recession, width of the recession, width of keratinized gingiva, relative attachment level were evaluated at baseline, 3 and 6 months post-surgery. Results The mean decrease in length of recession (LR) for Chorion site was 2.00±1.54mm and amnion site was 1.58±1.14mm. The gain in attachment level for amnion site was 2.17±1.53mm and for chorion site was 1.58±1.22mm. The total mean percentage of root coverage was 34% for chorion site and 22% for amnion site. Conclusion Both amnion membrane and chorion membrane has shown to be versatile allograft material to be used in the treatment of root coverage. PMID:26501023

Amnion and Chorion Membranes: Potential Stem Cell Reservoir with Wide Applications in Periodontics

PubMed Central

2015-01-01

The periodontal therapy usually aims at elimination of disease causing bacteria and resolution of inflammation. It involves either resective or regenerative surgery to resolve the inflammation associated defects. Over the years, several methods have been used for achievement of periodontal regeneration. One of the oldest biomaterials used for scaffolds is the fetal membrane. The amniotic membranes of developing embryo, that is, amnion (innermost lining) and chorion (a layer next to it), have the properties with significant potential uses in dentistry. This paper reviews the properties, mechanism of action, and various applications of these placental membranes in general and specifically in Periodontics. PMID:26770199

Amnion and Chorion Membranes: Potential Stem Cell Reservoir with Wide Applications in Periodontics.

PubMed

Gupta, Akanksha; Kedige, Suresh D; Jain, Kanu

2015-01-01

The periodontal therapy usually aims at elimination of disease causing bacteria and resolution of inflammation. It involves either resective or regenerative surgery to resolve the inflammation associated defects. Over the years, several methods have been used for achievement of periodontal regeneration. One of the oldest biomaterials used for scaffolds is the fetal membrane. The amniotic membranes of developing embryo, that is, amnion (innermost lining) and chorion (a layer next to it), have the properties with significant potential uses in dentistry. This paper reviews the properties, mechanism of action, and various applications of these placental membranes in general and specifically in Periodontics.

Organotypic culture of human amnion cells in air-liquid interface as a potential substitute for skin regeneration.

PubMed

Fatimah, Simat Siti; Chua, Kienhui; Tan, Geok Chin; Azmi, Tengku Ibrahim; Tan, Ay Eeng; Abdul Rahman, Hayati

2013-08-01

The aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture. HAECs at passage 1-2 were seeded onto a fibrin layer populated with human amnion mesenchymal cells to form the organotypic cultures. The organotypic HAECs were then cultured for 7, 14 and 21 d in two types of culture system: the submerged culture and the air-liquid interface culture. Cell morphogenesis was examined under the light and electron microscopes (transmission and scanning) and analyzed by immunohistochemistry. Organotypic HAECs formed a single layer epithelium after 3 wk in submerged as well as air-liquid interface cultures. Ultrastructurally, desmosomes were observed in organotypic HAECs cultured in the air-liquid interface but not in the submerged culture. The presence of desmosomes marked the onset of early epidermal differentiation. Organotypic HAECs were positive against anti-CK18 and anti-CK14 in both the submerged and the air-liquid interface cultures. The co-expression of CK14 and CK18 suggested that differentiation of HAECs into skin may follow the process of embryonic skin development. However, weak expression of CK14 was observed after 2 and 3 wk of culture in air-liquid interface. CK10, involucrin, type IV collagen and laminin-5 expression was absent in organotypic HAECs. This observation reflects the initial process of embryonic epidermal differentiation and stratification. Results from the present study suggest that the air-liquid interface could stimulate early differentiation of organotypic HAECs to epidermal cells, with a potential use for skin regeneration. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Eosinophilic/T-cell Chorionic Vasculitis: Histological and Clinical Correlations.

PubMed

Cheek, Bradley; Heinrich, Stephen; Ward, Kenneth; Craver, Randall

2015-04-01

Eosinophilic T-cell chorionic vasculitis (E/TCV) is composed of eosinophils and T-lymphocytes originating within chorionic vessels, radiating toward the intervillous space and away from the amnion in a fashion different from the fetal vascular response seen in amnionitis. Clinical significance and risk factors are not well established. We report four pregnancies (five infants, one triplet was spared) with E/TCV, gestational ranging from 23 weeks to term. All had concurrent acute chorioamnionitis, three had the typical acute fetal inflammatory response. One had placental fetal obstructive vasculopathy and an upper extremity reduction defect (radio-ulnar synostosis), the mother had pre-eclampsia. A second case involved 2 of 3 23 week previable triplets. Our third case had a metatarsus varus resistant to casting, the mother had gestational diabetes. The last case was a normal infant. We review the literature, discuss the clinical findings and present the histologic characteristics of this infrequently recognized lesion.

The forkhead transcription factor Foxf1 is required for differentiation of extra-embryonic and lateral plate mesoderm.

PubMed

Mahlapuu, M; Ormestad, M; Enerbäck, S; Carlsson, P

2001-01-01

The murine Foxf1 gene encodes a forkhead transcription factor expressed in extra-embryonic and lateral plate mesoderm and later in splanchnic mesenchyme surrounding the gut and its derivatives. We have disrupted Foxf1 and show that mutant embryos die at midgestation due to defects in mesodermal differentiation and cell adhesion. The embryos do not turn and become deformed by the constraints of a small, inflexible amnion. Extra-embryonic structures exhibit a number of differentiation defects: no vasculogenesis occurs in yolk sac or allantois; chorioallantoic fusion fails; the amnion does not expand with the growth of the embryo, but misexpresses vascular and hematopoietic markers. Separation of the bulk of yolk sac mesoderm from the endodermal layer and adherence between mesoderm of yolk sac and amnion, indicate altered cell adhesion properties and enhanced intramesodermal cohesion. A possible cause of this is misexpression of the cell-adhesion protein VCAM1 in Foxf1-deficient extra-embryonic mesoderm, which leads to co-expression of VCAM with its receptor, alpha(4)-integrin. The expression level of Bmp4 is decreased in the posterior part of the embryo proper. Consistent with this, mesodermal proliferation in the primitive streak is reduced and somite formation is retarded. Expression of Foxf1 and the homeobox gene Irx3 defines the splanchnic and somatic mesodermal layers, respectively. In Foxf1-deficient embryos incomplete separation of splanchnic and somatic mesoderm is accompanied by misexpression of Irx3 in the splanchnopleure, which implicates Foxf1 as a repressor of Irx3 and as a factor involved in coelom formation.

Cell recruitment by amnion chorion grafts promotes neovascularization.

PubMed

Maan, Zeshaan N; Rennert, Robert C; Koob, Thomas J; Januszyk, Michael; Li, William W; Gurtner, Geoffrey C

2015-02-01

Nonhealing wounds are a significant health burden. Stem and progenitor cells can accelerate wound repair and regeneration. Human amniotic membrane has demonstrated efficacy in promoting wound healing, though the underlying mechanisms remain unknown. A dehydrated human amnion chorion membrane (dHACM) was tested for its ability to recruit hematopoietic progenitor cells to a surgically implanted graft in a murine model of cutaneous ischemia. dHACM was subcutaneously implanted under elevated skin (ischemic stimulus) in either wild-type mice or mice surgically parabiosed to green fluorescent protein (GFP) + reporter mice. A control acellular dermal matrix, elevated skin without an implant, and normal unwounded skin were used as controls. Wound tissue was harvested and processed for histology and flow cytometric analysis. Implanted dHACMs recruited significantly more progenitor cells compared with controls (*P < 0.05) and displayed in vivo SDF-1 expression with incorporation of CD34 + progenitor cells within the matrix. Parabiosis modeling confirmed the circulatory origin of recruited cells, which coexpressed progenitor cell markers and were localized to foci of neovascularization within implanted matrices. In summary, dHACM effectively recruits circulating progenitor cells, likely because of stromal derived factor 1 (SDF-1) expression. The recruited cells express markers of "stemness" and localize to sites of neovascularization, providing a partial mechanism for the clinical efficacy of human amniotic membrane in the treatment of chronic wounds. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell recruitment by amnion chorion grafts promotes neovascularization

PubMed Central

Koob, Thomas J.; Januszyk, Michael; Li, William W.; Gurtner, Geoffrey C.

2015-01-01

Background Nonhealing wounds are a significant health burden. Stem and progenitor cells can accelerate wound repair and regeneration. Human amniotic membrane has demonstrated efficacy in promoting wound healing, though the underlying mechanisms remain unknown. A dehydrated human amnion chorion membrane (dHACM) was tested for its ability to recruit hematopoietic progenitor cells to a surgically implanted graft in a murine model of cutaneous ischemia. Methods dHACM was subcutaneously implanted under elevated skin (ischemic stimulus) in either wild-type mice or mice surgically parabiosed to green fluorescent protein (GFP) + reporter mice. A control acellular dermal matrix, elevated skin without an implant, and normal unwounded skin were used as controls. Wound tissue was harvested and processed for histology and flow cytometric analysis. Results Implanted dHACMs recruited significantly more progenitor cells compared with controls (*P < 0.05) and displayed in vivo SDF-1 expression with incorporation of CD34 + progenitor cells within the matrix. Parabiosis modeling confirmed the circulatory origin of recruited cells, which coexpressed progenitor cell markers and were localized to foci of neovascularization within implanted matrices. Conclusions In summary, dHACM effectively recruits circulating progenitor cells, likely because of stromal derived factor 1 (SDF-1) expression. The recruited cells express markers of “stemness” and localize to sites of neovascularization, providing a partial mechanism for the clinical efficacy of human amniotic membrane in the treatment of chronic wounds. PMID:25266600

Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing

PubMed Central

Koob, Thomas J; Rennert, Robert; Zabek, Nicole; Massee, Michelle; Lim, Jeremy J; Temenoff, Johnna S; Li, William W; Gurtner, Geoffrey

2013-01-01

Human amnion/chorion tissue derived from the placenta is rich in cytokines and growth factors known to promote wound healing; however, preservation of the biological activities of therapeutic allografts during processing remains a challenge. In this study, PURION® (MiMedx, Marietta, GA) processed dehydrated human amnion/chorion tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for the presence of growth factors, interleukins (ILs) and tissue inhibitors of metalloproteinases (TIMPs). Enzyme-linked immunosorbent assays (ELISA) were performed on samples of dHACM and showed quantifiable levels of the following growth factors: platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, transforming growth factor α (TGFα), TGFβ1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), placental growth factor (PLGF) and granulocyte colony-stimulating factor (GCSF). The ELISA assays also confirmed the presence of IL-4, 6, 8 and 10, and TIMP 1, 2 and 4. Moreover, the relative elution of growth factors into saline from the allograft ranged from 4% to 62%, indicating that there are bound and unbound fractions of these compounds within the allograft. dHACM retained biological activities that cause human dermal fibroblast proliferation and migration of human mesenchymal stem cells (MSCs) in vitro. An in vivo mouse model showed that dHACM when tested in a skin flap model caused mesenchymal progenitor cell recruitment to the site of implantation. The results from both the in vitro and in vivo experiments clearly established that dHACM contains one or more soluble factors capable of stimulating MSC migration and recruitment. In summary, PURION® processed dHACM retains its biological activities related to wound healing, including the potential to positively affect four distinct and pivotal physiological processes intimately involved in wound healing: cell proliferation, inflammation, metalloproteinase activity and recruitment of progenitor cells. This suggests a paracrine mechanism of action for dHACM when used for wound healing applications. PMID:23902526

Epidermal Growth Factor and Interleukin-1β Utilize Divergent Signaling Pathways to Synergistically Upregulate Cyclooxygenase-2 Gene Expression in Human Amnion-Derived WISH Cells1

PubMed Central

Ackerman, William E.; Rovin, Brad H.; Kniss, Douglas A.

2006-01-01

In human parturition, uterotonic prostaglandins (PGs) arise predominantly via increased expression of cyclooxygenase-2 (COX-2 [also known as prostaglandin synthase 2]) within intra-uterine tissues. Interleukin-1 (IL-1) and epidermal growth factor (EGF), both inducers of COX-2 transcription, are among numerous factors that accumulate within amniotic fluid with advancing gestation. It was previously demonstrated that EGF could potentiate IL-1β-driven PGE2 production in amnion and amnion-derived (WISH) cells. To define the mechanism for this observation, we hypothesized that EGF and IL-1β might exhibit synergism in regulating COX-2 gene expression. In WISH cells, combined treatment with EGF and IL-1β resulted in a greater-than-additive increase in COX-2 mRNA relative to challenge with either agent independently. Augmentation of IL-1β-induced transactivation by EGF was not observed in cells harboring reporter plasmids bearing nuclear factor-kappa B (NFκB) regulatory elements alone, but was evident when a fragment (−891/+9) of the COX-2 gene 5′-promoter was present. Both agents transiently activated intermediates of multiple signaling pathways potentially involved in the regulation of COX-2 gene expression. The 26 S proteasome inhibitor, MG-132, selectively abrogated IL-1β-driven NFκB activation and COX-2 mRNA expression. Only pharmacologic blockade of the p38 mitogen-activated protein kinase eliminated COX-2 expression following EGF stimulation. We conclude that EGF and IL-1β appear to signal through different signaling cascades leading to COX-2 gene expression. IL-1β employs the NFκB pathway predominantly, while the spectrum of EGF signaling is broader and includes p38 kinase. The synergism observed between IL-1β and EGF does not rely on augmented NFκB function, but rather, occurs through differential use of independent response elements within the COX-2 promoter. PMID:15329330

Amnion-Derived Multipotent Progenitor Cells Increase Gain of Incisional Breaking Strength and Decrease Incidence and Severity of Acute Wound Failure

PubMed Central

Xing, Liyu; Franz, Michael G.; Marcelo, Cynthia L.; Smith, Charlotte A.; Marshall, Vivienne S.; Robson, Martin C.

2007-01-01

Objective: Acute wound failure is a common complication following surgical procedures and trauma. Laparotomy wound failure leads to abdominal dehiscence and incisional hernia formation. Delayed recovery of wound-breaking strength is one mechanism for laparotomy wound failure. Early fascial wounds are relatively acellular, and there is a delay in the appearance of acute wound growth factors and cytokines. The objective of this study was to accelerate and improve laparotomy wound healing using amnion-derived multipotent cells (AMPs). AMPs' nonimmunogenic phenotype and relative abundance support its role as a cell therapy. Methods: AMPs were injected into the load-bearing layer of rat abdominal walls prior to laparotomy, and cell viability was confirmed. Wound mechanical properties were measured over 28 days. The incidence and severity of laparotomy wound failure was measured in an incisional hernia model. Results: AMP cells were viable in laparotomy wounds for at least 28 days and did not migrate to other tissues. Laparotomy wound-breaking strength was increased by postoperative day 7 following AMP therapy. AMP therapy reduced the incidence of hernia formation and the size of hernia defects. Histology suggested stimulated wound fibroplasia and angiogenesis. Conclusions: AMP cell therapy reduces the incidence of laparotomy wound failure by accelerating the recovery of wound-breaking strength. This results in fewer incisional hernias and smaller hernia defects. PMID:18091982

Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion

DTIC Science & Technology

2017-09-01

the ideal solution. Combined mixed allogeneic chimerism induction and kidney transplantation has been shown to induce robust tolerance to the kidney ...induction to kidney allografts in non-human primates and humans despite the transience of donor chimerism. However, evidence indicates that durable mixed...chimerism may be required for tolerance induction to tissues or organs other than kidney . Amnion-derived multipotent progenitor (AMP) cells possess

Properties of dehydrated human amnion/chorion composite grafts: Implications for wound repair and soft tissue regeneration.

PubMed

Koob, Thomas J; Lim, Jeremy J; Massee, Michelle; Zabek, Nicole; Denozière, Guilhem

2014-08-01

PURION(®) processed dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Marietta, GA) tissue products were analyzed for the effectiveness of the PURION(®) process in retaining the native composition of the amniotic membrane and preserving bioactivity in the resulting products. dHACM was analyzed for extracellular matrix (ECM) composition through histological staining and for growth factor content via multiplex ELISA arrays. Bioactivity was assessed by evaluating endogenous growth factor production by human dermal fibroblasts in response to dHACM and for thermal stability by mechanical tests and in vitro cell proliferation assays. Histology of dHACM demonstrated preservation of the native amnion and chorion layers with intact, nonviable cells, collagen, proteoglycan, and elastic fibers distributed in the individual layers. An array of 36 cytokines known to regulate processes involved in inflammation and wound healing were identified in dHACM. When treated with dHACM extracts, bioactivity was demonstrated through an upregulation of basic fibroblast growth factor, granulocyte colony-stimulating factor, and placental growth factor biosynthesis, three growth factors involved in wound healing, by dermal fibroblasts in vitro. After conditioning at temperatures ranging from -78.7 to +73.5°C, dHACM retained its tensile strength and ability to promote proliferation of dermal fibroblasts in vitro. Elution experiments demonstrated a soluble fraction of growth factors that eluted from the tissue and another fraction sequestered within the matrix. The PURION(®) process retains the native composition of ECM and signaling molecules and preserves bioactivity. The array of cytokines preserved in dHACM are in part responsible for its therapeutic efficacy in treating chronic wounds by orchestrating a "symphony of signals" to promote healing. © 2014 Wiley Periodicals, Inc.

Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing.

PubMed

Koob, Thomas J; Rennert, Robert; Zabek, Nicole; Massee, Michelle; Lim, Jeremy J; Temenoff, Johnna S; Li, William W; Gurtner, Geoffrey

2013-10-01

Human amnion/chorion tissue derived from the placenta is rich in cytokines and growth factors known to promote wound healing; however, preservation of the biological activities of therapeutic allografts during processing remains a challenge. In this study, PURION® (MiMedx, Marietta, GA) processed dehydrated human amnion/chorion tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for the presence of growth factors, interleukins (ILs) and tissue inhibitors of metalloproteinases (TIMPs). Enzyme-linked immunosorbent assays (ELISA) were performed on samples of dHACM and showed quantifiable levels of the following growth factors: platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, transforming growth factor α (TGFα), TGFβ1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), placental growth factor (PLGF) and granulocyte colony-stimulating factor (GCSF). The ELISA assays also confirmed the presence of IL-4, 6, 8 and 10, and TIMP 1, 2 and 4. Moreover, the relative elution of growth factors into saline from the allograft ranged from 4% to 62%, indicating that there are bound and unbound fractions of these compounds within the allograft. dHACM retained biological activities that cause human dermal fibroblast proliferation and migration of human mesenchymal stem cells (MSCs) in vitro. An in vivo mouse model showed that dHACM when tested in a skin flap model caused mesenchymal progenitor cell recruitment to the site of implantation. The results from both the in vitro and in vivo experiments clearly established that dHACM contains one or more soluble factors capable of stimulating MSC migration and recruitment. In summary, PURION® processed dHACM retains its biological activities related to wound healing, including the potential to positively affect four distinct and pivotal physiological processes intimately involved in wound healing: cell proliferation, inflammation, metalloproteinase activity and recruitment of progenitor cells. This suggests a paracrine mechanism of action for dHACM when used for wound healing applications. ©2013 The Authors. International Wound Journal published by John Wiley & Sons Ltd and Medicalhelplines.com Inc.

Amniotic mesenchymal tissue cells inhibit dendritic cell differentiation of peripheral blood and amnion resident monocytes.

PubMed

Magatti, Marta; De Munari, Silvia; Vertua, Elsa; Nassauto, Claudia; Albertini, Alberto; Wengler, Georg S; Parolini, Ornella

2009-01-01

Cells derived from the amniotic membranes of human term placenta have drawn much interest for their characteristics of multipotency and low immunogenicity, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously shown that cells derived from the mesenchymal region of human amnion (AMTC) can strongly inhibit T-lymphocyte proliferation. In this study, we demonstrate that AMTC can block differentiation and maturation of monocytes into dendritic cells (DC), preventing the expression of the DC marker CD1a and reducing the expression of HLA-DR, CD80, and CD83. The monocyte maturation block resulted in impaired allostimulatory ability of these cells on allogeneic T cells. In attempting to define the mechanisms responsible for these findings, we have observed that the presence of AMTC in differentiating DC cultures results in the arrest of the cells to the G(0) phase and abolishes the production of inflammatory cytokines such as TNF-alpha, CXCL10, CXCL9, and CCL5. Finally, we also demonstrate that the monocytic cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage. Taken together, our data suggest that the mechanisms by which AMTC exert immumodulatory effects do not only relate directly to T cells, but also include inhibition of the generation and maturation of antigen-presenting cells. In this context, AMTC represent a very attractive source of multipotent allogeneic cells that promise to be remarkably valuable for cell transplantation approaches, not only due to their low immunogenicity, but also because of the added potential of modulating immune responses, which could be fundamental both for controlling graft rejection after transplantation and also for controlling diseases characterized by inflammatory processes.

Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells.

PubMed

Wang, Yuli; Wu, Hongxia; Shen, Ming; Ding, Siyang; Miao, Jing; Chen, Ning

2017-01-01

Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2'-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro

PubMed Central

Massee, Michelle; Chinn, Kathryn; Lei, Jennifer; Lim, Jeremy J.; Young, Conan S.

2015-01-01

Abstract Human‐derived placental tissues have been shown in randomized clinical trials to be effective for healing chronic wounds, and have also demonstrated the ability to recruit stem cells to the wound site in vitro and in vivo. In this study, PURION® Processed dehydrated human amnion/chorion membrane allografts (dHACM, EpiFix®, MiMedx Group, Marietta, GA) were evaluated for their ability to alter stem cell activity in vitro. Human bone marrow mesenchymal stem cells (BM‐MSCs), adipose derived stem cells (ADSCs), and hematopoietic stem cells (HSCs) were treated with soluble extracts of dHACM tissue, and were evaluated for cellular proliferation, migration, and cytokine secretion. Stem cells were analyzed for cell number by DNA assay after 24 h, closure of an acellular zone using microscopy over 3 days, and soluble cytokine production in the medium of treated stem cells was analyzed after 3 days using a multiplex ELISA array. Treatment with soluble extracts of dHACM tissue stimulated BM‐MSCs, ADSCs, and HSCs to proliferate with a significant increase in cell number after 24 h. dHACM treatment accelerated closure of an acellular zone by ADSCs and BM‐MSCs after 3 days, compared to basal medium. BM‐MSCs, ADSCs, and HSCs also modulated endogenous production of a number of various soluble signals, including regulators of inflammation, mitogenesis, and wound healing. dHACM treatment promoted increased proliferation and migration of ADSCs, BM‐MSCs, and HSCs, along with modulation of secreted proteins from those cells. Therefore, dHACM may impact wound healing by amplifying host stem cell populations and modulating their responses in treated wound tissues. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1495–1503, 2016. PMID:26175122

Clinical application of amniotic membranes on a patient with epidermolysis bullosa.

PubMed

Martínez Pardo, M E; Reyes Frías, M L; Ramos Durón, L E; Gutiérrez Salgado, E; Gómez, J C; Marín, M A; Luna Zaragoza, D

1999-01-01

The case of a patient with dystrophic epidermolysis bullosa treated with radiosterilised amniotic membranes is presented. The disorder is a congenital disease characterised by a poor desmosomal junction in the keratinocyte membrane. After proper donor screening, amnios were collected at Hospital Central Sur de Alta Especialidad (HCSAE), PEMEX and microbiological analysis was performed at Universidad Nacional Autónoma de México, FQUNAM, (Biology Dept. of the Chemistry Faculty, National Autonomous University of Mexico), before and after radiation sterilisation. Processing, packaging and sterilisation were performed at Instituto Nacional de Investigaciones Nucleares, ININ, (National Nuclear Research Institute). The patient, a ten-year-old boy with severe malnutrition, extensive loss of skin and pseudomonad infection in the whole body, was treated with gentle debridement in a Hubbard bath. Later amnion application was performed with sterilised amnios by using two different processes, in one of which the amnion was sterilised with paracetic acid, preserved in glycerol, kindly donated by the German Institute for Tissue and Cell Replacement and applied by Dr. Johannes C. Bruck, IAEA visiting expert, and the other amnion was processed at ININ: air dried and sterilised by gamma radiation at dose of 30 kGy. After spontaneous epithelisation was successfully promoted for seven days, the pain was alleviated and mobility was improved in a few hours and the patient's general condition was so improved that in a month he was discharged. Unfortunately, because this disease is revertive and has malignant degeneration, the prognosis is not good.

Infections in the tissue material and their impact on the loss of transplants in the Laboratory of in vitro Cell and Tissue Culture with Tissue Bank in the years 2011-2015.

PubMed

Kitala, D; Klama-Baryła, A; Kawecki, M; Kraut, M; Łabuś, W; Glik, J; Ples, M; Tomanek, E; Nowak, M

2017-03-01

Radiation sterilization eliminates microbiological infections but causes the degradation of the cell factor. The negative result of microbiological examination for tissue transplants is one of the conditions for approval for distribution in patients. The study attempts to verify impact of the presence of microbes onto material for transplant loss. In the 2011-2015 period, we analyzed 293 donors of skin and amnion. Microbiological sampling was performed. The total of 21 strains of bacteria, molds and fungi was identified in collected tissue. The widest spectrum of strains was found in skin (17), followed by amnia (8). The total number of positive findings was 147 and was again highest in skin (129), while the number of positive findings in amnia was 18 only. The general percentage of fungal infections was very low. The presence of fungal strains was only observed in allogeneic skin (2%). Large number of microorganisms isolated from the skin before sterilization was observed, so it seems impossible to use allogeneic intravital skin. However, the intravital application of allogeneic amnion obtained from cesarean section remains to be considered.

Successful vitrification of human amnion-derived mesenchymal stem cells.

PubMed

Moon, Jeong Hee; Lee, Jung Ryeol; Jee, Byung Chul; Suh, Chang Suk; Kim, Seok Hyun; Lim, Hyun Jung; Kim, Hae Kwon

2008-08-01

A cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs. HAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs. The post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions. Our results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.

Sealing Penetrating Eye Injuries Using Photo-Activated Bonding

DTIC Science & Technology

2011-09-01

called PTB ) with the potential to decrease vision loss and ocular complications in warfighters sustaining penetrating eye injuries. Scope: In year 2...not competitive with PTB for sealing is amnion over penetrating cornea injuries, determined that two potential adverse effects (inhibition of...epithelial cell migration and keratocyte phototoxicity) are not significant problems, demonstrated that PTB can be used to seal lacerations in thin (e.g

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization.

PubMed

Mrázová, H; Koller, J; Kubišová, K; Fujeríková, G; Klincová, E; Babál, P

2016-06-01

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.

Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds.

PubMed

Ertl, Juliane; Pichlsberger, Melanie; Tuca, Alexandru-Cristian; Wurzer, Paul; Fuchs, Jakob; Geyer, Stefan H; Maurer-Gesek, Barbara; Weninger, Wolfgang J; Pfeiffer, Dagmar; Bubalo, Vladimir; Parvizi, Daryousch; Kamolz, Lars-Peter; Lang, Ingrid

2018-05-01

Mesenchymal stem/stromal cells derived from human term placentas (PMSCs) are novel therapeutic agents and more topical than ever. Here we evaluated the effects of three types of PMSCs on wound healing in an in vivo mouse model: Amnion-derived MSCs (AMSCs), blood vessel-derived MSCs (BV-MSCs) from the chorionic plate and Wharton's jelly-derived MSCs (WJ-MSCs) from the umbilical cord. We topically applied PMSCs onto skin wounds in mice using the dermal substitute Matriderm ® as carrier and evaluated wound healing parameters. In addition, we investigated the effects of all PMSC types under co-application with placental endothelial cells (PLECs). After 8 days, we compared the percent of wound closure and the angiogenic potential between all groups. AMSCs, BV-MSCs and WJ-MSCs significantly induced a faster healing and a higher number of blood vessels in the wound when compared to controls (Matriderm ® -alone). PLECs did not further improve the advantageous effects of PMSC-treatment. Quantitative data and 3D analysis by high resolution episcopic microscopy confirmed a lower density of vessels in Matriderm ® /PMSCs/PLECs co-application compared to Matriderm ® /PMSCs treatment. Results indicate that all three PMSC types exert similar beneficial effects on wound closure and neovascularization in our mouse model. Using Matriderm ® as carrier for PMSCs propagates rapid cell migration towards the wound area that allows a fast and clinically practicable method for stem cell application. These promising effects warrant further investigation in clinical trials. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Prostaglandin H2 synthase-1 and -2 expression in guinea pig gestational tissues during late pregnancy and parturition

PubMed Central

Welsh, Toni; Mitchell, Carolyn M; Walters, William A; Mesiano, Sam; Zakar, Tamas

2005-01-01

Increased intrauterine prostaglandin (PG) production is crucial for the initiation of parturition. To investigate the mechanisms controlling intrauterine PG synthesis, we examined the expression of the key PG biosynthetic isoenzymes, PG-H2 synthase (PTGS)-1 and -2, in the amnion, visceral yolk sac (VYS), placenta and myo-endometrium of pregnant guinea pigs. This animal model was chosen because the hormonal milieu of pregnancy and the role of PGs in the hormonal control of parturition are similar to those in the human. PTGS1 mRNA abundance, measured by real-time RT-PCR, increased in the amnion and the placenta during the last third of gestation. During labour, PTGS1 mRNA levels decreased precipitously in all four tissues. PTGS1 protein abundance, assessed by immunoblotting, increased to high levels in the amnion and the placenta by the end of pregnancy and remained high during labour. PTGS2 mRNA expression was higher in the placenta than in the other tissues, but did not change before and during labour. PTGS2 protein expression decreased in the placenta and remained low in the other tissues during labour. Immunohistochemistry showed pervasive PTGS1 protein expression in the amnion and strong expression in the parietal yolk sac membrane (PYS) covering the placenta. PTGS2 was expressed in the PYS and the endometrium. The PTGS inhibitor piroxicam, administered in doses that inhibited PTGS1 but not PTGS2, significantly prolonged gestation. These data suggest that PGs generated by intrauterine PTGS1 are involved in the timing of birth in guinea pigs. The induction of PTGS1 in the amnion and the PYS is a critical event leading to labour in guinea pigs and models analogous changes in the human gestational tissues before labour. PMID:16239272

Studies of hemidesmosomes in human amnion: the use of a detergent extraction protocol for compositional and ultrastructural analysis and preparation of a hemidesmosome-enriched fraction from tissue.

PubMed

Behzad, F; Jones, C J; Ball, S; Alvares, T; Aplin, J D

1995-01-01

A method is described for the sequential detergent and high ionic strength extraction of human amnion with the progressive enrichment of the intermediate filament (IF) cytoskeleton and its associated structures including hemidesmosomes (HD). TEM of the extracted epithelium in situ reveals IF bundles beneath the apical cell surface, around the nucleus and at the lateral edges of the cells where association with desmosomes occurs. IF bundles are also very prominent within basal cell processes where they loop through the cytoplasm adjacent to the HDs. A novel connecting filament network is observed running between the IFs and the hemidesmosomal dense plaque. The adjacent IF network contains both cytokeratin and vimentin, the latter revealed much more fully as a result of the extraction protocol. The hemidesmosomal plasma membrane contains integrin subunits alpha 6 and beta 4 and these are quantitatively retained as the basal cell surface during extraction, while nonjunctional plasma membrane is solubilised. Integrin beta 1 is found at the basolateral cell surface but, like actin, is extracted quantitatively and is not present in HDs. The extracted epithelial cells may be recovered by scraping and the IF network depolymerised to produce a particulate fraction containing short residual IFs, associated thin filaments and plaque material. This fraction contains immunoreactive cytokeratin and vimentin. Integrin alpha 6 beta 4 has been used as a biochemical criterion of the presence of HD material in the fraction. Both subunits are highly enriched. The fraction also contains the hemidesmosomal components HD1, BP230 and BP180. This method is likely to be useful in further characterisation of the HD.

Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro.

PubMed

Massee, Michelle; Chinn, Kathryn; Lei, Jennifer; Lim, Jeremy J; Young, Conan S; Koob, Thomas J

2016-10-01

Human-derived placental tissues have been shown in randomized clinical trials to be effective for healing chronic wounds, and have also demonstrated the ability to recruit stem cells to the wound site in vitro and in vivo. In this study, PURION(®) Processed dehydrated human amnion/chorion membrane allografts (dHACM, EpiFix(®) , MiMedx Group, Marietta, GA) were evaluated for their ability to alter stem cell activity in vitro. Human bone marrow mesenchymal stem cells (BM-MSCs), adipose derived stem cells (ADSCs), and hematopoietic stem cells (HSCs) were treated with soluble extracts of dHACM tissue, and were evaluated for cellular proliferation, migration, and cytokine secretion. Stem cells were analyzed for cell number by DNA assay after 24 h, closure of an acellular zone using microscopy over 3 days, and soluble cytokine production in the medium of treated stem cells was analyzed after 3 days using a multiplex ELISA array. Treatment with soluble extracts of dHACM tissue stimulated BM-MSCs, ADSCs, and HSCs to proliferate with a significant increase in cell number after 24 h. dHACM treatment accelerated closure of an acellular zone by ADSCs and BM-MSCs after 3 days, compared to basal medium. BM-MSCs, ADSCs, and HSCs also modulated endogenous production of a number of various soluble signals, including regulators of inflammation, mitogenesis, and wound healing. dHACM treatment promoted increased proliferation and migration of ADSCs, BM-MSCs, and HSCs, along with modulation of secreted proteins from those cells. Therefore, dHACM may impact wound healing by amplifying host stem cell populations and modulating their responses in treated wound tissues. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1495-1503, 2016. © 2015 The Authors. Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.

Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Tan, Ay Eeng; Nur Azurah, Abdul Ghani; Hayati, Abdul Rahman

2013-08-01

The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50 ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10 ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-β1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

Inflammatory Response of Human Gestational Membranes to Ureaplasma parvum Using a Novel Dual-Chamber Tissue Explant System.

PubMed

Potts, Lauren C; Feng, Liping; Seed, Patrick C; Jayes, Friederike L; Kuchibhatla, Maragatha; Antczak, Brian; Nazzal, Matthew K; Murtha, Amy P

2016-05-01

Preterm premature rupture of membranes (PPROM) is often associated with intra-amniotic inflammation and infection. Current understanding of the pathogenesis of PPROM includes activation of pro-inflammatory cytokines and proteolytic enzymes leading to compromise of membrane integrity. The impact of exposure to bacterial pathogens, including Ureaplasma parvum, on gestational membranes is poorly understood. Our objective was to develop a dual-chamber system to characterize the inflammatory response of gestational membranes to U. parvum in a directional nature. Full-thickness human gestational membrane explants, with either choriodecidua or amnion oriented superiorly, were suspended between two washers in a cylindrical device, creating two distinct compartments. Brilliant green dye was introduced into the top chamber to assess the integrity of the system. Tissue viability was evaluated after 72 h using a colorimetric cell proliferation assay. Choriodecidua or amnion was exposed to three doses of U. parvum and incubated for 24 h. Following treatment, media from each compartment were used for quantification of U. parvum (quantitative PCR), interleukin (IL)-8 (enzyme-linked immunosorbent assay), and matrix metalloproteinase (MMP)-2 and MMP-9 activity (zymography). We observed that system integrity and explant viability were maintained over 72 h. Dose-dependent increases in recovered U. parvum, IL-8 concentration, and MMP-2 activity were detected in both compartments. Significant differences in IL-8 concentration and MMP-9 activity were found between the choriodecidua and amnion. This tissue explant system can be used to investigate the inflammatory consequences of directional bacterial exposure for gestational membranes and provides insight into the pathogenesis of PPROM and infectious complications of pregnancy. © 2016 by the Society for the Study of Reproduction, Inc.

Comparison of cytomegalovirus (CMV)-specific neutralization capacity of hyperimmunoglobulin (HIG) versus standard intravenous immunoglobulin (IVIG) preparations: Impact of CMV IgG normalization.

PubMed

Schampera, Matthias Stefan; Schweinzer, Katrin; Abele, Harald; Kagan, Karl Oliver; Klein, Reinhild; Rettig, Ingo; Jahn, Gerhard; Hamprecht, Klaus

2017-05-01

Based on a non-randomized study of Nigro et al. (2005) the intravenous administration of hyperimmunoglobulins (HIGs) is applied frequently to women with primary CMV-infection as "off-label use" in Germany. In order to describe their CMV-specific neutralization-capacity in vitro, we analyzed the HIG preparations Cytotect ® , and Cytogam ® as well as the standard intravenous immunoglobulins (IVIG) Octagam ® , Gamunex ® , Kiovig ® . We performed short-term cell-free CMV neutralization assays (CFNT) and long-term cell-adapted neutralization-plaque-reduction assays (PRANT). Human retinal epithelial cells (ARPE-19) were used as target cells. A clinical CMV primary-isolate from amnion fluid propagated in epithelial cells without any initial fibroblast adaption was used. For calibration we previously generated serum-pools (N=100) from two cohorts of mothers at birth: seronegative and latently CMV-infected mothers. Biochemical analysis included total protein, albumin, Ig-class, and IgG-subclasses. Additionally, CMV antibody-reactivity was checked using recombinant immunoblotting. HIG and IVIG preparations showed differences in levels and patterns of protein, Ig-class and CMV-specific antibody concentrations. All IgG-preparations showed high in vitro NT-capacity and high IgG-avidity. The NT 90 -values for HIGs and IVIGs and our seropositive reference-pool showed similar NT-capacity at a dilution of (1:100) which corresponded well to 4.1 PEI-Units/ml. All HIG- and IVIG-preparations showed similar NT-capacity following CMV IgG-normalization. Our in vitro results are in strong contrast to former findings suggesting higher functional CMV NT titers in IVIG-preparations compared to HIGs. Copyright © 2017 Elsevier B.V. All rights reserved.

Type I and II Diabetic Adipose-Derived Stem Cells Respond In Vitro to Dehydrated Human Amnion/Chorion Membrane Allograft Treatment by Increasing Proliferation, Migration, and Altering Cytokine Secretion

PubMed Central

Massee, Michelle; Chinn, Kathryn; Lim, Jeremy J.; Godwin, Lisa; Young, Conan S.; Koob, Thomas J.

2016-01-01

Objective: Human amniotic membranes have been shown to be effective for healing diabetic foot ulcers clinically and to regulate stem cell activity in vitro and in vivo; however, diabetic stem cells may be impaired as a sequela of the disease. In this study, dehydrated human amnion/chorion membrane (dHACM) allografts (EpiFix®; MiMedx Group) were evaluated for their ability to regulate diabetic stem cells in vitro. Approach: Human adipose-derived stem cells (ADSCs) from normal, type I diabetic, and type II diabetic donors were treated with soluble extracts of dHACM and evaluated for proliferation after 3 days by DNA assay, chemotactic migration after 1 day by transwell assay, cytokine secretion after 3 days by multiplex ELISA, and gene expression after 5 days by reverse transcription–polymerase chain reaction. Results: Although diabetic ADSCs demonstrated decreased responses compared to normal ADSCs, dHACM treatment stimulated diabetic ADSCs to proliferate after 3 days and enhanced migration over 24 h, similar to normal ADSCs. dHACM-treated diabetic ADSCs modulated secretion of soluble signals, including regulators of inflammation, angiogenesis, and healing. All ADSCs evaluated also responded to dHACM treatment with altered expression of immunomodulatory genes, including interleukins (IL)-1α, IL-1β, and IL-1RA. Innovation: This is the first reported case demonstrating that diabetic ADSCs respond to novel amniotic membrane therapies, specifically treatment with dHACM. Conclusion: dHACM stimulated diabetic ADSCs to migrate, proliferate, and alter cytokine expression suggesting that, despite their diabetic origin, ADSCs may respond to dHACM to accelerate diabetic wound healing. PMID:26862462

Type I and II Diabetic Adipose-Derived Stem Cells Respond In Vitro to Dehydrated Human Amnion/Chorion Membrane Allograft Treatment by Increasing Proliferation, Migration, and Altering Cytokine Secretion.

PubMed

Massee, Michelle; Chinn, Kathryn; Lim, Jeremy J; Godwin, Lisa; Young, Conan S; Koob, Thomas J

2016-02-01

Objective: Human amniotic membranes have been shown to be effective for healing diabetic foot ulcers clinically and to regulate stem cell activity in vitro and in vivo ; however, diabetic stem cells may be impaired as a sequela of the disease. In this study, dehydrated human amnion/chorion membrane (dHACM) allografts (EpiFix ® ; MiMedx Group) were evaluated for their ability to regulate diabetic stem cells in vitro . Approach: Human adipose-derived stem cells (ADSCs) from normal, type I diabetic, and type II diabetic donors were treated with soluble extracts of dHACM and evaluated for proliferation after 3 days by DNA assay, chemotactic migration after 1 day by transwell assay, cytokine secretion after 3 days by multiplex ELISA, and gene expression after 5 days by reverse transcription-polymerase chain reaction. Results: Although diabetic ADSCs demonstrated decreased responses compared to normal ADSCs, dHACM treatment stimulated diabetic ADSCs to proliferate after 3 days and enhanced migration over 24 h, similar to normal ADSCs. dHACM-treated diabetic ADSCs modulated secretion of soluble signals, including regulators of inflammation, angiogenesis, and healing. All ADSCs evaluated also responded to dHACM treatment with altered expression of immunomodulatory genes, including interleukins (IL)-1α, IL-1β, and IL-1RA. Innovation: This is the first reported case demonstrating that diabetic ADSCs respond to novel amniotic membrane therapies, specifically treatment with dHACM. Conclusion: dHACM stimulated diabetic ADSCs to migrate, proliferate, and alter cytokine expression suggesting that, despite their diabetic origin, ADSCs may respond to dHACM to accelerate diabetic wound healing.

Stemness and angiogenic gene expression changes of serial-passage human amnion mesenchymal cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Fariha, Mohd Manzor Nur; Tan, Ay Eeng; Hayati, Abdul Rahman

2013-03-01

Particular attention has been directed towards human amnion mesenchymal stem cells (HAMCs) due to their accessibility, availability and immunomodulatory properties. Therefore, the aim of the present study was to determine the temporal changes of stemness and angiogenic gene expressions of serial-passage HAMCs. HAMCs were isolated from human term placenta and cultured in serial passages in culture medium supplemented with 10% fetal bovine serum. Morphological analysis, growth kinetic and CFU-F assay of HAMCs were assessed. In vitro differentiation and the immunophenotype of HAMCs at P5 were also analyzed. Quantitative PCR was used to determine the stemness, angiogenic and endothelial gene expression of cultured HAMCs after serial passage. Cultured HAMCs displayed intermediate epitheloid-fibroblastoid morphology at an initial culture and the fibroblastoid features became more pronounced in later passages. They showed high clonogenic activity and faster proliferation at later passages with colony forming efficiency of 0.88%. HAMCs were successfully differentiated into adipocytes, osteocytes and neuron-like cells. Most HAMCs expressed CD9, CD44, CD73, CD90 and HLA-A,B,C but negligibly expressed CD31, CD34, CD45, CD117 and HLA-DR,DP,DQ. After serial passage, stemness genes Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4 and FZD-9 expressions significantly decreased. Of the angiogenic genes PECAM-1, bFGF, eNOS, VEGFR-2, VEGF, and vWF expressions also decreased significantly except angiopoietin-1 which significantly increased. No significant differences were observed in ABCG-2, BST-1, nestin, PGF and HGF expressions after serial passage. These results suggested that cultured HAMCs could be an alternative source of stem cells and may have the potential for angiogenesis and hence its use in stem-cell based therapy. Copyright © 2012 Elsevier Inc. All rights reserved.

Human amnion-derived mesenchymal stem cells protect against UVA irradiation-induced human dermal fibroblast senescence, in vitro

PubMed Central

Zhang, Chunli; Yuchi, Haishen; Sun, Lu; Zhou, Xiaoli; Lin, Jinde

2017-01-01

The aim of the present study was to determine if human amnion-derived mesenchymal stem cells (HAMSCs) exert a protective effect on ultraviolet A (UVA) irradiation-induced human dermal fibroblast (HDF) senescence. A senescence model was constructed as follows: HDFs (104–106 cells/well) were cultured in a six-well plate in vitro and then exposed to UVA irradiation at 9 J/cm2 for 30 min. Following the irradiation period, HDFs were co-cultured with HAMSCs, which were seeded on transwells. A total of 72 h following the co-culturing, senescence-associated β-galactosidase staining was performed and reactive oxygen species (ROS) content and mitochondrial membrane potential (Δψm) were detected in the HDFs via flow cytometric analysis. The results demonstrated that the percentage of HDFs, detected via staining with X-gal, were markedly decreased when co-cultured with human HAMSCs, compared with the group that were not co-cultured. The ROS content was decreased and the mitochondrial membrane potential (Δψm) recovered in cells treated with UVA and HAMSCs, compared with that of cells treated with UVA alone. Reverse transcription-quantitative polymerase chain reaction revealed the significant effects of HAMSCs on the HDF senescence marker genes p53 and matrix metalloproteinase-1 mRNA expression. In addition to this, western blot analysis verified the effects of HAMSCs on UVA induced senescence, providing a foundation for novel regenerative therapeutic methods. Furthermore, the results suggested that activation of the extracellular-signal regulated kinase 1/2 mitogen activated protein kinase signal transduction pathway, is essential for the HAMSC-mediated UVA protective effects. The decrease in ROS content additionally indicated that HAMSCs may exhibit the potential to treat oxidative stress-mediated UVA skin senescence in the future. PMID:28627622

Myocytes of chorionic vessels from placentas with meconium-associated vascular necrosis exhibit apoptotic markers.

PubMed

King, Erin L; Redline, Raymond W; Smith, Steven D; Kraus, Frederick T; Sadovsky, Yoel; Nelson, D Michael

2004-04-01

Meconium-associated vascular necrosis (MAVN) is a histological abnormality of human placental chorionic vessels that is associated with poor neonatal outcome. We tested the hypothesis that MAVN shows apoptosis in the walls of chorionic vessels. Archival placental specimens with MAVN (n = 5) were compared with specimens from uncomplicated pregnancies at term (n = 5) and from placentas with intense chorionic vasculitis associated with acute chorioamnionitis with (n = 5) or without (n = 5) a clinical history of meconium in the amniotic fluid. Sections from all placentas were processed by the TUNEL method, and 2 observers who were blinded to specimen diagnosis quantified the immunofluorescent TUNEL staining in both the amnion-facing and villous-facing walls of the larger chorionic vessels in each specimen. Compared with the other 3 groups, only the amnion-facing wall of chorionic vessels in MAVN showed a significantly greater number of apoptotic cells. This was verified by morphological criteria and caspase 3 staining. There were limited or no detectable TUNEL-stained cells in either the villous-facing walls of vessels in the MAVN specimens or in any of the vessels of the placentas from uncomplicated pregnancies. There was a negligible level of apoptosis in chorionic vessels of placentas with intense chorionic vasculitis, with or without meconium, despite the inflammatory response or presence of meconium. We conclude that apoptosis contributes to the pathophysiology of MAVN.

A multicenter, randomized, controlled clinical trial evaluating the use of dehydrated human amnion/chorion membrane allografts and multilayer compression therapy vs. multilayer compression therapy alone in the treatment of venous leg ulcers.

PubMed

Serena, Thomas E; Carter, Marissa J; Le, Lam T; Sabo, Matthew J; DiMarco, Daniel T

2014-01-01

Venous leg ulcers produce significant clinical and economic burdens on society and often require advanced wound therapy. The purpose of this multicenter, randomized, controlled study is to evaluate the safety and efficacy of one or two applications of dehydrated human amnion/chorion membrane allograft and multilayer compression therapy vs. multilayer compression therapy alone in the treatment of venous leg ulcers. The primary study outcome was the proportion of patients achieving 40% wound closure at 4 weeks. Of the 84 participants enrolled, 53 were randomized to receive allograft and 31 were randomized to the control group of multilayer compression therapy alone. At 4 weeks, 62% in the allograft group and 32% in the control group showed a greater than 40% wound closure (p = 0.005), thus showing a significant difference between the allograft-treated groups and the multilayer compression therapy alone group at the 4-week surrogate endpoint. After 4 weeks, wounds treated with allograft had reduced in size a mean of 48.1% compared with 19.0% for controls. Venous leg ulcers treated with allograft had a significant improvement in healing at 4 weeks compared with multilayer compression therapy alone. © 2014 by the Wound Healing Society.

Brief communication: sliding displacement of amnion and chorion following controlled laser wounding suggests a mechanism for short-term sealing of ruptured membranes.

PubMed

Behzad, F; Dickinson, M R; Charlton, A; Aplin, J D

1994-10-01

The Erbium-YAG laser was used to produce narrow wounds of defined depth in term amniochorion. The charring effect of the laser meant that sites could be readily localized in histological sections. During brief post-wounding incubations, sliding displacement of the amnion relative to the chorion occurred through the plane of the spongy layer. This suggests a possible short-term mechanism whereby a spontaneous rupture could be sealed in vivo.

Phenotypic Screening Identifies Synergistically Acting Natural Product Enhancing the Performance of Biomaterial Based Wound Healing.

PubMed

Sivasubramanian, Srinivasan; Chandrasekar, Gayathri; Svensson Akusjärvi, Sara; Thangam, Ramar; Sathuvan, Malairaj; Kumar, R B S; Hussein, Hawraa; Vincent, Savariar; Madhan, Balaraman; Gunasekaran, Palani; Kitambi, Satish S

2017-01-01

The potential of multifunctional wound heal biomaterial relies on the optimal content of therapeutic constituents as well as the desirable physical, chemical, and biological properties to accelerate the healing process. Formulating biomaterials such as amnion or collagen based scaffolds with natural products offer an affordable strategy to develop dressing material with high efficiency in healing wounds. Using image based phenotyping and quantification, we screened natural product derived bioactive compounds for modulators of types I and III collagen production from human foreskin derived fibroblast cells. The identified hit was then formulated with amnion to develop a biomaterial, and its biophysical properties, in vitro and in vivo effects were characterized. In addition, we performed functional profiling analyses by PCR array to understand the effect of individual components of these materials on various genes such as inflammatory mediators including chemokines and cytokines, growth factors, fibroblast stimulating markers for collagen secretion, matrix metalloproteinases, etc., associated with wound healing. FACS based cell cycle analyses were carried out to evaluate the potential of biomaterials for induction of proliferation of fibroblasts. Western blot analyses was done to examine the effect of biomaterial on collagen synthesis by cells and compared to cells grown in the presence of growth factors. This work demonstrated an uncomplicated way of identifying components that synergistically promote healing. Besides, we demonstrated that modulating local wound environment using biomaterials with bioactive compounds could enhance healing. This study finds that the developed biomaterials offer immense scope for healing wounds by means of their skin regenerative features such as anti-inflammatory, fibroblast stimulation for collagen secretion as well as inhibition of enzymes and markers impeding the healing, hydrodynamic properties complemented with other features including non-toxicity, biocompatibility, and safety.

Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells

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

Wang, Yuli; Wu, Hongxia; Shen, Ming

Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assayingmore » reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling. - Highlights: • LPS inhibites osteogenic differentiation in HBMSCs via suppression of p38 MAPK signaling pathway. • HAMSCs promote LPS-induced HBMSCs osteogenic differentiation through p38 MAPK signaling pathway. • HAMSCs reverse LPS-induced oxidative stress in LPS-induced HBMSCs through p38 MAPK signaling pathway.« less

Phenotypic Screening Identifies Synergistically Acting Natural Product Enhancing the Performance of Biomaterial Based Wound Healing

PubMed Central

Sivasubramanian, Srinivasan; Chandrasekar, Gayathri; Svensson Akusjärvi, Sara; Thangam, Ramar; Sathuvan, Malairaj; Kumar, R. B. S.; Hussein, Hawraa; Vincent, Savariar; Madhan, Balaraman; Gunasekaran, Palani; Kitambi, Satish S.

2017-01-01

The potential of multifunctional wound heal biomaterial relies on the optimal content of therapeutic constituents as well as the desirable physical, chemical, and biological properties to accelerate the healing process. Formulating biomaterials such as amnion or collagen based scaffolds with natural products offer an affordable strategy to develop dressing material with high efficiency in healing wounds. Using image based phenotyping and quantification, we screened natural product derived bioactive compounds for modulators of types I and III collagen production from human foreskin derived fibroblast cells. The identified hit was then formulated with amnion to develop a biomaterial, and its biophysical properties, in vitro and in vivo effects were characterized. In addition, we performed functional profiling analyses by PCR array to understand the effect of individual components of these materials on various genes such as inflammatory mediators including chemokines and cytokines, growth factors, fibroblast stimulating markers for collagen secretion, matrix metalloproteinases, etc., associated with wound healing. FACS based cell cycle analyses were carried out to evaluate the potential of biomaterials for induction of proliferation of fibroblasts. Western blot analyses was done to examine the effect of biomaterial on collagen synthesis by cells and compared to cells grown in the presence of growth factors. This work demonstrated an uncomplicated way of identifying components that synergistically promote healing. Besides, we demonstrated that modulating local wound environment using biomaterials with bioactive compounds could enhance healing. This study finds that the developed biomaterials offer immense scope for healing wounds by means of their skin regenerative features such as anti-inflammatory, fibroblast stimulation for collagen secretion as well as inhibition of enzymes and markers impeding the healing, hydrodynamic properties complemented with other features including non-toxicity, biocompatibility, and safety. PMID:28769790

The fine structure of human germ layers in vivo: clues to the early differentiation of embryonic stem cells in vitro.

PubMed

Sathananthan, Henry; Selvaraj, Kamala; Clark, Joan

2011-08-01

The fine structure of the three germ layers in human ectopic embryos (stage 7) have been documented by digital light and electron microscopy. The formation of ectoderm, endoderm and mesoderm and notochordal cells, and also the extraembryonic membranes, amnion and yolk sac, are imaged. The germ layers give rise to all the cells and tissues of the human body. Possible clues to the early differentiation of embryonic stem cells (ESC) in vitro were obtained, since these events are more or less mimicked in cultures of ESC derived from the inner cell mass of human blastocysts. The findings are discussed with reference to previous studies on the fine structure of ESC using the same technique. Copyright © 2011. Published by Elsevier Ltd.

Grafting with Cryopreserved Amniotic Membrane versus Conservative Wound Care in Treatment of Pressure Ulcers: A Randomized Clinical Trial

PubMed Central

Dehghani, Mehdi; Azarpira, Negar; Mohammad Karimi, Vahid; Mossayebi, Hamid; Esfandiari, Elaheh

2017-01-01

Objective: To compare the healing process of pressure ulcers treated with cryopreserved human amniotic membrane allograft and routine pressure ulcer care in our hospital. Methods: From January 2012 to December 2013, in a prospective randomized clinical trial (IRCT201612041335N2), 24 patients with second and third stage of pressure ulcers were enrolled in this study. All patients needed split-thickness skin grafts for pressure ulcer-wound coverage. Selected patients had symmetric ulcers on both upper and lower extremities. The patients were randomly divided into two groups: amnion and control. In the amnion group, the ulcer was covered with cryopreserved amniotic membrane and in the control group it was treated with local Dilantin powder application. The duration and success rate of complete healing was compared between the two groups. Results: The study group was composed of 24 pressure ulcers in 24 patients (19 males and 5 females) with a mean age of 44±12.70 years. The demographic characteristics, ulcer area, and underlying diseases were similar in both groups. The early sign of response, such as decrease in wound discharge, was detected 12-14 days after biological dressing. Complete pressure ulcer healing occurred only in the amnion group (p< 0.001). Partial healing was significantly higher in the amnion group (p< 0.03). Healing time in this group was faster than that the control group (20 days versus 54 days). No major complication was recorded with amniotic membrane dressing Conclusion: Cryopreserved amniotic membrane is an effective biologic dressing that promotes re-epithelialization in pressure ulcers PMID:29177171

Rolling the Human Amnion to Engineer Laminated Vascular Tissues

PubMed Central

Amensag, Salma

2012-01-01

The prevalence of cardiovascular disease and the limited availability of suitable autologous transplant vessels for coronary and peripheral bypass surgeries is a significant clinical problem. A great deal of progress has been made over recent years to develop biodegradable materials with the potential to remodel and regenerate vascular tissues. However, the creation of functional biological scaffolds capable of withstanding vascular stress within a clinically relevant time frame has proved to be a challenging proposition. As an alternative approach, we report the use of a multilaminate rolling approach using the human amnion to generate a tubular construct for blood vessel regeneration. The human amniotic membrane was decellularized by agitation in 0.03% (w/v) sodium dodecyl sulfate to generate an immune compliant material. The adhesion of human umbilical vein endothelial cells (EC) and human vascular smooth muscle cells (SMC) was assessed to determine initial binding and biocompatibility (monocultures). Extended cultures were either assessed as flat membranes, or rolled to form concentric multilayered conduits. Results showed positive EC adhesion and a progressive repopulation by SMC. Functional changes in SMC gene expression and the constructs' bulk mechanical properties were concomitant with vessel remodeling as assessed over a 40-day culture period. A significant advantage with this approach is the ability to rapidly produce a cell-dense construct with an extracellular matrix similar in architecture and composition to natural vessels. The capacity to control physical parameters such as vessel diameter, wall thickness, shape, and length are critical to match vessel compliance and tailor vessel specifications to distinct anatomical locations. As such, this approach opens new avenues in a range of tissue regenerative applications that may have a much wider clinical impact. PMID:22616610

Inflammatory Gene Regulatory Networks in Amnion Cells Following Cytokine Stimulation: Translational Systems Approach to Modeling Human Parturition

PubMed Central

Summerfield, Taryn L.; Yu, Lianbo; Gulati, Parul; Zhang, Jie; Huang, Kun; Romero, Roberto; Kniss, Douglas A.

2011-01-01

A majority of the studies examining the molecular regulation of human labor have been conducted using single gene approaches. While the technology to produce multi-dimensional datasets is readily available, the means for facile analysis of such data are limited. The objective of this study was to develop a systems approach to infer regulatory mechanisms governing global gene expression in cytokine-challenged cells in vitro, and to apply these methods to predict gene regulatory networks (GRNs) in intrauterine tissues during term parturition. To this end, microarray analysis was applied to human amnion mesenchymal cells (AMCs) stimulated with interleukin-1β, and differentially expressed transcripts were subjected to hierarchical clustering, temporal expression profiling, and motif enrichment analysis, from which a GRN was constructed. These methods were then applied to fetal membrane specimens collected in the absence or presence of spontaneous term labor. Analysis of cytokine-responsive genes in AMCs revealed a sterile immune response signature, with promoters enriched in response elements for several inflammation-associated transcription factors. In comparison to the fetal membrane dataset, there were 34 genes commonly upregulated, many of which were part of an acute inflammation gene expression signature. Binding motifs for nuclear factor-κB were prominent in the gene interaction and regulatory networks for both datasets; however, we found little evidence to support the utilization of pathogen-associated molecular pattern (PAMP) signaling. The tissue specimens were also enriched for transcripts governed by hypoxia-inducible factor. The approach presented here provides an uncomplicated means to infer global relationships among gene clusters involved in cellular responses to labor-associated signals. PMID:21655103

RGC Neuroprotection Following Optic Nerve Trauma Mediated By Intranasal Delivery of Amnion Cell Secretome

PubMed Central

Grinblat, Gabriela A.; Khan, Reas S.; Dine, Kimberly; Wessel, Howard; Brown, Larry; Shindler, Kenneth S.

2018-01-01

Purpose Intranasally delivered ST266, the biological, proteinaceous secretome of amnion-derived multipotent progenitor cells, reduces retinal ganglion cell (RGC) loss, optic nerve inflammation, and demyelination in experimental optic neuritis. This unique therapy and novel administration route delivers numerous cytokines and growth factors to the eye and optic nerve, suggesting a potential to also treat other optic neuropathies. Thus, ST266-mediated neuroprotection was examined following traumatic optic nerve injury. Methods Optic nerve crush injury was surgically induced in C57BL/6J mice. Mice were treated daily with intranasal PBS or ST266. RGC function was assessed by optokinetic responses (OKRs), RGCs were counted, and optic nerve sections were stained with luxol fast blue and anti-neurofilament antibodies to assess myelin and RGC axon damage. Results Intranasal ST266 administered daily for 5 days, beginning at the time that a 1-second optic nerve crush was performed, significantly attenuated OKR decreases. Furthermore, ST266 treatment reduced damage to RGC axons and myelin within optic nerves, and blocked RGC loss. Following a 4-second optic nerve crush, intranasal ST266 increased RGC survival and showed a trend toward reduced RGC axon and myelin damage. Ten days following optic nerve crush, ST266 prevented myelin damage, while also inducing a trend toward increased RGC survival and visual function. Conclusions ST266 significantly attenuates traumatic optic neuropathy. Neuroprotective effects of this unique combination of biologic molecules observed here and previously in optic neuritis suggest potential broad application for preventing neuronal damage in multiple optic nerve disorders. Furthermore, results support intranasal delivery as a novel, noninvasive therapeutic modality for eyes and optic nerves. PMID:29847652

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

PubMed

Carta, M C; Mattana, A; Camici, M; Allegrini, S; Tozzi, M G; Sgarrella, F

2001-10-03

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

Expression of the cytokeratin endo A gene during early mouse embryogenesis.

PubMed Central

Duprey, P; Morello, D; Vasseur, M; Babinet, C; Condamine, H; Brûlet, P; Jacob, F

1985-01-01

Expression of cytokeratin endo A has been analyzed during mouse blastocyst formation and embryonal carcinoma cell differentiation. To study the regulation of endo A expression, nuclease S1 mapping experiments have been performed on RNA extracted from two-cell to 7.5-day embryos. Low levels of endo A mRNA begin to be detectable in eight-cell embryos. The amount of this mRNA increases at the blastocyst stage, suggesting that endo A expression is regulated at the mRNA level during blastocyst formation. At this stage, in situ hybridization studies show that endo A mRNA is present in the trophectoderm but not in the inner cell mass. In 7.5-day embryos, endo A mRNAs are also detectable in the endoderm layer and in the amnion. Images PMID:2417224

Amniotic therapeutic biomaterials in urology: current and future applications.

PubMed

Oottamasathien, Siam; Hotaling, James M; Craig, James R; Myers, Jeremy B; Brant, William O

2017-10-01

To examine the rationale and applications of amniotic tissue augmentation in urological surgery. Published literature in English-language was reviewed for basic science and clinical use of amniotic or amnion-chorionic tissue in genitourinary tissues. Basic science and animal studies support the likely benefit of clinical applications of amnion-derived tissues in a variety of urologic interventions. The broad number of properties found in amniotic membrane, coupled with its immunologically privileged status presents a number of future applications in the urological surgical realm. These applications are in their clinical infancy and suggest that further studies are warranted to investigate the use of these products in a systematic fashion.

Minimally Invasive Surgical Device for Precise Application of Bioadhesives to Prevent iPPROM.

PubMed

Devaud, Yannick Robert; Züger, Silvia; Zimmermann, Roland; Ehrbar, Martin; Ochsenbein-Kölble, Nicole

2018-06-19

The benefits of endoscopic fetal surgery are deteriorated by the high risk of iatrogenic preterm prelabor rupture of fetal membranes (iPPROM). While previous studies have reported good sealing candidates to prevent membrane rupture, the delivery of these materials to the location of membrane puncture remains unsolved. We describe an approach to apply sealing materials onto the amnion through the fetoscopy port. We developed a device composed of an umbrella-shaped polyester coated nitinol mesh and an applicator. The spontaneously unfolding umbrella is pushed through the port, pulled against the amnion, and glued onto the amnion defect site. We tested the adhesion strength of multiple glues and tested the feasibility and reproducibility of this fetal membrane sealing approach in an ex vivo model. The umbrella unfolded and was well positioned in all tests (n = 18). When applied via the fetoscopy port, umbrellas were successfully glued onto the fetal membrane, and all of them completely covered the defect (n = 5). The mean time needed for the whole procedure was 3 min. This study is a proof of concept presenting a potential future solution for the precise local application of bioadhesives for the prevention of iPPROM. © 2018 S. Karger AG, Basel.

Pregnant women have increased incidence of IgE autoantibodies reactive with the skin and placental antigen BP180 (type XVII collagen)

PubMed Central

Noe, Megan H.; Messingham, Kelly A.N.; Brandt, Debra S.; Andrews, Janet I.; Fairley, Janet A.

2016-01-01

BP180 (type XVII collagen) is a transmembrane protein expressed in a variety of cell types. It is also the target of autoantibodies in cutaneous autoimmune disease including bullous pemphigoid and pemphigoid gestationis, a disease unique to pregnancy. The purpose of this study was to determine the prevalence and specificity of cutaneous autoantibodies in a cohort of pregnant women. De-identified sera were collected from pregnant women (n = 299) and from non-pregnant controls (n = 134). Sera were analyzed by ELISA for the presence of IgG and IgE autoantibodies directed against several cutaneous autoantigens. IgE antibodies against the NC16A domain of BP180 were detected in 7.7% of pregnant women, compared to 2.2% of healthy controls (p = 0.01). No increase in total or cutaneous autoantigen specific IgG was seen. Total serum IgE was within the normal range. Full-length BP180 was detected by western immunoblot in epidermal, keratinocyte, placental and cytotrophoblast (CTB) cell lysates. Furthermore, flow cytometry and indirect immunofluorescence confirmed the expression of BP180 on the surface of cultured CTBs. Finally, it was demonstrated that IgE antibodies in the pregnancy sera labeled not only cultured CTBs, but also the placental amnion and cutaneous basement membrane zone using indirect immunofluorescence. We conclude that some pregnant women develop antibodies specific for BP180, and that these autoantibodies are capable of binding both CTB and the placental amnion, potentially affecting placental function. PMID:20471095

Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

PubMed

Kniss, Douglas A; Summerfield, Taryn L

2014-08-01

Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

Effect of Silibinin in Reducing Inflammatory Pathways in In Vitro and In Vivo Models of Infection-Induced Preterm Birth

PubMed Central

Lim, Ratana; Morwood, Carrington J.; Barker, Gillian; Lappas, Martha

2014-01-01

Infection-induced preterm birth is the largest cause of infant death and of neurological disabilities in survivors. Silibinin, from milk thistle, exerts potent anti-inflammatory activities in non-gestational tissues. The aims of this study were to determine the effect of silibinin on pro-inflammatory mediators in (i) human fetal membranes and myometrium treated with bacterial endotoxin lipopolysaccharide (LPS) or the pro-inflammatory cytokine IL-1β, and (ii) in preterm fetal membranes with active infection. The effect of silibinin on infection induced inflammation and brain injury in pregnant mice was also assessed. Fetal membranes and myometrium (tissue explants and primary cells) were treated with 200 μM silibinin in the presence or absence of 10 μg/ml LPS or 1 ng/ml IL-1β. C57BL/6 mice were injected with 70 mg/kg silibinin with or without 50 μg LPS on embryonic day 16. Fetal brains were collected after 6 h. In human fetal membranes, silibinin significantly decreased LPS-stimulated expression of IL-6 and IL-8, COX-2, and prostaglandins PGE2 and PGF2α. In primary amnion and myometrial cells, silibinin also decreased IL-1β-induced MMP-9 expression. Preterm fetal membranes with active infection treated with silibinin showed a decrease in IL-6, IL-8 and MMP-9 expression. Fetal brains from mice treated with silibinin showed a significant decrease in LPS-induced IL-8 and ninjurin, a marker of brain injury. Our study demonstrates that silibinin can reduce infection and inflammation-induced pro-labour mediators in human fetal membranes and myometrium. Excitingly, the in vivo results indicate a protective effect of silibinin on infection-induced brain injury in a mouse model of preterm birth. PMID:24647589

The advantages of the application of amnion membrane in the treatment of burns.

PubMed

Andonovska, D; Dzokic, Gj; Spasevska, L; Trajkovska, T; Popovska, K; Todorov, I; Petrovski, P; Kondov, G; Sapova, B; Marcikic, G; Atanasova, E; Obocki, E; Ugrinovska, J; Andonovski, D; Andonovski, D; Vasilevska, V; Mircevska-Zogovska, E

2008-07-01

A crucial and important factor for successful treatment of burns is the early covering of the burned area with skin substitutes. The covering of the burn requires material that restores the epidermal function and integrates itself into the process of healing. Biological dressings are the golden standard for the temporary covering of burns. All biological skin substitutes are susceptible to early graft reaction and the only exception is the amnion membrane. The importance of the amnion membrane as a biological dressing for burns amounts to: a barrier to bacterial colonization, hastens the epithelisation, and control of water loss. Amnioplasty is a method of application of amnion membrane on the recipient site. In this comparative study, 60 patients with dermal and sub-dermal burns were included. Research was made on an examination group of 30 patients with burns where the method of amnioplasty was applied, and for this amnion membrane conserved in 76% alcohol was used. The control group was made up of 30 patients with burns treated conventionally, and standard methods for the local treatment of burns were applied: exposition, occlusive dressing and initial excision with skin grafting. Pathohistological and microbiological analyses of the bioptical material were made. The degree of the burns was determined through a pathohistological analysis of the bioptical material taken the third day, and in some of the subjects where re-epithelialization was determined on the seventh day, the further re-epithelialization was observed clinically. Pathohistological examination enabled discrimination between bacterial colonization and the invasive bacterial infection. Furthermore, the type of bacterial colonization and infection was determined, which was confirmed with microbiological analysis. The analysis of the results from the microbiological and pathohistological researches of the bioptical material according to the bacterial colonization and infection showed that, although between the examined and the control group there was no statistically important difference, the value of p = 0.067 is close to the statistically important value of p < 0.05. The results of the pathohistological examination of the bioptical material taken the seventh day and analysed according to the re-epithelialization showed that there was a significant difference between the two groups of p < 0.035. It should be mentioned that, although according to the microbiological examinations of the bioptical material a statistically significant difference was not achieved, clinical significance was achieved. The obtained significance of p < 0.035 compared to the re-epithelialization in both groups approved the application of the method of amnioplasty. The histological analysis of the bioptical material not only determines the degree of the burns specifically, but facilitates the choice of method for further treatment, observes the speed of the re-epithelialization and plays an important part in the correct diagnosis and the early start of the specific therapy, important in preventing sepsis. The application of amnion membrane as a biological dressing speeds the re-epithelialization and prevents invasive bacterial infection. Pathohistological examination of the burns is recommended to be established as a standard method in clinical practice.

Transport and biodistribution of dendrimers across human fetal membranes: implications for intravaginal administration of dendrimer-drug conjugates.

PubMed

Menjoge, Anupa R; Navath, Raghavendra S; Asad, Abbas; Kannan, Sujatha; Kim, Chong J; Romero, Roberto; Kannan, Rangaramanujam M

2010-06-01

Dendrimers are emerging as promising topical antimicrobial agents, and as targeted nanoscale drug delivery vehicles. Topical intravaginal antimicrobial agents are prescribed to treat the ascending genital infections in pregnant women. The fetal membranes separate the extra-amniotic space and fetus. The purpose of the study is to determine if the dendrimers can be selectively used for local intravaginal application to pregnant women without crossing the membranes into the fetus. In the present study, the transport and permeability of PAMAM (poly (amidoamine)) dendrimers, across human fetal membrane (using a side by side diffusion chamber), and its biodistribution (using immunofluorescence) are evaluated ex-vivo. Transport across human fetal membranes (from the maternal side) was evaluated using Fluorescein (FITC), an established transplacental marker (positive control, size approximately 400 Da) and fluorophore-tagged G(4)-PAMAM dendrimers (approximately 16 kDa). The fluorophore-tagged G(4)-PAMAM dendrimers were synthesized and characterized using (1)H NMR, MALDI TOF MS and HPLC analysis. Transfer was measured across the intact fetal membrane (chorioamnion), and the separated chorion and amnion layers. Over a 5 h period, the dendrimer transport across all the three membranes was less than <3%, whereas the transport of FITC was relatively fast with as much as 49% transport across the amnion. The permeability of FITC (7.9 x 10(-7) cm(2)/s) through the chorioamnion was 7-fold higher than that of the dendrimer (5.8 x 10(-8) cm(2)/s). The biodistribution showed that the dendrimers were largely present in interstitial spaces in the decidual stromal cells and the chorionic trophoblast cells (in 2.5-4 h) and surprisingly, to a smaller extent internalized in nuclei of trophoblast cells and nuclei and cytoplasm of stromal cells. Passive diffusion and paracellular transport appear to be the major route for dendrimer transport. The overall findings further suggest that entry of drugs conjugated to dendrimers would be restricted across the human fetal membranes when administered topically by intravaginal route, suggesting new ways of selectively delivering therapeutics to the mother without affecting the fetus. (c) 2010 Elsevier Ltd. All rights reserved.

Transport and Biodistribution of Dendrimers Across Human Fetal Membranes: Implications for Intravaginal Administration of Dendrimers

PubMed Central

Menjoge, Anupa R.; Navath, Raghavendra S.; Asad, Abbas; Kannan, Sujatha; Kim, Chong Jai; Romero, Roberto; Kannan, Rangaramanujam M.

2010-01-01

Dendrimers are emerging as promising topical antimicrobial agents, and as targeted nanoscale drug delivery vehicles. Topical intravaginal antimicrobial agents are prescribed to treat the ascending genital infections in pregnant women. The fetal membranes separate the extra-amniotic space and fetus. The purpose of the study is to determine if the dendrimers can be selectively used for local intravaginal application to pregnant women without crossing the membranes into the fetus. In the present study, the transport and permeability of PAMAM (poly(amidoamine)) dendrimers, across human fetal membrane (using a side-by-side diffusion chamber), and its biodistribution (using immunofluorescence) are evaluated ex-vivo. Transport across human fetal membranes (from the maternal side) was evaluated using Fluorescein (FITC), an established transplacental marker (positive control, size~ 400 Da) and fluorophore-tagged G4-PAMAM dendrimers (~ 16 kDa). The fluorophore-tagged G4-PAMAM dendrimers were synthesized and characterized using 1H NMR, MALDI TOF-MS and HPLC analysis. Transfer was measured across the intact fetal membrane (chorioamnion), and the separated chorion and amnion layers. Over a five hour period, the dendrimer transport across all the three membranes was less than < 3 %, whereas the transport of FITC was relatively fast with as much as 49% transport across the amnion. The permeability of FITC (7.9 × 10-7 cm2/s) through the chorioamnion was 7-fold higher than that of the dendrimer (5.8 × 10-8 cm2/s). The biodistribution showed that the dendrimers were largely present in interstitial spaces in the decidual stromal cells and the chorionic trophoblast cells (in 2.5 to 4 h) and surprisingly, to a smaller extent internalized in nuclei of trophoblast cells and nuclei and cytoplasm of stromal cells. Passive diffusion and paracellular transport appear to be the major route for dendrimer transport. The overall findings further suggest that entry of drugs conjugated to dendrimers would be restricted across the human fetal membranes when administered topically by intravaginal route, suggesting new ways of selectively delivering therapeutics to the mother without affecting the fetus. PMID:20346497

Nitric oxide synthase mRNA expression in human fetal membranes: a possible role in parturition.

PubMed

Dennes, W J; Slater, D M; Bennett, P R

1997-04-07

Nitric oxide (NO) is a potent endogenous smooth-muscle relaxant. It is synthesised from 1-arginine by isoforms of nitric oxide synthase (NOS). Whilst it is clear that the uterus responds to NO by relaxation, NOS expression has not been investigated in fetal membranes or myometrium in human pregnancy. This study has shown, using semi-quantitative RT-PCR, expression of cNOS mRNA in human amnion, chorion-decidua, and placenta. iNOS mRNA expression was demonstrated in human amnion, chorion-decidua, and placenta. It is possible that NO synthesised in fetal membranes may act either directly to inhibit myometrial contractility or indirectly to interact with other labour-associated genes, such as cyclo-oxygenase, to coordinate the onset of labour.

A "two-objective, one-area" procedure in absorption microphotometry and its application using an inverted microscope.

PubMed

Chaubal, K A

1988-08-01

A 'two-objective, one-area' method and related equations are suggested to measure absorbance of microscopic stained objects. In such work, the measuring field invariably includes an image of the object and some clear area surrounding the image. The total intensity in the two areas is measured photometrically, using two different objectives, and substituted in the equation for absorbance. The equation is independent of the term representing intensity from the clear area and hence the error in the measurement of absorbance is reduced. The limitations of the 'two-objective, one-area' method are discussed and its pragmatic operation described with an experimental setup involving an inverted microscope. The method permits measurement of intensity in a part of a stained cell while the rest of the cell remains in the field of view. The method is applied to measure absorbance in Giemsa stained ascites cells and Feulgen stained liver and Human Amnion cells.

Quantitative mapping of intracellular cations in the human amniotic membrane

NASA Astrophysics Data System (ADS)

Moretto, Ph.; Llabador, Y.; Simonoff, M.; Razafindrabe, L.; Bara, M.; Guiet-Bara, A.

1993-05-01

The effect of magnesium and taurine on the permeability of cell membranes to monovalent cations has been investigated using the Bordeaux nuclear microprobe. PIXE and RBS techniques have been used to provide quantitative measurements and ion distributions in the isolated amniotic membrane. This physiological model for cellular exchanges allowed us to reveal the distribution of most elements involved in cellular pathways and the modifications under different experimental conditions of incubation in physiological fluids. The PIXE microanalysis provided an original viewpoint on these mechanisms. Following this first study, the amnion compact lamina was found to play a role which was not, up to now, taken into account in the interpretation of electrophysiological experimentations. The release of some ionic species, such as K +, from the epithelial cells, during immersion in isotonic fluids, could have been hitherto underestimated.

Fetal Membranes-Derived Stem Cells Microenvironment.

PubMed

Favaron, Phelipe Oliveira; Miglino, Maria Angelica

2017-01-01

Recently, the regenerative medicine has been trying to congregate different areas such as tissue engineering and cellular therapy, in order to offer effective treatments to overcome several human and veterinary medical problems. In this regard, fetal membranes have been proposed as a powerful source for obtainment of multipotent stem cells with low immunogenicity, anti-inflammatory properties and nontumorigenicity properties for the treatment of several diseases, including replacing cells lost due to tissue injuries or degenerative diseases. Morpho-physiological data have shown that fetal membranes, especially the yolk sac and amnion play different functions according to the gestational period, which are direct related to the features of the microenvironment that their cells are subject. The characteristics of the microenvironment affect or controls important cellular events involved with proliferation, division and maintenance of the undifferentiated stage or differentiation, especially acting on the extracellular matrix components. Considering the importance of the microenvironment and the diversity of embryonic and fetal membrane-derived stem cells, this chapter will addressed advances in the isolation, phenotyping, characteristics of the microenvironment, and applications of yolk sac and amniotic membrane-derived stem cells for human and veterinary regenerative medicine.

A prospective, randomised comparative study of weekly versus biweekly application of dehydrated human amnion/chorion membrane allograft in the management of diabetic foot ulcers

PubMed Central

Zelen, Charles M; Serena, Thomas E; Snyder, Robert J

2014-01-01

The aim of this study is to determine if weekly application of dehydrated human amnion/chorion membrane allograft reduce time to heal more effectively than biweekly application for treatment of diabetic foot ulcers. This was an institutional review board-approved, registered, prospective, randomised, comparative, non-blinded, single-centre clinical trial. Patients with non-infected ulcers of ≥ 4 weeks duration were included for the study. They were randomised to receive weekly or biweekly application of allograft in addition to a non-adherent, moist dressing with compressive wrapping. All wounds were offloaded. The primary study outcome was mean time to healing. Overall, during the 12-week study period, 92·5% (37/40) ulcers completely healed. Mean time to complete healing was 4·1 ± 2·9 versus 2·4 ± 1·8 weeks (P = 0·039) in the biweekly versus weekly groups, respectively. Complete healing occurred in 50% versus 90% by 4 weeks in the biweekly and weekly groups, respectively (P = 0·014). Number of grafts applied to healed wounds was similar at 2·4 ± 1·5 and 2·3 ± 1·8 for biweekly versus weekly groups, respectively (P = 0·841). These results validate previous studies showing that the allograft is an effective treatment for diabetic ulcers and show that wounds treated with weekly application heal more rapidly than with biweekly application. More rapid healing may decrease clinical operational costs and prevent long-term medical complications. PMID:24618401

Encapsulating Non-Human Primate Multipotent Stromal Cells in Alginate via High Voltage for Cell-Based Therapies and Cryopreservation

PubMed Central

Gryshkov, Oleksandr; Pogozhykh, Denys; Hofmann, Nicola; Pogozhykh, Olena; Mueller, Thomas; Glasmacher, Birgit

2014-01-01

Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15–30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application. PMID:25259731

FGF signalling controls anterior extraembryonic and embryonic fate in the beetle Tribolium.

PubMed

Sharma, Rahul; Beermann, Anke; Schröder, Reinhard

2013-09-01

Fibroblast growth factor (FGF) signalling plays a key role in early embryonic development and cell migration in vertebrates and in invertebrates. To gain novel insights into FGF signalling in an arthropod, we characterized the fgf1b ortholog in the beetle Tribolium that is not represented in the Drosophila genome. We found that FGF1b dependent signalling organizes the anterior to posterior axis of the early embryo. The loss of Tc-fgf1b function in Tribolium by RNA interference resulted in the reduction of the anteriormost extraembryonic fate, in an anterior shift of embryonic fate and in the loss or malformation of anterior embryonic structures. Without intact extraembryonic membranes the serosa and the amnion, Tc-fgf1b(RNAi) embryos did not undergo morphogenetic movements and remained posteriorly localized throughout embryogenesis. Only weakly affected embryos developed into a cuticle that show dorsally curved bodies with head defects and a dorsal opening. Except for the posterior dorsal amnion, the overall topology of the dorsal-ventral axis seemed unaffected. Moreover, FGF signalling was not required for the onset of mesoderm formation but for fine-tuning this tissue during later development. We also show that in affected embryos the dorsal epidermis was expanded and expressed Tc-dpp at a higher level. We conclude that in the Tribolium blastoderm embryo, FGF1-signalling organizes patterning along the AP-axis and also balances the expression level of Dpp in the dorsal epidermis, a tissue critically involved in dorsal closure. Copyright © 2013 Elsevier Inc. All rights reserved.

Amnioinfusion in preterm PROM: effects on amnion and cord histology.

PubMed

Locatelli, A; Andreani, M; Ghidini, A; Verderio, M; Pizzardi, A; Vergani, P; Salafia, C M

2008-02-01

To investigate the effects of transabdominal amnioinfusion (TA) on the histology of amnion (A) and umbilical cord (UC). From a cohort of 56 singleton pregnancies with premature rupture of membranes (PROM) at

Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration

PubMed Central

2014-01-01

Background Chronic wounds are associated with a number of deficiencies in critical wound healing processes, including growth factor signaling and neovascularization. Human-derived placental tissues are rich in regenerative cytokines and have been shown in randomized clinical trials to be effective for healing chronic wounds. In this study, PURION® Processed (MiMedx Group, Marietta, GA) dehydrated human amnion/chorion membrane tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for properties to support wound angiogenesis. Methods Angiogenic growth factors were identified in dHACM tissues using enzyme-linked immunosorbent assays (ELISAs), and the effects of dHACM extract on human microvascular endothelial cell (HMVEC) proliferation and production of angiogenic growth factors was determined in vitro. Chemotactic migration of human umbilical vein endothelial cells (HUVECs) toward pieces of dHACM tissue was determined using a standard in vitro transwell assay. Neovascularization of dHACM in vivo was determined utilizing a murine subcutaneous implant model. Results Quantifiable levels of the angiogenic cytokines angiogenin, angiopoietin-2 (ANG-2), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), platelet derived growth factor BB (PDGF-BB), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) were measured in dHACM. Soluble cues promoted HMVEC proliferation in vitro and increased endogenous production of over 30 angiogenic factors by HMVECs, including granulocyte macrophage colony-stimulating factor (GM-CSF), angiogenin, transforming growth factor β3 (TGF-β3), and HB-EGF. 6.0 mm disks of dHACM tissue were also found to recruit migration of HUVECs in vitro. Moreover, subcutaneous dHACM implants displayed a steady increase in microvessels over a period of 4 weeks, indicative of a dynamic intra-implant neovascular process. Conclusions Taken together, these results demonstrate that dHACM grafts: 1) contain angiogenic growth factors retaining biological activity; 2) promote amplification of angiogenic cues by inducing endothelial cell proliferation and migration and by upregulating production of endogenous angiogenic growth factors by endothelial cells; and 3) support the formation of blood vessels in vivo. dHACM grafts are a promising wound care therapy with the potential to promote revascularization and tissue healing within poorly vascularized, non-healing wounds. PMID:24817999

Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration.

PubMed

Koob, Thomas J; Lim, Jeremy J; Massee, Michelle; Zabek, Nicole; Rennert, Robert; Gurtner, Geoffrey; Li, William W

2014-01-01

Chronic wounds are associated with a number of deficiencies in critical wound healing processes, including growth factor signaling and neovascularization. Human-derived placental tissues are rich in regenerative cytokines and have been shown in randomized clinical trials to be effective for healing chronic wounds. In this study, PURION® Processed (MiMedx Group, Marietta, GA) dehydrated human amnion/chorion membrane tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for properties to support wound angiogenesis. Angiogenic growth factors were identified in dHACM tissues using enzyme-linked immunosorbent assays (ELISAs), and the effects of dHACM extract on human microvascular endothelial cell (HMVEC) proliferation and production of angiogenic growth factors was determined in vitro. Chemotactic migration of human umbilical vein endothelial cells (HUVECs) toward pieces of dHACM tissue was determined using a standard in vitro transwell assay. Neovascularization of dHACM in vivo was determined utilizing a murine subcutaneous implant model. Quantifiable levels of the angiogenic cytokines angiogenin, angiopoietin-2 (ANG-2), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), platelet derived growth factor BB (PDGF-BB), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) were measured in dHACM. Soluble cues promoted HMVEC proliferation in vitro and increased endogenous production of over 30 angiogenic factors by HMVECs, including granulocyte macrophage colony-stimulating factor (GM-CSF), angiogenin, transforming growth factor β3 (TGF-β3), and HB-EGF. 6.0 mm disks of dHACM tissue were also found to recruit migration of HUVECs in vitro. Moreover, subcutaneous dHACM implants displayed a steady increase in microvessels over a period of 4 weeks, indicative of a dynamic intra-implant neovascular process. TAKEN TOGETHER, THESE RESULTS DEMONSTRATE THAT DHACM GRAFTS: 1) contain angiogenic growth factors retaining biological activity; 2) promote amplification of angiogenic cues by inducing endothelial cell proliferation and migration and by upregulating production of endogenous angiogenic growth factors by endothelial cells; and 3) support the formation of blood vessels in vivo. dHACM grafts are a promising wound care therapy with the potential to promote revascularization and tissue healing within poorly vascularized, non-healing wounds.

Cell Therapy From Bench to Bedside Translation in CNS Neurorestoratology Era

PubMed Central

Huang, Hongyun; Chen, Lin; Sanberg, Paul

2010-01-01

Recent advances in cell biology, neural injury and repair, and the progress towards development of neurorestorative interventions are the basis for increased optimism. Based on the complexity of the processes of demyelination and remyelination, degeneration and regeneration, damage and repair, functional loss and recovery, it would be expected that effective therapeutic approaches will require a combination of strategies encompassing neuroplasticity, immunomodulation, neuroprotection, neurorepair, neuroreplacement, and neuromodulation. Cell-based restorative treatment has become a new trend, and increasing data worldwide have strongly proven that it has a pivotal therapeutic value in CNS disease. Moreover, functional neurorestoration has been achieved to a certain extent in the CNS clinically. Up to now, the cells successfully used in preclinical experiments and/or clinical trial/treatment include fetal/embryonic brain and spinal cord tissue, stem cells (embryonic stem cells, neural stem/progenitor cells, hematopoietic stem cells, adipose-derived adult stem/precursor cells, skin-derived precursor, induced pluripotent stem cells), glial cells (Schwann cells, oligodendrocyte, olfactory ensheathing cells, astrocytes, microglia, tanycytes), neuronal cells (various phenotypic neurons and Purkinje cells), mesenchymal stromal cells originating from bone marrow, umbilical cord, and umbilical cord blood, epithelial cells derived from the layer of retina and amnion, menstrual blood-derived stem cells, Sertoli cells, and active macrophages, etc. Proof-of-concept indicates that we have now entered a new era in neurorestoratology. PMID:21359168

Placenta-an alternative source of stem cells

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

Matikainen, Tiina; Laine, Jarmo

2005-09-01

The two most promising practical applications of human stem cells are cellular replacement therapies in human disease and toxicological screening of candidate drug molecules. Both require a source of human stem cells that can be isolated, purified, expanded in number and differentiated into the cell type of choice in a controlled manner. Currently, uses of both embryonic and adult stem cells are investigated. While embryonic stem cells are pluripotent and can differentiate into any specialised cell type, their use requires establishment of embryonic stem cell lines using the inner cell mass of an early pre-implantation embryo. As the blastocyst ismore » destroyed during the process, ethical issues need to be carefully considered. The use of embryonic stem cells is also limited by the difficulties in growing large numbers of the cells without inducing spontaneous differentiation, and the problems in controlling directed differentiation of the cells. The use of adult stem cells, typically derived from bone marrow, but also from other tissues, is ethically non-controversial but their differentiation potential is more limited than that of the embryonic stem cells. Since human cord blood, umbilical cord, placenta and amnion are normally discarded at birth, they provide an easily accessible alternative source of stem cells. We review the potential and current status of the use of adult stem cells derived from the placenta or umbilical cord in therapeutic and toxicological applications.« less

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Indonesia.

PubMed

Hilmy, Nazly; Manjas, Menkher; Ferdiansyah; Abbas, Basril; Morales Pedraza, Jorge

2009-05-01

In 1986, the National Nuclear Energy Agency (Batan) in Jakarta started the research and development for the setting up of a tissue bank (Batan Research Tissue Bank/BRTB) by preserving fresh amnion or fetal membranes by lyophilisation and then sterilising by gamma irradiation. During the period of 1990 and 2000, three more tissue banks were set up, i.e., Biomaterial Centre in Surabaya, Jamil Tissue Bank in Padang, and Sitanala Tissue Bank in Tangerang. In 1994, BRTB produced bone allografts. The banks established under the IAEA program concentrated its work on the production of amnion, bone and soft tissues allografts, as well as bone xenografts. These tissues (allografts and xenografts) were sterilised using gamma irradiation (about 90%) and the rest were sterilized by ETO and those products have been used in the treatment of patients at more than 50 hospitals in Indonesia. In 2004, those tissue banks produced 8,500 grafts and 5,000 of them were amnion grafts for eye treatment and wound dressing. All of those grafts were used for patients as well as for research. In 2006, the production increased to 9,000 grafts. Although the capacity of those banks can produce more grafts, we are facing problems on getting raw materials from suitable donors. To fulfill the demand of bone grafts we also produced bone xenografts. The impact of the IAEA program in tissue banking activities in Indonesia can be summarised as follows: to support the national program on importing substitutes for medical devices. The price of imported tissues are between US$ 50 and US$ 6,000 per graft. Local tissue bank can produce tissues with the same quality with the price for about 10-30% of the imported tissues.

Regional expression of prostaglandin E2 and F2alpha receptors in human myometrium, amnion, and choriodecidua with advancing gestation and labor.

PubMed

Grigsby, Peta L; Sooranna, Suren R; Adu-Amankwa, Bernice; Pitzer, Brad; Brockman, Diane E; Johnson, Mark R; Myatt, Leslie

2006-08-01

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.

Conditioned medium derived from rat amniotic epithelial cells confers protection against inflammation, cancer, and senescence.

PubMed

Di Germanio, Clara; Bernier, Michel; Petr, Michael; Mattioli, Mauro; Barboni, Barbara; de Cabo, Rafael

2016-06-28

Amniotic epithelial cells (AECs) are a class of fetal stem cells that derives from the epiblast and resides in the amnion until birth. AECs are suitable candidates for regenerative medicine because of the ease of collection, their low immunogenicity and inability to form tumors after transplantation. Even though human AECs have been widely investigated, the fact remains that very little is known about AECs isolated from rat, one of the most common animal models in medical testing. In this study, we showed that rat AECs retained stemness properties and plasticity, expressed the pluripotency markers Sox2, Nanog, and Oct4 and were able to differentiate toward the osteogenic lineage. The addition of conditioned medium collected from rat AECs to lipopolysaccharide-activated macrophages elicited anti-inflammatory properties through a decrease of Tnfa expression and slowed tumor cell proliferation in vitro and in vivo. The senescence-associated secretory phenotype was also significantly lower upon incubation of senescent human IMR-90 fibroblast cells with conditioned medium from rat AECs. These results confirm the potential of AECs in the modulation of inflammatory mechanisms and open new therapeutic possibilities for regenerative medicine and anti-aging therapies as well.

A tensile machine with a novel optical load cell for soft biological tissues application.

PubMed

Faturechi, Rahim; Hashemi, Ata; Abolfathi, Nabiollah

2014-11-01

The uniaxial tensile testing machine is the most common device used to measure the mechanical properties of industrial and biological materials. The need for a low-cost uniaxial tension testing device for small research centers has always been the subject of research. To address this need, a novel uniaxial tensile testing machine was designed and fabricated to measure the mechanical properties of soft biological tissues. The device is equipped with a new low-cost load cell which works based on the linear displacement/force relationship of beams. The deflection of the beam load cell is measured optically by a digital microscope with an accuracy of 1 µm. The stiffness of the designed load cell was experimentally and theoretically determined at 100 N mm(-1). The stiffness of the load cell can be easily adjusted according to the tissue's strength. The force-time behaviour of soft tissue specimens was obtained by an in-house image processing program. To demonstrate the efficiency of the fabricated device, the mechanical properties of amnion tissue was measured and compared with available data. The obtained results indicate a strong agreement with that of previous studies.

Two consecutive microtubule-based epithelial seaming events mediate dorsal closure in the scuttle fly Megaselia abdita.

PubMed

Fraire-Zamora, Juan Jose; Jaeger, Johannes; Solon, Jérôme

2018-03-14

Evolution of morphogenesis is generally associated with changes in genetic regulation. Here, we report evidence indicating that dorsal closure, a conserved morphogenetic process in dipterans, evolved as the consequence of rearrangements in epithelial organization rather than signaling regulation. In Drosophila melanogaster , dorsal closure consists of a two-tissue system where the contraction of extraembryonic amnioserosa and a JNK/Dpp-dependent epidermal actomyosin cable result in microtubule-dependent seaming of the epidermis. We find that dorsal closure in Megaselia abdita, a three-tissue system comprising serosa, amnion and epidermis, differs in morphogenetic rearrangements despite conservation of JNK/Dpp signaling. In addition to an actomyosin cable, M. abdita dorsal closure is driven by the rupture and contraction of the serosa and the consecutive microtubule-dependent seaming of amnion and epidermis. Our study indicates that the evolutionary transition to a reduced system of dorsal closure involves simplification of the seaming process without changing the signaling pathways of closure progression. © 2018, Fraire-Zamora et al.

Two consecutive microtubule-based epithelial seaming events mediate dorsal closure in the scuttle fly Megaselia abdita

PubMed Central

Jaeger, Johannes

2018-01-01

Evolution of morphogenesis is generally associated with changes in genetic regulation. Here, we report evidence indicating that dorsal closure, a conserved morphogenetic process in dipterans, evolved as the consequence of rearrangements in epithelial organization rather than signaling regulation. In Drosophila melanogaster, dorsal closure consists of a two-tissue system where the contraction of extraembryonic amnioserosa and a JNK/Dpp-dependent epidermal actomyosin cable result in microtubule-dependent seaming of the epidermis. We find that dorsal closure in Megaselia abdita, a three-tissue system comprising serosa, amnion and epidermis, differs in morphogenetic rearrangements despite conservation of JNK/Dpp signaling. In addition to an actomyosin cable, M. abdita dorsal closure is driven by the rupture and contraction of the serosa and the consecutive microtubule-dependent seaming of amnion and epidermis. Our study indicates that the evolutionary transition to a reduced system of dorsal closure involves simplification of the seaming process without changing the signaling pathways of closure progression. PMID:29537962

Intracellular Virus-Specific Structures and RNAs in Oncornavirus-Producing Human Cells

PubMed Central

Bukrinskaya, A. G.; Miller, G. G.; Lebedeva, E. N.; Zhdanov, V. M.

1974-01-01

Two kinds of virus-specific structures were isolated from the cytoplasm of Detroit-6 and human amnion cells producing oncornavirus-like particles. These structures represented A particles with the diameter of 70 to 80 nm and aggregated strands of nucleocapsids with the diameter of 3 and 6 nm. The structures were separated from cellular contaminants by isopycnic banding in linear sucrose gradients and subsequently further purified by sedimentation in velocity sucrose gradients. Their sedimentation coefficient was 250 and 150S, respectively. Both structures contain 60, 45, and 35S RNA species, and 150S structures also contained 20S RNA. The 35 and 20S RNA from the 150S structure formed hybrids with DNA enzymatically synthesized on extracellular virions. The structures displayed endogeneous polymerase activity, DNA product of the reaction being predominantly associated with 60S RNA. No 70S RNA was found in the cell structures of various densities. Also, the virions purified from tissue culture fluid contained 70S RNA. These findings are consistent with those on extracellular maturation of oncornavirus RNA. Images PMID:4810779

CYTOPATHIC EFFECT OF THE ATYPICAL PNEUMONIA ORGANISM IN CULTURES OF HUMAN TISSUE

PubMed Central

Eaton, Monroe D.; Farnham, Ann E.; Levinthal, Jeana D.; Scala, Anthony R.

1962-01-01

Eaton, Monroe D. (Harvard Medical School, Boston, Mass.), Ann E. Farnham, Jeana D. Levinthal, and Anthony R. Scala. Cytopathic effect of the atypical pneumonia organism in cultures of human tissue. J. Bacteriol. 84:1330–1337. 1962.—Three strains of the atypical pneumonia agent were adapted to grow in continuous cell cultures of human amnion or human embryonic lung, with production of initial increased acidity followed by destruction of the cells. Evidence is presented that cytopathic effects of the organism were associated with intracellular growth and formation of microcolonies. Clumps of organisms stained specifically with fluorescein-labeled antibody, and showed distinctive tinctorial reactions with the May Grünwald-Giemsa stain. The cytopathic effect was prevented by fresh serum from a rabbit immunized with an egg-passage strain of the atypical pneumonia agent. Heating the immune serum to 56 C for 30 min abolished the neutralizing effect. The significance of heat-labile serum constituents in killing or inhibition of mycoplasma is discussed. Images PMID:16561984

Progestin treatment does not affect expression of cytokines, steroid receptors, oxytocin receptor, and cyclooxygenase 2 in fetal membranes and endometrium from pony mares at parturition.

PubMed

Palm, F; Walter, I; Nowotny, N; Budik, S; Helmreich, M; Aurich, C

2013-01-01

In most mammalian species, progestins have a major function in maintaining pregnancy. In humans, the physiologic initiation of parturition bears similarities with inflammatory processes and anti-inflammatory effects of progestins have been suggested to postpone birth until term. To examine if comparable effects exist in the horse, mares were treated with the synthetic progestin altrenogest from day 280 of gestation until parturition (N = 5) or were left untreated as controls (N = 7). Tissue from the amnion (AMN), allantochorion (AC), and endometrium (EM) was collected at foaling and mRNA expression of interleukin (IL)-6 and -8, cyclooxygenase 2 (COX2), estrogen receptor (ER) α, progesterone receptor, and oxytocin receptor (OTR) was analyzed. Leukocytes, steroid receptors, COX2, and OTR were also investigated by histology and immunohistochemistry. Expression of mRNA for IL-6 was higher in AMN and EM versus AC (P < 0.01). Expression of IL-8 was higher in AMN than AC and EM (P < 0.001). Steroid receptors and OTR were highly expressed in EM but not in AMN and AC (P < 0.001). Expression of COX2 was most pronounced in AC whereas IL expression was not upregulated in AC. No differences in mRNA expression existed between altrenogest-treated and control animals. Endometrial polymorphonuclear leukocytes were increased in altrenogest-treated mares. Epithelial cells of all tissues, except AC chorionic villi stained progesterone receptor-positive. Staining for ER was more pronounced in the amnion facing epithelium of the AC in altrenogest-treated versus control animals (P < 0.01). In conclusion, COX2 is highly expressed in the AC. The fetal membranes thus might play a role in the onset of labor in the horse. Altrenogest did not affect gene expression in the AMN, AC, and EM but had localized effects on inflammatory cells and ER expression. No anti-inflammatory effects of altrenogest in healthy, late pregnant pony mares could be detected. Copyright © 2013 Elsevier Inc. All rights reserved.

Biological wound dressings sterilized with gamma radiation: Mexican clinical experience

NASA Astrophysics Data System (ADS)

Martínez-Pardo, M. E.; Ley-Chávez, E.; Reyes-Frías, M. L.; Rodríguez-Ferreyra, P.; Vázquez-Maya, L.; Salazar, M. A.

2007-11-01

Biological wound dressings sterilized with gamma radiation, such as amnion and pig skin, are a reality in Mexico. These tissues are currently processed in the tissue bank and sterilized in the Gamma Industrial Irradiation Plant; both facilities belong to the Instituto Nacional de Investigaciones Nucleares (ININ) (National Institute of Nuclear Research). With the strong support of the International Atomic Energy Agency, the bank was established at the ININ and the Mexican Ministry of Health issued its sanitary license on July 7, 1999. The Quality Management System of the bank was certified by ISO 9001:2000 on August 1, 2003; the scope of the system is "Research, Development and Processing of Biological Tissues Sterilized with Gamma Radiation". At present, more than 150 patients from 16 hospitals have been successfully treated with these tissues. This paper presents a brief description of the tissue processing, as well as the present Mexican clinical experience with children and adult patients who underwent medical treatment with radiosterilized amnion and pig skin, used as biological wound dressings on burns and ocular surface disorders.

Experimental infection of pregnant goats with swine fever virus.

PubMed

Shimizu, M; Kumagai, T

1989-07-01

Thirteen pregnant goats were inoculated intravenously with the ALD strain of virulent swine fever (SF) virus on Days 64-84 of gestation. Dams showed transient and mild viremia, and produced high serum neutralizing (SN) antibody after inoculation. Six inoculated dams were reared until parturition occurred and bore six apparently normal, one apparently normal but dead, one mummified and three edematous kids. Neutralizing antibody was demonstrated in the pre-colostral sera obtained from all normal kids, but no SF virus was isolated from any of them. The other seven dams were killed on post-inoculation days (PID) 5-61, and fetuses, placenta and amnion were tested for the virus and SN antibody. All fetuses of five dams examined within PID 40 were positive for SF virus, but negative for SN antibody. SF virus was also isolated from one of three fetuses examined on PID 61. Conversely, the other two fetuses examined on PID 61 were negative for SF virus, but positive for SN antibody. Furthermore, SF virus was isolated from the placenta and amnion of all the dams.

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

PubMed Central

Sthanam, Lakshmi Kavitha; Srivastava, Rohit; Basu, Bhakti; Dutta, Suryendu; Sen, Shamik; Modi, Deepak

2016-01-01

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death. PMID:27583406

Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways.

PubMed

Zhao, Bin; Liu, Jia-Qi; Zheng, Zhao; Zhang, Jun; Wang, Shu-Yue; Han, Shi-Chao; Zhou, Qin; Guan, Hao; Li, Chao; Su, Lin-Lin; Hu, Da-Hai

2016-07-01

Wound healing is a highly orchestrated physiological process consisting in a complex interaction of cellular and biochemical events. Human amniotic epithelial stem cells (HAESCs) have been shown to be an attractive resource for wound healing because they are primitive stem cells. However, the exact effects of amnion-derived stem cells on the migration or proliferation of keratinocytes and their potential mechanism are not fully understood. We have found that HAESCs accelerate the migration of keratinocytes and induce a remarkable increase in the activity of phospho-ERK, phospho-JNK, and phospho-AKT, the blockade of which by their specific inhibitors significantly inhibits migration induced by HAESC-conditioned medium (CM). Furthermore, the co-culture of keratinocytes with HAESCs up-regulates the expression levels of cell proliferation proteins Cyclin D1, Cyclin D3 and Mdm2. In vivo animal experiments have shown that HAESC-CM improves wound healing, whereas blockade with ERK, JNK and AKT inhibitors significantly impairs wound healing. Taken together, these results reveal, for the first time, that HAESCs promote wound healing by facilitating the migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways and might be a potential therapy in skin wound healing.

Chorionicity and Heritability Estimates from Twin Studies: The Prenatal Environment of Twins and Their Resemblance Across a Large Number of Traits.

PubMed

van Beijsterveldt, C E M; Overbeek, L I H; Rozendaal, L; McMaster, M T B; Glasner, T J; Bartels, M; Vink, J M; Martin, N G; Dolan, C V; Boomsma, D I

2016-05-01

There are three types of monozygotic (MZ) twins. MZ twins can either share one chorion and one amnion, each twin can have its own amnion, or MZ twins can-like dizygotic twins-each have their own chorion and amnion. Sharing the same chorion may create a more similar/dissimilar prenatal environment and bias heritability estimates, but most twin studies do not distinguish between these three types of MZ twin pairs. The aim of this paper is to investigate the effect of chorion sharing on the similarity within MZ twin pairs for a large number of traits. Information on chorion status was obtained for the Netherlands twin register (NTR) by linkage to the records from the database of the dutch pathological anatomy national automated archive (PALGA). Record linkage was successful for over 9000 pairs. Effect of chorion type was tested by comparing the within-pair similarity between monochorionic (MC) and dichorionic (DC) MZ twins on 66 traits including weight, height, motor milestones, child problem behaviors, cognitive function, wellbeing and personality. For only 10 traits, within-pair similarity differed between MCMZ and DCMZ pairs. For traits influenced by birth weight (e.g. weight and height in young children) we expected that MC twins would be more discordant. This was found for 5 out of 13 measures. When looking at traits where blood supply is important, we saw MCMZ twins to be more concordant than DCMZ's for 3 traits. We conclude that the influence on the MZ twin correlation of the intra-uterine prenatal environment, as measured by sharing a chorion type, is small and limited to a few phenotypes. This implies that the assumption of equal prenatal environment of mono- and DC MZ twins, which characterizes the classical twin design, is largely tenable.

Involvement of human decidual cell-expressed tissue factor in uterine hemostasis and abruption.

PubMed

Lockwood, C J; Paidas, M; Murk, W K; Kayisli, U A; Gopinath, A; Huang, S J; Krikun, G; Schatz, F

2009-11-01

Vascular injury increases access and binding of plasma-derived factor VII to perivascular cell membrane-bound tissue factor (TF). The resulting TF/VIIa complex promotes hemostasis by cleaving pro-thrombin to thrombin leading to the fibrin clot. In human pregnancy, decidual cell-expressed TF prevents decidual hemorrhage (abruption). During placentation, trophoblasts remodel decidual spiral arteries into high conductance vessels. Shallow trophoblast invasion impedes decidual vascular conversion, producing an inadequate uteroplacental blood flow that elicits abruption-related placental ischemia. Thrombin induces several biological effects via cell surface protease activated receptors. In first trimester human DCs thrombin increases synthesis of sFlt-1, which elicits placental ischemia by impeding angiogenesis-related decidual vascular remodeling. During pregnacy, the fibrillar collagen-rich amnion and choriodecidua extracellular matrix (ECM) provides greater than additive tensile strength and structural integrity. Thrombin acts as an autocrine/paracrine mediator that degrades these ECMs by augmenting decidual cell expression of: 1) matrix metalloproteinases and 2) interleukin-8, a key mediator of abruption-associated decidual infiltration of neutrophils, which express several ECM degrading proteases. Among the cell types at the maternal fetal interface at term, TF expression is highest in decidual cells indicating that this TF meets the hemostatic demands of labor and delivery. TF expression in cultured term decidual cells is enhanced by progestin and thrombin suggesting that the maintenance of elevated circulating progesterone provides hemostatic protection and that abruption-generated thrombin acts in an autocrine/paracrine fashion on decidual cells to promote hemostasis via enhanced TF expression.

PRODUCTION AND CHARACTERIZATION OF MULTIPLE-LAYERED POPULATIONS OF ANIMAL CELLS

PubMed Central

Kruse, Paul F.; Miedema, Ed

1965-01-01

Dense populations containing 129 x 106 Jensen sarcoma, 134 x 106 DON Chinese hamster, 28.9 x 106 WI-38 human diploid, 61.8 x 106 HEp-2 human carcinoma, and 67.4 x 106 WISH human amnion cells were produced from dilute inocula, 0.85 to 5.33 x 106, in 7 to 8 days in a perfusion system using replicate T-60 flasks. Perfusion rates as high as 560 ml medium/day/T-60 were required to maintain pH (to ca ±0.1 unit) and adequate nutrient supplies. The cell densities encountered are described by the term "monolayer equivalents" (M.E.), defined as number of cells per culture divided by number of cells in a monolayer. The M.E.'s for T-60 cultures containing unusually dense populations of 40 x 106 WI-38 and 250 x 106 DON cells (9-day perfusion) were 5 and 17, respectively, and numbers of cells in illustrations of stained cross-sections of membranes from these cultures were in excellent agreement. Threshold M.E.'s exist below which proliferation is the chief cellular activity and above which one or more cell functions may predominate even though proliferation persists. Cellular nutrition and metabolism may change with changes in M.E., as illustrated in different patterns of glutamic acid, proline, and glycine utilization or production in dense vs. dilute WI-38 cell populations. The results indicated that the role of contact inhibition phenomena in arresting cellular proliferation was diminished in perfusion system environments. PMID:5884626

Mesenchymal Stem Cells from Human Amniotic Membrane and Umbilical Cord Can Diminish Immunological Response in an in vitro Allograft Model.

PubMed

Dabrowski, Filip A; Burdzinska, Anna; Kulesza, Agnieszka; Chlebus, Marcin; Kaleta, Beata; Borysowski, Jan; Zolocinska, Aleksandra; Paczek, Leszek; Wielgos, Miroslaw

2017-01-01

Mesenchymal stem cells (MSCs) are gaining rising interest in gynecology and obstetrics. MSCs immunomodulatory properties are suitable enough to reduce perinatal morbidity caused by inflammation in premature neonates. The aim of this study was to evaluate and compare the ability to inhibit allo-activated lymphocytes proliferation by MSCs derived from different sources: amniotic membrane (AM), umbilical cord (UC) and adipose tissue (AT). MSCs were isolated from AM (n = 7) and UC (n = 6) and AT (n = 6) of healthy women. Cells were characterized by flow cytometry and differentiation assay. To evaluate the potential of fetal and adult MSCs to diminish immunological response, mixed lymphocytes reaction (MLR) was performed. Amnion and UC-derived cells displayed typical MSCs characteristics. Addition of MSCs to MLR significantly inhibited the proliferation of stimulated lymphocytes. The effect was observed regardless of the MSCs type used (p < 0.01 in all groups). Comparative analysis revealed no significant differences in this action between tested MSCs types. Additionally, no type of MSCs significantly stimulated allogeneic lymphocytes. The results prove the immunosuppressive capacities of fetal-derived MSCs in vitro. In the future, they may be potentially used to treat premature newborn as well as in immunomodulation in post-transplant therapy. © 2016 S. Karger AG, Basel.

Injectible candidate sealants for fetal membrane repair: Bonding and toxicity in vitro

PubMed Central

Bilic, Grozdana; Brubaker, Carrie; Messersmith, Phillip B.; Mallik, Ajit S.; Quinn, Thomas M.; Haller, Claudia; Done, Elisa; Gucciardo, Leonardo; Zeisberger, Steffen M.; Zimmermann, Roland; Deprest, Jan; Zisch, Andreas H.

2010-01-01

Objective This study was undertaken to test injectible surgical sealants that are biocompatible with fetal membranes, eventually for closure of iatrogenic membrane defects. Study Design Dermabond, Histoacryl, Tissucol fibrin glue, and three types of in situ forming poly(ethylene glycol)-based polymer hydrogels were tested for acute toxicity upon direct contact with fetal membranes for 24h. For determination of elution toxicity, extracts of sealants were incubated on amnion cell cultures for 72h. Bonding and toxicity was assessed through morphological and/or biochemical analysis. Results Extracts of all adhesives were non-toxic for cultured cells. However, only Tissucol and one type of poly(ethylene glycol)-based hydrogel, mussel-mimetic tissue adhesive, showed efficient, non-disruptive, non-toxic bonding to fetal membranes. Mussel-mimetic tissue adhesive applied over membrane defects created with a 3.5 mm trocar accomplished leak-proof closure that withstood membrane stretch in an in vitro model. Conclusion A synthetic hydrogel-type tissue adhesive emerged as potential sealing modality for iatrogenic membrane defects that merits further evaluation in vivo. PMID:20096254

Human Decidua-Derived Mesenchymal Cells Are a Promising Source for the Generation and Cell Banking of Human Induced Pluripotent Stem Cells

PubMed Central

Shofuda, Tomoko; Kanematsu, Daisuke; Fukusumi, Hayato; Yamamoto, Atsuyo; Bamba, Yohei; Yoshitatsu, Sumiko; Suemizu, Hiroshi; Nakamura, Masato; Sugimoto, Yoshikazu; Furue, Miho Kusuda; Kohara, Arihiro; Akamatsu, Wado; Okada, Yohei; Okano, Hideyuki; Yamasaki, Mami; Kanemura, Yonehiro

2013-01-01

Placental tissue is a biomaterial with remarkable potential for use in regenerative medicine. It has a three-layer structure derived from the fetus (amnion and chorion) and the mother (decidua), and it contains huge numbers of cells. Moreover, placental tissue can be collected without any physical danger to the donor and can be matched with a variety of HLA types. The decidua-derived mesenchymal cells (DMCs) are highly proliferative fibroblast-like cells that express a similar pattern of CD antigens as bone marrow-derived mesenchymal cells (BM-MSCs). Here we demonstrated that induced pluripotent stem (iPS) cells could be efficiently generated from DMCs by retroviral transfer of reprogramming factor genes. DMC-hiPS cells showed equivalent characteristics to human embryonic stem cells (hESCs) in colony morphology, global gene expression profile (including human pluripotent stem cell markers), DNA methylation status of the OCT3/4 and NANOG promoters, and ability to differentiate into components of the three germ layers in vitro and in vivo. The RNA expression of XIST and the methylation status of its promoter region suggested that DMC-iPSCs, when maintained undifferentiated and pluripotent, had three distinct states: (1) complete X-chromosome reactivation, (2) one inactive X-chromosome, or (3) an epigenetic aberration. Because DMCs are derived from the maternal portion of the placenta, they can be collected with the full consent of the adult donor and have considerable ethical advantages for cell banking and the subsequent generation of human iPS cells for regenerative applications. PMID:26858858

Placental stem cell correction of murine intermediate maple syrup urine disease.

PubMed

Skvorak, Kristen J; Dorko, Kenneth; Marongiu, Fabio; Tahan, Veysel; Hansel, Marc C; Gramignoli, Roberto; Gibson, K Michael; Strom, Stephen C

2013-03-01

There is improved survival and partial metabolic correction of a mouse intermediate maple syrup urine disease (iMSUD) model after allogenic hepatocyte transplantation, confirming that a small number of enzyme-proficient liver-engrafted cells can improve phenotype. However, clinical shortages of suitable livers for hepatocyte isolation indicate a need for alternative cell sources. Human amnion epithelial cells (hAECs) share stem cell characteristics without the latter's safety and ethical concerns and differentiate to hepatocyte-like cells. Eight direct hepatic hAEC transplantations were performed in iMSUD mice over the first 35 days beginning at birth; animals were provided a normal protein diet and sacrificed at 35 and 100 days. Treatment at the neonatal stage is clinically relevant for MSUD and may offer a donor cell engraftment advantage. Survival was significantly extended and body weight was normalized in iMSUD mice receiving hAEC transplantations compared with untreated iMSUD mice, which were severely cachectic and died ≤28 days after birth. Branched chain α-keto acid dehydrogenase enzyme activity was significantly increased in transplanted livers. The branched chain amino acids leucine, isoleucine, valine, and alloisoleucine were significantly improved in serum and brain, as were other large neutral amino acids. Placental-derived stem cell transplantation lengthened survival and corrected many amino acid imbalances in a mouse model of iMSUD. This highlights the potential for their use as a viable alternative clinical therapy for MSUD and other liver-based metabolic diseases. Copyright © 2012 American Association for the Study of Liver Diseases.

Placental Stem Cell Correction of Murine Intermediate Maple Syrup Urine Disease

PubMed Central

Skvorak, Kristen J.; Dorko, Kenneth; Marongiu, Fabio; Tahan, Veysel; Hansel, Marc C.; Gramignoli, Roberto; Gibson, K. Michael; Strom, Stephen C.

2012-01-01

We previously reported improved survival and partial metabolic correction of a mouse intermediate maple syrup urine disease (iMSUD) model post allogenic hepatocyte transplant, confirming that a small number of enzyme proficient liver-engrafted cells can improve phenotype. However, clinical shortages of suitable livers for hepatocyte isolation indicate a need for alternative cell sources. Human amnion epithelial cells (hAEC) share stem cell characteristics while lacking many safety and ethical concerns, and differentiate to hepatocyte-like cells. Eight direct hepatic hAEC transplants were administered to iMSUD mice over the first 35 days beginning at birth; animals were provided a normal protein diet and sacrificed at days 35 and 100. Treatment at the neonatal stage is clinically relevant for MSUD, and may offer a donor cell engraftment advantage. Survival was significantly extended and body weight was normalized in iMSUD mice receiving hAEC transplants compared to iMSUD (severely cachectic; dead ≤28 days). Branched chain α-keto acid dehydrogenase enzyme activity was significantly increased in transplanted livers. Branched chain amino acids leucine, isoleucine, valine, and alloisoleucine were significantly improved in the sera and brain, as were other large neutral amino acids. Conclusion: Placental-derived stem cell transplantation lengthened survival and corrected many amino acid imbalances in a mouse model of iMSUD. This highlights the potential for their use as a viable alternative clinical therapy for MSUD and other liver-based metabolic diseases. PMID:23175463

PLACENTA OF THE INDIAN ELEPHANT, ELEPHAS INDICUS.

PubMed

COOPER, R A; CONNELL, R S; WELLINGS, S R

1964-10-16

The placenta of the Indian elephant is incompletely annular and zonary macroscopically and occupies the equator of an ovoid chorioallantoic sac. The amnion is fused with the chorion over the zone. Microscopically, the placenta is labyrinthine and endotheliochorial with a rudimentary marginal hematoma. Both macroscopically and microscopically it resembles the placentas of thecarnivores, particularly the racoon, the cat, and the dog.

Effect of Fetal Membrane-Derived Mesenchymal Stem Cell Transplantation in Rats With Acute and Chronic Pancreatitis.

PubMed

Kawakubo, Kazumichi; Ohnishi, Shunsuke; Fujita, Hirotoshi; Kuwatani, Masaki; Onishi, Reizo; Masamune, Atsushi; Takeda, Hiroshi; Sakamoto, Naoya

2016-01-01

Mesenchymal stem cells (MSCs) are a valuable cell source in regenerative medicine and can be isolated from fetal membranes (FMs), particularly amniotic membranes. We investigated the effect of rat FM-derived MSCs (rFM-MSCs) and human amnion-derived MSCs (hAMSCs) on the inflammatory reaction in vitro and therapeutic effects in rats with acute and chronic pancreatitis. Effect of rFM-MSCs or hAMSC-conditioned medium was investigated in vitro. Acute pancreatitis was induced by intraductal injection of 4% taurocholate, and rFM-MSCs were transplanted intravenously. Chronic pancreatitis was induced by intravenous injection of 5 mg/kg dibutyltin dichloride, and hAMSCs were transplanted intravenously. The inflammatory reaction of macrophages induced by lipopolysaccharide and trypsin was significantly suppressed by rFM-MSC coculture. Pancreatic acinar cell injury induced by cerulein was significantly ameliorated by hAMSC-conditioned medium. Pancreatic stellate cell activation induced by tumor necrosis factor-α was significantly decreased by hAMSC-conditioned medium. Transplantation of rFM-MSCs significantly reduced the histological score and infiltration of CD68-positive macrophages in the rat pancreas. The hAMSC transplantation significantly decreased the expression of MCP-1 and attenuated the downregulation of amylase expression in the pancreas. Transplantation of FM-MSCs and AMSCs suppressed the inflammatory reaction of acute and chronic pancreatitis in rats.

Large Extremity Peripheral Nerve Repair

DTIC Science & Technology

2013-10-01

approaching that of conventional microsurgery and that the PTB repair is unlikely to be disturbed in vivo. The results in Figure 5 were obtained with...with large nerve deficit reconstruction and long nerve grafts. As with the human amnion nerve wraps, it was important for us to confirm that, in...xHAM wrap compared to standard (suture) of care microsurgery . Demonstrated improved nerve regeneration in a muscle mass retention model

Large Extremity Peripheral Nerve Repair

DTIC Science & Technology

2013-10-01

show that the PTB method can provide fixation strengths approaching that of conventional microsurgery and that the PTB repair is unlikely to be...biomaterial during long periods of recovery associated with large nerve deficit reconstruction and long nerve grafts. As with the human amnion nerve...functional recovery model (SFI, sciatic function index) using PTB/xHAM wrap compared to standard (suture) of care microsurgery . Demonstrated improved nerve

Corneal Protection for Burn Patients

DTIC Science & Technology

2011-07-01

enzymatic degradation The methods used to harvest AM, to treat the amnion with proteolytic enzymes and to measure the extent of enzymatic...Multiple enzymatic cleavages of the membrane proteins degrade the AM and release peptide fragments containing free amino groups. We measured the level...appropriate modified AM for the goals of this study. Although these measurements were not part of the original proposal, we will soon be able quantify

Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells.

PubMed

Kehl, Debora; Generali, Melanie; Görtz, Sabrina; Geering, Diego; Slamecka, Jaroslav; Hoerstrup, Simon P; Bleul, Ulrich; Weber, Benedikt

2017-10-01

Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras

PubMed Central

Karagenç, Levent; Sandikci, Mustafa

2010-01-01

The objective of the current study was to determine the tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. Quail-chick chimeras were constructed by transferring dissociated cells from the area opaca of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in embryonic as well as extra-embryonic tissues of the recipient embryo were examined using the QCPN monoclonal antibody after 6 days of incubation in serial sections taken at 100-μm intervals. Data gathered in the present study demonstrated that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently the mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in tissues derived from the neural ectoderm and the surface ectoderm, including the optic cup, diencephalon and lens. Although the possibility of random incorporation of transplanted cells into these embryonic structures cannot be excluded, these results would suggest that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types in the recipient chick embryo, including those derived from the surface ectoderm and neural ectoderm. PMID:19900180

Production and characterization of multiple-layered populations of animal cells.

PubMed

Kruse, P F; Miedema, E

1965-11-01

Dense populations containing 129 x 10(6) Jensen sarcoma, 134 x 10(6) DON Chinese hamster, 28.9 x 10(6) WI-38 human diploid, 61.8 x 10(6) HEp-2 human carcinoma, and 67.4 x 10(6) WISH human amnion cells were produced from dilute inocula, 0.85 to 5.33 x 10(6), in 7 to 8 days in a perfusion system using replicate T-60 flasks. Perfusion rates as high as 560 ml medium/day/T-60 were required to maintain pH (to ca +/-0.1 unit) and adequate nutrient supplies. The cell densities encountered are described by the term "monolayer equivalents" (M.E.), defined as number of cells per culture divided by number of cells in a monolayer. The M.E.'s for T-60 cultures containing unusually dense populations of 40 x 10(6) WI-38 and 250 x 10(6) DON cells (9-day perfusion) were 5 and 17, respectively, and numbers of cells in illustrations of stained cross-sections of membranes from these cultures were in excellent agreement. Threshold M.E.'s exist below which proliferation is the chief cellular activity and above which one or more cell functions may predominate even though proliferation persists. Cellular nutrition and metabolism may change with changes in M.E., as illustrated in different patterns of glutamic acid, proline, and glycine utilization or production in dense vs. dilute WI-38 cell populations. The results indicated that the role of contact inhibition phenomena in arresting cellular proliferation was diminished in perfusion system environments.

Enhanced differentiation potential of human amniotic mesenchymal stromal cells by using three-dimensional culturing.

PubMed

Lin, Xue; Li, Hao Yu; Chen, Lian Feng; Liu, Bo Jiang; Yao, Yian; Zhu, Wen Ling

2013-06-01

The therapeutic potential of human amniotic mesenchymal stromal cells (hAMSCs) remains limited because of their differentiation towards mesenchymal stem cells (MSCs) following adherence. The aim of this study was to develop a three-dimensional (3-D) culture system that would permit hAMSCs to differentiate into cardiomyocyte-like cells. hAMSCs were isolated from human amnions of full-term births collected after Cesarean section. Immunocytochemistry, immunofluorescence and flow cytometry analyses were undertaken to examine hAMSC marker expression for differentiation status after adherence. Membrane currents were determined by patch clamp analysis of hAMSCs grown with or without cardiac lysates. Freshly isolated hAMSCs were positive for human embryonic stem-cell-related markers but their marker profile significantly shifted towards that of MSCs following adherence. hAMSCs cultured in the 3-D culture system in the presence of cardiac lysate expressed cardiomyocyte-specific markers, in contrast to those maintained in standard adherent cultures or those in 3-D cultures without cardiac lysate. hAMSCs cultured in 3-D with cardiac lysate displayed a cardiomyocyte-like phenotype as observed by membrane currents, including a calcium-activated potassium current, a delayed rectifier potassium current and a Ca(2+)-resistant transient outward K(+) current. Thus, although adherence limits the potential of hAMSCs to differentiate into cardiomyocyte-like cells, the 3-D culture of hAMSCs represents a more effective method of their culture for use in regenerative medicine.

Involvement of human decidual cell-expressed tissue factor in uterine hemostasis and abruption

PubMed Central

Lockwood, C.J.; Paidas, M.; Murk, W.K.; Kayisli, U.A.; Gopinath, A.; Krikun, G.; Huang, S.J.; Schatz, F.

2009-01-01

Vascular injury increases access and binding of plasma-derived factor VII to perivascular cell membrane-bound tissue factor (TF). The resulting TF/VIIa complex promotes hemostasis by cleaving pro-thrombin to thrombin leading to the fibrin clot. In human pregnancy, decidual cell-expressed TF prevents decidual hemorrhage (abruption). During placentation, trophoblasts remodel decidual spiral arteries into high conductance vessels. Shallow trophoblast invasion impedes decidual vascular conversion, producing an inadequate uteroplacental blood flow that elicits abruption-related placental ischemia. Thrombin induces several biological effects via cell surface protease activated receptors. In first trimester human DCs thrombin increases synthesis of sFlt-1, which elicits placental ischemia by impeding angiogenesis-related decidual vascular remodeling. During pregnacy, the fibrillar collagen-rich amnion and choriodecidua extracellular matrix (ECM) provides greater than additive tensile strength and structural integrity. Thrombin acts as an autocrine/paracrine mediator that degrades these ECMs by augmenting decidual cell expression of: 1) matrix metalloproteinases and 2) interleukin-8, a key mediator of abruption-associated decidual infiltration of neutrophils, which express several ECM degrading proteases. Our recent observations that: 1) among the cell types at the maternal fetal interface at term TF expression is highest in decidual cells indicates that this TF meets the hemostatic demands of labor and delivery; 2) TF expression in cultured term decidual cells is enhanced by progestin and thrombin suggest that maintenance of elevated circulating progesterone at term provides hemostatic protection, whereas abruption-generated thrombin can act in autocrine/paracrine fashion on DCs to promote hemostasis via enhanced TF expression. PMID:19720393

Sealing Penetrating Eye Injuries With Photoactivated Bonding

DTIC Science & Technology

2013-09-01

penetrating eye injuries. Scope: To establish, in ex vitro and in vivo animal models, the treatment parameters for sealing corneal and scleral...cornea surface was compared to that produced by our conventional, bare fiber system using ex vivo rabbit eyes and the standard treatment protocol...Identified the PTB treatment parameters that produce strong bonding of an amnion patch over corneal wounds in ex vivo rabbit eyes . • Determined that a

Experiences using IAEA Code of practice for radiation sterilization of tissue allografts: Validation and routine control

NASA Astrophysics Data System (ADS)

Hilmy, N.; Febrida, A.; Basril, A.

2007-11-01

Problems of tissue allografts in using International Standard (ISO) 11137 for validation of radiation sterilization dose (RSD) are limited and low numbers of uniform samples per production batch, those are products obtained from one donor. Allograft is a graft transplanted between two different individuals of the same species. The minimum number of uniform samples needed for verification dose (VD) experiment at the selected sterility assurance level (SAL) per production batch according to the IAEA Code is 20, i.e., 10 for bio-burden determination and the remaining 10 for sterilization test. Three methods of the IAEA Code have been used for validation of RSD, i.e., method A1 that is a modification of method 1 of ISO 11137:1995, method B (ISO 13409:1996), and method C (AAMI TIR 27:2001). This paper describes VD experiments using uniform products obtained from one cadaver donor, i.e., cancellous bones, demineralized bone powders and amnion grafts from one life donor. Results of the verification dose experiments show that RSD is 15.4 kGy for cancellous and demineralized bone grafts and 19.2 kGy for amnion grafts according to method A1 and 25 kGy according to methods B and C.

The Use of a Dehydrated Amnion/Chorion Membrane Allograft in Patients Who Subsequently Undergo Reexploration after Posterior Lumbar Instrumentation

PubMed Central

Subach, Brian R.; Copay, Anne G.

2015-01-01

Background Context. Products that can reduce development of epidural fibrosis may reduce risk for ongoing pain associated with development of scar tissue and make subsequent epidural reexploration easier. Purpose. To evaluate the use of dehydrated human amnion/chorion membrane (dHACM) on the formation of soft tissue scarring in the epidural space. Study Design. Case series. Patient Sample. Five patients having transforaminal lumbar interbody lumbar fusion (TLIF) with posterior instrumentation and implantation of dHACM in the epidural space and subsequent epidural reexploration. Outcome Measures. Degree of scar tissue adjacent to the epidural space at reexploration. Intraoperative and postoperative complications related to dHACM and patient reported outcomes. Methods. The degree of scar tissue adjacent to the epidural space was assessed during the reexploration surgery. Patients' outcomes were collected using standard validated questionnaires. Results. Four of 5 cases had easily detachable tissue during epidural reexploration. Angiolipoma of 10% was noted in 1 case and 5% in 2 cases. Significant improvements in patient reported outcomes were observed. No intraoperative or postoperative complications occurred. Conclusions. Our findings suggest that dHACM implant during TLIF may have favorable effects on epidural fibrosis and is well tolerated. Further studies with larger cohorts are required to prove our results. PMID:25653880

Angiogenin distribution in human term placenta, and expression by cultured trophoblastic cells

PubMed Central

Pavlov, Nadine; Hatzi, Elissavet; Bassaglia, Yann; Frendo, Jean-Louis; Evain-Brion, Danièle; Badet, Josette

2003-01-01

Human angiogenin is a 14-kDa secreted protein with angiogenic and ribonucleolytic activities. Angiogenin is associated with tumour development but is also present in normal biological fluids and tissues. To further address the physiological role of angiogenin, we studied its expression in situ and in vitro, using the human term placenta as a model of physiological angiogenesis. Angiogenin was immunodetected by light and transmission electron microscopy, and its cellular distribution was established by double immunolabelling with cell markers including von Willebrand factor, platelet/endothelial cell adhesion molecule-1 (PECAM-1), CD34, Tie-2, vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGF-R2), erythropoeitin receptor (Epo-R), alpha-smooth muscle actin, CD45, cytokeratin 7, and Ki-67. Angiogenin immunoreactivity was detected in villous and extravillous trophoblasts, the trophoblast basement membrane, the endothelial basal lamina, foetal blood vessels, foetal and maternal red blood cells, and amnionic cells. Its expression was confirmed by in situ hybridisation with a digoxygenin-labelled cDNA probe and reverse transcriptase-polymerase chain reaction amplification. Villous cytotrophoblasts, isolated and differentiated in vitro into a functional syncytiotrophoblast, expressed and secreted angiogenin. Given its known biological activities in vitro and its observed pattern of expression, these data suggest that, in human placenta, angiogenin has a role not only in angiogenesis but also in vascular and tissue homeostasis, maternal immune tolerance of the foetus, and host defences. PMID:15166501

Anti-Inflammatory Effects of Adult Stem Cells in Sustained Lung Injury: A Comparative Study

PubMed Central

Moodley, Yuben; Vaghjiani, Vijesh; Chan, James; Baltic, Svetlana; Ryan, Marisa; Tchongue, Jorge; Samuel, Chrishan S.; Murthi, Padma; Parolini, Ornella; Manuelpillai, Ursula

2013-01-01

Lung diseases are a major cause of global morbidity and mortality that are treated with limited efficacy. Recently stem cell therapies have been shown to effectively treat animal models of lung disease. However, there are limitations to the translation of these cell therapies to clinical disease. Studies have shown that delayed treatment of animal models does not improve outcomes and that the models do not reflect the repeated injury that is present in most lung diseases. We tested the efficacy of amnion mesenchymal stem cells (AM-MSC), bone marrow MSC (BM-MSC) and human amniotic epithelial cells (hAEC) in C57BL/6 mice using a repeat dose bleomycin-induced model of lung injury that better reflects the repeat injury seen in lung diseases. The dual bleomycin dose led to significantly higher levels of inflammation and fibrosis in the mouse lung compared to a single bleomycin dose. Intravenously infused stem cells were present in the lung in similar numbers at days 7 and 21 post cell injection. In addition, stem cell injection resulted in a significant decrease in inflammatory cell infiltrate and a reduction in IL-1 (AM-MSC), IL-6 (AM-MSC, BM-MSC, hAEC) and TNF-α (AM-MSC). The only trophic factor tested that increased following stem cell injection was IL-1RA (AM-MSC). IL-1RA levels may be modulated by GM-CSF produced by AM-MSC. Furthermore, only AM-MSC reduced collagen deposition and increased MMP-9 activity in the lung although there was a reduction of the pro-fibrogenic cytokine TGF-β following BM-MSC, AM-MSC and hAEC treatment. Therefore, AM-MSC may be more effective in reducing injury following delayed injection in the setting of repeated lung injury. PMID:23936322

A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

PubMed

Nogami, Makiko; Kimura, Tomoatsu; Seki, Shoji; Matsui, Yoshito; Yoshida, Toshiko; Koike-Soko, Chika; Okabe, Motonori; Motomura, Hiraku; Gejo, Ryuichi; Nikaido, Toshio

2016-04-01

Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

SUBCLINICAL INFECTION OF DOGS BY CANINE-ADAPTED MEASLES VIRUS EVIDENCED BY THEIR SUBSEQUENT IMMUNITY TO CANINE DISTEMPER VIRUS

PubMed Central

Moura, Roberto A.; Warren, Joel

1961-01-01

Moura, Roberto A. (Chas. Pfizer and Company, Inc., Terre Haute, Ind.) and Joel Warren. Subclinical infection of dogs by canine-adapted measles virus evidenced by their subsequent immunity to canine distemper virus. J. Bacteriol. 82:702–705. 1961.—Young dogs were inoculated with virulent measles virus which had been adapted to canine kidney or human amnion cell culture. None of the animals showed any clinical symptoms nor could virus be isolated from the blood, although measles-neutralizing and complement-fixing antibodies developed during convalescence. All dogs failed to develop antibody to canine distemper. However, when these and normal control animals were subsequently inoculated intracerebrally with virulent distemper virus, each of the controls succumbed to typical symptoms, whereas all of the measles-immune dogs survived. These results suggest that the cross-protection conferred by measles against canine distemper virus infection involves factors other than humoral antibody. The immunity persists for a considerable length of time. PMID:14476677

Sealing Penetrating Eye Injuries Using Photoactivated Bonding

DTIC Science & Technology

2011-09-01

of the rabbit pushed the viscoeastic out of the eye during the laser phase of the treatment . This interfered with the bonding of the amnion...ANSI Std. Z39.18 Sealing Penetrating Eye Injuries Using Photoactivated Bonding W81XWH-09-2-0059 1 Sep 2010 - 31 Aug 2011Annual01-09-2011 Anthony...called PTB) with the potential to decrease vision loss and ocular complications in warfighters sustaining penetrating eye injuries. Scope: In

[The protective effect of dexpanthenol in nasal sprays. First results of cytotoxic and ciliary-toxic studies in vitro].

PubMed

Klöcker, N; Verse, T; Rudolph, P

2003-03-01

In Germany more than 60 million units of nasal decongestants are prescribed or sold over the counter. The cytotoxic and ciliary-toxic potential of alpha-sympathomimetic decongestants is well established. Furthermore, in many of the marketed products preservatives are added, predominantly benzalchonium-chloride, which can lead to a further alteration of cell- and ciliary function. Recently a protective effect of dexpanthenol was found for the human nasal mucosa. The objective of the present studies was to prove the hypothesis that dexpanthenol is able to neutralise the toxic effects of both alpha-sympathomimetic decongestants, in particular those of xylometazoline, and those of benzalconium-chloride. Therefore, systematic cytotoxic and ex vivo in vitro ciliary-toxic studies were performed. After exposition to xylometazoline in concentrations of 0.1 % and 0.05 %, the influence of dexpanthenol (5 %) and benzalconium-chloride (0,01 %) was assessed by determination of a) cell growth of FL-cells of human amnion origin, and b) ciliary beat frequency of human nasal mucosa. All tests were performed placebo-controlled. Both hypotheses were confirmed. Dexpanthenol (5 %) reduces statistically significantly the concentration-dependent toxic effects of xylometazoline, and benzalchonium-cloride regarding cell growth and ciliary beat frequency (p < 0.001). The combination of xylometazoline with dexpanthenol, while benzalconium-chloride is eliminated, resulted in a further significant increase of cell growth and ciliary beat frequency (p < 0.001), similar to control. The additive application of dexpanthenol (5 %) with nasal decongestants and/or with preserved nasal sprays seems to be able to reduce the cell- and ciliary-toxic effects of these substances.

Interleukin-33 in the Human Placenta

PubMed Central

Topping, Vanessa; Romero, Roberto; Than, Nandor Gabor; Tarca, Adi L.; Xu, Zhonghui; Kim, Sun Young; Wang, Bing; Yeo, Lami; Kim, Chong Jai; Hassan, Sonia S.; Kim, Jung-Sun

2012-01-01

Objective Interleukin-33 (IL-33) is the newest member of the IL-1 cytokine family, a group of key regulators of inflammation. The purpose of this study was to determine whether IL-33 is expressed in the human placenta and to investigate its expression in the context of acute and chronic chorioamnionitis. Methods Placental tissues were obtained from five groups of patients: (1) normal pregnancy at term without labor (n=10); (2) normal pregnancy at term in labor (n=10); (3) preterm labor without inflammation (n=10); (4) preterm labor with acute chorioamnionitis (n=10); and (5) preterm labor with chronic chorioamnionitis (n=10). Immunostaining was performed to determine IL-33 protein expression patterns in the placental disk, chorioamniotic membranes, and umbilical cord. mRNA expression of IL-33 and its receptor IL1RL1 (ST2) was measured in primary amnion epithelial and mesenchymal cells (AECs and AMCs, n=4) and human umbilical vein endothelial cells (HUVECs, n=4) treated with IL-1β (1ng/ml and 10ng/ml) and CXCL10 (0.5ng/ml and 1ng/ml or 5ng/ml). Results 1) Nuclear IL-33 expression was found in endothelial and smooth muscle cells in the placenta, chorioamniotic membranes, and umbilical cord; 2) IL-33 was detected in the nucleus of CD14+ macrophages in the chorioamniotic membranes, chorionic plate, and umbilical cord, and in the cytoplasm of myofibroblasts in the Wharton’s jelly; 3) acute (but not chronic) chorioamnionitis was associated with the presence of IL-33+ macrophages in the chorioamniotic membranes and umbilical cord; 4) expression of IL-33 or IL1RL1 (ST2) mRNA in AECs was undetectable; 5) IL-33 mRNA expression increased in AMCs and HUVECs after IL-1β treatment but did not change with CXCL10 treatment; and 6) IL1RL1 (ST2) expression decreased in AMCs and increased in HUVECs after IL-1β but not CXCL10 treatment. Conclusions IL-33 is expressed in the nucleus of placental endothelial cells, CD14+ macrophages, and myofibroblasts in the Wharton’s jelly. IL-1β can induce the expression of IL-33 and its receptor. Protein expression of IL-33 is detectable in macrophages of the chorioamniotic membranes in acute (but not chronic) chorioamnionitis. PMID:23039129

Damage-associated molecular patterns (DAMPs) in preterm labor with intact membranes and preterm PROM: a study of the alarmin HMGB1

PubMed Central

Romero, Roberto; Chaiworapongsa, Tinnakorn; Savasan, Zeynep Alpay; Xu, Yi; Hussein, Youssef; Dong, Zhong; Kusanovic, Juan Pedro; Kim, Chong Jai; Hassan, Sonia S

2012-01-01

Objective Preterm parturition is a syndrome caused by multiple etiologies. Although intra-amniotic infection is causally linked with intrauterine inflammation and the onset of preterm labor, other patients have preterm labor in the absence of demonstrable infection. It is now clear that inflammation may be elicited by activation of the Damage-Associated Molecular Patterns (DAMPs), which include pathogen-associated molecular patterns (PAMPs) as well as “alarmins” (endogenous molecules that signal tissue and cellular damage). A prototypic alarmin is high-mobility group box-1 (HMGB1) protein, capable of inducing inflammation and tissue repair when it reaches the extracellular environment. HMGB1 is a late-mediator of sepsis, and blockade of HMGB1 activity reduces mortality in an animal model of endotoxemia, even if administered late during the course of the disorder. The objectives of this study were to: 1) determine whether intra-amniotic infection/inflammation (IAI) is associated with changes in amniotic fluid concentrations of HMGB1; and 2) localize immunoreactivity of HMGB1 in the fetal membranes and umbilical cord of patients with chorioamnionitis. Methods Amniotic fluid samples were collected from the following groups: 1) preterm labor with intact membranes (PTL) with (n=42) and without IAI (n=84); and 2) preterm prelabor rupture of membranes (PROM) with (n=38) and without IAI (n=35). IAI was defined as either a positive amniotic fluid culture or amniotic fluid concentration of interleukin-6 (IL-6) ≥2.6 ng/mL. HMGB1 concentrations in amniotic fluid were determined by ELISA. Immunofluorescence staining for HMGB1 was performed in the fetal membranes and umbilical cord of pregnancies with acute chorioamnionitis. Results Amniotic fluid HMGB1 concentrations were higher in patients with IAI than in those without IAI in both the PTL and preterm PROM groups (PTL IAI: median 3.1 ng/mL vs. without IAI; median 0.98 ng/mL; p<0.001; and preterm PROM with IAI median 7.3 ng/mL vs. without IAI median 2.6 ng/mL; p=0.002); patients with preterm PROM without IAI had a higher median amniotic fluid HMGB1 concentration than those with PTL and intact membranes without IAI (p<0.001); and HMGB1 was immunolocalized to amnion epithelial cells and stromal cells in the Wharton’s jelly (prominent in the nuclei and cytoplasm). Myofibroblasts and macrophages of the chorioamniotic connective tissue layer and infiltrating neutrophils showed diffuse cytoplasmic HMGB1 immunoreactivity. Conclusions Intra-amniotic infection/inflammation is associated with elevated amniotic fluid HMGB1 concentrations regardless of membrane status; preterm PROM was associated with a higher amniotic fluid HMGB1 concentration than PTL with intact membranes, suggesting that rupture of membranes is associated with an elevation of alarmins; immunoreactive HMGB1 was localized to amnion epithelial cells, Wharton’s jelly and cells involved in the innate immune response; and we propose that HMGB1 released from stress or injured cells into amniotic fluid may be responsible, in part, for intra-amniotic inflammation due to non-microbial insults. PMID:21958433

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion.

PubMed

Smeringaiova, Ingrida; Trosan, Peter; Mrstinova, Miluse Berka; Matecha, Jan; Burkert, Jan; Bednar, Jan; Jirsova, Katerina

2017-09-01

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Mexico.

PubMed

Martínez-Pardo, María Esther; Morales Pedraza, Jorge; Sánchez Ramírez, Omar

2009-05-01

Tissue banking started in Mexico in 1948-1949, when two bone banks were established, one at the Infantile Hospital of Mexico and other at the Central Military Hospital. Mexico has benefited for the implementation of the IAEA program since through it has been able to settle down and to consolidate the Tissue Bank at the Instituto Nacional de Investigaciones Nucleares ININ (National Institute for Nuclear Research). This is the only bank in Latin America that has a Quality Management System in force, certified under ISO 9001:2000 since August 1, 2003. The first tissue processed was amnion. The main products of the BTR are amnion and pig skin. Both are biological tissues which their main use is as a wound dressing in patients with burns, scars, diabetic ulcers, epidermolysis bullosa, damaged ocular surface, etc. The General Health Law, published in 1984 and reformed in June 19, 2007, describes the procedure for the disposal of organs, tissues and human cadavers in its fourteenth title and in the Regulation for Sanitary Control. During the period 2001-2005, the ININ Tissue Bank produced 292 sterilised tissues (amnion, 86,668 cm(2), and frozen pig skin, 164,220 cm(2), at an estimated cost of 1,012,668 Mexican pesos. Until 2006, one hundred eighty five (185) patients have been treated with the use of sterilised tissues produced by the ININ Tissue Bank. The radiation source used for sterilisation of tissues is an industrial Cobalt-60 irradiator model JS-6500 AECL, which belongs to ININ. This equipment is located in other building, close to the BTR, in the Centro Nuclear de México "Dr. Nabor Carrillo Flores" (Nuclear Center of Mexico). Until 2006, six hospitals use in a routine way the sterilised tissues produced by the ININ Tissue Bank, for the treatment of burns originated by diverse agents like flame, electricity, liquids in boil, chemical reagents, as well as for the reconstruction of the ocular surface. Two of these hospitals treat patients of very low economic incomes, mainly needy individuals, who cannot afford to pay this type of treatments in other hospitals due to their high cost. The results obtained up to now are highly promising.

Uterine overdistention induces preterm labor mediated by inflammation: observations in pregnant women and nonhuman primates

PubMed Central

Waldorf, Kristina M. Adams; Singh, Natasha; Mohan, Aarthi R.; Young, Roger C.; Ngo, Lisa; Das, Ananya; Tsai, Jesse; Bansal, Aasthaa; Paolella, Louis; Herbert, Bronwen R.; Sooranna, Suren R.; Gough, G. Michael; Astley, Cliff; Vogel, Keith; Baldessari, Audrey E.; Bammler, Theodor K.; MacDonald, James; Gravett, Michael G.; Rajagopal, Lakshmi; Johnson, Mark R.

2015-01-01

OBJECTIVE Uterine overdistention is thought to induce preterm labor in women with twin and multiple pregnancies, but the pathophysiology remains unclear. We investigated for the first time the pathogenesis of preterm birth associated with rapid uterine distention in a pregnant nonhuman primate model. STUDY DESIGN A nonhuman primate model of uterine overdistention was created using preterm chronically catheterized pregnant pigtail macaques (Macaca nemestrina) by inflation of intraamniotic balloons (N = 6), which were compared to saline controls (N = 5). Cesarean delivery was performed due to preterm labor or at experimental end. Microarray, quantitative reverse transcriptase polymerase chain reaction, Luminex (Austin, TX), and enzyme-linked immunosorbent assay were used to measure messenger RNA (mRNA) and/or protein levels from monkey (amniotic fluid, myometrium, maternal plasma) and human (amniocytes, amnion, myometrium) tissues. Statistical analysis employed analysis of covariance and Wilcoxon rank sum. Biomechanical forces were calculated using the law of Laplace. RESULTS Preterm labor occurred in 3 of 6 animals after balloon inflation and correlated with greater balloon volume and uterine wall stress. Significant elevations of inflammatory cytokines and prostaglandins occurred following uterine overdistention in an “inflammatory pulse” that correlated with preterm labor (interleukin [IL]-1β, tumor necrosis factor [TNF]-α, IL-6, IL-8, CCL2, prostaglandin E2, prostaglandin F2α, all P < .05). A similar inflammatory response was observed in amniocytes in vitro following mechanical stretch (IL1β, IL6, and IL8 mRNA multiple time points, P < .05), in amnion of women with polyhydramnios (IL6 and TNF mRNA, P < .05) and in amnion (TNF-α) and myometrium of women with twins in early labor (IL6, IL8, CCL2, all P < .05). Genes differentially expressed in the nonhuman primate after balloon inflation and in women with polyhydramnios and twins are involved in tissue remodeling and muscle growth. CONCLUSION Uterine overdistention by inflation of an intraamniotic balloon is associated with an inflammatory pulse that precedes and correlates with preterm labor. Our results indicate that inflammation is an early event after a mechanical stress on the uterus and leads to preterm labor when the stress is sufficiently great. Further, we find evidence of uterine tissue remodeling and muscle growth as a common, perhaps compensatory, response to uterine distension. PMID:26284599

In Amnio MRI of Mouse Embryos

PubMed Central

Roberts, Thomas A.; Norris, Francesca C.; Carnaghan, Helen; Savery, Dawn; Wells, Jack A.; Siow, Bernard; Scambler, Peter J.; Pierro, Agostino; De Coppi, Paolo; Eaton, Simon; Lythgoe, Mark F.

2014-01-01

Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community. PMID:25330230

Extraction Site Preservation Using FDBA With Bioxclude vs. FDBA With Bio-Gide: A Randomized Clinical Trial

DTIC Science & Technology

2015-06-01

neovascularization. J Surgical Res 2015; 193(2): 953-962 48. Zelen CM, Gould L, Serena TE, et al . A prospective, randomized, controlled, multi-center...tissue repair and regeneration. Tiss Eng 2002; 8(2): 295-308. 24. Niknejad H, Peirovi H, Jmjani M, et al . Properties of the amniotic membrane for...Guler R, Ercan MT, Ulutuncel M, et al . Measurement of blood flow by the 133Xe clearance technique to grafts of amnion used in vestibuloplasty. Brit J

Evidence for increased SOX3 dosage as a risk factor for X-linked hypopituitarism and neural tube defects.

PubMed

Bauters, Marijke; Frints, Suzanna G; Van Esch, Hilde; Spruijt, Liesbeth; Baldewijns, Marcella M; de Die-Smulders, Christine E M; Fryns, Jean-Pierre; Marynen, Peter; Froyen, Guy

2014-08-01

Genomic duplications of varying lengths at Xq26-q27 involving SOX3 have been described in families with X-linked hypopituitarism. Using array-CGH we detected a 1.1 Mb microduplication at Xq27 in a large family with three males suffering from X-linked hypopituitarism. The duplication was mapped from 138.7 to 139.8 Mb, harboring only two annotated genes, SOX3 and ATP11C, and was shown to be a direct tandem copy number gain. Unexpectedly, the microduplication did not fully segregate with the disease in this family suggesting that SOX3 duplications have variable penetrance for X-linked hypopituitarism. In the same family, a female fetus presenting with a neural tube defect was also shown to carry the SOX3 copy number gain. Since we also demonstrated increased SOX3 mRNA levels in amnion cells derived from an unrelated t(X;22)(q27;q11) female fetus with spina bifida, we propose that increased levels of SOX3 could be a risk factor for neural tube defects. © 2014 Wiley Periodicals, Inc.

The tissue bank at the national nuclear research institute in Mexico.

PubMed

Esther Martínez-Pardo, María; Lourdes Reyes-Frías, Ma

2003-01-01

The Instituto Nacional de Investigaciones Nucleares (ININ, The National Nuclear Research Institute) received during 1997-1998 strong support of the International Atomic Energy Agency (IAEA), to establish the first and only one tissue bank (BTR ININ tissue bank) in Mexico that uses ionising radiation as sterilising agent. In that time, the BTR staff was trained in different tissue banks in several countries. Basic equipment for tissue processing donated by the IAEA was received in 1998. In July, 1999 the Mexican Health Secretariat gave the Sanitary License No. 1062000001 to the BTR to operate as an official organ and tissue bank. In August, 2001 the ININ and the Hospital Materno Infantil (HMI-ISSEMYM) signed an agreement to collaborate in amnion processing. The hospital is responsible for donor selection, serology tests, tissue procurement and washing, since this hospital is the BTR amnion supplier. The tissues are collected by ININ weekly with complete documentation. The BTR is responsible for processing: cleaning, air drying, packaging, labelling, microbiological control and sterilisation by gamma irradiation. The sterilised tissue is kept under quarantine for 6 months to obtain the results of the donor second serology test. From March to June, 2002 the BTR has processed 347.86 units (50 cm(2) each), is say, 17,393 cm(2). In addition, the pig skin xenograft process has been implemented and a protocol for clinical applications of it is running at the Hospital Central Sur de Alta Especialidad (PEMEX). Also the ININ tissue bank present status and perspectives are described.

The evolution and impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Asia and the Pacific region.

PubMed

Morales Pedraza, Jorge; Phillips, Glyn O

2009-05-01

The Asia and the Pacific region was within the IAEA program on radiation and tissue banking, the most active region. Most of the tissue banks in the Asia and the Pacific region were developed during the late 1980s and 1990s. The initial number of tissue banks established or supported by the IAEA program in the framework of the RCA Agreement for Asia and the Pacific region was 18. At the end of 2006, the number of tissue banks participating, in one way or another in the IAEA program was 59. Since the beginning of the implementation of the IAEA program in Asia and the Pacific region 63,537 amnion and 44,282 bone allografts were produced and 57,683 amnion and 36,388 bone allografts were used. The main impact of the IAEA program in the region was the following: the establishment or consolidation of at least 59 tissue banks in 15 countries in the region (the IAEA supported directly 16 of these banks); the improvement on the quality and safety of tissues procured and produced in the region reaching international standards; the implementation of eight national projects, two regional projects and two interregional projects; the elaboration of International Standards, a Code of Practice and a Public Awareness Strategies and, the application of quality control and quality assurances programs in all participating tissue banks.

N-methyl-N'-nitro-N-nitrosoguanidine interferes with the epidermal growth factor receptor-mediated signaling pathway.

PubMed

Gao, Zhihua; Yang, Jun; Huang, Yun; Yu, Yingnian

2005-03-01

Many environmental factors, such as ultraviolet (UV) and arsenic, can induce the clustering of cell surface receptors, including epidermal growth factor receptor (EGFR). This is accompanied by the phosphorylation of the receptors and the activation of ensuing cellular signal transduction pathways, which are implicated in the various cellular responses caused by the exposure to these factors. In this study, we have shown that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, also induced the clustering of EGFR in human amnion FL cells, which was similar in morphology to that of epidermal growth factor treatment. However, MNNG treatment did not activate Ras, the downstream mediator in EGFR signaling pathway, as compared to EGF treatment. The autophosphorylation of tyrosine residues Y1068 and Y1173 at the intracellular domain of EGFR, which is related to Ras activation under EGF treatment, was also not observed by MNNG exposure. Interestingly, although MNNG did not affect the binding of EGF to EGFR, MNNG can interfere with EGF function. For instance, pre-incubating FL cells with MNNG inhibited the autophosphorylation of EGFR by EGF treatment, as well as the activation of Ras. In addition, the phosphorylation of Y845 on EGFR by EGF, which is mediated through c-Src or related kinases but not autophosphorylation, was also affected by MNNG. Therefore, MNNG may influence the tyrosine kinase activity as well as the phosphorylation of EGFR through its interaction with EGFR.

Induction of Suppressor of Cytokine Signaling-3 by Herpes Simplex Virus Type 1 Contributes to Inhibition of the Interferon Signaling Pathway

PubMed Central

Yokota, Shin-ichi; Yokosawa, Noriko; Okabayashi, Tamaki; Suzutani, Tatsuo; Miura, Shunsuke; Jimbow, Kowichi; Fujii, Nobuhiro

2004-01-01

We showed previously that herpes simplex virus type 1 (HSV-1) suppresses the interferon (IFN) signaling pathway during the early infection stage in the human amnion cell line FL. HSV-1 inhibits the IFN-induced phosphorylation of Janus kinases (JAK) in infected FL cells. In the present study, we showed that the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, is rapidly induced in FL cells after HSV-1 infection. Maximal levels of SOCS3 protein were detected at around 1 to 2 h after infection. This is consistent with the occurrence of HSV-1-mediated inhibition of IFN-induced JAK phosphorylation. The HSV-1 wild-type strain VR3 induced SOCS3 more efficiently than did mutants that are defective in UL41 or UL13 and that are hyperresponsive to IFN. Induction of the IRF-7 protein and transcriptional activation of IFN-α4, which occur in a JAK/STAT pathway-dependent manner, were poorly induced by VR3 but efficiently induced by the mutant viruses. In contrast, phosphorylation of IRF-3 and transcriptional activation of IFN-β, which are JAK/STAT pathway-independent process, were equally well induced by the wild-type strain and the mutants. In conclusion, the SOCS3 protein appears to be mainly responsible for the suppression of IFN signaling and IFN production that occurs during HSV-1 infection. PMID:15163721

Alpha-fetoprotein and its value for predicting pregnancy outcomes – a re-evaluation

PubMed Central

Darouich, Ayham Alhaj; Liehr, Thomas; Weise, Anja; Schlembach, Dietmar; Schleußner, Ekkehart; Kiehntopf, Michael; Schreyer, Isolde

2015-01-01

Introduction Alpha-fetoprotein (AFP) concentrations can be determined framing others from invasively acquired amnion fluid (AF-AFP). While the biological role of AFP remains unclear it is well known that AFP-levels can be altered in connection with specific clinical and/or genetic alterations of the fetus. Materials and Method here a retrospective study based on 3,119 singleton and 56 twin pregnancies is presented. The standard levels of amnion fluid derived alpha-fetoprotein level (AF-AFP) between 12th and 36th weeks of gestation were determined. Additionally, acetylcholinesterase (AChE) test results for 63 cases, ultrasonography results for 32 cases and abnormal karyotypic findings for 100 cases were available for selected cases. Results and Discussion according to the present data the AF-AFP test is reliable and provides expected test results in terms of population studies. However, individual AF-AFP test results can be subject to high individual variations. In this study AF-AFP multiple of medians (MoM) over 1.7 were indicative for neuronal tube defects and/or omphalocele in only 6.3% of the cases, while such AF-AFP values were hints on severe sonographic signs in 62% of the cases. Also, altered AF-AFP concentrations were present in 82% of cytogenetically abnormal cases. Overall, even though predicative value of the AF-AFP-test is matter of discussion it continues to be widely applied in invasive prenatal diagnostics. This study indicates that it only can be applied reliably in combination with other tests like banding cytogenetics, ultrasonography and all embedded in well-established genetic counseling. PMID:27358693

Optimal treatment of partial thickness burns in children: a systematic review.

PubMed

Vloemans, A F P M; Hermans, M H E; van der Wal, M B A; Liebregts, J; Middelkoop, E

2014-03-01

A large part of the patient population of a burn centre consists of children, most of whom are younger than four years. The majority of these young children suffer from superficial and deep partial thickness scald burns that may easily deepen to full thickness burns. A proper wound therapy, that prevents infection and ensures a moist wound condition, might prevent the deterioration of the wound. Therefore, we performed a systematic review of wound management and dressing materials to select the best treatment option for children with burns. A search in Medline and Embase revealed 51 articles for a critical appraisal. The articles were divided into randomized controlled trials, cohort studies and a group of case-reports. Total appraisal did not differ much amongst the groups; the level of evidence was highest in the randomized controlled trials and lowest in the case-reports. In 16 out of 34 comparative studies, silver sulfadiazine or a silver sulfadiazine/chlorhexidine-gluconate combination was the standard of wound care treatment. The competitor dressing was Biobrane(®) in six studies and amnion membrane in three. Tulle gauze, or tulle gauze impregnated with an antibacterial addition were the standard of care treatment in seven studies. In general, membranous dressings like Biobrane(®) and amnion membrane performed better than the standard of care on epithelialization rate, length of hospital stay and pain for treatment of partial thickness burns in children. However, hardly any of the studies investigated long-term results like scar formation. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Amniotic membranes as prosthetic material: experimental utilization data of a rat model.

PubMed

Zachariou, Z

1997-10-01

Prosthetic materials are applied for closing big tissue defects, the repair of traumatized organs, or hernias. Because nonabsorbable synthetic materials are rigid, possess a defined and unchangeable size, and foreign body reaction (FBR) may occur, biological materials may be an alternative. In experimental studies in rats the authors implanted the fetal parts of the human amniotic membranes and examined the utilization and FBR induced in a standardized model. In addition amnion (AM) was combined with vicryl-net (VN) for higher implant stability. Fifteen, 30 and 90 days after implantation, macroscopic appearance was examined, and light microscopy and immunohistology testing of the specimens were performed. Adhesions to parenchymal organs and omentum were present irrespective of the side facing the abdominal cavity. AM induced a rapid FBR, which diminished with time. Chorion (CH) and parts of the AM were resorbed within the examined period after infiltration with recipient cells and neovascularisation. The combined implant, AM, and VN showed best results because disadvantages of one material could be compensated for by the advantages of the other. The studies show that AM, in its anatomic definition, combined with VN proves to be a safe and reliable prosthetic material for the use in tissue defects.

Intra-articular injection of micronized dehydrated human amnion/chorion membrane attenuates osteoarthritis development

PubMed Central

2014-01-01

Introduction Micronized dehydrated human amnion/chorion membrane (μ-dHACM) is derived from donated human placentae and has anti-inflammatory, low immunogenic and anti-fibrotic properties. The objective of this study was to quantitatively assess the efficacy of μ-dHACM as a disease modifying intervention in a rat model of osteoarthritis (OA). It was hypothesized that intra-articular injection of μ-dHACM would attenuate OA progression. Methods Lewis rats underwent medial meniscal transection (MMT) surgery to induce OA. Twenty four hours post-surgery, μ-dHACM or saline was injected intra-articularly into the rat joint. Naïve rats also received μ-dHACM injections. Microstructural changes in the tibial articular cartilage were assessed using equilibrium partitioning of an ionic contrast agent (EPIC-μCT) at 21 days post-surgery. The joint was also evaluated histologically and synovial fluid was analyzed for inflammatory markers at 3 and 21 days post-surgery. Results There was no measured baseline effect of μ-dHACM on cartilage in naïve animals. Histological staining of treated joints showed presence of μ-dHACM in the synovium along with local hypercellularity at 3 and 21 days post-surgery. In MMT animals, development of cartilage lesions at 21 days was prevented and number of partial erosions was significantly reduced by treatment with μ-dHACM. EPIC-μCT analysis quantitatively showed that μ-dHACM reduced proteoglycan loss in MMT animals. Conclusions μ-dHACM is rapidly sequestered in the synovial membrane following intra-articular injection and attenuates cartilage degradation in a rat OA model. These data suggest that intra-articular delivery of μ-dHACM may have a therapeutic effect on OA development. PMID:24499554

Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane

PubMed Central

Lei, Jennifer; Priddy, Lauren B.; Lim, Jeremy J.; Massee, Michelle; Koob, Thomas J.

2017-01-01

Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION® Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek® activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair. PMID:28224047

Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane.

PubMed

Lei, Jennifer; Priddy, Lauren B; Lim, Jeremy J; Massee, Michelle; Koob, Thomas J

2017-02-01

Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION ® Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek ® activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair.

Influence of the antiseptic agents polyhexanide and octenidine on FL cells and on healing of experimental superficial aseptic wounds in piglets. A double-blind, randomised, stratified, controlled, parallel-group study.

PubMed

Kramer, A; Roth, B; Müller, G; Rudolph, P; Klöcker, N

2004-01-01

The main target of the combination of octenidine with phenoxyethanol (Octenisept) is the antisepsis of acute wounds, whereas polyhexanide combined with polyethylene glycol in Ringer solution (Lavasept) is the agent of choice for antisepsis of chronic wounds and burns. Because comparative data for both agents on the effects on wound healing are lacking, we investigated the influence of preparations based on polyhexanide and octenidine versus placebo (Ringer solution) in experimental superficial aseptic skin wounds (n = 108) of 20 mm diameter, using a double-blind, randomised, stratified, controlled, parallel-group design in piglets. Computerised planimetry and histopathological methods were used for the assessment of wound healing. Histologically, no significant differences could be verified at any time between the 3 groups. However, in the early phase (day 9 after wounding), the octenidine-based product retarded wound contraction to a significantly greater extent than placebo and polyhexanide, whereas in the later phase (days 18 and 28), polyhexanide promoted contraction significantly more than did placebo and octenidine. The consequence is complete wound closure after 22.9 days using polyhexanide, in comparison to the placebo after 24.1 days (p < 0.05) and octenidine after 28.3 days (no statistical difference to placebo). This may be explained by the better tolerance of polyhexanide in vitro, which was demonstrated with dose and time dependence in cytotoxicity tests on human amnion cells. Copyright 2004 S. Karger AG, Basel

Role of ultrasonography in the management of twin gestation.

PubMed

Smith, Jessica; Treadwell, Marjorie C; Berman, Deborah R

2018-06-01

Twins represent 1%-2% of all pregnancies, yet continue to account for a disproportionate share of neonatal adverse events including neonatal intensive care admission, morbidity, and mortality. Ultrasonography is central to the proper diagnosis of the type of twinning. Ideally, ultrasonography is performed before 14 weeks of gestation to determine chorionicity and amnionicity. Correct identification of the chorionicity in a twin pregnancy facilitates proper counseling and management of the gestation, including ultrasonography follow-up. Herein, the different types of twinning are reviewed, together with the implications for ultrasonography monitoring of each specific type of twin gestation. © 2018 International Federation of Gynecology and Obstetrics.

Response of human limbal epithelial cells to wounding on 3D RAFT tissue equivalents: effect of airlifting and human limbal fibroblasts.

PubMed

Massie, Isobel; Levis, Hannah J; Daniels, Julie T

2014-10-01

Limbal epithelial stem cell deficiency can cause blindness but may be treated by human limbal epithelial cell (hLE) transplantation, normally on human amniotic membrane. Clinical outcomes using amnion can be unreliable and so we have developed an alternative tissue equivalent (TE), RAFT (Real Architecture for 3D Tissue), which supports hLE expansion, and stratification when airlifted. Human limbal fibroblasts (hLF) may be incorporated into RAFT TEs, where they support overlying hLE and improve phenotype. However, the impact of neither airlifting nor hLF on hLE function has been investigated. hLE on RAFT TEs (±hLF and airlifting) were wounded using heptanol and re-epithelialisation (fluorescein diacetate staining), and percentage putative stem cell marker p63α and proliferative marker Ki67 expression (wholemount immunohistochemistry), measured. Airlifted, hLF- RAFT TEs were unable to close the wound and p63α expression was 7 ± 0.2% after wounding. Conversely, non-airlifted, hLF- RAFT TEs closed the wound within 9 days and p63α expression was higher at 22 ± 5% (p < 0.01). hLE on both hLF- and hLF+ RAFT TEs (non-airlifted) closed the wound and p63α expression was 26 ± 8% and 36 ± 3% respectively (ns). Ki67 expression by hLE increased from 1.3 ± 0.5% before wounding to 7.89 ± 2.53% post-wounding for hLF- RAFT TEs (p < 0.01), and 0.8 ± 0.08% to 17.68 ± 10.88% for hLF+ RAFT TEs (p < 0.05), suggesting that re-epithelialisation was a result of proliferation. These data suggest that neither airlifting nor hLF are necessarily required to maintain a functional epithelium on RAFT TEs, thus simplifying and shortening the production process. This is important when working towards clinical application of regenerative medicine products. Copyright © 2014 Elsevier Ltd. All rights reserved.

Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure.

PubMed

Massie, Isobel; Dale, Sarah B; Daniels, Julie T

2015-06-01

Limbal epithelial stem cell deficiency can cause blindness, but transplantation of these cells on a carrier such as human amniotic membrane can restore vision. Unfortunately, clinical graft manufacture using amnion can be inconsistent. Therefore, we have developed an alternative substrate, Real Architecture for 3D Tissue (RAFT), which supports human limbal epithelial cells (hLE) expansion. Epithelial organization is improved when human limbal fibroblasts (hLF) are incorporated into RAFT tissue equivalent (TE). However, hLF have the potential to transdifferentiate into a pro-scarring cell type, which would be incompatible with therapeutic transplantation. The aim of this work was to assess the scarring phenotype of hLF in RAFT TEs in hLE+ and hLE- RAFT TEs and in nonairlifted and airlifted RAFT TEs. Diseased fibroblasts (dFib) isolated from the fibrotic conjunctivae of ocular mucous membrane pemphigoid (Oc-MMP) patients were used as a pro-scarring positive control against which hLF were compared using surrogate scarring parameters: matrix metalloproteinase (MMP) activity, de novo collagen synthesis, α-smooth muscle actin (α-SMA) expression, and transforming growth factor-β (TGF-β) secretion. Normal hLF and dFib maintained different phenotypes in RAFT TE. MMP-2 and -9 activity, de novo collagen synthesis, and α-SMA expression were all increased in dFib cf. normal hLF RAFT TEs, although TGF-β1 secretion did not differ between normal hLF and dFib RAFT TEs. Normal hLF do not progress toward a scarring-like phenotype during culture in RAFT TEs and, therefore, may be safe to include in therapeutic RAFT TE, where they can support hLE, although in vivo work is required to confirm this. dFib RAFT TEs (used in this study as a positive control) may be useful toward the development of an ex vivo disease model of Oc-MMP.

Limbal Fibroblasts Maintain Normal Phenotype in 3D RAFT Tissue Equivalents Suggesting Potential for Safe Clinical Use in Treatment of Ocular Surface Failure

PubMed Central

Dale, Sarah B.; Daniels, Julie T.

2015-01-01

Limbal epithelial stem cell deficiency can cause blindness, but transplantation of these cells on a carrier such as human amniotic membrane can restore vision. Unfortunately, clinical graft manufacture using amnion can be inconsistent. Therefore, we have developed an alternative substrate, Real Architecture for 3D Tissue (RAFT), which supports human limbal epithelial cells (hLE) expansion. Epithelial organization is improved when human limbal fibroblasts (hLF) are incorporated into RAFT tissue equivalent (TE). However, hLF have the potential to transdifferentiate into a pro-scarring cell type, which would be incompatible with therapeutic transplantation. The aim of this work was to assess the scarring phenotype of hLF in RAFT TEs in hLE+ and hLE− RAFT TEs and in nonairlifted and airlifted RAFT TEs. Diseased fibroblasts (dFib) isolated from the fibrotic conjunctivae of ocular mucous membrane pemphigoid (Oc-MMP) patients were used as a pro-scarring positive control against which hLF were compared using surrogate scarring parameters: matrix metalloproteinase (MMP) activity, de novo collagen synthesis, α-smooth muscle actin (α-SMA) expression, and transforming growth factor-β (TGF-β) secretion. Normal hLF and dFib maintained different phenotypes in RAFT TE. MMP-2 and -9 activity, de novo collagen synthesis, and α-SMA expression were all increased in dFib cf. normal hLF RAFT TEs, although TGF-β1 secretion did not differ between normal hLF and dFib RAFT TEs. Normal hLF do not progress toward a scarring-like phenotype during culture in RAFT TEs and, therefore, may be safe to include in therapeutic RAFT TE, where they can support hLE, although in vivo work is required to confirm this. dFib RAFT TEs (used in this study as a positive control) may be useful toward the development of an ex vivo disease model of Oc-MMP. PMID:25380529

Regulation of amniotic fluid volume: mathematical model based on intramembranous transport mechanisms

PubMed Central

Anderson, Debra F.; Cheung, Cecilia Y.

2014-01-01

Experimentation in late-gestation fetal sheep has suggested that regulation of amniotic fluid (AF) volume occurs primarily by modulating the rate of intramembranous transport of water and solutes across the amnion into underlying fetal blood vessels. In order to gain insight into intramembranous transport mechanisms, we developed a computer model that allows simulation of experimentally measured changes in AF volume and composition over time. The model included fetal urine excretion and lung liquid secretion as inflows into the amniotic compartment plus fetal swallowing and intramembranous absorption as outflows. By using experimental flows and solute concentrations for urine, lung liquid, and swallowed fluid in combination with the passive and active transport mechanisms of the intramembranous pathway, we simulated AF responses to basal conditions, intra-amniotic fluid infusions, fetal intravascular infusions, urine replacement, and tracheoesophageal occlusion. The experimental data are consistent with four intramembranous transport mechanisms acting in concert: 1) an active unidirectional bulk transport of AF with all dissolved solutes out of AF into fetal blood presumably by vesicles; 2) passive bidirectional diffusion of solutes, such as sodium and chloride, between fetal blood and AF; 3) passive bidirectional water movement between AF and fetal blood; and 4) unidirectional transport of lactate into the AF. Further, only unidirectional bulk transport is dynamically regulated. The simulations also identified areas for future study: 1) identifying intramembranous stimulators and inhibitors, 2) determining the semipermeability characteristics of the intramembranous pathway, and 3) characterizing the vesicles that are the primary mediators of intramembranous transport. PMID:25186112

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Peru.

PubMed

Gamero, Emma Castro; Morales Pedraza, Jorge

2009-05-01

The tissue bank "Rosa Guerzoni Chambergo" (RGCTB) located at the Child's Health Institute was inaugurated in 1996, with the financial and technical support of the IAEA program on radiation and tissue banking. Since 1998, the biological bandage of fresh and lyophilised pigskin, amnion and bone tissue is processed routinely in this bank. In all cases, the tissue is sterilised with the use of Cobalt-60 radiation, process carried out at the Laboratories of Irradiation of the Peruvian Institute of Nuclear Energy (IPEN). The tissue bank in the Child's Health Institute helped to save lives in an accident occurred in Lima, when a New Year's fireworks celebration ran out of control in January 2002. Nearly 300 people died in the tragic blaze and hundreds more were seriously burned and injured. Eight Lima hospitals and clinics suddenly were faced with saving the lives of severely burned men, women and children. Fortunately, authorities were ready to respond to the emergency. More than 1,600 dressings were sterilised and supplied to Lima surgeons. The efforts helped save the lives of patients who otherwise might not have survived the Lima fire. Between 1998 and September 2007, 35,012 tissue grafts were produced and irradiated. Radiation sterilised tissues are used by 20 national medical institutions as well as 17 private health institutions. The tissue bank established in Peru with the support of the IAEA is now producing the following tissues: pigskin dressings, fresh and freeze-dried; bone allografts, chips, wedges and powdered, and amnion dressings air-dried. It is also now leading the elaboration of national standards, assignment being entrusted by ONDT (Organización Nacional de Donación y Transplantes; National Organisation on Donation and Transplant). This among other will permit the accreditation of the tissue bank. In this task is also participating IPEN.

Regulation of amniotic fluid volume: mathematical model based on intramembranous transport mechanisms.

PubMed

Brace, Robert A; Anderson, Debra F; Cheung, Cecilia Y

2014-11-15

Experimentation in late-gestation fetal sheep has suggested that regulation of amniotic fluid (AF) volume occurs primarily by modulating the rate of intramembranous transport of water and solutes across the amnion into underlying fetal blood vessels. In order to gain insight into intramembranous transport mechanisms, we developed a computer model that allows simulation of experimentally measured changes in AF volume and composition over time. The model included fetal urine excretion and lung liquid secretion as inflows into the amniotic compartment plus fetal swallowing and intramembranous absorption as outflows. By using experimental flows and solute concentrations for urine, lung liquid, and swallowed fluid in combination with the passive and active transport mechanisms of the intramembranous pathway, we simulated AF responses to basal conditions, intra-amniotic fluid infusions, fetal intravascular infusions, urine replacement, and tracheoesophageal occlusion. The experimental data are consistent with four intramembranous transport mechanisms acting in concert: 1) an active unidirectional bulk transport of AF with all dissolved solutes out of AF into fetal blood presumably by vesicles; 2) passive bidirectional diffusion of solutes, such as sodium and chloride, between fetal blood and AF; 3) passive bidirectional water movement between AF and fetal blood; and 4) unidirectional transport of lactate into the AF. Further, only unidirectional bulk transport is dynamically regulated. The simulations also identified areas for future study: 1) identifying intramembranous stimulators and inhibitors, 2) determining the semipermeability characteristics of the intramembranous pathway, and 3) characterizing the vesicles that are the primary mediators of intramembranous transport. Copyright © 2014 the American Physiological Society.

Dystocia following prolonged retention of a dead fetus in an Asian elephant (Elephas maximus).

PubMed

Thitaram, Chatchote; Pongsopawijit, Pornsawan; Thongtip, Nikorn; Angkavanich, Taweepoke; Chansittivej, Sarun; Wongkalasin, Waroot; Somgird, Chalermchart; Suwankong, Niyada; Prachsilpchai, Worapat; Suchit, Kittiphat; Clausen, Bjarne; Boonthong, Pinich; Nimtrakul, Kannika; Niponkit, Charawee; Siritepsongklod, Somchai; Roongsri, Ronnachit; Mahasavankul, Sittidet

2006-09-15

A 32-year-old nulliparous female Asian elephant (Elephas maximus) showed signs of parturition 8 months later than predicted from the breeding records. However, while serosanguineous fluid, necrotic tissue and pieces of amnion were expelled, second-stage labor did not progress. Since the fetus was not found during an endoscopic examination of the vestibule, it was assumed that the elephant had calved unseen and she was returned to the forest to recuperate. Twelve months later, the elephant showed clear signs of second-stage labor accompanied by a bulge in the perineum and passage of keratinized nail through the vulva. A 35 cm episiotomy incision was made in the perineum just below the anus, via which chains were attached to the forelimbs of the fetus. Traction on the forelimbs alone proved insufficient to achieve delivery because the fetal head kept rotating and impacting in the pelvis. However, traction applied via a hook inserted behind the mandibular symphysis allowed the head to be elevated and extended, and the fetus to be delivered. The episiotomy wound was sutured in two layers and although the skin did not heal during primary closure it subsequently healed uneventfully by second intention. Retrospective evaluation of the elephant's serum progestagens profile demonstrated a fall to baseline at the suspected onset of parturition, supporting the supposition that the fetus was retained in the uterus for 12 months after parturition began. It is suggested that serum progestagens concentrations should be monitored regularly in mated elephant cows to verify the establishment of pregnancy and to better estimate the expected timing, and the onset of calving.

Placental sulfatase deficiency: maternal and fetal expression of steroid sulfatase deficiency and X-linked ichthyosis.

PubMed

Bradshaw, K D; Carr, B R

1986-07-01

PSD-X-linked ichthyosis are manifestations of a similar disorder of an inborn error of metabolism characterized by a deficiency of steroid sulfatase. The decreased enzyme activity is due to the absence of the expression of enzyme (steroid sulfatase) protein. Affected individuals with this disorder are males (X-linked inheritance) with a frequency of 1/2000 to 1/6000 births. Homozygous females from cosanguineous marriages have been reported with this disorder. The diagnosis is suspected and confirmed by: Low estriol excretion; Negative DHEAS loading test Increased DHEAS in amnionic fluid; Normal DHEAS in cord plasma; Possible delayed or abnormal labor patterns; Decreased sulfatase activity in the placenta, fibroblast, erythrocytes, lymphocytes or leukocytes of affected individuals; Development of ichthyosis in male infants at 2 to 3 months of age.

Constriction band syndrome occurring in the setting of in vitro fertilization and advanced maternal age

PubMed Central

Rinker, Brian; Vasconez, Henry C

2006-01-01

The debate as to the pathogenesis of constriction band syndrome began with Hippocrates and continues today. The exogenous theory attributes the condition to entanglement of the fetus in the amniotic remnants following premature rupture of the amnion, which is in contrast to the endogenous, or genetic, mechanism. A case of constriction band syndrome in the setting of in vitro fertilization, where the child was genetically unrelated to the birth mother, is presented. Constriction band syndrome has been reported following amniocentesis and chorionic villus sampling, but it has not heretofore been presented in the setting of in vitro fertilization. In addition, the present case presents an opportunity to separate maternal from genetic factors and, possibly, shed some light on the etiology of the condition. PMID:19554231

Differences in placental telomere length suggest a link between racial disparities in birth outcomes and cellular aging

PubMed Central

JONES, Christopher W.; GAMBALA, Cecilia; ESTEVES, Kyle C.; WALLACE, Maeve; SCHLESINGER, Reid; O’QUINN, Marguerite; KIDD, Laura; THEALL, Katherine P.; DRURY, Stacy S.

2017-01-01

BACKGROUND Health disparities begin early in life and persist across the life course. Despite current efforts Black women exhibit greater risk for pregnancy complications and negative perinatal outcomes compared to White women. The placenta, a complex multi-tissue organ, serves as the primary transducer of bidirectional information between the mother and fetus. Altered placental function is linked to multiple racially disparate pregnancy complications, however little is known about racial differences in molecular factors within the placenta. Several pregnancy complications, including preeclampsia and fetal growth restriction, exhibit racial disparities and are associated with shorter placental telomere length, an indicator of cellular stress and aging. Cellular senescence and telomere dynamics are linked to the molecular mechanisms associated with the onset of labor and parturition. Further, racial differences in telomere length are found in a range of different peripheral tissues. Together these factors suggest that exploration of racial differences in telomere length of the placenta may provide novel mechanistic insight into racial disparities in birth outcomes. OBJECTIVE This study examined whether telomere length measured in four distinct fetally-derived tissues were significantly different between Blacks and Whites. The study had two hypotheses: (1) that telomere length measured in different placental tissue types would be correlated and (2) that across all sampled tissues telomere length would differ by race. STUDY DESIGN In a prospective study, placental tissue samples were collected from the amnion, chorion, villus, and umbilical cord from Black and White singleton pregnancies (N=46). Telomere length was determined using monochrome multiplex quantitative real-time polymerase chain reaction in each placental tissue. Demographic and pregnancy-related data were also collected. Descriptive statistics characterized the sample overall and among Black and White women separately. The overall impact of race was assessed by multilevel mixed-effects linear regression models that included empirically relevant covariates. RESULTS Telomere length was significantly correlated across all placental tissues. Pairwise analyses of placental tissue telomere length revealed significantly longer telomere length in the amnion compared to the chorion (t=−2.06, p=0.043). Overall telomere length measured in placenta samples from Black mothers were significantly shorter than those from White mothers (β=−0.09, p=0.04). Controlling for relevant maternal and infant characteristics strengthened the significance of the observed racial differences (β=−0.12, p=0.02). Within tissue analyses revealed that the greatest difference by race was found in chorionic telomere length (t=−2.81, p=0.007). CONCLUSION These findings provide the first evidence of racial differences in placental telomere length. Telomere length was significantly shorter in placental samples derived from Black mothers compared to White. Given previous studies reporting that telomere length, cellular senescence, and telomere dynamics are molecular factors contributing to the rupture of the amniotic sac, onset of labor, and parturition, our findings of shorter telomere length in placentas from Black mothers suggests that accelerated cellular aging across placental tissues may be relevant to the increased risk of preterm delivery in Blacks. Our results suggest that racial differences in cellular aging in the placenta contribute to the earliest roots of health disparities. PMID:27865975

Using Amniotic Membrane as a Novel Method to Reduce Post-burn Hypertrophic Scar Formation: A Prospective Follow-up Study

PubMed Central

Mohammadi, Ali Akbar; Eskandari, Shima; Johari, Hamed Ghoddusi; Rajabnejad, Ata'ollah

2017-01-01

Background: Several studies have shown that the application of amniotic membrane as a biological dressing in the management of burns is accompanied by rapid re-epithelialisation. In this follow-up study, we aimed to evaluate the possible role of amniotic membrane as an adjunct to split thickness skin grafting on reducing itching and severity of hypertrophic scar formation. Materials and Methods: From October 2013 to January 2015, in a prospective follow-up study, 54 patients (108 limbs) with second and third degree burns, covering 4%–15% of total body surface area (TBSA), were included in the study. All patients needed split-thickness skin grafts for burn-wound coverage. Selected patients had symmetric burns on two (upper or lower) extremities. Then, in every patient, the extremities were randomly divided into two groups: In one limb, the skin graft was traditionally fixed with skin staples (control group) and in the other limb, the skin graft was covered with an amniotic membrane (amnion group). Therefore, in every patient, the graft was covered with an amniotic membrane in one extremity and fixed with skin staples in the other extremity. Finally, after 6 months, the degree of itching and hypertrophic scar formation was compared between the two groups. Results: The study group was composed of 108 limbs in 54 patients (27 males and 27 females) with a mean age of 23.54 ± 4.9 years and burn 9.03 ± 2.69% TBSA. The patients were divided into two groups: 54 limbs in amnion group and 54 limbs in control group. In 59.25% of the cases, patient had less itching in the extremity covered with amniotic membrane. Furthermore, in 64.81% of the cases, patients had less hypertrophic scar formation in the extremity covered with amniotic membrane. These differences were statistically significant (P < 0.001). Conclusions: Amniotic membrane used as an adjunct in split thickness skin grafting is a novel modality which significantly reduces scar formation and itching that can be greatly distressing to burn patients. However, still more prospective well designed studies are needed to prove it. PMID:28529415

Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

PubMed

Dunand, C; Hoffmann, P; Sapin, V; Blanchon, L; Salomon, A; Sergent, F; Benharouga, M; Sabra, S; Guibourdenche, J; Lye, S J; Feige, J J; Alfaidy, N

2014-09-01

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors. © 2014 by the Society for the Study of Reproduction, Inc.

Copanlisib and Nivolumab in Treating Participants With Recurrent or Refractory Diffuse Large B-cell Lymphoma or Primary Mediastinal Large B-cell Lymphoma

ClinicalTrials.gov

2018-06-11

Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Primary Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Refractory Diffuse Large B-Cell Lymphoma; Refractory Primary Mediastinal (Thymic) Large B-Cell Cell Lymphoma

Double-hit or dual expression of MYC and BCL2 in primary cutaneous large B-cell lymphomas.

PubMed

Menguy, Sarah; Frison, Eric; Prochazkova-Carlotti, Martina; Dalle, Stephane; Dereure, Olivier; Boulinguez, Serge; Dalac, Sophie; Machet, Laurent; Ram-Wolff, Caroline; Verneuil, Laurence; Gros, Audrey; Vergier, Béatrice; Beylot-Barry, Marie; Merlio, Jean-Philippe; Pham-Ledard, Anne

2018-03-26

In nodal diffuse large B-cell lymphoma, the search for double-hit with MYC and BCL2 and/or BCL6 rearrangements or for dual expression of BCL2 and MYC defines subgroups of patients with altered prognosis that has not been evaluated in primary cutaneous large B-cell lymphoma. Our objectives were to assess the double-hit and dual expressor status in a cohort of 44 patients with primary cutaneous large B-cell lymphoma according to the histological subtype and to evaluate their prognosis relevance. The 44 cases defined by the presence of more than 80% of large B-cells in the dermis corresponded to 21 primary cutaneous follicle centre lymphoma with large cell morphology and 23 primary cutaneous diffuse large B-cell lymphoma, leg type. Thirty-one cases (70%) expressed BCL2 and 29 (66%) expressed MYC. Dual expressor profile was observed in 25 cases (57%) of either subtypes (n = 6 or n = 19, respectively). Only one primary cutaneous follicle centre lymphoma, large-cell case had a double-hit status (2%). Specific survival was significantly worse in primary cutaneous diffuse large B-cell lymphoma, leg type than in primary cutaneous follicle centre lymphoma, large cell (p = 0.021) and for the dual expressor primary cutaneous large B-cell lymphoma group (p = 0.030). Both overall survival and specific survival were worse for patients belonging to the dual expressor primary cutaneous diffuse large B-cell lymphoma, leg type subgroup (p = 0.001 and p = 0.046, respectively). Expression of either MYC and/or BCL2 negatively impacted overall survival (p = 0.017 and p = 0.018 respectively). As the differential diagnosis between primary cutaneous follicle centre lymphoma, large cell and primary cutaneous diffuse large B-cell lymphoma, leg type has a major impact on prognosis, dual-expression of BCL2 and MYC may represent a new diagnostic criterion for primary cutaneous diffuse large B-cell lymphoma, leg type subtype and further identifies patients with impaired survival. Finally, the double-hit assessment does not appear clinically relevant in primary cutaneous large B-cell lymphoma.

Primary cilium - antenna-like structure on the surface of most mammalian cell types

NASA Astrophysics Data System (ADS)

Dvorak, J.; Sitorova, V.; Hadzi Nikolov, D.; Mokry, J.; Richter, I.; Kasaova, L.; Filip, S.; Ryska, A.; Petera, J.

2011-12-01

The primary cilium is a sensory solitary non-motile microtubule-based organelle protruding in the quiescent phase of the cell cycle from the surface of the majority of human cells, including embryonic cells, stem cells and stromal cells of malignant tumors. The presence of a primary cilium on the surface of a cell is transient, limited to the quiescent G1(G0) phase and the beginning of the S phase of the cell cycle. The primary cilium is formed from the mother centriole. Primary cilia are key coordinators of signaling pathways during development and tissue homeostasis and, when deffective, they are a major cause of human diseases and developmental disorders, now commonly referred to as ciliopathies. Most cancer cells do not possess a primary cilium. The loss of the primary cilium is a regular feature of neoplastic transformation in the majority of solid tumors. The primary cilium could serve as a tumor suppressor organelle. The aim of this paper was to provide a review of the current knowledge of the primary cilium.

Comparison of Adaptive Dose Painting by Numbers With Standard Radiotherapy for Head and Neck Cancer.

ClinicalTrials.gov

2018-05-17

Primary Non-operated Squamous Cell Carcinoma of Oral Cavity; Primary Non-operated Squamous Cell Carcinoma of Oropharynx; Primary Non-operated Squamous Cell Carcinoma of Hypopharynx; Primary Non-operated Squamous Cell Carcinoma of Larynx

Differences in placental telomere length suggest a link between racial disparities in birth outcomes and cellular aging.

PubMed

Jones, Christopher W; Gambala, Cecilia; Esteves, Kyle C; Wallace, Maeve; Schlesinger, Reid; O'Quinn, Marguerite; Kidd, Laura; Theall, Katherine P; Drury, Stacy S

2017-03-01

Health disparities begin early in life and persist across the life course. Despite current efforts, black women exhibit greater risk for pregnancy complications and negative perinatal outcomes compared with white women. The placenta, which is a complex multi-tissue organ, serves as the primary transducer of bidirectional information between the mother and fetus. Altered placental function is linked to multiple racially disparate pregnancy complications; however, little is known about racial differences in molecular factors within the placenta. Several pregnancy complications, which include preeclampsia and fetal growth restriction, exhibit racial disparities and are associated with shorter placental telomere length, which is an indicator of cellular stress and aging. Cellular senescence and telomere dynamics are linked to the molecular mechanisms that are associated with the onset of labor and parturition. Further, racial differences in telomere length are found in a range of different peripheral tissues. Together these factors suggest that exploration of racial differences in telomere length of the placenta may provide novel mechanistic insight into racial disparities in birth outcomes. This study examined whether telomere length measured in 4 distinct fetally derived tissues were significantly different between black and white women. The study had 2 hypotheses: (1) that telomere length that is measured in different placental tissue types would be correlated and (2) that across all sampled tissues telomere length would differ by race. In a prospective study, placental tissue samples were collected from the amnion, chorion, villus, and umbilical cord from black and white singleton pregnancies (N=46). Telomere length was determined with the use of monochrome multiplex quantitative real-time polymerase chain reaction in each placental tissue. Demographic and pregnancy-related data were also collected. Descriptive statistics characterized the sample overall and among black and white women separately. The overall impact of race was assessed by multilevel mixed-effects linear regression models that included empirically relevant covariates. Telomere length was correlated significantly across all placental tissues. Pairwise analyses of placental tissue telomere length revealed significantly longer telomere length in the amnion compared with the chorion (t=-2.06; P=.043). Overall telomere length measured in placenta samples from black mothers were significantly shorter than those from white mothers (β=-0.09; P=.04). Controlling for relevant maternal and infant characteristics strengthened the significance of the observed racial differences (β=-0.12; P=.02). Within tissue analyses revealed that the greatest difference by race was found in chorionic telomere length (t=-2.81; P=.007). These findings provide the first evidence of racial differences in placental telomere length. Telomere length was significantly shorter in placental samples from black mothers compared with white mothers. Given previous studies that have reported that telomere length, cellular senescence, and telomere dynamics are molecular factors that contribute to the rupture of the amniotic sac, onset of labor, and parturition, our findings of shorter telomere length in placentas from black mothers suggest that accelerated cellular aging across placental tissues may be relevant to the increased risk of preterm delivery in black pregnancies. Our results suggest that racial differences in cellular aging in the placenta contribute to the earliest roots of health disparities. Copyright © 2016 Elsevier Inc. All rights reserved.

Significance of fibrotic bands in utero--Amniotic band sequence with limb body wall complex: A rare case of fetal autopsy.

PubMed

Devi, P Lekshmi; Cicy, P J; Thambi, Renu; Poothiode, Usha

2015-01-01

Amniotic band sequence (ABS) includes a wide spectrum of abnormalities resulting from entrapment of various fetal parts from a disrupted amnion, ranging from a mere constriction ring affecting a finger to a fatal form called limb body wall complex (LBWC). Reported cases of ABS with LBWC are very few. The spectrum of anomalies depends on which part gets entrapped and at what point of gestation. Hence, the clinical presentation can be extremely variable. Early detection of such cases using sonology is really challenging due to the small size of the fibrotic bands. Here, we present a case of amniotic band syndrome with LBWC in a fetus at 24 weeks of gestation, which was referred for an autopsy. The fetus also showed scoliosis, gastroschisis, lumbosacral meningocele, congenital talipes equinovarus, and cleft palate, thus having features of placenta cranial and placenta abdominal phenotype which is very rare.

Fetal-to-maternal signaling to initiate parturition

PubMed Central

Reinl, Erin L.; England, Sarah K.

2015-01-01

Multiple processes are capable of activating the onset of parturition; however, the specific contributions of the mother and the fetus to this process are not fully understood. In this issue of the JCI, Gao and colleagues present evidence that steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2) regulate surfactant protein-A (SP-A) and platelet-activating factor (PAF) expression, which increases in the developing fetal lung. WT dams crossed with males deficient for both SRC-1 and SRC-2 had suppressed myometrial inflammation, increased serum progesterone, and delayed parturition, which could be reconciled by injection of either SP-A or PAF into the amnion. Together, the results of this study demonstrate that the fetal lungs produce signals to initiate labor in the mouse. This work underscores the importance of the fetus as a contributor to the onset of murine, and potentially human, parturition. PMID:26098207

Chorioamnionitis caused by Serratia marcescens in a non-immunocompromised host

PubMed Central

Shimizu, S; Kojima, H; Yoshida, C; Suzukawa, K; Mukai, H Y; Hasegawa, Y; Hitomi, S; Nagasawa, T

2003-01-01

A 26 year old pregnant woman with antithrombin III deficiency developed recurrent septicaemia with Serratia marcescens. In spite of the administration of antibiotics, high grade fever persisted. She subsequently manifested lower abdominal pain, and spontaneous abortion occurred. After the abortion, she became completely afebrile. The amnion was turbid, and microscopic examination of the placenta showed haemorrhage and massive infiltration of neutrophils, suggestive of infectious chorioamnionitis. Pulsed field gel electrophoresis showed that isolates from the blood, urine, and vaginal discharge were genetically identical. Intravenous pyelography revealed that she had a bilateral completed double ureter. It was thought that a urinary tract anomaly caused infection with S marcescens, and the pathogen spread to the chorioamnion via the bloodstream. This is the first report of chorioamnionitis caused by S marcescens in a non-immunocompromised host. In addition, these findings indicate that the chorioamnion can serve as a site for persistent infection in normal pregnancies. PMID:14600137

Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA

PubMed Central

Chakrabortty, Sudipto K.; Prakash, Ashwin; Nechooshtan, Gal; Hearn, Stephen; Gingeras, Thomas R.

2015-01-01

Extracellular vesicles (EVs) have been proposed as a means to promote intercellular communication. We show that when human primary cells are exposed to cancer cell EVs, rapid cell death of the primary cells is observed, while cancer cells treated with primary or cancer cell EVs do not display this response. The active agents that trigger cell death are 29- to 31-nucleotide (nt) or 22- to 23-nt processed fragments of an 83-nt primary transcript of the human RNY5 gene that are highly likely to be formed within the EVs. Primary cells treated with either cancer cell EVs, deproteinized total RNA from either primary or cancer cell EVs, or synthetic versions of 31- and 23-nt fragments trigger rapid cell death in a dose-dependent manner. The transfer of processed RNY5 fragments through EVs may reflect a novel strategy used by cancer cells toward the establishment of a favorable microenvironment for their proliferation and invasion. PMID:26392588

Nivolumab With or Without Varlilumab in Treating Patients With Relapsed or Refractory Aggressive B-cell Lymphomas

ClinicalTrials.gov

2018-06-11

ALK-Positive Large B-Cell Lymphoma; Atypical Burkitt/Burkitt-Like Lymphoma; Burkitt-Like Lymphoma With 11q Aberration; Diffuse Large B-Cell Lymphoma Activated B-Cell Type; Diffuse Large B-Cell Lymphoma Associated With Chronic Inflammation; Diffuse Large B-Cell Lymphoma Germinal Center B-Cell Type; Diffuse Large B-Cell Lymphoma, Not Otherwise Specified; EBV-Positive Diffuse Large B-Cell Lymphoma, Not Otherwise Specified; EBV-Positive Mucocutaneous Ulcer; High-Grade B-Cell Lymphoma With MYC, BCL2, and BCL6 Rearrangements; Human Herpesvirus 8-Positive Neoplastic Cells Present; Intravascular Large B-Cell Lymphoma; Large B-Cell Lymphoma With IRF4 Rearrangement; Plasmablastic Lymphoma; Primary Cutaneous Diffuse Large B-Cell Lymphoma; Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type; Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System; Primary Effusion Lymphoma; Recurrent B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Classic Hodgkin Lymphoma; Recurrent Burkitt Lymphoma; Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Lymphomatoid Granulomatosis; Recurrent Primary Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Refractory B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Classic Hodgkin Lymphoma; Refractory Burkitt Lymphoma; Refractory Diffuse Large B-Cell Lymphoma; Refractory Primary Mediastinal (Thymic) Large B-Cell Cell Lymphoma; Small Intestinal High Grade B-Cell Lymphoma, Not Otherwise Specified; T-Cell/Histiocyte-Rich Large B-Cell Lymphoma

Comparison of gene expression profiles in primary and immortalized human pterygium fibroblast cells.

PubMed

Hou, Aihua; Voorhoeve, P Mathijs; Lan, Wanwen; Tin, Minqi; Tong, Louis

2013-11-01

Pterygium is a fibrovascular growth on the ocular surface with corneal tissue destruction, matrix degradation and varying extents of chronic inflammation. To facilitate investigation of pterygium etiology, we immortalized pterygium fibroblast cells and profiled their global transcript levels compared to primary cultured cells. Fibroblast cells were cultured from surgically excised pterygium tissue using the explant method and propagated to passage number 2-4. We hypothesized that intervention with 3 critical molecular intermediates may be necessary to propage these cells. Primary fibroblast cells were immortalized sequentially by a retroviral construct containing the human telomerase reverse transcriptase gene and another retroviral expression vector expressing p53/p16 shRNAs. Primary and immortalized fibroblast cells were evaluated for differences in global gene transcript levels using an Agilent Genechip microarray. Light microscopic morphology of immortalized cells was similar to primary pterygium fibroblast at passage 2-4. Telomerase reverse transcriptase was expressed, and p53 and p16 levels were reduced in immortalized pterygium fibroblast cells. There were 3308 significantly dysregulated genes showing at least 2 fold changes in transcript levels between immortalized and primary cultured cells (2005 genes were up-regulated and 1303 genes were down-regulated). Overall, 13.58% (95% CI: 13.08-14.10) of transcripts in immortalized cells were differentially expressed by at least 2 folds compared to primary cells. Pterygium primary fibroblast cells were successfully immortalized to at least passage 11. Although a variety of genes are differentially expressed between immortalized and primary cells, only genes related to cell cycle are significantly changed, suggesting that the immortalized cells may be used as an in vitro model for pterygium pathology. © 2013 Elsevier Inc. All rights reserved.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis).

PubMed

Jager, Martine J; Magner, J Antonio Bermudez; Ksander, Bruce R; Dubovy, Sander R

2016-08-01

To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

PubMed Central

Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

2016-01-01

Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

Primary Sjögren's syndrome is characterized by distinct phenotypic and transcriptional profiles of IgD+ unswitched memory B cells.

PubMed

Roberts, Mustimbo E P; Kaminski, Denise; Jenks, Scott A; Maguire, Craig; Ching, Kathryn; Burbelo, Peter D; Iadarola, Michael J; Rosenberg, Alexander; Coca, Andreea; Anolik, Jennifer; Sanz, Iñaki

2014-09-01

The significance of distinct B cell abnormalities in primary Sjögren's syndrome (SS) remains to be established. We undertook this study to analyze the phenotype and messenger RNA (mRNA) transcript profiles of B cell subsets in patients with primary SS and to compare them with those in sicca syndrome patients and healthy controls. CD19+ B cells from 26 patients with primary SS, 27 sicca syndrome patients, and 22 healthy controls were analyzed by flow cytometry. Gene expression profiles of purified B cell subsets (from 3-5 subjects per group per test) were analyzed using Affymetrix gene arrays. Patients with primary SS had lower frequencies of CD27+IgD- switched memory B cells and CD27+IgD+ unswitched memory B cells compared with healthy controls. Unswitched memory B cell frequencies were also lower in sicca syndrome patients and correlated inversely with serologic hyperactivity in both disease states. Further, unswitched memory B cells in primary SS had lower expression of CD1c and CD21. Gene expression analysis of CD27+ memory B cells separated patients with primary SS from healthy controls and identified a subgroup of sicca syndrome patients with a primary SS-like transcript profile. Moreover, unswitched memory B cell gene expression analysis identified 187 genes differentially expressed between patients with primary SS and healthy controls. A decrease in unswitched memory B cells with serologic hyperactivity is characteristic of both established primary SS and a subgroup of sicca syndrome, which suggests the value of these B cells both as biomarkers of future disease progression and for understanding disease pathogenesis. Overall, the mRNA transcript analysis of unswitched memory B cells suggests that their activation in primary SS takes place through innate immune pathways in the context of attenuated antigen-mediated adaptive signaling. Thus, our findings provide important insight into the mechanisms and potential consequences of decreased unswitched memory B cells in primary SS. Copyright © 2014 by the American College of Rheumatology.

9 CFR 113.51 - Requirements for primary cells used for production of biologics.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from normal...

9 CFR 113.51 - Requirements for primary cells used for production of biologics.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from normal...

9 CFR 113.51 - Requirements for primary cells used for production of biologics.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from normal...

9 CFR 113.51 - Requirements for primary cells used for production of biologics.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from normal...

9 CFR 113.51 - Requirements for primary cells used for production of biologics.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from normal...

Brentuximab Vedotin and Lenalidomide in Treating Patients With Stage IB-IVB Relapsed or Refractory T-Cell Lymphoma

ClinicalTrials.gov

2018-03-19

Lymphomatoid Papulosis; Primary Cutaneous Anaplastic Large Cell Lymphoma; Recurrent Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Recurrent T-Cell Non-Hodgkin Lymphoma; Refractory Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Stage I Cutaneous T-Cell Non-Hodgkin Lymphoma; Stage II Cutaneous T-Cell Non-Hodgkin Lymphoma; Stage III Cutaneous T-Cell Non-Hodgkin Lymphoma; Stage IV Cutaneous T-Cell Non-Hodgkin Lymphoma

Heterogeneity in cancer cells: variation in drug response in different primary and secondary colorectal cancer cell lines in vitro.

PubMed

Arul, Melanie; Roslani, April Camilla; Cheah, Swee Hung

2017-05-01

Tumor heterogeneity may give rise to differential responses to chemotherapy drugs. Therefore, unraveling tumor heterogeneity has an implication for biomarker discovery and cancer therapeutics. To test this phenomenon, we investigated the differential responses of three secondary colorectal cancer cell lines of different origins (HCT116, HT29, and SW620 cells) and four novel primary cell lines obtained from different colorectal cancer patients to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP) and explored the differences in gene expression among the primary cell lines in response to exposure to cytotoxic drugs. Cells were exposed to different doses of 5-FU and L-OHP separately or in combinations of equitoxic drug or equimolar drug ratios (median effect of Chou-Talalay principle). Cell viability was assessed using MTT assay and the respective IC 50 values were determined. Changes in gene expression in primary cell lines after exposure to the same drug doses were compared using real-time PCR array. The sensitivities (IC 50 ) of different cell lines, both secondary and primary, to 5-FU and L-OHP were significantly different, whether in monotherapy or combined treatment. Primary cell lines needed higher doses to reach IC 50 . There were variations in gene expression among the primary cell lines of different chemosensitivities to the challenge of the same combined dose of 5-FU and L-OHP. The results confirm the heterogeneous nature of colorectal cancer cells from different patient tumors. Studies using primary cancer cells established from patient's tumors rather than secondary cell lines will more closely reflect the actual character of the disease.

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.

Imaging Nuclear-Cytoplasmic Dynamics in Primary and Metastatic Colon Cancer in Nude Mice.

PubMed

Hasegawa, Kosuke; Suetsugu, Atsushi; Nakamura, Miki; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Bouvet, Michael; Shimizu, Masahito; Hoffman, Robert M

2016-05-01

Colon cancer frequently results in metastasis to the liver, where it becomes the main cause of death. However, the cell cycle in primary tumors and metastases is poorly understood. We developed a mouse model of liver metastasis using the human colon cancer cell line HCT-116, which expresses green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP cells were injected into the spleen of nu/nu nude mice. HCT-116-GFP-RFP cells subsequently formed primary tumors in the spleen, as well as metastatic colonies in the liver and retroperitoneum by 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, it was possible to clearly image mitosis of the dual-colored colon cancer cells in the primary tumor as well as liver and other metastases. Multi-nucleate cancer cells, in addition to mono-nucleate cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nucleate HCT-116-GFP-RFP cells were also observed after culture of the primary and metastatic tumors. A similar ratio of mono-nucleate, multi-nucleate, and mitotic cells grew from the primary and metastatic tumors in culture, suggesting similarity of the nuclear-cytoplasmic dynamics of primary and metastatic cancer cells, further emphasizing the stochastic nature of metastasis. Our results demonstrate a similar heterogeneity of nuclear-cytoplasmic dynamics within primary tumors and metastases, which may be an important factor in the stochastic nature of metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Primary signet ring cell adenocarcinoma of the uterine cervix - A rare neoplasm that raises the question of metastasis to the cervix.

PubMed

Cracchiolo, Bernadette; Kuhn, Theresa; Heller, Debra

2016-04-01

Primary signet ring cell adenocarcinoma is extremely rare. Signet ring cell carcinoma is more commonly primary in the stomach or breast, and the more likely metastatic disease to the cervix needs to be ruled out. We present a case of primary signet ring cell carcinoma of the cervix and review the literature.

Multicentric primary extramammary Paget disease: a Toker cell disorder?

PubMed

Hashemi, Pantea; Kao, Grace F; Konia, Thomas; Kauffman, Lisa C; Tam, Christine C; Sina, Bahram

2014-07-01

Toker cells are epithelial clear cells found in the areolar and nipple areas of the breast, vulvar region, and other apocrine gland-bearing areas of the skin. Toker cells have been implicated in the pathogenesis of clear cell papulosis, cutaneous hamartoma with pagetoid cells, and rare cases of primary extramammary Paget disease (EMPD) but not in secondary EMPD with underlying adenocarcinoma. The pathogenesis of primary EMPD is not well defined. We report a case of multicentric primary EMPD with evidence of Toker cell proliferation and nonaggressive biologic behavior in a 63-year-old white man. A detailed description of the morphologic and biologic features of Toker cells and their possible carcinogenetic links also are discussed. Based on the observation and follow-up of our patient, we hypothesize that multicentric primary EMPD starts with Toker cell hyperplasia and can potentially evolve to carcinoma in the genital region.

49 CFR 171.24 - Additional requirements for the use of the ICAO Technical Instructions.

Code of Federal Regulations, 2011 CFR

2011-10-01

.... (ii) Primary lithium batteries and cells. Primary lithium batteries and cells are forbidden for transportation aboard passenger-carrying aircraft. Equipment containing or packed with primary lithium batteries... containing primary lithium batteries and cells transported in accordance with Special Provision A45 of the...

49 CFR 171.24 - Additional requirements for the use of the ICAO Technical Instructions.

Code of Federal Regulations, 2013 CFR

2013-10-01

.... (ii) Primary lithium batteries and cells. Primary lithium batteries and cells are forbidden for transportation aboard passenger-carrying aircraft. Equipment containing or packed with primary lithium batteries... containing primary lithium batteries and cells transported in accordance with Special Provision A45 of the...

49 CFR 171.24 - Additional requirements for the use of the ICAO Technical Instructions.

Code of Federal Regulations, 2012 CFR

2012-10-01

.... (ii) Primary lithium batteries and cells. Primary lithium batteries and cells are forbidden for transportation aboard passenger-carrying aircraft. Equipment containing or packed with primary lithium batteries... containing primary lithium batteries and cells transported in accordance with Special Provision A45 of the...

Can Tissue Cilia Lengths and Urine Cilia Proteins Be Markers of Kidney Diseases?

PubMed

Park, Kwon Moo

2018-05-01

The primary cilium is an organelle which consists of a microtubule in the core and a surrounding cilia membrane, and has long been recognized as a "vestigial organelle". However, new evidence demonstrates that the primary cilium has a notable effect on signal transduction in the cell and is associated with some genetic and non-genetic diseases. In the kidney, the primary cilium protrudes into the Bowman's space and the tubular lumen from the apical side of epithelial cells. The length of primary cilia is dynamically altered during the normal cell cycle, being shortened by retraction into the cell body at the entry of cell division and elongated at differentiation. Furthermore, the length of primary cilia is also dynamically changed in the cells, as a result and/or cause, during the progression of various kidney diseases including acute kidney injury and chronic kidney disease. Notably, recent data has demonstrated that the shortening of the primary cilium in the cell is associated with fragmentation, apart from retraction into the cell body, in the progression of diseases and that the fragmented primary cilia are released into the urine. This data reveals that the alteration of primary cilia length could be related to the progression of diseases. This review will consider if primary cilia length alteration is associated with the progression of kidney diseases and if the length of tissue primary cilia and the presence or increase of cilia proteins in the urine is indicative of kidney diseases.

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

PubMed Central

Kornete, Mara

2018-01-01

Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing–mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells. PMID:29445007

Metformin and Chemotherapy in Treating Patients With Stage III-IV Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-04-17

Brenner Tumor; Malignant Ascites; Malignant Pleural Effusion; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mixed Epithelial Carcinoma; Ovarian Serous Cystadenocarcinoma; Ovarian Undifferentiated Adenocarcinoma; Recurrent Fallopian Tube Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Recurrent Primary Peritoneal Cavity Cancer; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Epithelial Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIA Primary Peritoneal Cavity Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Epithelial Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIB Primary Peritoneal Cavity Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Epithelial Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IIIC Primary Peritoneal Cavity Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Primary Peritoneal Cavity Cancer

The correlation between NK cell and liver function in patients with primary hepatocellular carcinoma.

PubMed

Sha, Wei Hong; Zeng, Xiao Hui; Min, Lu

2014-05-01

This study aimed to detect the expression of natural killer (NK) cell receptor natural killer group 2D (NKG2D) in the peripheral blood of patients with primary hepatocellular carcinoma and to discuss the correlation between NK cell cytotoxicity and liver function. The number of NK cells and the expression of NK cell receptor NKG2D in peripheral blood were determined by flow cytometry in patients with primary hepatocellular carcinoma, hepatitis B cirrhosis, chronic hepatitis B, and healthy controls. When compared with patients in the healthy and the chronic hepatitis B groups, the primary hepatocellular carcinoma group showed significant decreases in all parameters, including the cytotoxicity of NK cells on K562 cells, expression rate of NKG2D in NK cells, number of NKG2D(+) NK cells, expression level of NKG2D, and number of NK cells (p<0.05). The activity of NK cells showed a positive correlation, whereas the Child-Pugh scores in the primary hepatocellular carcinoma and the hepatitis B cirrhosis groups showed a negative correlation with all parameters detected above. The decrease of NK cell activity in patients with primary hepatocellular carcinoma is closely related to their lower expression of NKG2D. Liver function affects the expression of NKG2D and the activity of NK cells.

Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines.

PubMed

Hearst, Scoty M; Gilder, Andrew S; Negi, Sandeep S; Davis, Misty D; George, Eric M; Whittom, Angela A; Toyota, Cory G; Husedzinovic, Alma; Gruss, Oliver J; Hebert, Michael D

2009-06-01

Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells.

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

PubMed

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

2017-03-01

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

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

PubMed Central

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

2017-01-01

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

Constitutive expression of HCA(2) in human retina and primary human retinal pigment epithelial cells.

PubMed

Yu, Alice L; Birke, Kerstin; Lorenz, Reinhard L; Welge-Lussen, Ulrich

2014-05-01

HCA2, a receptor of β-hydroxybutyrate and niacin, has recently been described in mouse retina and immortalized human retinal pigment epithelial (RPE) cell lines. As HCA2 might be a pharmacologic target, e.g. in diabetic retinopathy, we studied its expression in human retina and primary human RPE cells. Paraffin sections of human retina and primary human RPE cells were obtained from human donor eyes. Expression of HCA2 in human retina was investigated by immunohistochemistry of paraffin sections and by RT-PCR. HCA2 expression in primary human RPE cells was examined by immunocytochemistry and by Western-blot analysis. Positive immunohistochemical staining for HCA2 was found in paraffin sections of human retina, and positive immunocytochemical staining for HCA2 in primary human RPE cells. RT-PCR analysis detected mRNA expression of HCA2 in human retina. The expression of HCA2 protein was found in primary human RPE cells. Based on these results, HCA2 appears to be constitutively expressed in human retina and in primary human RPE cells. Although its functional role is still unknown, HCA2 may be potentially involved in the pathogenesis of various retinopathies and may offer a new therapeutic target.

Resveratrol Differentially Regulates NAMPT and SIRT1 in Hepatocarcinoma Cells and Primary Human Hepatocytes

PubMed Central

Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Petzold-Quinque, Stefanie; Damm, Georg; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

2014-01-01

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells. PMID:24603648

Experimental characterization of recurrent ovarian immature teratoma cells after optimal surgery.

PubMed

Tanaka, Tetsuji; Toujima, Saori; Utsunomiya, Tomoko; Yukawa, Kazunori; Umesaki, Naohiko

2008-07-01

Minimal optimal surgery without chemotherapy is often performed for patients with ovarian immature teratoma, which frequently occurs in young women who hope for future pregnancies. If tumors recur after the operation, anticancer drug chemotherapy is often administered, although few studies have highlighted differences between the recurrent and the primary tumor cells. Therefore, we have established experimental animal models of recurrent ovarian immature teratoma cells after optimal surgery and characterized the anticancer drug sensitivity and antigenicity of the recurrent tumors. Surgically-excised tumor cells of a grade II ovarian immature teratoma were cultured in vitro and transplanted into nude mice to establish stable cell lines. Differential drug sensitivity and antigenicity of the tumor cells were compared between the primary and the nude mouse tumors. Nude mouse tumor cells showed a normal 46XX karyotype. Cultured primary cells showed a remarkably high sensitivity to paclitaxel, docetaxel, adriamycin and pirarubicin, compared to peritoneal cancer cells obtained from a patient with ovarian adenocarcinomatous peritonitis. The drug sensitivity of teratoma cells to 5-fluorouracil, bleomycin or peplomycin was also significantly higher. However, there was no significant difference in sensitivity to platinum drugs between the primary teratoma and the peritoneal adenocarcinoma cells. As for nude mouse tumor cells, sensitivity to 12 anticancer drugs was significantly lower than that of the primary tumor cells, while there was little difference in sensitivity to carboplatin or peplomycin between the primary and nude mouse tumor cells. Flow cytometry showed that the expression of smooth muscle actin (SMA) significantly decreased in nude mouse tumor cells when compared to cultured primary cells. In conclusion, ovarian immature teratomas with normal karyotypes have a malignant potential to recur after minimal surgery. During nude mouse transplantation, SMA-overexpressing cells appeared to be selectively excluded and nude mouse tumor cells were less sensitive to the majority of anticancer drugs than the primary tumor cells. These results indicate that after optimal surgery for ovarian immature teratoma, recurrent cells can be more resistant to anticancer drugs than the primary tumors. Therefore, it is likely that adjuvant chemotherapy lowers the risk of ovarian immature teratomas recurring after optimal surgery. BEP and PBV regimens are frequently given to teratoma patients. However, paclitaxel/carboplatin or docetaxel/carboplatin, which are the most effective chemotherapy treatments for epithelial ovarian cancer patients, are considered to be an alternative regimen, especially in the prevention of reproductive toxicity.

Establishment and characterization of three immortal bovine muscular epithelial cell lines.

PubMed

Jin, Xun; Lee, Joong-Seob; Kwak, Sungwook; Lee, Soo-Yeon; Jung, Ji-Eun; Kim, Tae-Kyung; Xu, Chenxiong; Hong, Zhongshan; Li, Zhehu; Kim, Sun-Myung; Pian, Xumin; Lee, Dong-Hee; Yoon, Jong-Taek; You, Seungkwon; Choi, Yun-Jaie; Kim, Huunggee

2006-02-28

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.

A morphometric analysis of cellular differentiation in caps of primary and lateral roots of Helianthus annuus

NASA Technical Reports Server (NTRS)

Moore, R.

1985-01-01

In order to determine if patterns of cell differentiation are similar in primary and lateral roots, I performed a morphometric analysis of the ultrastructure of calyptrogen, columella, and peripheral cells in primary and lateral roots of Helianthus annuus. Each cell type is characterized by a unique ultrastructure, and the ultrastructural changes characteristic of cellular differentiation in root caps are organelle specific. No major structural differences exist in the structures of the composite cell types, or in patterns of cell differentiation in caps of primary vs. lateral roots.

A case of robot-assisted laparoscopic radical prostatectomy in primary small cell prostate cancer.

PubMed

Kim, Ki Hong; Park, Sang Un; Jang, Jee Young; Park, Won Kyu; Oh, Chul Kyu; Rha, Koon Ho

2010-12-01

Primary small cell carcinoma of the prostate is a rare and very aggressive disease with a poor prognosis, even in its localized form. We managed a case of primary small cell carcinoma of the prostate. The patient was treated with robot-assisted laparoscopic radical prostatectomy and adjuvant chemotherapy. Herein we report this first case of robot-assisted laparoscopic radical prostatectomy performed in a patient with primary small cell carcinoma of the prostate.

Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines

PubMed Central

Hearst, Scoty M.; Gilder, Andrew S.; Negi, Sandeep S.; Davis, Misty D.; George, Eric M.; Whittom, Angela A.; Toyota, Cory G.; Husedzinovic, Alma; Gruss, Oliver J.; Hebert, Michael D.

2009-01-01

Summary Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells. PMID:19435804

Cellular basis for neonatally induced T-suppressor activity. Primary B cell maturation is blocked by suppressor-helper interactions restricted by loci on chromosome 12

PubMed Central

1985-01-01

The cellular mechanism and genetic restriction of neonatally induced HA- specific suppressor T (Ts) cells have been examined. The in vivo effect of these Ts cells on antibody production, primary B cell proliferation, B cell surface marker changes, and helper T (Th) cell priming during primary responses to HA have been determined. The results indicate that, although antigen-induced B cell proliferative responses and surface marker changes occur in the presence of Ts cells, differentiation to Ig secretion, and long-lived memory B cell production are prevented. Further, antigen-specific Th cell priming is completely ablated by Ts cells, suggesting that Ts act by preventing the delivery of Th signals required for both the later stages of primary B cell maturation, and the formation of memory B cell populations. Finally, in vivo cell mixing experiments using congenic mice indicate that this Ts-Th interaction is restricted by loci on mouse chromosome 12. PMID:2580040

Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.

PubMed

Pierfelice, Tarran J; Gaiano, Nicholas

2010-11-13

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Interleukin 18 messenger RNA and proIL-18 protein expression in chorioamniotic membranes from pregnant women with preterm prelabor rupture of membranes.

PubMed

Polettini, Jossimara; Vieira, Eliane Passarelli; Santos, Mariana Perlati dos; Peraçoli, José Carlos; Witkin, Steven S; da Silva, Márcia Guimarães

2012-04-01

To quantify the expression of IL-18 mRNA and protein in the chorioamniotic membranes of pregnant women with PPROM and correlate expression with histological chorioamnionitis. A case control study that included 42 pregnant women not in labor in the following groups: PPROM (n=28) and controls with intact membranes submitted to selective cesarean section at term (n=14). Expression of IL-18 mRNA in chorioamniotic membranes was determined by real-time polymerase chain reaction, and IL-18 protein expression was measured by western blot. Histopathological analyses and immunolocalization of IL-18 by immunohistochemistry were also performed. Analyses were performed using the Mann-Whitney or Fisher's exact tests and the group effect was considered significant if the adjusted p-values were <0.05 and the magnitude of change was greater than 2-fold for mRNA expression. IL-18 mRNA was present in 100% of samples and no difference in expression was observed between term vs. PPROM membranes (fold-change 0.12; p=0.88). In the PPROM group, no difference was observed in IL-18 mRNA regarding gestational age (fold-change 0.11; p=0.42) or the presence of histological chorioamnionitis (fold-change 0.26; p=0.15). ProIL-18 was present in all samples. IL-18 was immunolocalized to amnion, chorion and decidua cells, with intense immunohistochemical staining at the choriodecidual junction. Chorioamniotic membranes are sources of IL-18 mRNA and proIL-18, and their expression is unrelated to PPROM or histological chorioamnionitis. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

PubMed Central

Magaldi, Thomas G.; Almstead, Laura L.; Bellone, Stefania; Prevatt, Edward G.; Santin, Alessandro D.; DiMaio, Daniel

2011-01-01

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells. PMID:22056390

Evolution and development of fetal membranes and placentation in amniote vertebrates.

PubMed

Ferner, Kirsten; Mess, Andrea

2011-08-31

We review aspects of fetal membrane evolution and patterns of placentation within amniotes, the most successful land vertebrates. Special reference is given to embryonic gas supply. The evolution of fetal membranes is a prerequisite for reproduction independent from aquatic environments. Starting from a basically similar repertoire of fetal membranes - the amnion, chorion, allantois and yolk sac, which form the cleidoic egg - different structural solutions for embryonic development have evolved. In oviparous amniotes the chorioallantoic membrane is the major site for the exchange of respiratory gases between fetus and outer environment. The richly vascularised yolk sac and allantois in concert with the chorion play an important role in the evolution of placentation in various viviparous amniotes. Highly complex placentas have evolved independently among squamate sauropsids and in marsupial and placental mammals. In conclusion, there seems to be a natural force to improve gas exchange processes in intrauterine environments by reducing the barrier between the blood systems and optimising the exchange areas. Copyright © 2011 Elsevier B.V. All rights reserved.

The earliest known reptile

NASA Astrophysics Data System (ADS)

Smithson, T. R.

1989-12-01

AMNIOTES (reptiles, birds and mammals) are distinguished from non-amniote tetrapods (amphibians) by the presence of complex embryonic membranes. One of these, the amnion, gives its name to the group. Very few skeletal characters distinguish amniotes from amphibians1, making it difficult to recognize early amniotes in the fossil record. The earliest amniote fossil identified so far is Hylonomus from the Westphalian (Upper Carboniferous) of Joggins, Nova Scotia2,3, (~300 Myr). I report here the discovery of a much earlier amniote skeleton from the Brigantian (Lower Carboniferous) of Scotland (~338 Myr) 4, which thus represents the earliest occurrence of amniotes in the fossil record. The specimen was collected from the East Kirkton Limestone, near Bathgate, West Lothian4-8, and is part of a unique terrestrial fauna that includes eurypterids, myriapods, scorpions and the earliest-known harvestman spider7,9, together with the earliest known temno-spondyls, a group that may include the ancestors of all living amphibians10. It will make an important contribution to our knowledge of early amniote morphology and the interrelationships of tetrapods.

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

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

Mortimer, Jenny C.; Faria-Blanc, Nuno; Yu, Xiaolan

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays,more » and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. Lastly, the differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.« less

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

DOE PAGES

Mortimer, Jenny C.; Faria-Blanc, Nuno; Yu, Xiaolan; ...

2015-06-04

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays,more » and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. Lastly, the differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.« less

Metabolic and structural integrity of magnetic nanoparticle-loaded primary endothelial cells for targeted cell therapy.

PubMed

Orynbayeva, Zulfiya; Sensenig, Richard; Polyak, Boris

2015-05-01

To successfully translate magnetically mediated cell targeting from bench to bedside, there is a need to systematically assess the potential adverse effects of magnetic nanoparticles (MNPs) interacting with 'therapeutic' cells. Here, we examined in detail the effects of internalized polymeric MNPs on primary rat endothelial cells' structural intactness, metabolic integrity and proliferation potential. The intactness of cytoskeleton and organelles was studied by fluorescent confocal microscopy, flow cytometry and high-resolution respirometry. MNP-loaded primary endothelial cells preserve intact cytoskeleton and organelles, maintain normal rate of proliferation, calcium signaling and mitochondria energy metabolism. This study provides supportive evidence that MNPs at doses necessary for targeting did not induce significant adverse effects on structural integrity and functionality of primary endothelial cells - potential cell therapy vectors.

Psychosexual Intervention in Patients With Stage I-III Gynecologic or Breast Cancer

ClinicalTrials.gov

2018-05-25

Ovarian Sarcoma; Ovarian Stromal Cancer; Stage I Uterine Sarcoma; Stage I Vaginal Cancer; Stage I Vulvar Cancer; Stage IA Cervical Cancer; Stage IA Endometrial Carcinoma; Stage IA Fallopian Tube Cancer; Stage IA Ovarian Epithelial Cancer; Stage IA Ovarian Germ Cell Tumor; Stage IA Primary Peritoneal Cavity Cancer; Stage IB Cervical Cancer; Stage IB Endometrial Carcinoma; Stage IB Fallopian Tube Cancer; Stage IB Ovarian Epithelial Cancer; Stage IB Ovarian Germ Cell Tumor; Stage IB Primary Peritoneal Cavity Cancer; Stage IC Fallopian Tube Cancer; Stage IC Ovarian Epithelial Cancer; Stage IC Ovarian Germ Cell Tumor; Stage IC Primary Peritoneal Cavity Cancer; Stage II Endometrial Carcinoma; Stage II Gestational Trophoblastic Tumor; Stage II Uterine Sarcoma; Stage II Vaginal Cancer; Stage II Vulvar Cancer; Stage IIA Cervical Cancer; Stage IIA Fallopian Tube Cancer; Stage IIA Ovarian Epithelial Cancer; Stage IIA Ovarian Germ Cell Tumor; Stage IIA Primary Peritoneal Cavity Cancer; Stage IIB Cervical Cancer; Stage IIB Fallopian Tube Cancer; Stage IIB Ovarian Epithelial Cancer; Stage IIB Ovarian Germ Cell Tumor; Stage IIB Primary Peritoneal Cavity Cancer; Stage IIC Fallopian Tube Cancer; Stage IIC Ovarian Epithelial Cancer; Stage IIC Ovarian Germ Cell Tumor; Stage IIC Primary Peritoneal Cavity Cancer; Stage III Gestational Trophoblastic Tumor; Stage III Uterine Sarcoma; Stage III Vaginal Cancer; Stage III Vulvar Cancer; Stage IIIA Cervical Cancer; Stage IIIA Endometrial Carcinoma; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Epithelial Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIA Primary Peritoneal Cavity Cancer; Stage IIIB Cervical Cancer; Stage IIIB Endometrial Carcinoma; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Epithelial Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIB Primary Peritoneal Cavity Cancer; Stage IIIC Endometrial Carcinoma; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Epithelial Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IIIC Primary Peritoneal Cavity Cancer; Breast Cancer

Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line.

PubMed

Chu, Van Trung; Graf, Robin; Wirtz, Tristan; Weber, Timm; Favret, Jeremy; Li, Xun; Petsch, Kerstin; Tran, Ngoc Tung; Sieweke, Michael H; Berek, Claudia; Kühn, Ralf; Rajewsky, Klaus

2016-11-01

Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.

Vaginal type-II mucosa is an inductive site for primary CD8+ T-cell mucosal immunity

PubMed Central

Wang, Yichuan; Sui, Yongjun; Kato, Shingo; Hogg, Alison E.; Steel, Jason C.; Morris, John C.; Berzofsky, Jay A.

2014-01-01

The structured lymphoid tissues are considered the only inductive sites where primary T cell immune responses occur. The naïve T cells in structured lymphoid tissues, once being primed by antigen -bearing dendritic cells, differentiate into memory T cells and traffic back to the mucosal sites through the bloodstream. Contrary to this belief, here we show that the vaginal type-II mucosa itself, despite lack of structured lymphoid tissues, can act as an inductive site during primary CD8+ T cell immune responses. We provide evidence that the vaginal mucosa supports both the local immune priming of naïve CD8+ T cells and the local expansion of antigen-specific CD8+ T cells, thereby demonstrating a different paradigm for primary mucosal T cell immune induction. PMID:25600442

Thalidomide distinctly affected TNF-α, IL-6 and MMP secretion by an ovarian cancer cell line (SKOV-3) and primary ovarian cancer cells.

PubMed

Piura, Benjamin; Medina, Liat; Rabinovich, Alex; Dyomin, Victor; Huleihel, Mahmoud

2013-01-01

Thalidomide inhibits TNF-α production in lipopolysaccharide-stimulated monocytes. The aim of this study was to evaluate the effect of thalidomide on TNF-α, IL-6 and MMP secretion in epithelial ovarian carcinoma cells. SKOV-3 cells and primary epithelial ovarian carcinoma cells were cultured in the presence of various concentrations of thalidomide. Cell proliferation was examined by MTT proliferation assay. TNF-α and IL-6 levels were determined in the supernatants of the cell cultures by ELISA, and MMP activity was examined by gelatin zymography. Thalidomide did not significantly affect the proliferation and growth of SKOV-3 cells. However, it decreased significantly the capacity of SKOV-3 cells and primary epithelial ovarian carcinoma cells to secrete TNF-α. Thalidomide also significantly decreased the capacity of SKOV-3 cells, but not primary epithelial ovarian carcinoma cells, to secrete MMP-9 and MMP-2. However, thalidomide did not affect IL-6 secretion in SKOV-3 cells or primary epithelial ovarian carcinoma cells. Our study suggests that thalidomide distinctly affected TNF-α, IL-6 and MMPs secretion by an ovarian carcinoma cell line (SKOV-3) and primary ovarian cancer cells. This might suggest a different susceptibility of these two types of cells to thalidomide, and/or that the mechanisms of secretion of the factors examined are differently regulated in these cells. Our results may deepen our understanding the mechanism/s of action of thalidomide in ovarian carcinoma cells. The results might have important implications in future therapeutic strategies that will incorporate thalidomide and other cytokine inhibitors in the treatment of epithelial ovarian carcinoma.

Efficient Immortalization of Primary Nasopharyngeal Epithelial Cells for EBV Infection Study

PubMed Central

Yip, Yim Ling; Pang, Pei Shin; Deng, Wen; Tsang, Chi Man; Zeng, Musheng; Hau, Pok Man; Man, Cornelia; Jin, Yuesheng; Yuen, Anthony Po Wing; Tsao, Sai Wah

2013-01-01

Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma. PMID:24167620

Investigating the cell death mechanisms in primary prostate cancer cells using low-temperature plasma treatment

NASA Astrophysics Data System (ADS)

O'Connell, Deborah; Hirst, A. M.; Packer, J. R.; Simms, M. S.; Mann, V. M.; Frame, F. M.; Maitland, N. J.

2016-09-01

Atmospheric pressure plasmas have shown considerable promise as a potential cancer therapy. An atmospheric pressure plasma driven with kHz kV excitation, operated with helium and oxygen admixtures is used to investigate the interaction with prostate cancer cells. The cytopathic effect was verified first in two commonly used prostate cancer cell lines (BPH-1 and PC-3 cells) and further extended to examine the effects in paired normal and tumour prostate epithelial cells cultured directly from patient tissues. Through the formation of reactive species in cell culture media, and potentially other plasma components, we observed high levels of DNA damage, together with reduced cell viability and colony-forming ability. We observed differences in response between the prostate cell lines and primary cells, particularly in terms of the mechanism of cell death. The primary cells ultimately undergo necrotic cell death in both the normal and tumour samples, in the complete absence of apoptosis. In addition, we provide the first evidence of an autophagic response in primary cells. This work highlights the importance of studying primary cultures in order to gain a more realistic insight into patient efficacy. EPSRC EP/H003797/1 & EP/K018388/1, Yorkshire Cancer Research: YCR Y257PA.

Vaccine Therapy in Treating Patients With Stage IIIC-IV Ovarian Epithelial, Fallopian Tube, or Primary Peritoneal Cavity Cancer Following Surgery and Chemotherapy

ClinicalTrials.gov

2017-10-12

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Tumor; Fallopian Tube Mucinous Neoplasm; Fallopian Tube Serous Neoplasm; Fallopian Tube Transitional Cell Carcinoma; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Cystadenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Cystadenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Impact of host cell variation on the neutralization of HIV-1 in vitro.

PubMed

Polonis, Victoria R; Schuitemaker, Hanneke; Bunnik, Evelien M; Brown, Bruce K; Scarlatti, Gabriella

2009-09-01

In this review we present current advances in our understanding of HIV-1 neutralization assays that employ primary cell types, as compared with those that utilize cell lines and the newer, more standardized pseudovirus assays. A commentary on the challenges of standardizing in-vitro neutralization assays using primary cells is included. The data from reporter cell line neutralization assays may agree with results observed in primary cells; however, exceptions have recently been reported. Multiple variables exist in primary cell assays using peripheral blood mononuclear cells from HIV-seronegative donors; in-vitro neutralization titers can vary significantly based on the donor cells used for assay targets and for virus propagation. Thus, more research is required to achieve validated primary cell neutralization assays. HIV-vaccine-induced antibody performance in the current neutralization assays may function as a 'gatekeeper' for HIV-1 subunit vaccine advancement. Development of standardized platforms for reproducible measurement of in-vitro neutralization is therefore a high priority. Given the considerable variation in results obtained from some widely applied HIV neutralization platforms, parallel evaluation of new antibodies using different host cells for assay targets, as well as virus propagation, is recommended until immune correlates of protection are identified.

Asymmetric Distribution of Primary Cilia Allocates Satellite Cells for Self-Renewal.

PubMed

Jaafar Marican, Nur Hayati; Cruz-Migoni, Sara B; Borycki, Anne-Gaëlle

2016-06-14

Regeneration of vertebrate skeletal muscles requires satellite cells, a population of stem cells that are quiescent in normal conditions and divide, differentiate, and self-renew upon activation triggered by exercise, injury, and degenerative diseases. Satellite cell self-renewal is essential for long-term tissue homeostasis, and previous work has identified a number of external cues that control this process. However, little is known of the possible intrinsic control mechanisms of satellite cell self-renewal. Here, we show that quiescent satellite cells harbor a primary cilium, which is rapidly disassembled upon entry into the cell cycle. Contrasting with a commonly accepted belief, cilia reassembly does not occur uniformly in cells exiting the cell cycle. We found that primary cilia reassemble preferentially in cells committed to self-renew, and disruption of cilia reassembly causes a specific deficit in self-renewing satellite cells. These observations indicate that primary cilia provide an intrinsic cue essential for satellite cell self-renewal. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research

PubMed Central

Stewart, Ceri E.; Torr, Elizabeth E.; Mohd Jamili, Nur H.; Bosquillon, Cynthia; Sayers, Ian

2012-01-01

The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions. PMID:22287976

Primary Cilium-Regulated EG-VEGF Signaling Facilitates Trophoblast Invasion.

PubMed

Wang, Chia-Yih; Tsai, Hui-Ling; Syu, Jhih-Siang; Chen, Ting-Yu; Su, Mei-Tsz

2017-06-01

Trophoblast invasion is an important event in embryo implantation and placental development. During these processes, endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is the key regulator mediating the crosstalk at the feto-maternal interface. The primary cilium is a cellular antenna receiving environmental signals and is crucial for proper development. However, little is known regarding the role of the primary cilium in early human pregnancy. Here, we demonstrate that EG-VEGF regulates trophoblast cell invasion via primary cilia. We found that EG-VEGF activated ERK1/2 signaling and subsequent upregulation of MMP2 and MMP9, thereby facilitating cell invasion in human trophoblast HTR-8/SVneo cells. Inhibition of ERK1/2 alleviated the expression of MMPs and trophoblast cell invasion after EG-VEGF treatment. In addition, primary cilia were observed in all the trophoblast cell lines tested and, more importantly, in human first-trimester placental tissue. The receptor of EG-VEGF, PROKR1, was detected in primary cilia. Depletion of IFT88, the intraflagellar transporter required for ciliogenesis, inhibited primary cilium growth, thereby ameliorating ERK1/2 activation, MMP upregulation, and trophoblast cell invasion promoted by EG-VEGF. These findings demonstrate a novel function of primary cilia in controlling EG-VEGF-regulated trophoblast invasion and reveal the underlying molecular mechanism. J. Cell. Physiol. 232: 1467-1477, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Establishment and characterization of fetal and maternal mesenchymal stem/stromal cell lines from the human term placenta.

PubMed

Qin, Sharon Q; Kusuma, Gina D; Al-Sowayan, Batla; Pace, Rishika A; Isenmann, Sandra; Pertile, Mark D; Gronthos, Stan; Abumaree, Mohamed H; Brennecke, Shaun P; Kalionis, Bill

2016-03-01

Human placental mesenchymal stem/stromal cells (MSC) are an attractive source of MSC with great therapeutic potential. However, primary MSC are difficult to study in vitro due to their limited lifespan and patient-to-patient variation. Fetal and maternal MSC were prepared from cells of the chorionic and basal plates of the placenta, respectively. Fetal and maternal MSC were transduced with the human telomerase reverse transcriptase (hTERT). Conventional stem cell assays assessed the MSC characteristics of the cell lines. Functional assays for cell proliferation, cell migration and ability to form colonies in soft agar were used to assess the whether transduced cells retained properties of primary MSC. Fetal chorionic and maternal MSC were successfully transduced with hTERT to create the cell lines CMSC29 and DMSC23 respectively. The lifespans of CMSC29 and DMSC23 were extended in cell culture. Both cell lines retained important MSC characteristics including cell surface marker expression and multipotent differentiation potential. Neither of the cell lines was tumourigenic in vitro. Gene expression differences were observed between CMSC29 and DMSC23 cells and their corresponding parent, primary MSC. Both cell lines show similar migration potential to their corresponding primary, parent MSC. The data show that transduced MSC retained important functional properties of the primary MSC. There were gene expression and functional differences between cell lines CMSC29 and DMSC23 that reflect their different tissue microenvironments of the parent, primary MSC. CMSC29 and DMSC23 cell lines could be useful tools for optimisation and functional studies of MSC. Copyright © 2016 Elsevier Ltd. All rights reserved.

Talimogene Laherparepvec and Nivolumab in Treating Patients With Refractory Lymphomas or Advanced or Refractory Non-melanoma Skin Cancers

ClinicalTrials.gov

2018-06-25

Adenoid Cystic Carcinoma; Adnexal Carcinoma; Apocrine Carcinoma; Eccrine Porocarcinoma; Extraocular Cutaneous Sebaceous Carcinoma; Hidradenocarcinoma; Keratoacanthoma; Malignant Sweat Gland Neoplasm; Merkel Cell Carcinoma; Microcystic Adnexal Carcinoma; NK-Cell Lymphoma, Unclassifiable; Non-Melanomatous Lesion; Paget Disease; Papillary Adenocarcinoma; Primary Cutaneous Mucinous Carcinoma; Refractory Anaplastic Large Cell Lymphoma; Refractory Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Refractory Mycosis Fungoides; Refractory Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Refractory T-Cell Non-Hodgkin Lymphoma; Sezary Syndrome; Signet Ring Cell Carcinoma; Skin Basal Cell Carcinoma; Skin Basosquamous Cell Carcinoma; Skin Squamous Cell Carcinoma; Spiradenocarcinoma; Squamous Cell Carcinoma of Unknown Primary Origin; Stage III Skin Cancer; Stage IV Skin Cancer; Sweat Gland Carcinoma; Trichilemmocarcinoma; Vulvar Squamous Cell Carcinoma

Low-density lipoproteins modulate endothelial cells to secrete endothelin-1 in a polarized pattern: a study using a culture model system simulating arterial intima.

PubMed

Unoki, H; Fan, J; Watanabe, T

1999-01-01

We investigated the structural and functional properties of human umbilical vein endothelial cells (HUVECs) cultured on a two-chamber culture model system using an amnion membrane. Compared to HUVECs cultured on a plastic dish, HUVECs cultured on the model system exhibited several features similar to those of in vivo vessels, including formation of the intercellular junctional devices and expression of tight junction-associated protein ZO-1 and adherence junction-associated protein alpha-catenin. Furthermore, we found that HUVECs had a property of polar secretion of endothelin-1 (ET-1). About 90% of the total amount of synthesized ET-1 was found in the lower well, designated as the basal side. When HUVECs were incubated with either native low-density lipoproteins (nLDLs) or oxidized LDLs (oxLDLs) at a concentration of 100 microgram/ml, ET-1 secretion was significantly increased, dependent on the cell side (apical vs basal) on which the nLDLs or oxLDLs were loaded. When the LDLs were loaded on the apical side, the secretion of ET-1 from HUVECs on the apical side was increased by 48% (nLDL) and 61% (oxLDL), whereas it was accompanied by a concomitant decrease of ET-1 on the basal side (45% by nLDLs and 38% by oxLDLs). When loaded on the basal side, however, ET-1 was increased by 23% (nLDLs) and 53% (oxLDLs) on the basal side, with a 26% simultaneous decrease of ET-1 on the opposite side for both nLDLs and oxLDLs. On the contrary, high-density lipoproteins (HDLs) inhibited ET-1 secretion from HUVECs on the opposite side of the well on which HDLs were loaded; there was a 57% decrease on the basal side when HDLs were loaded on the apical side, and a 46% decrease on the apical side when loaded on the basal side. These results indicate that modulation of ET-1 secretion from ECs by lipoproteins is virtually dependent on the place (apical vs basal) where these proteins are present. The finding that nLDLs and oxLDLs enhance ET-1 secretion by ECs in a polarized pattern suggests that ET-1 may be involved in pathophysiological processes such as atherogenesis.

Primary Lung Signet Ring Cell Carcinoma Presenting as a Cavitary Pancoast Tumor in a 32-Year-Old Man.

PubMed

Corvini, Michael; Koorji, Alysha; Sgroe, Erica; Nguyen, Uyen

2018-06-01

Signet ring cell carcinoma, a subtype of adenocarcinoma, is a rare cause of primary lung cancer. The authors report a case of primary lung signet ring cell carcinoma presenting as a cavitary Pancoast tumor in a 32-year-old male smoker. Beyond the rarity of primary lung signet ring cell carcinoma itself, the youth of the patient, his smoking status, the presence of cavitation, and the location of the tumor in the superior sulcus make it especially atypical.

Primary cultures of human colon cancer as a model to study cancer stem cells.

PubMed

Koshkin, Sergey; Danilova, Anna; Raskin, Grigory; Petrov, Nikolai; Bajenova, Olga; O'Brien, Stephen J; Tomilin, Alexey; Tolkunova, Elena

2016-09-01

The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

PubMed

Lapointe, Jason F; Dunphy, Gary B; Giannoulis, Paschalis; Mandato, Craig A; Nardi, James B; Gharib, Osama H; Niven, Donald F

2011-11-01

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions. Copyright © 2011 Elsevier Inc. All rights reserved.

Patient-centered evaluation of microsurgical management of gingival recession using coronally advanced flap with platelet-rich fibrin or amnion membrane: A comparative analysis

PubMed Central

Agarwal, Sumit Kumar; Jhingran, Rajesh; Bains, Vivek Kumar; Srivastava, Ruchi; Madan, Rohit; Rizvi, Iram

2016-01-01

Objective: To evaluate the efficacy of coronally advanced flap (CAF) procedure under microsurgical approach for the management of Miller's Class I and II gingival recession defects with the use of either platelet-rich fibrin (PRF) or amnion membrane (AM) in comparison to CAF alone. Materials and Methods: A total of 45 sites with Miller's Class I or II gingival recession defect were randomly distributed for: Experimental Group I (CAF with PRF) sites (n = 15) which were treated with the microsurgical approach using CAF along with PRF; experimental Group II (CAF with AM) sites (n = 15) were treated with the microsurgical approach using CAF along with AM; control Group III (CAF alone) sites (n = 15) were treated with the microsurgical approach using CAF alone. Vertical gingival recession (VGR), horizontal gingival recession (HGR), gingival thickness (GT) (using transgingival probing [TGP] and ultrasonography [USG]) and patients’ response and acceptance were documented at baseline, 3 months and 6 months after surgical interventions. Results: CAF alone and in combination with PRF or AM, were effective techniques for root coverage with average VGR values of 1.47 ± 0.92 mm (56%), 0.67 ± 1.23 mm (36%) and 0.60 ± 1.06 mm (33%) in Group I (CAF with PRF), Group II (CAF with AM), and Group III (CAF alone), respectively. Complete coverage (100%) was obtained in 33.3% sites of Group I (CAF with PRF), 26.6% sites of Group II (CAF with AM) and 13.3% in Group III (CAF alone). Patients’ response and acceptance for surgical treatment modality in terms of patient esthetic score and decrease in hypersensitivity score was highest for Group I (CAF with PRF), whereas patient comfort score was highest for Group II (CAF with AM). At 6 months follow-up, significant increase in GT measurements (using TGP and USG) in Group I (CAF with PRF), whereas, nonsignificant increase for Group II (CAF with AM) and no change or decrease for Group III (CAF alone) as compared to baseline was observed. Conclusion: The present study observed enhancement in root coverage when PRF or AM are used in conjunction with CAF as compared to CAF alone. These results are based on 6-month follow-up. Therefore, the long-term evaluation may be necessary to appreciate the clinical effect of autologous PRF and AM. PMID:27011751

Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis.

PubMed

Desterke, Christophe; Martinaud, Christophe; Guerton, Bernadette; Pieri, Lisa; Bogani, Costanza; Clay, Denis; Torossian, Frederic; Lataillade, Jean-Jacques; Hasselbach, Hans C; Gisslinger, Heinz; Demory, Jean-Loup; Dupriez, Brigitte; Boucheix, Claude; Rubinstein, Eric; Amsellem, Sophie; Vannucchi, Alessandro M; Le Bousse-Kerdilès, Marie-Caroline

2015-06-01

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the "bad seed in bad soil" hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis. Copyright© Ferrata Storti Foundation.

Carboplatin and Paclitaxel With or Without Bevacizumab in Treating Patients With Stage III or Stage IV Ovarian Epithelial, Primary Peritoneal, or Fallopian Tube Cancer

ClinicalTrials.gov

2017-10-23

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Malignant Ovarian Mixed Epithelial Tumor; Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Acrylamide-induced apoptosis in rat primary astrocytes and human astrocytoma cell lines.

PubMed

Lee, Jiann-Gwu; Wang, Yan-Shiu; Chou, Chin-Cheng

2014-06-01

This study aimed to evaluate the acrylamide (ACR)-induced apoptotic effects on rat primary astrocytes and three human astrocytoma-derived cell lines (U-1240 MG, U-87 MG, and U-251 MG). As determined through the MTT assay, treatment with 1 and 2 mM ACR for 24-72 h resulted in decreased cell viability in all cells. Decreases in cell viability could be blocked in all cells with the exception of U-251 MG cells by Z-DEVD FMK. ACR-induced dose-dependent apoptotic effects were also demonstrated by increases in the sub-G1 phase cell population in all cells. The decreased expressions of pro-caspase 3, 8, and 9 and the interruption of the mitochondrial membrane potential were observed in all cells. Exposure to 2 mM ACR for 48 h resulted in increased Bax/Bcl-2 ratios in primary astrocytes and U-87 MG cells, whereas the overexpression of Bcl-2 was observed in U-1240 MG and U-251 MG cells. The ACR-induced increases in the levels of p53 and pp53 in primary astrocytes could be attenuated by caffeine. These results suggest the existence of a common apoptotic pathway among all cell types and that U-87 MG cells may be a suitable substitute in vitro model for primary astrocytes in future studies on ACR-induced neurotoxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Carcinogens induce loss of the primary cilium in human renal proximal tubular epithelial cells independently of effects on the cell cycle

PubMed Central

Radford, Robert; Slattery, Craig; Jennings, Paul; Blacque, Oliver; Pfaller, Walter; Gmuender, Hans; Van Delft, Joost; Ryan, Michael P.

2012-01-01

The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line. Using both scanning electron microscopy and immunofluorescent labeling of the ciliary markers acetylated tubulin and Arl13b, we confirmed that RPTEC/TERT1 cells express primary cilium upon reaching confluence. Treatment with the carcinogens ochratoxin A (OTA) and potassium bromate (KBrO3) caused a significant reduction in the number of ciliated cells, while exposure to nifedipine, a noncarcinogenic renal toxin, had no effect on primary cilium expression. Flow cytometric analysis of the effects of all three compounds on the cell cycle revealed that only KBrO3 resulted in an increase in the proportion of cells entering the cell cycle. Microarray analysis revealed dysregulation of multiple pathways affecting ciliogenesis and ciliary maintenance following OTA and KBrO3 exposure, which were unaffected by nifedipine exposure. The primary cilium represents a unique physical checkpoint with relevance to carcinogenesis. We have shown that the renal carcinogens OTA and KBrO3 cause significant deciliation in a model of the proximal tubule. With KBrO3, this was followed by reentry into the cell cycle; however, deciliation was not found to be associated with reentry into the cell cycle following OTA exposure. Transcriptomic analysis identified dysregulation of Wnt signaling and ciliary trafficking in response to OTA and KBrO3 exposure. PMID:22262483

Type I collagen-induced YAP nuclear expression promotes primary cilia growth and contributes to cell migration in confluent mouse embryo fibroblast 3T3-L1 cells.

PubMed

Xu, Qian; Liu, Xiaoling; Liu, Weiwei; Hayashi, Toshihiko; Yamato, Masayuki; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2018-05-30

The extracellular matrix (ECM) is a major biomechanical environment for all cells in vivo, and tightly controls wound healing and cancer progression. Type I collagen (Col I) is the most abundant component in ECM and plays an essential role for cell motility control and migration beyond structural support. Our previous results showed that Col I increased the length of primary cilia and the expression of primary cilia-associated proteins in 3T3-L1 cells. The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes for the development and maintenance of tissue functions. In this study, we investigated the role of Hippo/YAP signaling in primary cilia growth of cells cultured on Col I-coated plate, as well as the potential link between primary cilia and migration. At 2-day post-confluence, YAP localization in the nucleus was dramatically increased when the cells were cultured on Col I-coated plate, accompanied by cilia growth. YAP inhibitor verteporfin repressed the growth of primary cilia as well as the expressions of ciliogenesis-associated proteins in confluent 3T3-L1 cells cultured on Col I-coated plate. Moreover, knockdown of either YAP or IFT88, one of the ciliogenesis-associated proteins, reversed the migration of confluent 3T3-L1 cells promoted by Col I-coating. In conclusion, activation of YAP pathway by Col I-coating of culture plate for confluent 3T3-L1 cells is positively associated with the primary cilia growth, which eventually results in promoted migration.

FoxP3+ CD25+ CD8+ T-Cell Induction during Primary Simian Immunodeficiency Virus Infection in Cynomolgus Macaques Correlates with Low CD4+ T-Cell Activation and High Viral Load▿

PubMed Central

Karlsson, Ingrid; Malleret, Benoît; Brochard, Patricia; Delache, Benoît; Calvo, Julien; Le Grand, Roger; Vaslin, Bruno

2007-01-01

The early immune response fails to prevent the establishment of chronic human immunodeficiency virus (HIV) infection but may influence viremia during primary infection, thereby possibly affecting long-term disease progression. CD25+ FoxP3+ regulatory T cells may contribute to HIV/simian immunodeficiency virus (SIV) pathogenesis by suppressing efficient antiviral responses during primary infection, favoring high levels of viral replication and the establishment of chronic infection. In contrast, they may decrease immune activation during chronic infection. CD4+ regulatory T cells have been studied in the most detail, but CD8+ CD25+ FoxP3+ T cells also have regulatory properties. We monitored the dynamics of CD25+ FoxP3+ T cells during primary and chronic SIVmac251 infection in cynomolgus macaques. The number of peripheral CD4+ CD25+ FoxP3+ T cells paralleled that of memory CD4+ T cells, with a rapid decline during primary infection followed by a rebound to levels just below baseline and gradual depletion during the course of infection. No change in the proportion of CD25+ FoxP3+ T cells was observed in peripheral lymph nodes. A small number of CD4+ CD25+ FoxP3+ T cells at set point was associated with a high plasma viral load. In contrast, peripheral CD8+ CD25+ FoxP3+ T cells were induced a few days after peak plasma viral load during primary infection. The number of these cells was positively correlated with viral load and negatively correlated with CD4+ T-cell activation, SIV antigen-specific proliferative responses during primary infection, and plasma viral load at set point, with large numbers of CD8+ CD25+ FoxP3+ T cells being indicative of a poor prognosis. PMID:17898053

Defective ciliogenesis in thyroid hürthle cell tumors is associated with increased autophagy

PubMed Central

Lee, Junguee; Yi, Shinae; Kang, Yea Eun; Chang, Joon Young; Kim, Jung Tae; Sul, Hae Joung; Kim, Jong Ok; Kim, Jin Man; Kim, Joon; Porcelli, Anna Maria; Kim, Koon Soon; Shong, Minho

2016-01-01

Primary cilia are found in the apical membrane of thyrocytes, where they may play a role in the maintenance of follicular homeostasis. In this study, we examined the distribution of primary cilia in the human thyroid cancer to address the involvement of abnormal ciliogenesis in different thyroid cancers. We examined 92 human thyroid tissues, including nodular hyperplasia, Hashimoto's thyroiditis, follicular tumor, Hürthle cell tumor, and papillary carcinoma to observe the distribution of primary cilia. The distribution and length of primary cilia facing the follicular lumen were uniform across variable-sized follicles in the normal thyroid gland. However, most Hürthle cells found in benign and malignant thyroid diseases were devoid of primary cilia. Conventional variant of papillary carcinoma (PTC) displayed longer primary cilia than those of healthy tissue, whereas both the frequency and length of primary cilia were decreased in oncocytic variant of PTC. In addition, ciliogenesis was markedly defective in primary Hürthle cell tumors, including Hürthle cell adenomas and carcinomas, which showed higher level of autophagosome biogenesis. Remarkably, inhibition of autophagosome formation by Atg5 silencing or treatment with pharmacological inhibitors of autophagosome formation restored ciliogenesis in the Hürthle cell carcinoma cell line XTC.UC1 which exhibits a high basal autophagic flux. Moreover, the inhibition of autophagy promoted the accumulation of two factors critical for ciliogenesis, IFT88 and ARL13B. These results suggest that abnormal ciliogenesis, a common feature of Hürthle cells in diseased thyroid glands, is associated with increased basal autophagy. PMID:27816963

Mixed microencapsulation of rat primary hepatocytes and Sertoli cells improves the metabolic function in a D-galactosamine and lipopolysaccharide-induced rat model of acute liver failure.

PubMed

Zheng, Ming-Hua; Lin, Hai-Long; Qiu, Li-Xin; Cui, Yao-Li; Sun, Qing-Feng; Chen, Yong-Ping

2009-01-01

Hepatocyte transplantation is an alternative to transplantation of the whole liver. Compared with xenogeneic hepatocytes, primary hepatocytes have some advantages, such as a more powerful function and a smaller frequency of rejection caused by the host. Cell microencapsulation prevents direct access of host cells to the graft but cannot impede transfer of transplant-derived peptides, which can cross the physical barrier. Sertoli cells are central to the immune privilege demonstrated in the testis, and their actions have been utilized to protect cell transplants. Co-microencapsulating Sertoli cells with HepG2 cells has proved to be a valuable strategy in hepatocyte transplantation. Thus mixed microcapsules of primary rat hepatocytes and primary Sertoli cells may improve metabolic function in a d-galactosamine and lipopolysaccharide-induced rat model of acute liver failure.

Primary renal diffuse large B-cell lymphoma with central nervous system involvement: a rare case report and literature review.

PubMed

Wang, Ying; Guo, Shuangshuang

2015-01-01

Primary renal lymphoma is a rare entity. Of these, diffuse large B-cell lymphoma is the most common pathological type and, R-CHOP regimen was the preferred chemotherapy for it. Here we present an adult case of primary renal diffuse large B-cell lymphoma.

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

PubMed Central

Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

2016-01-01

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

Novel concepts on pregnancy clocks and alarms: redundancy and synergy in human parturition

PubMed Central

Menon, Ramkumar; Bonney, Elizabeth A.; Condon, Jennifer; Mesiano, Sam; Taylor, Robert N.

2016-01-01

The signals and mechanisms that synchronize the timing of human parturition remain a mystery and a better understanding of these processes is essential to avert adverse pregnancy outcomes. Although our insights into human labor initiation have been informed by studies in animal models, the timing of parturition relative to fetal maturation varies among viviparous species, indicative of phylogenetically different clocks and alarms; but what is clear is that important common pathways must converge to control the birth process. For example, in all species, parturition involves the transition of the myometrium from a relaxed to a highly excitable state, where the muscle rhythmically and forcefully contracts, softening the cervical extracellular matrix to allow distensibility and dilatation and thus a shearing of the fetal membranes to facilitate their rupture. We review a number of theories promulgated to explain how a variety of different timing mechanisms, including fetal membrane cell senescence, circadian endocrine clocks, and inflammatory and mechanical factors, are coordinated as initiators and effectors of parturition. Many of these factors have been independently described with a focus on specific tissue compartments. In this review, we put forth the core hypothesis that fetal membrane (amnion and chorion) senescence is the initiator of a coordinated, redundant signal cascade leading to parturition. Whether modified by oxidative stress or other factors, this process constitutes a counting device, i.e. a clock, that measures maturation of the fetal organ systems and the production of hormones and other soluble mediators (including alarmins) and that promotes inflammation and orchestrates an immune cascade to propagate signals across different uterine compartments. This mechanism in turn sensitizes decidual responsiveness and eventually promotes functional progesterone withdrawal in the myometrium, leading to increased myometrial cell contraction and the triggering of parturition. Linkage of these processes allows convergence and integration of the gestational clocks and alarms, prompting a timely and safe birth. In summary, we provide a comprehensive synthesis of the mediators that contribute to the timing of human labor. Integrating these concepts will provide a better understanding of human parturition and ultimately improve pregnancy outcomes. PMID:27363410

Leech segmental repeats develop normally in the absence of signals from either anterior or posterior segments

NASA Technical Reports Server (NTRS)

Seaver, E. C.; Shankland, M.

2000-01-01

We have investigated whether the development of segmental repeats is autonomous in the embryo of the leech Helobdella robusta. The segmental tissues of the germinal band arise from progeny of five stem cells called teloblasts. Asymmetric divisions of the teloblasts form chains of segment founder cells (called primary blast cells) that divide in a stereotypical manner to produce differentiated descendants. Using two distinct techniques, we have looked for potential interactions between neighboring blast cell clones along the anterior-posterior axis. In one technique, we prevented the birth of primary blast cells by injection of DNase I into the teloblast, thereby depriving the last blast cell produced before the ablation of its normal posterior neighbors. We also ablated single blast cells with a laser microbeam, which allowed us to assess potential signals acting on either more anterior or more posterior primary blast cell clones. Our results suggest that interactions along the anterior-posterior axis between neighboring primary blast cell clones are not required for development of normal segmental organization within the blast cell clone. We also examined the possibility that blast cells receive redundant signals from both anterior and posterior neighboring clones and that either is sufficient for normal development. Using double blast cell laser ablations to isolate a primary blast cell clone by removal of both its anterior and its posterior neighbor, we found that the isolated clone still develops normally. These results reveal that the fundamental segmental repeat in the leech embryo, the primary blast cell clone, can develop normally in the apparent absence of signals from adjacent repeats along the anterior-posterior axis.

Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells.

PubMed

Fink, R D; McClay, D R

1985-01-01

At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.

Establishment of immortal swine kidney epithelial cells.

PubMed

Kwak, Sungwook; Jung, Ji-Eun; Jin, Xun; Kim, Sun-Myung; Kim, Tae-Kyung; Lee, Joong-Seob; Lee, Soo-Yeon; Pian, Xumin; You, Seungkwon; Kim, Hyunggee; Choi, Yun-Jaie

2006-01-01

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.

Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells.

PubMed

Seki, Akiko; Rutz, Sascha

2018-03-05

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell-based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies. © 2018 Genentech.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

PubMed

Fernando, Elizabeth H; Dicay, Michael; Stahl, Martin; Gordon, Marilyn H; Vegso, Andrew; Baggio, Cristiane; Alston, Laurie; Lopes, Fernando; Baker, Kristi; Hirota, Simon; McKay, Derek M; Vallance, Bruce; MacNaughton, Wallace K

2017-11-01

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner. Copyright © 2017 the American Physiological Society.

rhEPO Enhances Cellular Anti-oxidant Capacity to Protect Long-Term Cultured Aging Primary Nerve Cells.

PubMed

Wang, Huqing; Fan, Jiaxin; Chen, Mengyi; Yao, Qingling; Gao, Zhen; Zhang, Guilian; Wu, Haiqin; Yu, Xiaorui

2017-08-01

Erythropoietin (EPO) may protect the nervous system of animals against aging damage, making it a potential anti-aging drug for the nervous system. However, experimental evidence from natural aging nerve cell models is lacking, and the efficacy of EPO and underlying mechanism of this effect warrant further study. Thus, the present study used long-term cultured primary nerve cells to successfully mimic the natural aging process of nerve cells. Starting on the 11th day of culture, cells were treated with different concentrations of recombinant human erythropoietin (rhEPO). Using double immunofluorescence labeling, we found that rhEPO significantly improved the morphology of long-term cultured primary nerve cells and increased the total number of long-term cultured primary cells. However, rhEPO did not improve the ratio of nerve cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure nerve cell activity and showed that rhEPO significantly improved the activity of long-term cultured primary nerve cells. Moreover, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double immunofluorescence labeling flow cytometry revealed that rhEPO reduced the apoptotic rate of long-term cultured primary nerve cells. Senescence-associated β-galactosidase (SA-β-gal) immunohistochemistry staining showed that rhEPO significantly reduced the aging rate of long-term cultured primary nerve cells. Immunochemistry revealed that rhEPO enhanced intracellular superoxide dismutase (SOD) activity and glutathione (GSH) abundance and reduced the intracellular malondialdehyde (MDA) level. In addition, this effect depended on the dose, was maximized at a dose of 100 U/ml and was more pronounced than that of vitamin E. In summary, this study finds that rhEPO protects long-term cultured primary nerve cells from aging in a dose-dependent manner. The mechanism of this effect may be associated with the enhancement of the intracellular anti-oxidant capacity. These findings provide a theoretical basis to further the anti-aging mechanism of EPO in the nervous system, and they provide experimental evidence at the cellular level for the clinical application of EPO to protect the nervous system from aging.

Brentuximab Vedotin and Lenalidomide in Treating Patients With Relapsed or Refractory T-Cell Lymphomas

ClinicalTrials.gov

2018-06-15

CD30-Positive Neoplastic Cells Present; Folliculotropic Mycosis Fungoides; Recurrent Mycosis Fungoides; Recurrent Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Refractory Mycosis Fungoides; Refractory Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Sezary Syndrome

The activation pattern of macrophages in giant cell (temporal) arteritis and primary angiitis of the central nervous system.

PubMed

Mihm, Bernhard; Bergmann, Markus; Brück, Wolfgang; Probst-Cousin, Stefan

2014-06-01

To determine if the pattern of macrophage activation reflects differences in the pathogenesis and clinical presentation of giant cell arteritis and primary angiitis of the central nervous system, specimens of 10 patients with giant cell arteritis and five with primary angiitis of the central nervous system were immunohistochemically studied and the expression of the macrophage activation markers 27E10, MRP14, MRP8 and 25F9 was determined in the vasculitic infiltrates. Thus, a partly different expression pattern of macrophage activation markers in giant cell arteritis and primary angiitis of the central nervous system was observed. The group comparison revealed that giant cell arteritis cases had significantly higher numbers of acute activated MRP14-positive macrophages, whereas primary angiitis of the central nervous system is characterized by a tendency toward more MRP8-positive intermediate/late activated macrophages. Furthermore, in giant cell arteritis comparably fewer CD8-positive lymphocytes were observed. These observations suggest, that despite their histopathological similarities, giant cell arteritis and primary angiitis of the central nervous system appear to represent either distinct entities within the spectrum of granulomatous vasculitides or different stages of similar disease processes. Their discrete clinical presentation is reflected by different activation patterns of macrophages, which may characterize giant cell arteritis as a more acute process and primary angiitis of the central nervous system as a more advanced inflammatory process. © 2013 Japanese Society of Neuropathology.

[Regulation of age-dependent phenomena. Influence of C6-substituted purines on cell aggregation and cell migration in primary cultures of lense epithelial cells].

PubMed

Glässer, D; Iwig, M; Weber, E

1975-01-01

The existence of an age dependent latent period of cell emigration has been proved in the primary culture of epithelial cells of bovine lenses. The previously described aggregation phenomenon as well as the latent period of the cell emigration increase with the age of the sponsor animals. Extracellular adenine and other C6-substituted purines, isolated from the cells themselves and added to the medium, act the same way on the lens cells in the primary culture as the increasing age of the sponsor animals. Adenine stimulates cell aggregation and inhibits the adhesion of the cells to the substratum, the cell flattening and the cell migration. The adenine action has been proved down to a concentration of 3 X 10(-6) M. During the primary culture, the lens cells gradually los the adenine sensitivity. The adenine action also occurs on single cells, isolated by trypsination, it differs from the reaction of ouabain and can be removed at low concentration by washing procedures. The results favour the suggestion C6-substituted purines to be involved in cell ageing.

Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin.

PubMed

Koval, Olga A; Sakaeva, Galiya R; Fomin, Alexander S; Nushtaeva, Anna A; Semenov, Dmitry V; Kuligina, Elena V; Gulyaeva, Ludmila F; Gerasimov, Alexey V; Richter, Vladimir A

2015-01-01

Endometrial carcinoma is the most common gynecologic malignancy which is associated with a poor prognosis when diagnosed at an advanced stage; therefore, the discovery of efficacious new drugs is required to reinforce conventional chemotherapy. Short-term cultures of primary cells from endometrial tumors could be used for testing new anticancer therapeutics as well as for the development of personalized cancer therapy strategy. Here, the antitumor effect of a recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, was examined against primary human endometrial cancer cells. Primary cell cultures of malignant and normal human endometrium were performed by enzymatic digestion of endometrial tissue from biopsy material. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the messenger ribonucleic acid (mRNA) state of estrogen (ERs) and progesterone (PRs) hormone receptors and aromatase (Cyp 19) in cell cultures. Dynamic monitoring of cell adhesion and proliferation was made using the iCELLigence system (ASEA Biosciences). The sensitivity of cell cultures to conventional anticancer drugs and the lactaptin analog was estimated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and the iCELLligence system. Established short-term primary cultures of endometrial cancer cells were ERα/ERβ/PR-positive and sensitive for RL2. The IC 50 values of doxorubicin and cisplatin were determined for all of the primary cultures designed. KE normal cells displaying low Cyp19 mRNA levels and high ERβ and PR mRNA levels were more resistant to RL2 treatment as well as to cisplatin and doxorubicin. Our results indicate that the recombinant analog of lactaptin, RL2, exerts cytotoxic effects against primary hormone-dependent endometrial tumor cells in vitro with features of apoptosis.

Squamous Cell Cancer of The Lung with Synchronous Renal Cell Carcinoma

PubMed Central

Ateş, İhsan; Yazıcı, Ozan; Ateş, Hale; Yazılıtaş, Doğan; Özcan, Ayşe Naz; Ağaçkıran, Yetkin; Zengin, Nurullah

2016-01-01

Coexistence of two or more primary cancers is a relatively rare case. Not with standing that the coexistence of multiple primary cancers is often discussed in the literature, there is a small number of publications concerning the coexistence of squamous cell lung carcinoma and renal cancer. In this case report, detection of both squamous cell lung carcinoma and primary renal cancer in one male patient is going to be discussed. PMID:29404140

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

Magaldi, Thomas G.; Almstead, Laura L.; Bellone, Stefania

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, amore » cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.« less

The rapid destabilization of p53 mRNA in immortal chicken embryo fibroblast cells.

PubMed

Kim, H; You, S; Foster, L K; Farris, J; Foster, D N

2001-08-23

The steady-state levels of p53 mRNA were dramatically lower in immortal chicken embryo fibroblast (CEF) cell lines compared to primary CEF cells. In the presence of cycloheximide (CHX), the steady-state levels of p53 mRNA markedly increased in immortal CEF cell lines, similar to levels found in primary cells. The de novo synthetic rates of p53 mRNA were relatively similar in primary and immortal cells grown in the presence or absence of CHX. Destabilization of p53 mRNA was observed in the nuclei of immortal, but not primary, CEF cells. The half-life of p53 mRNA in primary cells was found to be a relatively long 23 h compared to only 3 h in immortal cells. The expression of transfected p53 cDNA was inhibited in immortal cells, but restored upon CHX treatment. The 5'-region of the p53 mRNA was shown to be involved in the rapid p53 mRNA destabilization in immortal cells by expression analysis of 5'- and 3'-deleted p53 cDNAs as well as fusion mRNA constructs of N-terminal p53 and N-terminal deleted LacZ genes. Together, it is suggestive that the downregulation of p53 mRNA in immortal CEF cells occurs through a post-transcriptional destabilizing mechanism.

Paclitaxel, Polyglutamate Paclitaxel, or Observation in Treating Patients With Stage III or Stage IV Ovarian Epithelial, Peritoneal Cancer, or Fallopian Tube Cancer

ClinicalTrials.gov

2017-05-03

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

RNA interference mediated in human primary cells via recombinant baculoviral vectors.

PubMed

Nicholson, Linda J; Philippe, Marie; Paine, Alan J; Mann, Derek A; Dolphin, Colin T

2005-04-01

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described. However, the use of such human viral vectors is inherently problematic, e.g., Class 2 status and requirement of secondary helper functions. Although insect cells are their natural host, baculoviruses also transduce a range of vertebrate cell lines and primary cells with high efficiency. The inability of baculoviral vectors to replicate in mammalian cells, their Class 1 status, and the simplicity of their construction make baculovirus an attractive alternative gene delivery vector. We have developed a baculoviral-based RNAi system designed to express shRNAs and GFP from U6 and CMV promoters, respectively. Transduction of Saos2, HepG2, Huh7, and primary human hepatic stellate cells with a baculoviral construct expressing shRNAs targeting lamin A/C resulted in effective knockdown of the corresponding mRNA and protein. Development of this baculoviral-based system provides an additional shRNA delivery option for RNAi-based investigations in mammalian cells.

Characterization of ibrutinib-sensitive and -resistant mantle lymphoma cells.

PubMed

Ma, Jiao; Lu, Pin; Guo, Ailin; Cheng, Shuhua; Zong, Hongliang; Martin, Peter; Coleman, Morton; Wang, Y Lynn

2014-09-01

Ibrutinib inhibits Bruton tyrosine kinase (BTK), a key component of early B-cell receptor (BCR) signalling pathways. A multicentre phase 2 trial of ibrutinib in patients with relapsed/refractory mantle cell lymphoma (MCL) demonstrated a remarkable response rate. However, approximately one-third of patients have primary resistance to the drug while other patients appear to lose response and develop secondary resistance. Understanding the molecular mechanisms underlying ibrutinib sensitivity is of paramount importance. In this study, we investigated cell lines and primary MCL cells that display differential sensitivity to ibrutinib. We found that the primary cells display a higher BTK activity than normal B cells and MCL cells show differential sensitivity to BTK inhibition. Genetic knockdown of BTK inhibits the growth, survival and proliferation of ibrutinib-sensitive but not resistant MCL cell lines, suggesting that ibrutinib acts through BTK to produce its anti-tumour activities. Interestingly, inhibition of ERK1/2 and AKT, but not BTK phosphorylation per se, correlates well with cellular response to BTK inhibition in cell lines as well as in primary tumours. Our study suggests that, to prevent primary resistance or to overcome secondary resistance to BTK inhibition, a combinatory strategy that targets multiple components or multiple pathways may represent the most effective approach. © 2014 John Wiley & Sons Ltd.

Matrix metalloproteinase inhibitors disrupt spicule formation by primary mesenchyme cells in the sea urchin embryo.

PubMed

Ingersoll, E P; Wilt, F H

1998-04-01

The primary mesenchyme cells of the sea urchin embryo construct an elaborate calcareous endoskeletal spicule beginning at gastrulation. This process begins by ingression of prospective primary mesenchyme cells into the blastocoel, after which they migrate and then fuse to form a syncytium. Skeleton deposition occurs in spaces enclosed by the cytoplasmic cables between the cell bodies. Experiments are described which probe the role of proteases in these early events of spicule formation and their role in the continued elaboration of the spicule during later stages of embryogenesis. We find that several inhibitors of metalloproteinases inhibit the continuation of spiculogenesis, an effect first reported by Roe et al. (Exp. Cell Res. 181, 542-550, 1989). A detailed study of one of these inhibitors, BB-94, shows that fusion of primary mesenchyme cells still occurs in the presence of the inhibitor and the formation of the first calcite granule is not impeded. Continued elaboration of the spicule, however, is completely stopped; addition of the inhibitor during the active elongation of the spicule stops further elongation immediately. Removal of the inhibitor allows resumption of spicule growth. The inhibition is accompanied by almost complete cessation of massive Ca ion transport via the primary mesenchyme cells to the spicule. The inhibitor does not prevent the continued synthesis of several spicule matrix proteins. Electron microscopic examination of inhibited primary mesenchyme cells shows an accumulation of characteristic vesicles in the cytoplasm. Gel zymography demonstrates that although most proteases in homogenates of primary mesenchyme cells are not sensitive to the inhibitor in vitro, a protease of low abundance detectable in the medium of cultured primary mesenchyme cells is inhibited by BB-94. We propose that the inhibitor is interfering with the delivery of precipitated calcium carbonate and matrix proteins to the site(s) of spicule growth. Copyright 1998 Academic Press.

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

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

PubMed Central

Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

2016-01-01

Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

Primary Cilia Are Not Calcium-Responsive Mechanosensors

PubMed Central

Delling, M.; Indzhykulian, A. A.; Liu, X.; Liu, Y.; Xie, T.; Corey, D. P.; Clapham, D. E.

2016-01-01

Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Their antenna-like structure leads naturally to the assumption that they sense the surrounding environment, the most common hypothesis being sensation of mechanical force through calcium-permeable ion channels within the cilium1. This Ca2+- Responsive MechanoSensor (CaRMS) hypothesis for primary cilia has been invoked to explain a large range of biological responses, from control of left-right axis determination in embryonic development to adult progression of polycystic kidney disease and some cancers2,3. Here, we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. First, we developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator (GECI) in all primary cilia. We then measured responses to flow in primary cilia of cultured kidney epithelial cells, kidney thick ascending tubules, crown cells of the embryonic node, kinocilia of inner ear hair cells, and several cell lines. Cilia-specific Ca2+ influxes were not observed in physiological or even highly supraphysiological levels of fluid flow. We conclude that mechanosensation, if it originates in primary cilia, is not via calcium signaling. PMID:27007841

Immortalized Mouse Floxed Fam20c Dental Papillar Mesenchymal and Osteoblast Cell Lines Retain Their Primary Characteristics

PubMed Central

Liu, Chao; Wang, Xiaofang; Zhang, Hua; Xie, Xiaohua; Liu, Peihong; Liu, Ying; Jani, Priyam H.; Lu, Yongbo; Chen, Shuo; Qin, Chunlin

2016-01-01

Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T-antigen (SV40 T-Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long-term passages and genomic integration of SV40 T-Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. PMID:25833681

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes.

PubMed

Speiseder, Thomas; Hofmann-Sieber, Helga; Rodríguez, Estefanía; Schellenberg, Anna; Akyüz, Nuray; Dierlamm, Judith; Spruss, Thilo; Lange, Claudia; Dobner, Thomas

2017-01-01

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells. Copyright © 2016 American Society for Microbiology.

Anthocyanins extracted from black soybean seed coat protect primary cortical neurons against in vitro ischemia.

PubMed

Bhuiyan, Mohammad Iqbal Hossain; Kim, Joo Youn; Ha, Tae Joung; Kim, Seong Yun; Cho, Kyung-Ok

2012-01-01

The present study investigated the neuroprotective effects of anthocyanins extracted from black soybean (cv. Cheongja 3, Glycine max (L.) MERR.) seed coat against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays were employed to assess cell membrane damage and viability of primary neurons, respectively. OGD-induced cell death in 7 d in vitro primary cortical neurons was found to be OGD duration-dependent, and approximately 3.5 h of OGD resulted in ≈60% cell death. Treatment with black soybean anthocyanins dose-dependently prevented membrane damage and increased the viability of primary neurons that were exposed to OGD. Glutamate-induced neuronal cell death was dependent on the glutamate concentration at relatively low concentrations and the number of days the cells remained in culture. Interestingly, black soybean anthocyanins did not protect against glutamate-induced neuronal cell death. They did, however, inhibit the excessive generation of reactive oxygen species (ROS) and preserve mitochondrial membrane potential (MMP) in primary neurons exposed to OGD. In agreement with the neuroprotective effect of crude black soybean anthocyanins, purified cyanidin-3-glucoside (C3G), the major component of anthocyanins, also offered dose-dependent neuroprotection against OGD-induced neuronal cell death. Moreover, black soybean C3G markedly prevented excessive generation of ROS and preserved MMP in primary neurons that were exposed to OGD. Collectively, these results suggest that the neuroprotection of primary rat cortical neurons by anthocyanins that were extracted from black soybean seed coat might be mediated through oxidative stress inhibition and MMP preservation but not through glutamate-induced excitotoxicity attenuation.

Primary Intra-aortic Epstein-Barr Virus-Positive Large B-Cell Lymphoma Presenting as Aortic Mural Thrombosis: An Entity Distinct From Intravascular Large B-Cell Lymphoma.

PubMed

Nakao, Ryuta; Sakashita, Aki; Omoto, Atsushi; Sato, Osamu; Hino, Yoko; Yanagisawa, Akio; Urata, Yoji

2017-12-01

Intravascular selective growth of neoplastic B lymphocytes is a characteristic finding of intravascular large B-cell lymphoma (IVLBCL). However, because neoplastic B cells of IVLBCL grow merely in the lumina of capillaries or small vessels, primary IVLBCL of the great vessels is considered exceptional. To our knowledge, only 2 primary B-cell lymphomas in the lumina of the vena cava have been reported. However, there has been no report of primary B-cell lymphoma with intra-aortic growth. We describe a novel manifestation of primary Epstein-Barr virus-positive large B-cell lymphoma mainly affecting the lumina of the aorta and its major branches in a 76-year-old man. He had a long-term fever that was refractory to antibiotics and aortic mural thrombosis with visceral embolization. Because he had no detectable mass suggesting a malignancy, it was difficult to diagnose while he was alive. He died without anticancer treatment, and the confirmed diagnosis was made at autopsy.

[A method for the primary culture of fibroblasts isolated from human airway granulation tissues].

PubMed

Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu-ling; Pei, Ying-hua

2013-04-01

To establish a feasible method to culture primary fibroblasts isolated from human airway granulation tissues, and therefore to provide experimental data for the investigation of the pathogenesis of benign airway stenosis. The granulation tissues were collected from 6 patients during routine bronchoscopy at our department of Beijing Tiantan Hospital from April to June 2011. Primary fibroblasts were obtained by culturing the explanted tissues. Cell growth was observed under inverted microscope. All of these 6 primary cultures were successful. Fibroblast-like cells were observed to migrate from the tissue pieces 3 d after inoculation. After 9-11 d of culture, cells reached to 90% confluence and could be sub-cultured. After passage, the cells were still in a typical elongated spindle-shape and grew well. The cells could be sub-cultured further when they formed a monolayer. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissues.

Dysregulation of fatty acid synthesis and glycolysis in non-Hodgkin lymphoma

PubMed Central

Bhatt, Aadra P.; Jacobs, Sarah R.; Freemerman, Alex J.; Makowski, Liza; Rathmell, Jeffrey C.; Dittmer, Dirk P.; Damania, Blossom

2012-01-01

The metabolic differences between B-NHL and primary human B cells are poorly understood. Among human B-cell non-Hodgkin lymphomas (B-NHL), primary effusion lymphoma (PEL) is a unique subset that is linked to infection with Kaposi's sarcoma-associated herpesvirus (KSHV). We report that the metabolic profiles of primary B cells are significantly different from that of PEL. Compared with primary B cells, both aerobic glycolysis and fatty acid synthesis (FAS) are up-regulated in PEL and other types of nonviral B-NHL. We found that aerobic glycolysis and FAS occur in a PI3K-dependent manner and appear to be interdependent. PEL overexpress the fatty acid synthesizing enzyme, FASN, and both PEL and other B-NHL were much more sensitive to the FAS inhibitor, C75, than primary B cells. Our findings suggest that FASN may be a unique candidate for molecular targeted therapy against PEL and other B-NHL. PMID:22752304

Isolation of Mouse Primary Gastric Epithelial Cells to Investigate the Mechanisms of Helicobacter pylori Associated Disease.

PubMed

Tran, Le Son; Ferrero, Richard L

2018-01-01

The gastrointestinal epithelium provides the first line of defense against invading pathogens, among which Helicobacter pylori is linked to numerous gastric pathologies, including chronic gastritis and cancer. Primary gastric epithelial cells represent a useful model for the investigation of the underlying molecular and cellular mechanisms involved in these H. pylori associated diseases. In this chapter, we describe a method for the isolation of primary gastric epithelial cells from mice and detection of epithelial cell adhesion molecule (EpCAM) expression in the isolated cells.

Differences in the Importance of Mast Cells, Basophils, IgE, and IgG versus That of CD4+ T Cells and ILC2 Cells in Primary and Secondary Immunity to Strongyloides venezuelensis.

PubMed

Mukai, Kaori; Karasuyama, Hajime; Kabashima, Kenji; Kubo, Masato; Galli, Stephen J

2017-05-01

There is evidence that mast cells, basophils, and IgE can contribute to immune responses to parasites; however, the relative levels of importance of these effector elements in parasite immunity are not fully understood. Previous work in Il3 -deficient and c- kit mutant Kit W / W-v mice indicated that interleukin-3 and c-Kit contribute to expulsion of the intestinal nematode Strongyloides venezuelensis during primary infection. Our findings in mast cell-deficient Kit W-sh / W-sh mice and two types of mast cell-deficient mice that have normal c- kit ("Hello Kit ty" and MasTRECK mice) confirmed prior work in Kit W / W-v mice that suggested that mast cells play an important role in S. venezuelensis egg clearance in primary infections. We also assessed a possible contribution of basophils in immune responses to S. venezuelensis By immunohistochemistry, we found that numbers of basophils and mast cells were markedly increased in the jejunal mucosa during primary infections with S. venezuelensis Studies in basophil-deficient Mcpt8 DTR mice revealed a small but significant contribution of basophils to S. venezuelensis egg clearance in primary infections. Studies in mice deficient in various components of immune responses showed that CD4 + T cells and ILC2 cells, IgG, FcRγ, and, to a lesser extent, IgE and FcεRI contribute to effective immunity in primary S. venezuelensis infections. These findings support the conclusion that the hierarchy of importance of immune effector mechanisms in primary S. venezuelensis infection is as follows: CD4 + T cells/ILC2 cells, IgG, and FcRγ>mast cells>IgE and FcεRI>basophils. In contrast, in secondary S. venezuelensis infection, our evidence indicates that the presence of CD4 + T cells is of critical importance but mast cells, antibodies, and basophils have few or no nonredundant roles. Copyright © 2017 American Society for Microbiology.

Nuclear DNA Methylation and Chromatin Condensation Phenotypes Are Distinct Between Normally Proliferating/Aging, Rapidly Growing/Immortal, and Senescent Cells

PubMed Central

Gertych, Arkadiusz; Tajbakhsh, Jian

2013-01-01

This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns — visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis — in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors. PMID:23562889

Nuclear DNA methylation and chromatin condensation phenotypes are distinct between normally proliferating/aging, rapidly growing/immortal, and senescent cells.

PubMed

Oh, Jin Ho; Gertych, Arkadiusz; Tajbakhsh, Jian

2013-03-01

This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns - visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis - in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors.

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

PubMed Central

Degrassi, A; Hilbert, D M; Rudikoff, S; Anderson, A O; Potter, M; Coon, H G

1993-01-01

Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma cells, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmacytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion. Images Fig. 1 Fig. 2 PMID:8446628

Primary intraosseous squamous cell carcinoma of the mandible arising de novo.

PubMed

Shamim, Thorakkal

2009-07-01

Primary intraosseous squamous cell carcinoma is an odontogenic tumour with aggressive behaviour usually noticed in 6th to 7th decades of life. The tumour is characterized by progressive swelling of the jaw, pain and loosening of teeth. Microscopically, the lesion is showing foci of keratinising cells separated by collagenous connective tissue stroma. A case of primary intraosseous squamous cell carcinoma of mandible arising de novo in a 40-year-old man is reported.

YKL-40 in Serum Samples From Patients With Newly Diagnosed Stage III-IV Ovarian Epithelial, Primary Peritoneal Cavity, or Fallopian Tube Cancer Receiving Chemotherapy

ClinicalTrials.gov

2018-05-21

Fallopian Tube Adenocarcinoma; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Malignant Ovarian Brenner Tumor; Malignant Ovarian Clear Cell Tumor; Malignant Ovarian Endometrioid Tumor; Malignant Ovarian Mixed Epithelial Tumor; Malignant Ovarian Mucinous Tumor; Malignant Ovarian Neoplasm; Malignant Ovarian Serous Tumor; Malignant Ovarian Transitional Cell Tumor; Ovarian Adenocarcinoma; Primary Peritoneal Serous Adenocarcinoma; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Defined, serum/feeder-free conditions for expansion and drug screening of primary B-acute lymphoblastic leukemia.

PubMed

Jiang, Zhiwu; Wu, Di; Ye, Wei; Weng, Jianyu; Lai, Peilong; Shi, Pengcheng; Guo, Xutao; Huang, Guohua; Deng, Qiuhua; Tang, Yanlai; Zhao, Hongyu; Cui, Shuzhong; Lin, Simiao; Wang, Suna; Li, Baiheng; Wu, Qiting; Li, Yangqiu; Liu, Pentao; Pei, Duanqing; Du, Xin; Yao, Yao; Li, Peng

2017-12-05

Functional screening for compounds represents a major hurdle in the development of rational therapeutics for B-acute lymphoblastic leukemia (B-ALL). In addition, using cell lines as valid models for evaluating responses to novel drug therapies raises serious concerns, as cell lines are prone to genotypic/phenotypic drift and loss of heterogeneity in vitro . Here, we reported that OP9 cells, not OP9-derived adipocytes (OP9TA), support the growth of primary B-ALL cells in vitro . To identify the factors from OP9 cells that support the growth of primary B-ALL cells, we performed RNA-Seq to analyze the gene expression profiles of OP9 and OP9TA cells. We thus developed a defined, serum/feeder-free condition (FI76V) that can support the expansion of a range of clinically distinct primary B-ALL cells that still maintain their leukemia-initiating ability. We demonstrated the suitability of high-throughput drug screening based on our B-ALL cultured conditions. Upon screening 378 kinase inhibitors, we identified a cluster of 17 kinase inhibitors that can efficiently kill B-ALL cells in vitro . Importantly, we demonstrated the synergistic cytotoxicity of dinaciclib/BTG226 to B-ALL cells. Taken together, we developed a defined condition for the ex vivo expansion of primary B-ALL cells that is suitable for high-throughput screening of novel compounds.

Assessment of Cell Line Models of Primary Human Cells by Raman Spectral Phenotyping

PubMed Central

Swain, Robin J.; Kemp, Sarah J.; Goldstraw, Peter; Tetley, Teresa D.; Stevens, Molly M.

2010-01-01

Abstract Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures. PMID:20409492

Circulating CXCR5+CD4+ T cells assist in the survival and growth of primary diffuse large B cell lymphoma cells through interleukin 10 pathway

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

Cha, Zhanshan; Qian, Guangfang; Zang, Yan

Diffuse large B cell lymphoma (DLBCL) is a common and aggressive cancer caused by the malignant transformation of B cells. Although it has been established that the follicular helper T (Tfh) cells play a central role in B cell development, little information is available on their involvement in DLBCL pathogenesis. We studied the role of the peripheral Tfh equivalent, the CXCR5{sup +} CD4{sup +} T cells, in DLBCL. Data showed that compared to CXCR5{sup -} CD4{sup +} T cells, CXCR5{sup +} CD4{sup +} T cells were significantly more effective at promoting the proliferation as well as inhibiting the apoptosis ofmore » primary autologous DLBCL tumor cells. Surprisingly, we found that at equal cell numbers, CXCR5{sup +} CD4{sup +} T cells in DLBCL patients secreted significantly less interleukin (IL)-21 than CXCR5{sup -} CD4{sup +} T cells, while the level of IL-10 secretion was significant elevated in the CXCR5{sup +} compartment compared to the CXCR5{sup -} compartment. Neutralization of IL-10 in the primary DLBCL-CXCR5{sup +} CD4{sup +} T cell coculture compromised the CXCR5{sup +} CD4{sup +} T cell-mediated pro-tumor effects, in a manner that was dependent on the concentration of anti-IL-10 antibodies. The CXCR5{sup +} compartment also contained significantly lower frequencies of cytotoxic CD4{sup +} T cells than the CXCR5{sup -} compartment. In conclusion, our investigations discovered a previously unknown pro-tumor role of CXCR5-expressing circulating CD4{sup +} T cells, which assisted the survival and proliferation of primary DLBCL cells through IL-10. - Highlights: • We studied the role of the peripheral Tfh in DLBCL. • Tfh were effective at promoting the proliferation of primary DLBCL tumor cells. • Tfh were effective at inhibiting the apoptosis of primary DLBCL tumor cells. • IL-10 secretion in Tfh was significant elevated in DLBCL. • Neutralization of IL-10 compromised Tfh-mediated pro-tumor effects.« less

Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

PubMed

Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

2015-08-01

Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

[Comparative analysis of the susceptibility and productivity of respiratory tract target cells of mice and rats exposed to inflienza virus in vitro].

PubMed

Zhukov, V A; Shishkina, L N; Sergeev, A A; Malkova, E M; Riabchikova, E I; Petrishchenko, V A; Sergeev, A N; Ustiuzhanina, N V; Nesvizhskiĭ, Iu V; Vorob'ev, A A

2008-01-01

The levels of susceptibility to influenza virus A/Aichi/2/68 H3N2 and the virus yield were determined using primary cells of the trachea and lungs of CD-1 mice and Wistar rats, and for 3 sets of cells obtained from primary lung cells of the both species by centrifugation in the gradient of density and by sedimentation on a surface. The values of ID50 virus dose for 10(6) cells and virus yield per 1 infected cell determined for primary mice cells were 4.0+/-0.47 and 3.2+/-0.27 IgEID50 (lung cells), 3.8+/-0.17 and 3.3+/-0.20 IgEID50 (tracheal cells), and those determined for primary rat cells were 4.0+/-0.35 and 2.1+/-0.24 IgEID50 (lung cells), 3.7+/-0.27 and 2.2+/-0.46 IgEID50 (tracheal cells). The values of ID50 and yield measured for mixtures of cells obtained from primary lung cells by centrifugation in gradient of density and by sedimentation on a surface differed insignificantly (p = 0.05) from the values of the corresponding parameters measured for lung and tracheal cells for both rats and mice. The analysis of data on the variation of the concentrations of different cell types in the experimental cell mixtures shows that type 1 and 2 alveolocytes possess significantly lower (p = 0.05) susceptibility and productivity vs. ciliated cells of the both species. The investigation was conducted within the frame of the ISTC/DARPA#450p project.

Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the aryl hydrocarbon receptor (AHR)-mediated impairment of immunoglobulin secretion in human primary B cells.

PubMed

Zhou, Jiajun; Zhang, Qiang; Henriquez, Joseph E; Crawford, Robert B; Kaminski, Norbert E

2018-05-31

The aryl hydrocarbon receptor (AHR) is a cytosolic ligand-activated transcription factor involved in xenobiotic sensing, cell cycle regulation and cell development. In humans, the activation of AHR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AHR-ligand, impairs the secretion of immunoglobulin M (IgM) to suppress humoral immunity. However, the mechanisms bridging the activation of AHR and the impairment of IgM secretion by human primary B cells remain poorly understood. Recent transcriptomic analysis revealed upregulation of lymphocyte-specific protein tyrosine kinase (LCK) in AHR activated human primary B cells. LCK is a well-characterized tyrosine kinase that phosphorylates critical signaling proteins involved in activation and cytokine production in T cells. Conversely, the role of LCK in human primary B cells is not well understood. In the current studies, we have verified the transcriptomic finding by detecting AHR-mediated upregulation of LCK protein in human primary B cells. We also confirmed the role of AHR in the upregulation of LCK by using a specific AHR antagonist, which abolished the AHR-mediated increase of LCK. Furthermore, we have confirmed the role of LCK in the AHR-mediated suppression of IgM by using LCK specific inhibitors, which restored IgM secretion by human B cells in the presence of TCDD. Collectively, the current studies demonstrate a novel role of LCK in IgM secretion and provide new insights into the mechanism for AHR-mediated impairment of immunoglobulin secretion by human primary B cells.

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

PubMed Central

Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; Wang, Hong; Fu, Hongyong; Zhou, Fan; Yao, Chencheng; Wang, Xiaobo; Li, Zheng; He, Zuping

2017-01-01

Sertoli cells are required for normal spermatogenesis and they can be reprogrammed to other types of functional cells. However, the number of primary Sertoli cells is rare and human Sertoli cell line is unavailable. In this study, we have for the first time reported a stable human Sertoli cell line, namely hS1 cells, by overexpression of human telomerase. The hS1 cells expressed a number of hallmarks for human Sertoli cells, including SOX9, WT1, GDNF, SCF, BMP4, BMP6, GATA4, and VIM, and they were negative for 3β-HSD, SMA, and VASA. Higher levels of AR and FSHR were observed in hS1 cells compared to primary human Sertoli cells. Microarray analysis showed that 70.4% of global gene profiles of hS1 cells were similar to primary human Sertoli cells. Proliferation assay demonstrated that hS1 cells proliferated rapidly and they could be passaged for more than 30 times in 6 months. Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line and 90% normal karyotypes existed in hS1 cells. Collectively, we have established the first human Sertoli cell line with phenotype of primary human Sertoli cells, an unlimited proliferation potential and high safety, which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine. PMID:28152522

GE-17ALTERATION OF THE p53 PATHWAY AND ANCESTRAL PROGENITORS ARE ASSOCIATED WITH TUMOR RECURRENCE IN GLIOBLASTOMA

PubMed Central

Kim, Hoon; Zheng, Siyuan; Amini, Seyed; Virk, Selene; Mikkelsen, Tom; Brat, Daniel; Sougnez, Carrie; Muller, Florian; Hu, Jian; Sloan, Andrew; Cohen, Mark; Van Meir, Erwin; Scarpace, Lisa; Lander, Eric; Gabriel, Stacey; Getz, Gad; Meyerson, Matthew; Chin, Lynda; Barnholtz-Sloan, Jill; Verhaak, Roel

2014-01-01

To evaluate evolutionary patterns of GBM recurrence, we analyzed whole genome sequencing (WGS) and multi-sector exome sequencing data from pairs of primary and posttreatment GBM. WGS on ten primary-recurrent pairs detected a median number of 12,214 mutations which we utilized to uncover clonal structures, by analyzing the distribution of mutation cellular frequencies (the fraction of tumor cells harboring a mutation). On average, 41 % of the mutations were shared by primary and recurrence. The majority of shared mutations were clonal in both primary and recurrence, but we also observed many clonal mutations that were uniquely detected in either the primary or the recurrence. This raises the intriguing possibility that major tumor clones in the primary tumor and disease relapse both evolved from a shared ancestral tumor cell population. At least one subclone was identified in the majority of WGS samples, and we observed groups of mutations that were at low cancer cell fractions in both primary and recurrence, suggesting that both subclones evolved from the same ancestral tumor cells separate from the major clone ancestral cells. To address the possibility that the lack of overlap between subsequent tumors was due to intratumoral heterogeneity, we analyzed exome sequencing from a second tumor sector of seven primary and six recurrent tumors. We found that the majority of "second biopsy" mutations were not conserved between time points, suggesting that intratumoral heterogeneity did not explain the large number of mutations uniquely detected in primary and recurrence. The limited overlap of mutations in primary and recurrence provides evidence for ancestral tumor cell populations that could not be eradicated by therapy, while offspring cell populations contained unique mutations, were selectively killed by treatment and could therefore no longer be detected after disease relapse. This study has provided new insights into patterns and dynamics of tumor evolution.

Comparative study of the primary cilia in thyrocytes of adult mammals

PubMed Central

Utrilla, J C; Gordillo-Martínez, F; Gómez-Pascual, A; Fernández-Santos, J M; Garnacho, C; Vázquez-Román, V; Morillo-Bernal, J; García-Marín, R; Jiménez-García, A; Martín-Lacave, I

2015-01-01

Since their discovery in different human tissues by Zimmermann in 1898, primary cilia have been found in the vast majority of cell types in vertebrates. Primary cilia are considered to be cellular antennae that occupy an ideal cellular location for the interpretation of information both from the environment and from other cells. To date, in mammalian thyroid gland, primary cilia have been found in the thyrocytes of humans and dogs (fetuses and adults) and in rat embryos. The present study investigated whether the existence of this organelle in follicular cells is a general event in the postnatal thyroid gland of different mammals, using both immunolabeling by immunofluorescence and electron microscopy. Furthermore, we aimed to analyse the presence of primary cilia in various thyroid cell lines. According to our results, primary cilia are present in the adult thyroid gland of most mammal species we studied (human, pig, guinea pig and rabbit), usually as a single copy per follicular cell. Strikingly, they were not found in rat or mouse thyroid tissues. Similarly, cilia were also observed in all human thyroid cell lines tested, both normal and neoplastic follicular cells, but not in cultured thyrocytes of rat origin. We hypothesize that primary cilia could be involved in the regulation of normal thyroid function through specific signaling pathways. Nevertheless, further studies are needed to shed light on the permanence of these organelles in the thyroid gland of most species during postnatal life. PMID:26228270

Characterization of primary cilia in human airway smooth muscle cells.

PubMed

Wu, Jun; Du, Hui; Wang, Xiangling; Mei, Changlin; Sieck, Gary C; Qian, Qi

2009-08-01

Considerable evidence indicates a key role for primary cilia of mammalian cells in mechanochemical sensing. Dysfunctions of primary cilia have been linked to the pathogenesis of several human diseases. However, cilia-related research has been limited to a few cell and tissue types; to our knowledge, no literature exists on primary cilia in airway smooth muscle (ASM). The aim of this study was to characterize primary cilia in human ASM. Primary cilia of human bronchial smooth muscle cells (HBSMCs) were examined using immunofluorescence confocal microscopy, and scanning and transmission electron microscopy. HBSMC migration and injury repair were examined by scratch-wound and epidermal growth factor (EGF)-induced migration assays. Cross-sectional images of normal human bronchi revealed that primary cilia of HBSMCs within each ASM bundle aggregated at the same horizontal level, forming a "cilium layer." Individual cilia of HBSMCs projected into extracellular matrix and exhibited varying degrees of deflection. Mechanochemical sensing molecules, polycystins, and alpha2-, alpha5-, and beta1-integrins were enriched in cilia, as was EGF receptor, known to activate jointly with integrins during cell migration. Migration assays demonstrated a ciliary contribution to HBSMC migration and wound repair. The primary cilia of ASM cells exert a role in sensing and transducing extracellular mechanochemical signals and in ASM injury repair. Defects in ASM ciliary function could potentially affect airway wall maintenance and/or remodeling, possibly relating to the genesis of bronchiectasis in autosomal dominant polycystic kidney disease, a disease of ciliopathy.

Cediranib Maleate and Olaparib or Standard Chemotherapy in Treating Patients With Recurrent Platinum-Resistant or -Refractory Ovarian, Fallopian Tube, or Primary Peritoneal Cancer

ClinicalTrials.gov

2018-06-15

Deleterious BRCA1 Gene Mutation; Deleterious BRCA2 Gene Mutation; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Elesclomol Sodium and Paclitaxel in Treating Patients With Recurrent or Persistent Ovarian Epithelial Cancer, Fallopian Tube Cancer, or Primary Peritoneal Cancer

ClinicalTrials.gov

2017-05-02

Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Tumor; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Changes in Brain Function in Patients With Stage I, Stage II, Stage III, or Stage IV Ovarian, Primary Peritoneal, or Fallopian Tube Cancer Who Are Receiving Chemotherapy

ClinicalTrials.gov

2018-04-11

Cognitive Side Effects of Cancer Therapy; Malignant Ovarian Epithelial Tumor; Ovarian Brenner Tumor; Ovarian Carcinosarcoma; Ovarian Choriocarcinoma; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Dysgerminoma; Ovarian Embryonal Carcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mixed Germ Cell Tumor; Ovarian Mucinous Cystadenocarcinoma; Ovarian Polyembryoma; Ovarian Sarcoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Cystadenocarcinoma; Ovarian Teratoma; Ovarian Yolk Sac Tumor; Stage I Ovarian Cancer; Stage IA Fallopian Tube Cancer; Stage IA Ovarian Cancer; Stage IA Ovarian Germ Cell Tumor; Stage IB Fallopian Tube Cancer; Stage IB Ovarian Cancer; Stage IB Ovarian Germ Cell Tumor; Stage IC Fallopian Tube Cancer; Stage IC Ovarian Cancer; Stage IC Ovarian Germ Cell Tumor; Stage II Ovarian Cancer; Stage IIA Fallopian Tube Cancer; Stage IIA Ovarian Cancer; Stage IIA Ovarian Germ Cell Tumor; Stage IIB Fallopian Tube Cancer; Stage IIB Ovarian Cancer; Stage IIB Ovarian Germ Cell Tumor; Stage IIC Fallopian Tube Cancer; Stage IIC Ovarian Cancer; Stage IIC Ovarian Germ Cell Tumor; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Primary Peritoneal Cancer; Undifferentiated Ovarian Carcinoma

Identification and Targeting of Candidate Preexisting Lurker Cells That Give Rise to Castration-Resistant Prostate Cancer

DTIC Science & Technology

2016-10-01

cells as the pre-existing “lurker” cells in primary prostate tumors, to evaluate potential therapeutic targets in intermediate luminal progenitor cells...intermediate luminal progenitor cells as the pre-existing “lurker” cells in primary prostate tumors, to evaluate potential therapeutic targets in...candidate target expressed in CD38-lo cells and evaluated the role of CD38 in cell proliferation. No prior Hormonal *** No prior therapy

Viscoelastic Properties Measurement of Human Lymphocytes by Atomic Force Microscopy Based on Magnetic Beads Cell Isolation.

PubMed

Mi Li; Lianqing Liu; Xiubin Xiao; Ning Xi; Yuechao Wang

2016-07-01

Cell mechanics has been proved to be an effective biomarker for indicating cellular states. The advent of atomic force microscopy (AFM) provides an exciting instrument for measuring the mechanical properties of single cells. However, current AFM single-cell mechanical measurements are commonly performed on cell lines cultured in vitro which are quite different from the primary cells in the human body. Investigating the mechanical properties of primary cells from clinical environments can help us to better understand cell behaviors. Here, by combining AFM with magnetic beads cell isolation, the viscoelastic properties of human primary B lymphocytes were quantitatively measured. B lymphocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and CD19 magnetic beads cell isolation. The activity and specificity of the isolated cells were confirmed by fluorescence microscopy. AFM imaging revealed the surface topography and geometric parameters of B lymphocytes. The instantaneous modulus and relaxation time of living B lymphocytes were measured by AFM indenting technique, showing that the instantaneous modulus of human normal B lymphocytes was 2-3 kPa and the relaxation times were 0.03-0.06 s and 0.35-0.55 s. The differences in cellular visocoelastic properties between primary B lymphocytes and cell lines cultured in vitro were analyzed. The study proves the capability of AFM in quantifying the viscoelastic properties of individual specific primary cells from the blood sample of clinical patients, which will improve our understanding of the behaviors of cells in the human body.

Immortalized Mouse Floxed Fam20c Dental Papillar Mesenchymal and Osteoblast Cell Lines Retain Their Primary Characteristics.

PubMed

Liu, Chao; Wang, Xiaofang; Zhang, Hua; Xie, Xiaohua; Liu, Peihong; Liu, Ying; Jani, Priyam H; Lu, Yongbo; Chen, Shuo; Qin, Chunlin

2015-11-01

Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T-antigen (SV40 T-Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long-term passages and genomic integration of SV40 T-Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. © 2015 Wiley Periodicals, Inc.

Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions

PubMed Central

Gerlini, Gianni; Tun-Kyi, Adrian; Dudli, Christa; Burg, Günter; Pimpinelli, Nicola; Nestle, Frank O.

2004-01-01

CD1 molecules are expressed by antigen-presenting cells such as dendritic cells and mediate primary immune responses to lipids and glycolipids which have been shown to be expressed by various tumors. Glycolipids are expressed by melanoma cells but, despite their immunogenicity, no efficient spontaneous immune responses are elicited. As IL-10 has previously been shown to down-regulate CD1a on dendritic cells and is known to be expressed by various melanoma cell lines, we investigated if melanoma-derived IL-10 could down-regulate CD1 molecule expression on dendritic cells as a possible way to circumvent immune recognition. We found that CD1a, CD1b, CD1c, and CD1d were significantly down-regulated on dendritic cells in metastatic (n = 10) but not in primary melanoma lesions (n = 10). We further detected significantly higher IL-10 protein levels in metastatic than in primary melanomas. Moreover, supernatants from metastatic melanomas were significantly more effective in down-regulating CD1 molecules on dendritic cells than supernatants from primary melanoma cultures. This effect was blocked using a neutralizing IL-10 antibody in a dose dependent manner. Our findings suggest that metastatic but not primary melanomas can down-regulate CD1 molecules on infiltrating dendritic cells by secreting IL-10 which may represent a novel way to escape the immune response directed against the tumor. PMID:15579430

Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay.

PubMed

Olschläger, Veronika; Schrader, Andreas; Hockertz, Stefan

2009-01-01

Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phenotype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells.

CD4(+) T-cell help amplifies innate signals for primary CD8(+) T-cell immunity.

PubMed

Bedoui, Sammy; Heath, William R; Mueller, Scott N

2016-07-01

CD8(+) T cells provide an important component of protection against intracellular infections and cancer. Immune responses by these T cells involve a primary phase of effector expansion and differentiation, followed by a contraction phase leading to memory formation and, if antigen is re-encountered, a secondary expansion phase with more rapid differentiation. Both primary and secondary phases of CD8(+) T-cell immunity have been shown to depend on CD4(+) T-cell help, although during certain infections the primary phase is variable in this requirement. One explanation for such variability relates to the strength of associated inflammatory signals, with weak signals requiring help. Here, we focus on our studies that have dissected the requirements for help in the primary phase of the CTL response to herpes simplex virus, elucidating intricate interactions and communications between CD4(+) T cells, various dendritic cell subsets, and CD8(+) T cells. We place our studies in the context of others and describe a simple model of help where CD40 signaling amplifies innate signals to enable efficient CD8(+) T-cell expansion and differentiation. This model facilitates CTL induction to various different agents, without altering the qualitative innate signals that direct other important arms of immunity. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Reduction of CD147 surface expression on primary T cells leads to enhanced cell proliferation.

PubMed

Biegler, Brian; Kasinrerk, Watchara

2012-12-01

CD147 is a ubiquitously expressed membrane glycoprotein that has numerous functional associations in health and disease. However, the molecular mechanisms by which CD147 participates in these processes are unclear. Establishing physiologically relevant silencing of CD147 in primary T cells could provide clues essential for elucidating some aspects of CD147 biology. To date, achieving the knockdown of CD147 in primary T cells has remained elusive. Utilizing RNA interference and the Nucleofector transfection system, we were able to reduce the expression of CD147 in primary T cells. Comparison of basic functions, such as proliferation and CD25 expression, were then made between control populations and populations with reduced expression. Up-regulation of CD147 was found upon T-cell activation, indicating a role in T-cell responses. To better understand the possible importance of this up-regulation, we knocked down the expression of CD147 using RNA interference. When compared to control populations the CD147 knockdown populations exhibited increased proliferation. This alteration of cell proliferation, however, was not linked to a change in CD25 expression. We achieved reduction of CD147 surface expression in primary T cells by siRNA-mediated gene silencing. Our results point to CD147 having a possible negative regulatory role in T cell-mediated immune responses.

Linking the Primary Cilium to Cell Migration in Tissue Repair and Brain Development

PubMed Central

Veland, Iben Rønn; Lindbæk, Louise; Christensen, Søren Tvorup

2014-01-01

Primary cilia are unique sensory organelles that coordinate cellular signaling networks in vertebrates. Inevitably, defects in the formation or function of primary cilia lead to imbalanced regulation of cellular processes that causes multisystemic disorders and diseases, commonly known as ciliopathies. Mounting evidence has demonstrated that primary cilia coordinate multiple activities that are required for cell migration, which, when they are aberrantly regulated, lead to defects in organogenesis and tissue repair, as well as metastasis of tumors. Here, we present an overview on how primary cilia may contribute to the regulation of the cellular signaling pathways that control cyclic processes in directional cell migration. PMID:26955067

Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors.

PubMed

Burgess, J K; Ketheson, A; Faiz, A; Limbert Rempel, K A; Oliver, B G; Ward, J P T; Halayko, A J

2018-01-16

Asthma is an obstructive respiratory disease characterised by chronic inflammation with airway hyperresponsiveness. In asthmatic airways, there is an increase in airway smooth muscle (ASM) cell bulk, which differs from non-asthmatic ASM in characteristics. This study aimed to assess the usefulness of hTERT immortalisation of human ASM cells as a research tool. Specifically we compared proliferative capacity, inflammatory mediator release and extracellular matrix (ECM) production in hTERT immortalised and parent primary ASM cells from asthmatic and non-asthmatic donors. Our studies revealed no significant differences in proliferation, IL-6 and eotaxin-1 production, or CTGF synthesis between donor-matched parent and hTERT immortalised ASM cell lines. However, deposition of ECM proteins fibronectin and fibulin-1 was significantly lower in immortalised ASM cells compared to corresponding primary cells. Notably, previously reported differences in proliferation and inflammatory mediator release between asthmatic and non-asthmatic ASM cells were retained, but excessive ECM protein deposition in asthmatic ASM cells was lost in hTERT ASM cells. This study shows that hTERT immortalised ASM cells mirror primary ASM cells in proliferation and inflammatory profile characteristics. Moreover, we demonstrate both strengths and weaknesses of this immortalised cell model as a representation of primary ASM cells for future asthma pathophysiological research.

First siRNA library screening in hard-to-transfect HUVEC cells

PubMed Central

Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole UE; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra BS; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

2010-01-01

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection. PMID:20628494

Gelatin for purification and proliferation of primary keratinocyte culture for use in chronic wounds and burns.

PubMed

Rahsaz, Marjan; Geramizadeh, Bita; Kaviani, Maryam; Marzban, Saeed

2015-04-01

Human epidermal keratinocytes are currently established as a treatment for burns and wounds and have laboratory applications. Keratinocyte culture contamination by unwanted cells and inhibition of cell proliferation are barriers in primary keratinocyte culture. According to the recent literature, these cells are hard to culture. The present study was conducted to evaluate the efficacy of gelatin-coated surfaces in keratinocyte cultures. After enzymatic isolation of keratinocytes from normal epidermis by trypsin, the cells were cultured on gelatin-coated flasks in serum-free medium. Another group of cells were cultured as a control group without gelatin coating. We showed positive effects of surface coating with gelatin on the primary culture of keratinocytes. Culture of these cells on a gelatincoated surface showed better proliferation with suitable morphology. By using gelatin, adhesion of these cells to the surface was more efficient and without contamination by small round cells. Successful primary culture of keratinocytes on a gelatin-coated surface may provide better yield and optimal number of cells for research and clinical applications.

Advances and Remaining Challenges in the Study of Influenza and Anthrax Infection in Lung Cell Culture

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

Powell, Joshua D.; Straub, Timothy M.

For over thirty years immortalized lung cells have enabled researchers to elucidate lung-pathogen molecular interactions. However, over the last five years numerous commercial companies are now providing affordable, ready-to-use primary lung cells for use in research laboratories. Despite advances in primary cell culture, studies using immortalized lung cells still dominate the recent scientific literature. In this paper, we highlight recent influenza and anthrax studies using in vitro primary lung tissue models and how these models are providing better predictive outcomes for when extrapolated to in vivo observations.

Advances and Remaining Challenges in the Study of Influenza and Anthrax Infection in Lung Cell Culture

DOE PAGES

Powell, Joshua D.; Straub, Timothy M.

2018-01-17

For over thirty years immortalized lung cells have enabled researchers to elucidate lung-pathogen molecular interactions. However, over the last five years numerous commercial companies are now providing affordable, ready-to-use primary lung cells for use in research laboratories. Despite advances in primary cell culture, studies using immortalized lung cells still dominate the recent scientific literature. In this paper, we highlight recent influenza and anthrax studies using in vitro primary lung tissue models and how these models are providing better predictive outcomes for when extrapolated to in vivo observations.

A 3D Human Renal Cell Carcinoma-on-a-Chip for the Study of Tumor Angiogenesis.

PubMed

Miller, Chris P; Tsuchida, Connor; Zheng, Ying; Himmelfarb, Jonathan; Akilesh, Shreeram

2018-06-01

Tractable human tissue-engineered 3D models of cancer that enable fine control of tumor growth, metabolism, and reciprocal interactions between different cell types in the tumor microenvironment promise to accelerate cancer research and pharmacologic testing. Progress to date mostly reflects the use of immortalized cancer cell lines, and progression to primary patient-derived tumor cells is needed to realize the full potential of these platforms. For the first time, we report endothelial sprouting induced by primary patient tumor cells in a 3D microfluidic system. Specifically, we have combined primary human clear cell renal cell carcinoma (ccRCC) cells from six independent donors with human endothelial cells in a vascularized, flow-directed, 3D culture system ("ccRCC-on-a-chip"). The upregulation of key angiogenic factors in primary human ccRCC cells, which exhibited unique patterns of donor variation, was further enhanced when they were cultured in 3D clusters. When embedded in the matrix surrounding engineered human vessels, these ccRCC tumor clusters drove potent endothelial cell sprouting under continuous flow, thus recapitulating the critical angiogenic signaling axis between human ccRCC cells and endothelial cells. Importantly, this phenotype was driven by a primary tumor cell-derived biochemical gradient of angiogenic growth factor accumulation that was subject to pharmacological blockade. Our novel 3D system represents a vascularized tumor model that is easy to image and quantify and is fully tunable in terms of input cells, perfusate, and matrices. We envision that this ccRCC-on-a-chip will be valuable for mechanistic studies, for studying tumor-vascular cell interactions, and for developing novel and personalized antitumor therapies. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

PubMed

Kono, Ken; Hiruma, Hitomi; Kobayashi, Shingo; Sato, Yoji; Tanaka, Masaru; Sawada, Rumi; Niimi, Shingo

2016-01-01

Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

EPA Science Inventory

Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

Simultaneous Presence of Non- and Highly Mutated Keyhole Limpet Hemocyanin (KLH)-Specific Plasmablasts Early after Primary KLH Immunization Suggests Cross-Reactive Memory B Cell Activation.

PubMed

Giesecke, Claudia; Meyer, Tim; Durek, Pawel; Maul, Jochen; Preiß, Jan; Jacobs, Joannes F M; Thiel, Andreas; Radbruch, Andreas; Ullrich, Reiner; Dörner, Thomas

2018-06-15

There are currently limited insights into the progression of human primary humoral immunity despite numerous studies in experimental models. In this study, we analyzed a primary and related secondary parenteral keyhole limpet hemocyanin (KLH) immunization in five human adults. The primary challenge elicited discordant KLH-specific serum and blood effector B cell responses (i.e., dominant serum KLH-specific IgG and IgM levels versus dominant KLH-specific IgA plasmablast frequencies). Single-cell IgH sequencing revealed early appearance of highly (>15 mutations) mutated circulating KLH-specific plasmablasts 2 wk after primary KLH immunization, with simultaneous KLH-specific plasmablasts carrying non- and low-mutated IgH sequences. The data suggest that the highly mutated cells might originate from cross-reactive memory B cells (mBCs) rather than from the naive B cell repertoire, consistent with previous reported mutation rates and the presence of KLH-reactive mBCs in naive vaccinees prior to immunization. Whereas upon secondary immunization, serum Ab response kinetics and plasmablast mutation loads suggested the exclusive reactivation of KLH-specific mBCs, we, however, detected only little clonal overlap between the peripheral KLH-specific secondary plasmablast IgH repertoire and the primary plasmablast and mBC repertoire, respectively. Our data provide novel mechanistic insights into human humoral immune responses and suggest that primary KLH immunization recruits both naive B cells and cross-reactive mBCs, whereas secondary challenge exclusively recruits from a memory repertoire, with little clonal overlap with the primary response. Copyright © 2018 by The American Association of Immunologists, Inc.

Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine.

PubMed

Lobo, Nazleen C; Gedye, Craig; Apostoli, Anthony J; Brown, Kevin R; Paterson, Joshua; Stickle, Natalie; Robinette, Michael; Fleshner, Neil; Hamilton, Robert J; Kulkarni, Girish; Zlotta, Alexandre; Evans, Andrew; Finelli, Antonio; Moffat, Jason; Jewett, Michael A S; Ailles, Laurie

2016-07-16

Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The ability to establish primary cultures of ccRCC cells and matched normal kidney epithelial cells from almost every patient provides a resource for future development of novel therapies and personalized medicine for ccRCC patients.

Interaction of primary mast cells with Borrelia burgdorferi (sensu stricto): role in transmission and dissemination in C57BL/6 mice.

PubMed

Bernard, Quentin; Wang, Zhenping; Di Nardo, Anna; Boulanger, Nathalie

2017-06-27

Borrelia burgdorferi (sensu lato), the causative agent of Lyme borreliosis is a bacterium transmitted by hard ticks, Ixodes spp. Bacteria are injected into the host skin during the tick blood meal with tick saliva. There, Borrelia and saliva interact together with skin cells such as keratinocytes, fibroblasts, mast cells and other specific immune cells before disseminating to target organs. To study the role of mast cells in the transmission of Lyme borreliosis, we isolated mouse primary mast cells from bone marrow and incubated them in the presence of Borrelia burgdorferi (sensu stricto) and tick salivary gland extract. We further analyzed their potential role in vivo, in a mouse model of deficient in mast cells (Kit wsh-/- mice). To our knowledge, we report here for the first time the bacteria ability to induce the inflammatory response of mouse primary mast cells. We show that OspC, a major surface lipoprotein involved in the early transmission of Borrelia, induces the degranulation of primary mast cells but has a limited effect on the overall inflammatory response of these cells. In contrast, whole bacteria have an opposite effect. We also show that mast cell activation is significantly inhibited by tick salivary gland extract. Finally, we demonstrate that mast cells are likely not the only host cells involved in the early transmission and dissemination of Borrelia since the use of mast cell deficient Kit wsh-/- mice shows a limited impact on these two processes in the context of this mouse genetic background. The absence of mast cells did not change the replication rate of Borrelia in the skin. However, in the absence of mast cells, Borrelia dissemination to the joints was faster. Mast cells do not control skin bacterial proliferation during primary infection and the establishment of the primary infection, as shown in the C57BL/6 mouse model studied. Nevertheless, the Borrelia induced cytotokine modulation on mast cells might be involved in long term and/or repeated infections and protect from Lyme borreliosis due to the development of a hypersensitivity to tick saliva.

Primary Mouse Myoblast Purification using Magnetic Cell Separation.

PubMed

Sincennes, Marie Claude; Wang, Yu Xin; Rudnicki, Michael A

2017-01-01

Primary myoblasts can be isolated from mouse muscle cell extracts and cultured in vitro. Muscle cells are usually dissociated manually by mincing with razor blades or scissors in a collagenase/dispase solution. Primary myoblasts are then gradually enriched by pre-plating on collagen-coated plates, based on the observation that mouse fibroblasts attach quickly to collagen-coated plates, and are less adherent. Here, we describe an automated muscle dissociation protocol. We also propose an alternative to pre-plating using magnetic bead separation of primary myoblasts, which improve myoblast purity by minimizing fibroblast contamination.

Features of morfological changes in primary thyroid gland CTLL cultures of rats descendants prenatally exposed by radioisotopes of iodine-131.

PubMed

Boiko, O A; Lavrenchuk, H Yo; Lypska, A I; Talko, V V; Asmolkov, V S

2017-12-01

to investigate morphological changes in the primary thyroid cell culture of rat infants whose parents were prenatally exposed by radioisotope iodine 131. obtaining and culturing of thyroid tissue primary cell cultures of newborn rats, cytological (receipt and analysis of cell cultures agents for optical microscopy), biophysical (flow cytometry), statistics. It was shown that cells in thyroid primary culture of offspring rats prenatally exposed by radioisotopes of iodine 131 signs of destructive degenerative changes were observed mostly when animals of both sexes were irra diated. Increased number of two and three nuclear cells and induction of ring like cells is an evidence of signifi cant genotoxic violation and points to the genome instability in offspring of animals exposed by radioisotope iodine 131. Analysis and quantitative morphological parameters of cells in thyroid primary culture of newborn rats whose parents were exposed prenatally by radioisotopes of iodine 131 showed that upon exposure to radiation thy roid undergoes destructive changes at the cellular level and, even in the second generation of offspring, leads to disruption of its functions. O. A. Boiko, H. Yo. Lavrenchuk, A. I. Lypska, V. V. Talko, V. S. Asmolkov.

Toxicity modelling of Plk1-targeted therapies in genetically engineered mice and cultured primary mammalian cells

PubMed Central

Raab, Monika; Kappel, Sven; Krämer, Andrea; Sanhaji, Mourad; Matthess, Yves; Kurunci-Csacsko, Elisabeth; Calzada-Wack, Julia; Rathkolb, Birgit; Rozman, Jan; Adler, Thure; Busch, Dirk H.; Esposito, Irene; Fuchs, Helmut; Gailus-Durner, Valérie; Klingenspor, Martin; Wolf, Eckhard; Sänger, Nicole; Prinz, Florian; Angelis, Martin Hrabě de; Seibler, Jost; Yuan, Juping; Bergmann, Martin; Knecht, Rainald; Kreft, Bertolt; Strebhardt, Klaus

2011-01-01

High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions. PMID:21772266

Cytotoxicity, oxidative stress and expression of adhesion molecules in human umbilical vein endothelial cells exposed to dust from paints with or without nanoparticles.

PubMed

Mikkelsen, Lone; Jensen, Keld A; Koponen, Ismo K; Saber, Anne T; Wallin, Håkan; Loft, Steffen; Vogel, Ulla; Møller, Peter

2013-03-01

Nanoparticles in primary form and nanoproducts might elicit different toxicological responses. We compared paint-related nanoparticles with respect to effects on endothelial oxidative stress, cytotoxicity and cell adhesion molecule expression. Primary human umbilical vein endothelial cells were exposed to primary nanoparticles (fine, photocatalytic or nanosized TiO(2), aluminium silicate, carbon black, nano-silicasol or axilate) and dust from sanding reference- or nanoparticle-containing paints. Most of the samples increased cell surface expressions of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), but paint sanding dust samples generally generated less response than primary particles of TiO(2) and carbon black. We found no relationship between the expression of adhesion molecules, cytotoxicity and production of reactive oxygen species. In conclusion, sanding dust from nanoparticle-containing paint did not generate more oxidative stress or expression of cell adhesion molecules than sanding dust from paint without nanoparticles, whereas the primary particles had the largest effect on mass basis.

T cell-B cell interactions in primary immunodeficiencies.

PubMed

Tangye, Stuart G; Deenick, Elissa K; Palendira, Umaimainthan; Ma, Cindy S

2012-02-01

Regulated interactions between cells of the immune system facilitate the generation of successful immune responses, thereby enabling efficient neutralization and clearance of pathogens and the establishment of both cell- and humoral-mediated immunological memory. The corollary of this is that impediments to efficient cell-cell interactions, normally necessary for differentiation and effector functions of immune cells, underly the clinical features and disease pathogenesis of primary immunodeficiencies. In affected individuals, these defects manifest as impaired long-term humoral immunity and susceptibility to infection by specific pathogens. In this review, we discuss the importance of, and requirements for, effective interactions between B cells and T cells during the formation of CD4(+) T follicular helper cells and the elicitation of cytotoxic function of virus-specific CD8(+) T cells, as well as how these processes are abrogated in primary immunodeficiencies due to loss-of-function mutations in defined genes. © 2012 New York Academy of Sciences.

Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy.

PubMed

Guo, Haiying; Xing, Yizhan; Zhang, Yiming; He, Long; Deng, Fang; Ma, Xiaogen; Li, Yuhong

2018-01-01

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

PubMed Central

Zhang, Yiming; He, Long; Deng, Fang; Ma, Xiaogen

2018-01-01

Dermal papilla (DP) plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells. PMID:29383288

Cell Adhesion Molecule and Lymphocyte Activation Marker Expression during Experimental Vaginal Candidiasis

PubMed Central

Wormley, Floyd L.; Chaiban, Joseph; Fidel, Paul L.

2001-01-01

Cell-mediated immunity by Th1-type CD4+ T cells is the predominant host defense mechanism against mucosal candidiasis. However, studies using an estrogen-dependent murine model of vaginal candidiasis have demonstrated little to no change in resident vaginal T cells during infection and no systemic T-cell infiltration despite the presence of Candida-specific systemic Th1-type responses in infected mice. The present study was designed to further investigate these observations by characterizing T-cell activation and cell adhesion molecule expression during primary and secondary C. albicans vaginal infections. While flow cytometry analysis of activation markers showed some evidence for activation of CD3+ draining lymph node and/or vaginal lymphocytes during both primary and secondary vaginal Candida infection, CD3+ cells expressing the homing receptors and integrins α4β7, αM290β7, and α4β1 in draining lymph nodes of mice with primary and secondary infections were reduced compared to results for uninfected mice. At the local level, few vaginal lymphocytes expressed integrins, with only minor changes observed during both primary and secondary infections. On the other hand, immunohistochemical analysis of vaginal cell adhesion molecule expression showed increases in mucosal addressin cell adhesion molecule 1 and vascular cell adhesion molecule 1 expression during both primary and secondary infections. Altogether, these data suggest that although the vaginal tissue is permissive to cellular infiltration during a vaginal Candida infection, the reduced numbers of systemic cells expressing the reciprocal cellular adhesion molecules may preempt cellular infiltration, thereby limiting Candida-specific T-cell responses against infection. PMID:11447188

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

Primary Cilia: Highly Sophisticated Biological Sensors

PubMed Central

Abou Alaiwi, Wissam A.; Lo, Shao T.; Nauli, Surya M.

2009-01-01

Primary cilia, thin hair-like structures protruding from the apical surface of most mammalian cells, have gained the attention of many researchers over the past decade. Primary cilia are microtubule-filled sensory organelles that are enclosed within the ciliary membrane. They originate at the cell surface from the mother centriole that becomes the mature basal body. In this review, we will discuss recent literatures on the roles of cilia as sophisticated sensory organelles. With particular emphasis on vascular endothelia and renal epithelia, the mechanosensory role of cilia in sensing fluid shear stress will be discussed. Also highlighted is the ciliary involvement in cell cycle regulation, development, cell signaling and cancer. Finally, primary cilia-related disorders will be briefly described. PMID:22423203

A Method for the Immortalization of Newborn Mouse Skin Keratinocytes

PubMed Central

Hammiller, Brianna O.; El-Abaseri, Taghrid Bahig; Dlugosz, Andrzej A.; Hansen, Laura A.

2015-01-01

Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-rasHa infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation. PMID:26284198

Regulation of CD4 T cells and their effects on immunopathological inflammation following viral infection.

PubMed

Bhattacharyya, Mitra; Madden, Patrick; Henning, Nathan; Gregory, Shana; Aid, Malika; Martinot, Amanda J; Barouch, Dan H; Penaloza-MacMaster, Pablo

2017-10-01

CD4 T cells help immune responses, but knowledge of how memory CD4 T cells are regulated and how they regulate adaptive immune responses and induce immunopathology is limited. Using adoptive transfer of virus-specific CD4 T cells, we show that naive CD4 T cells undergo substantial expansion following infection, but can induce lethal T helper type 1-driven inflammation. In contrast, memory CD4 T cells exhibit a biased proliferation of T follicular helper cell subsets and were able to improve adaptive immune responses in the context of minimal tissue damage. Our analyses revealed that type I interferon regulates the expansion of primary CD4 T cells, but does not seem to play a critical role in regulating the expansion of secondary CD4 T cells. Strikingly, blockade of type I interferon abrogated lethal inflammation by primary CD4 T cells following viral infection, despite that this treatment increased the numbers of primary CD4 T-cell responses. Altogether, these data demonstrate important aspects of how primary and secondary CD4 T cells are regulated in vivo, and how they contribute to immune protection and immunopathology. These findings are important for rational vaccine design and for improving adoptive T-cell therapies against persistent antigens. © 2017 John Wiley & Sons Ltd.

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

PubMed Central

Phelps, Edward A.; Cianciaruso, Chiara; Santo-Domingo, Jaime; Pasquier, Miriella; Galliverti, Gabriele; Piemonti, Lorenzo; Berishvili, Ekaterine; Burri, Olivier; Wiederkehr, Andreas; Hubbell, Jeffrey A.; Baekkeskov, Steinunn

2017-01-01

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells. PMID:28401888

Mesenchymal Stem Cells Retain Their Defining Stem Cell Characteristics After Exposure to Ionizing Radiation

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

Nicolay, Nils H., E-mail: n.nicolay@dkfz.de; Department of Molecular and Radiation Oncology, German Cancer Research Center, Heidelberg; Sommer, Eva

2013-12-01

Purpose: Mesenchymal stem cells (MSCs) have the ability to migrate to lesion sites and undergo differentiation into functional tissues. Although this function may be important for tissue regeneration after radiation therapy, the influence of ionizing radiation (IR) on cellular survival and the functional aspects of differentiation and stem cell characteristics of MSCs have remained largely unknown. Methods and Materials: Radiation sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells was examined, and cellular morphology, cell cycle effects, apoptosis, and differentiation potential after exposure to IR were assessed. Stem cell gene expression patterns after exposure to IRmore » were studied using gene arrays. Results: MSCs were not more radiosensitive than human primary fibroblasts, whereas there were considerable differences regarding radiation sensitivity within individual MSCs. Cellular morphology, cytoskeletal architecture, and cell motility were not markedly altered by IR. Even after high radiation doses up to 10 Gy, MSCs maintained their differentiation potential. Compared to primary fibroblast cells, MSCs did not show an increase in irradiation-induced apoptosis. Gene expression analyses revealed an upregulation of various genes involved in DNA damage response and DNA repair, but expression of established MSC surface markers appeared only marginally influenced by IR. Conclusions: These data suggest that human MSCs are not more radiosensitive than differentiated primary fibroblasts. In addition, upon photon irradiation, MSCs were able to retain their defining stem cell characteristics both on a functional level and regarding stem cell marker expression.« less

[Primary culture of human malignant meningioma cells and its intracranial orthotopic transplantation in nude mice].

PubMed

Hu, Mei-Xin; Liu, Jia-le; Chen, Xuan-Bo; Xu, An-Qi; Shu, Song-Ren; Wang, Chao-Hu; Liu, Yi

2018-03-20

To obtain stable primary cultures of human malignant meningioma cells and establish an intracranial in-situ tumor model in nude mice. Ten surgical specimens of highly suspected malignant meningioma were obtained with postoperative pathological confirmation. Primary malignant meningioma cells were cultured from the tissues using a modified method and passaged. After identification with cell immunofluorescence, the cultured cells were inoculated into the right parietal lobe of 6 nude mice using stereotaxic apparatus and also transplanted subcutaneously in another 6 nude mice. The nude mice were executed after 6 weeks, and HE staining and immunohistochmistry were used to detect tumor growth and the invasion of the adjacent brain tissues. The primary malignant meningioma cells were cultured successfully, and postoperative pathology reported anaplastic malignant meningioma. Cell immunofluorescence revealed positivity for vimentin and EMA in the cells, which showed a S-shaped growth curve in culture. Flow cytometry revealed a cell percentage in the Q3 area of (95.99∓2.58)%. Six weeks after transplantation, tumor nodules occurred in the subcutaneous tumor group, and the nude mice bearing the in situ tumor showed obvious body weight loss. The xenografts in both groups contained a mean of (36∓5.35)% cells expressing Ki-67, and the intracranial in situ tumor showed obvious invasion of the adjacent peripheral brain tissues. We obtained stable primary cultures of malignant meningioma cells and successfully established a nude mouse model bearing in situ human malignant meningioma.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats.

PubMed

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-05-10

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function.

The Effect of VPA on Increasing Radiosensitivity in Osteosarcoma Cells and Primary-Culture Cells from Chemical Carcinogen-Induced Breast Cancer in Rats

PubMed Central

Liu, Guochao; Wang, Hui; Zhang, Fengmei; Tian, Youjia; Tian, Zhujun; Cai, Zuchao; Lim, David; Feng, Zhihui

2017-01-01

This study explored whether valproic acid (VPA, a histone deacetylase inhibitor) could radiosensitize osteosarcoma and primary-culture tumor cells, and determined the mechanism of VPA-induced radiosensitization. The working system included osteosarcoma cells (U2OS) and primary-culture cells from chemical carcinogen (DMBA)-induced breast cancer in rats; and clonogenic survival, immunofluorescence, fluorescent in situ hybridization (FISH) for chromosome aberrations, and comet assays were used in this study. It was found that VPA at the safe or critical safe concentration of 0.5 or 1.0 mM VPA could result in the accumulation of more ionizing radiation (IR)-induced DNA double strand breaks, and increase the cell radiosensitivity. VPA-induced radiosensitivity was associated with the inhibition of DNA repair activity in the working systems. In addition, the chromosome aberrations including chromosome breaks, chromatid breaks, and radial structures significantly increased after the combination treatment of VPA and IR. Importantly, the results obtained by primary-culture cells from the tissue of chemical carcinogen-induced breast cancer in rats further confirmed our findings. The data in this study demonstrated that VPA at a safe dose was a radiosensitizer for osteosarcoma and primary-culture tumor cells through suppressing DNA-double strand breaks repair function. PMID:28489060

Toxicity of nutritionally available selenium compounds in primary and transformed hepatocytes.

PubMed

Weiller, Markus; Latta, Markus; Kresse, Matthias; Lucas, Rudolf; Wendel, Albrecht

2004-09-01

The essential trace element selenium is also toxic at low doses. Since supplementation of selenium is discussed as cancer prophylaxis, we investigated whether or not bioavailable selenium compounds are selectively toxic on malignant cells by comparing primary and transformed liver cells as to the extent and mode of cell death. Sodium selenite and selenate exclusively induced necrosis in a concentration-dependent manner in all cell types investigated. In primary murine hepatocytes, the EC50 was 20 microM for selenite, 270 microM for selenate, and 30 microM for Se-methionine. In the human carcinoma cell line HepG2, the EC50 for selenite was 40 microM, and for selenate 1.1 mM, whereas Se-methionine was essentially non-toxic up to 10 mM. Similar results were found in murine Hepa1-6 cells. Exposure of primary murine cells to selenate or selenite resulted in increased lipid peroxidation. Toxicity was inhibited by superoxide dismutase plus catalase, indicating an important role for reactive oxygen intermediates. In primary hepatocytes, metabolical depletion of intracellular ATP by the ketohexose tagatose, significantly decreased the cytotoxicity of Se-methionine, while the one of selenite was increased. These data do not provide any in vitro evidence that bioavailable selenium compounds induce preferentially apoptotic cell death or selectively kill transformed hepatocytes.

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio).

PubMed

Eide, Marta; Rusten, Marte; Male, Rune; Jensen, Knut Helge Midtbø; Goksøyr, Anders

2014-02-01

The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses. Copyright © 2013 Elsevier B.V. All rights reserved.

Presence of S100A8/Gr1-Positive Myeloid-Derived Suppressor Cells in Primary Tumors and Visceral Organs Invaded by Breast Carcinoma Cells.

PubMed

Tanriover, Gamze; Eyinc, Mehmet Berk; Aliyev, Elnur; Dilmac, Sayra; Erin, Nuray

2018-04-26

Increased S100A8/A9 expression in Gr1-positive cells has been shown in myeloid-derived suppressor cells and may play a role in the formation of a metastatic milieu. We aimed to determine S100A8/A9 expression alone and with coexpression of Gr1 (a myeloid marker) in primary tumor and visceral tissues invaded by metastatic breast carcinoma. Female BALB/c mice were injected with 4TLM, 4THM, and 67NR orthotopically. Confluent cells (75%-80%) were used. Primary tumor, lung, liver, and spleen tissue samples were removed 26 days after injection. Peripheral blood smears and metastasis assay were performed, as was immunohistochemistry and staining. S100A8/A9 immunoreactivity alone or coexpressed with Gr1 was found in primary tumors formed by 4TLM and 4THM cells, which was markedly higher than in primary tumors formed by nonmetastatic 67NR cells. Similarly, liver and lung tissues obtained from mice injected with 4TLM or 4THM cells were invaded by S100A8/A9-positive and Gr1-positive cells. Double-positive cells were markedly fewer in liver and lung tissues of animals injected with 67NR cells. S100A8/A9-positive cells were mostly localized in red pulp of spleens. We observed an increased number of neutrophils in the peripheral blood of mice injected with metastatic breast carcinoma cells. Tumor-derived factors may increase S100A8/A9-positive cells locally and systemically, and S100A8/A9-positive cells may provide an appropriate milieu for the formation of metastasis. Copyright © 2018 Elsevier Inc. All rights reserved.

Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

PubMed

Yamazaki, Taisuke; Wakai, Mariko; Enosawa, Shin; Tokiwa, Takayoshi

2017-06-01

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

Genetics Home Reference: Joubert syndrome

MedlinePlus

... sensing the physical environment and in chemical signaling. Primary cilia are important for the structure and function of many types of cells, including brain cells (neurons) and certain cells in the kidneys and liver. Primary cilia are also necessary for the perception ...

Site-Specific Expression of Polycomb-Group Genes Encoding the HPC-HPH/PRC1 Complex in Clinically Defined Primary Nodal and Cutaneous Large B-Cell Lymphomas

PubMed Central

Raaphorst, Frank M.; Vermeer, Maarten; Fieret, Elly; Blokzijl, Tjasso; Dukers, Danny; Sewalt, Richard G.A.B.; Otte, Arie P.; Willemze, Rein; Meijer, Chris J.L.M.

2004-01-01

Polycomb-group (PcG) genes preserve cell identity by gene silencing, and contribute to regulation of lymphopoiesis and malignant transformation. We show that primary nodal large B-cell lymphomas (LBCLs), and secondary cutaneous deposits from such lymphomas, abnormally express the BMI-1, RING1, and HPH1 PcG genes in cycling neoplastic cells. By contrast, tumor cells in primary cutaneous LBCLs lacked BMI-1 expression, whereas RING1 was variably detected. Lack of BMI-1 expression was characteristic for primary cutaneous LBCLs, because other primary extranodal LBCLs originating from brain, testes, and stomach were BMI-1-positive. Expression of HPH1 was rarely detected in primary cutaneous LBCLs of the head or trunk and abundant in primary cutaneous LBCLs of the legs, which fits well with its earlier recognition as a distinct clinical pathological entity with different clinical behavior. We conclude that clinically defined subclasses of primary LBCLs display site-specific abnormal expression patterns of PcG genes of the HPC-HPH/PRC1 PcG complex. Some of these patterns (such as the expression profile of BMI-1) may be diagnostically relevant. We propose that distinct expression profiles of PcG genes results in abnormal formation of HPC-HPH/PRC1 PcG complexes, and that this contributes to lymphomagenesis and different clinical behavior of clinically defined LBCLs. PMID:14742259

Primary central nervous system B-cell lymphoma in a young dog

PubMed Central

Kim, Na-Hyun; Ciesielski, Thomas; Kim, Jung H.; Yhee, Ji-Young; Im, Keum-Soon; Nam, Hae-Mi; Kim, Il-Hwan; Kim, Jong-Hyuk; Sur, Jung-Hyang

2012-01-01

This report describes a primary central nervous system B-cell lymphoma in a 3-year-old intact female Maltese dog. Canine primary central nervous system lymphomas constitute about 4% of all intracranial primary neoplasms, but comprehensive histopathologic classifications have rarely been carried out. This is the first report of this disease in a young adult dog. PMID:23115372

Culture conditions defining glioblastoma cells behavior: what is the impact for novel discoveries?

PubMed

Ledur, Pítia Flores; Onzi, Giovana Ravizzoni; Zong, Hui; Lenz, Guido

2017-09-15

In cancer research, the use of established cell lines has gradually been replaced by primary cell cultures due to their better representation of in vivo cancer cell behaviors. However, a major challenge with primary culture involves the finding of growth conditions that minimize alterations in the biological state of the cells. To ensure reproducibility and translational potentials for research findings, culture conditions need to be chosen so that the cell population in culture best mimics tumor cells in vivo . Glioblastoma (GBM) is one of the most aggressive and heterogeneous tumor types and the GBM research field would certainly benefit from culture conditions that could maintain the original plethora of phenotype of the cells. Here, we review culture media and supplementation options for GBM cultures, the rationale behind their use, and how much those choices affect drug-screening outcomes. We provide an overview of 120 papers that use primary GBM cultures and discuss the current predominant conditions. We also show important primary research data indicating that "mis-cultured" glioma cells can acquire unnatural drug sensitivity, which would have devastating effects for clinical translations. Finally, we propose the concurrent test of four culture conditions to minimize the loss of cell coverage in culture.

Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

PubMed Central

2010-01-01

Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival. PMID:20144189

A comparison of oncological outcomes between transoral surgical and non-surgical treatment protocols in the management of oropharyngeal squamous cell carcinoma.

PubMed

Kao, S S; Micklem, J; Ofo, E; Edwards, S; Dhatrak, D; Foreman, A; Krishnan, S; Hodge, J-C

2018-04-01

The incidence of oropharyngeal squamous cell carcinoma in the Western world is increasing, with the human papillomavirus epidemic implicated in this observed trend. The optimal treatment modality is yet undetermined regarding oncological outcomes. This study comprised 98 patients with oropharyngeal squamous cell carcinoma, treated with either primary transoral surgery with adjuvant therapy or primary chemoradiotherapy with curative intent, between 2008 and 2012. Clinicopathological characteristics including tumour-node-metastasis stage, human papillomavirus status, treatment modality, recurrence and overall survival were collated. Five per cent of primary surgical patients had locoregional recurrences compared with 25 per cent of primary chemoradiotherapy patients. A lower rate of locoregional recurrence was observed in the human papillomavirus positive group. This paper reports higher rates of overall survival and local control for oropharyngeal squamous cell carcinoma treated with primary surgery compared with primary chemoradiotherapy. This reflects overall lower tumour stage and higher human papillomavirus status in this group.

Expression and function of Allergin-1 on human primary mast cells.

PubMed

Nagai, Kei; Tahara-Hanaoka, Satoko; Morishima, Yuko; Tokunaga, Takahiro; Imoto, Yoshimasa; Noguchi, Emiko; Kanemaru, Kazumasa; Imai, Masamichi; Shibayama, Shiro; Hizawa, Nobuyuki; Fujieda, Shigeharu; Yamagata, Kunihiro; Shibuya, Akira

2013-01-01

Mast cells (MC) play an important role in allergic and non-allergic immune responses. Activation of human MC is modulated by several cell surface inhibitory receptors, including recently identified Allergin-1 expressed on both human and mouse MC. Although Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice, the expression profile and function of Allergin-1 on human primary MC remains undetermined. Here, we established a seven-color flow cytometry method for assessing expression and function of a very small number of human primary MC. We show that Allergin-1S1, a splicing isoform of Allergin-1, is predominantly expressed on human primary MC in both bronchoalveolar lavage (BAL) fluid and nasal scratching specimens. Moreover, Allergin-1S1 inhibits IgE-mediated activation from human primary MC in BAL fluid. These results indicate that Allergin-1 on human primary MC exhibits similar characteristics as mouse Allergin-1 in the expression profile and function.

Challenges in validating the sterilisation dose for processed human amniotic membranes

NASA Astrophysics Data System (ADS)

Yusof, Norimah; Hassan, Asnah; Firdaus Abd Rahman, M. N.; Hamid, Suzina A.

2007-11-01

Most of the tissue banks in the Asia Pacific region have been using ionising radiation at 25 kGy to sterilise human tissues for save clinical usage. Under tissue banking quality system, any dose employed for sterilisation has to be validated and the validation exercise has to be a part of quality document. Tissue grafts, unlike medical items, are not produced in large number per each processing batch and tissues relatively have a different microbial population. A Code of Practice established by the International Atomic Energy Agency (IAEA) in 2004 offers several validation methods using smaller number of samples compared to ISO 11137 (1995), which is meant for medical products. The methods emphasise on bioburden determination, followed by sterility test on samples after they were exposed to verification dose for attaining of sterility assurance level (SAL) of 10 -1. This paper describes our experience in using the IAEA Code of Practice in conducting the validation exercise for substantiating 25 kGy as sterilisation dose for both air-dried amnion and those preserved in 99% glycerol.

Dehydrated human amnion/chorion tissue in difficult-to-heal DFUs: a case series.

PubMed

Penny, H; Rifkah, M; Weaver, A; Zaki, P; Young, A; Meloy, G; Flores, R

2015-03-01

Diabetic foot ulcers (DFUs) occur as a result of multifactorial complications and are commonly found in the diabetic community. Underlying disease states such as neuropathy and peripheral vascular disease can slow healing rates, potentially leading to recurrence, amputation, and increased mortality. As with many other disease processes, DFUs have several treatment options, such as debriding agents, alginate seaweed extract, hydrocolloid gels, and amniotic membrane allografts. The presented cases all used a dehydrated human amniotic/chorionic membrane allograft (dHACM; EpiFix) to aid the healing process. Human amniotic epithelial membranes have seen increased usage due to their ability to enhance the healing process and accelerate cellular regeneration. The DFUs healed in all of the five patients treated, and patients saw a full recovery in 2.5-11 weeks. In addition, the healing time decreased in spite of the non-adherence seen in three of the patients. These results suggest another possible use for dHACM; however, further studies are required to confirm these data. This project was self-funded and had no influences outside the fact that Dr Penny is a speaker for MiMedx.

Regulation of intramembranous absorption and amniotic fluid volume by constituents in fetal sheep urine

PubMed Central

Jonker, Sonnet S.; Louey, Samantha; Cheung, Cecilia Y.; Brace, Robert A.

2013-01-01

Our objective was to test the hypothesis that fetal urine contains a substance(s) that regulates amniotic fluid volume by altering the rate of intramembranous absorption of amniotic fluid. In late gestation ovine fetuses, amniotic fluid volumes, urine, and lung liquid production rates, swallowed volumes and intramembranous volume and solute absorption rates were measured over 2-day periods under control conditions and when urine was removed and continuously replaced at an equal rate with exogenous fluid. Intramembranous volume absorption rate decreased by 40% when urine was replaced with lactated Ringer solution or lactated Ringer solution diluted 50% with water. Amniotic fluid volume doubled under both conditions. Analysis of the intramembranous sodium and chloride fluxes suggests that the active but not passive component of intramembranous volume absorption was altered by urine replacement, whereas both active and passive components of solute fluxes were altered. We conclude that fetal urine contains an unidentified substance(s) that stimulates active intramembranous transport of amniotic fluid across the amnion into the underlying fetal vasculature and thereby functions as a regulator of amniotic fluid volume. PMID:23824958

Endoscopy, histology and electron microscopy analysis of foetal membranes in pregnant South American plains vizcacha reveal unusual excrescences on the yolk sac.

PubMed

Giacchino, Mariela; Inserra, Pablo I F; Lange, Fernando D; Gariboldi, María C; Ferraris, Sergio R; Vitullo, Alfredo D

2018-06-01

The South American hystricognathe Lagostomus maximus is a fossorial rodent whose females show unique reproductive characteristics. They have a 155-day long gestation, show massive polyovulation and a selective process of embryonic resorption in the first half of gestation. In order to explore and perform an in-situ characterization of the reproductive tract, we visualized internal structures through ultrasonography and video-endoscopy in pregnant and non-pregnant females. We describe the finding of protruding structures that lie on the yolk sac and their histological and ultrastructural characterization. The placenta was covered with whitish, small pearl-shaped structures. These structures were also seen on the extra-embryonic space, being the amnion and the umbilical cord free of them. Pearl-shaped structures were composed with loose connective tissue, lacked blood vessels, and showed collagen fibers organized in a spiral form. They were anchored by pedicles to the villous surface of the extraembryonic membrane. We discuss the biological and evolutionary meaning of the pearl-shaped structures that relate L. maximus to the African origin of the South American hystricognathe fauna.

Mechanisms leading to increased risk of preterm birth in growth-restricted guinea pig pregnancies.

PubMed

Palliser, Hannah K; Kelleher, Meredith A; Welsh, Toni N; Zakar, Tamas; Hirst, Jonathan J

2014-02-01

Intrauterine growth restriction (IUGR) is a risk factor for preterm labor; however, the mechanisms of the relationship remain unknown. Prostaglandin (PG), key stimulants of labor, availability is regulated by the synthetic enzymes, prostaglandin endoperoxidases 1 and 2 (PTGS1 and 2), and the metabolizing enzyme, 15-hydroxyprostaglandin dehydrogenase (HPGD). We hypothesized that IUGR increases susceptibility to preterm labor due to the changing balance of synthetic and metabolizing enzymes and hence greater PG availability. We have tested this hypothesis using a surgically induced IUGR model in guinea pigs, which results in significantly shorter gestation. Myometrium, amnion, chorion, and placentas were collected from sham operated or IUGR pregnancies, and PTGS1 and HPGD protein expression were quantified throughout late gestation (>62 days) and labor. The PTGS1 expression was significantly upregulated in the myometrium of IUGR animals, and chorionic HPGD expression was markedly decreased (P < .01 and P < .001, respectively). These findings suggest a shift in the balance of PG production over metabolism in IUGR pregnancies leads to a greater susceptibility to preterm birth.

The role of placental alpha microglobulin-1 amnisure in determining the status of the fetal membranes; its association with preterm birth. Traditions … traditions ….

PubMed

Mariona, Federico G; Roura, Lluis Cabero

2016-03-01

The integrity of the fetal amnion-chorion is an imperative for the preservation of a normal pregnancy in the human. The diagnosis of the status of the fetal membranes has traditionally been reduced to either intact or ruptured. In the last decades, evidence has accumulated demonstrating that this clinical approach may well be an over simplification. Practically, all maternal organs experienced physiologic or eventually pathologic changes during the length of the gestational period. We propose that the fetal membranes are also significantly impacted by those changes. The accurate, specific, simplified and low-cost diagnosis of the status of the fetal membranes is of critical importance for the assessment of risk to the pregnancy followed by efficient and prompt treatment. The presence of placental alpha macroglobulin-1 in the vagina specifically indicates a disruption in the integrity of the fetal membranes and may indirectly mean increased risk for preterm birth. Further research to properly characterize this marker and its importance in the care of pregnant woman at risk for preterm birth is strongly recommended.

Establishment and characterization of a goat synovial membrane cell line susceptible to small ruminant lentivirus infection.

PubMed

Rolland, Morgane; Chauvineau, Cécile; Valas, Stephen; Mamoun, Robert Z; Perrin, Gérard

2004-06-15

Primary goat synovial membrane (GSM) cells are widely used to study small ruminant lentiviruses (SRLV), i.e. maedi visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV), but their limited life-span of 15-20 passages in vitro is problematic. Here, we report that ectopic expression of the catalytic subunit of human telomerase (hTERT) was sufficient to immortalize primary GSM cells. Cultures of hTERT-transfected GSM cells have been passaged for 2 years without showing any phenotypic difference from the original primary GSM cells. The hTERT-transfected cells continued to grow beyond a population doubling number of 250, while no net telomere lengthening was observed for these cells. Moreover, the immortalized GSM cells were susceptible to infection by both CAEV and MVV and were able to propagate theses viruses. Such cell line provides a useful source of standard and robust cells for both research and veterinary purposes.

Fuel Cell Research and Development for Future NASA Missions

NASA Technical Reports Server (NTRS)

Manzo, Michelle A.; Hoberecht, Mark; Loyselle, Patricia; Burke, Kenneth; Bents, David; Farmer, Serene; Kohout, Lisa

2006-01-01

NASA has been using fuel cell systems since the early days of space flight. Polymer Exchange Membrane Fuel cells provided the primary power for the Gemini and Apollo missions and more recently, alkaline fuel cells serve as the primary power source for the Space Shuttle. NASA's current investments in fuel cell technology support both Exploration and Aeronautics programs. This presentation provides an overview of NASA's fuel cell development programs.

Identification of genuine primary pulmonary NK cell lymphoma via clinicopathologic observation and clonality assay.

PubMed

Gong, Li; Wei, Long-Xiao; Huang, Gao-Sheng; Zhang, Wen-Dong; Wang, Lu; Zhu, Shao-Jun; Han, Xiu-Juan; Yao, Li; Lan, Miao; Li, Yan-Hong; Zhang, Wei

2013-08-19

Extranodal natural killer (NK)/T-cell lymphoma, nasal type, is an uncommon lymphoma associated with the Epstein-Barr virus (EBV). It most commonly involves the nasal cavity and upper respiratory tract. Primary pulmonary NK/T cell lymphoma is extremely rare. If a patient with a NK or T-cell tumor has an unusual reaction to treatment or an unusual prognosis, it is wise to differentiate NK from T-cell tumors. The clinicopathologic characteristics, immunophenotype, EBV in situ hybridization, and T cell receptor (TCR) gene rearrangement of primary pulmonary NK cell lymphoma from a 73-year-old Chinese woman were investigated and the clonal status was determined using female X-chromosomal inactivation mosaicism and polymorphisms at the phosphoglycerate kinase (PGK) gene. The lesion showed the typical histopathologic characteristics and immunohistochemical features of NK/T cell lymphoma. However, the sample was negative for TCR gene rearrangement. A clonality assay demonstrated that the lesion was monoclonal. It is concluded that this is the first recorded case of genuine primary pulmonary NK cell lymphoma. The purpose of the present work is to recommend that pathologists carefully investigate the whole lesion to reduce the likelihood that primary pulmonary NK cell lymphoma will be misdiagnosed as an infectious lesion. In addition, TCR gene rearrangement and clonal analysis, which is based on female X-chromosomal inactivation mosaicism and polymorphisms at PGK and androgen receptor (AR) loci, were found to play important roles in differentiating NK cell lymphoma from T cell lymphoma. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5205300349457729.

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+ Cell Populations

PubMed Central

Albright, Emily R.

2013-01-01

Human cytomegalovirus (HCMV) is a significant human pathogen that achieves lifelong persistence by establishing latent infections in undifferentiated cells of the myeloid lineage, such as CD34+ hematopoietic progenitor cells. When latency is established, viral lytic gene expression is silenced in part by a cellular intrinsic defense consisting of Daxx and histone deacetylases (HDACs) because pp71, the tegument transactivator that travels to the nucleus and inactivates this defense at the start of a lytic infection in differentiated cells, remains in the cytoplasm. Because the current in vitro and ex vivo latency models have physiological and practical limitations, we evaluated two CD34+ myeloblastic cell lines, KG-1 and Kasumi-3, for their ability to establish, maintain, and reactivate HCMV experimental latent infections. Tegument protein pp71 was cytoplasmic, and immediate-early (IE) genes were silenced as in primary CD34+ cells. However, in contrast to what occurs in primary CD34+ cells ex vivo or in NT2 and THP-1 in vitro model systems, viral IE gene expression from the laboratory-adapted AD169 genome was not induced in the presence of HDAC inhibitors in either KG-1 or Kasumi-3 cells. Furthermore, while the clinical strain FIX was able to reactivate from Kasumi-3 cells, AD169 was not, and neither strain reactivated from KG-1 cells. Thus, KG-1 and Kasumi-3 experimental latent infections differ in important parameters from those in primary CD34+ cell populations. Aspects of latency illuminated through the use of these myeloblastoid cell lines should not be considered independently but integrated with results obtained in primary cell systems when paradigms for HCMV latency are proposed. PMID:23824798

Alternative functional in vitro models of human intestinal epithelia

PubMed Central

Kauffman, Amanda L.; Gyurdieva, Alexandra V.; Mabus, John R.; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J.

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport. PMID:23847534

Alternative functional in vitro models of human intestinal epithelia.

PubMed

Kauffman, Amanda L; Gyurdieva, Alexandra V; Mabus, John R; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

Hematopoietic Stem Cell Transplantation in Primary Immunodeficiency Patients in the Black Sea Region of Turkey.

PubMed

Yıldıran, Alişan; Çeliksoy, Mehmet Halil; Borte, Stephan; Güner, Şükrü Nail; Elli, Murat; Fışgın, Tunç; Özyürek, Emel; Sancak, Recep; Oğur, Gönül

2017-12-01

Hematopoietic stem cell transplantation is a promising curative therapy for many combined primary immunodeficiencies and phagocytic disorders. We retrospectively reviewed pediatric cases of patients diagnosed with primary immunodeficiencies and scheduled for hematopoietic stem cell transplantation. We identified 22 patients (median age, 6 months; age range, 1 month to 10 years) with various diagnoses who received hematopoietic stem cell transplantation. The patient diagnoses included severe combined immunodeficiency (n=11), Chediak-Higashi syndrome (n=2), leukocyte adhesion deficiency (n=2), MHC class 2 deficiency (n=2), chronic granulomatous syndrome (n=2), hemophagocytic lymphohistiocytosis (n=1), Wiskott-Aldrich syndrome (n=1), and Omenn syndrome (n=1). Of the 22 patients, 7 received human leukocyte antigen-matched related hematopoietic stem cell transplantation, 12 received haploidentical hematopoietic stem cell transplantation, and 2 received matched unrelated hematopoietic stem cell transplantation. The results showed that 5 patients had graft failure. Fourteen patients survived, yielding an overall survival rate of 67%. Screening newborn infants for primary immunodeficiency diseases may result in timely administration of hematopoietic stem cell transplantation.

[Primary peripheral T-cell lymphoma of the penis: a case report and review of the literature].

PubMed

Shi, Yan-Lin; Yin, Hong-Lin; Zhou, Xiao-Jun; Zhou, Hang-Bo; Lu, Zhen-Feng

2008-11-01

To report a case of primary peripheral T-cell lymphoma of the penis. We analyzed the clinicopathological characteristics of the case of primary peripheral T-cell lymphoma using histological, cytochemical and immunohistochemical methods and by review of the literature. The patient was a 65 years old man and presented with a diffuse enlargement of the penis as the initial sign, followed by erosive ulcer in the caput penis and inguinal lymphadenectasis. The tumor was pathohistologically manifested as an epidermal ulcer, with tumorous necrosis around the capillary, infiltrative growth and atypical changes of the neoplastic cells and proliferation of capillaries. Immunohistochemically, the tumor cells were positive for CD43 and CD3, but negative for CD20, CD79a, CD34, CD30, CD56 and CD34. Clinically it responded to the chemotherapy designed for peripheral T-cell lymphoma. Primary peripheral T-cell lymphoma of the penis is an extremely rare malignant tumor, the diagnosis of which relies on histopathological examination, immunohistochemical staining and differentiation between squamous cell carcinoma and other types of lymphoma.

Single-cell sequencing deciphers a convergent evolution of copy number alterations from primary to circulating tumor cells.

PubMed

Gao, Yan; Ni, Xiaohui; Guo, Hua; Su, Zhe; Ba, Yi; Tong, Zhongsheng; Guo, Zhi; Yao, Xin; Chen, Xixi; Yin, Jian; Yan, Zhao; Guo, Lin; Liu, Ying; Bai, Fan; Xie, X Sunney; Zhang, Ning

2017-08-01

Copy number alteration (CNA) is a major contributor to genome instability, a hallmark of cancer. Here, we studied genomic alterations in single primary tumor cells and circulating tumor cells (CTCs) from the same patient. Single-nucleotide variants (SNVs) in single cells from both samples occurred sporadically, whereas CNAs among primary tumor cells emerged accumulatively rather than abruptly, converging toward the CNA in CTCs. Focal CNAs affecting the MYC gene and the PTEN gene were observed only in a minor portion of primary tumor cells but were present in all CTCs, suggesting a strong selection toward metastasis. Single-cell structural variant (SV) analyses revealed a two-step mechanism, a complex rearrangement followed by gene amplification, for the simultaneous formation of anomalous CNAs in multiple chromosome regions. Integrative CNA analyses of 97 CTCs from 23 patients confirmed the convergence of CNAs and revealed single, concurrent, and mutually exclusive CNAs that could be the driving events in cancer metastasis. © 2017 Gao et al.; Published by Cold Spring Harbor Laboratory Press.

49 CFR 171.12 - North American Shipments.

Code of Federal Regulations, 2010 CFR

2010-10-01

...) Primary lithium batteries and cells. Packages containing primary lithium batteries and cells that meet the exception in § 172.102, Special Provision 188 or 189 of this subchapter must be marked “PRIMARY LITHIUM BATTERIES—FORBIDDEN FOR TRANSPORT ABOARD PASSENGER AIRCRAFT” or “LITHIUM METAL BATTERIES—FORBIDDEN FOR...

49 CFR 171.12 - North American Shipments.

Code of Federal Regulations, 2012 CFR

2012-10-01

...) Primary lithium batteries and cells. Packages containing primary lithium batteries and cells that meet the exception in § 172.102, Special Provision 188 or 189 of this subchapter must be marked “PRIMARY LITHIUM BATTERIES—FORBIDDEN FOR TRANSPORT ABOARD PASSENGER AIRCRAFT” or “LITHIUM METAL BATTERIES—FORBIDDEN FOR...

49 CFR 171.12 - North American Shipments.

Code of Federal Regulations, 2011 CFR

2011-10-01

...) Primary lithium batteries and cells. Packages containing primary lithium batteries and cells that meet the exception in § 172.102, Special Provision 188 or 189 of this subchapter must be marked “PRIMARY LITHIUM BATTERIES—FORBIDDEN FOR TRANSPORT ABOARD PASSENGER AIRCRAFT” or “LITHIUM METAL BATTERIES—FORBIDDEN FOR...

49 CFR 171.12 - North American Shipments.

Code of Federal Regulations, 2013 CFR

2013-10-01

...) Primary lithium batteries and cells. Packages containing primary lithium batteries and cells that meet the exception in § 172.102, Special Provision 188 or 189 of this subchapter must be marked “PRIMARY LITHIUM BATTERIES—FORBIDDEN FOR TRANSPORT ABOARD PASSENGER AIRCRAFT” or “LITHIUM METAL BATTERIES—FORBIDDEN FOR...

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation.

PubMed

Watanabe, Yoichiro; Schneider, Rene; Barkwill, Sarah; Gonzales-Vigil, Eliana; Hill, Joseph L; Samuels, A Lacey; Persson, Staffan; Mansfield, Shawn D

2018-06-05

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs. Copyright © 2018 the Author(s). Published by PNAS.

Bacillus anthracis spores germinate extracellularly at air-liquid interface in an in vitro lung model under serum-free conditions

DOE PAGES

Powell, Joshua D.; Hutchison, Janine R.; Hess, Becky M.; ...

2015-07-30

Aims: To better understand the parameters that govern spore dissemination after lung exposure using in vitro cell systems. Methods and Results: We evaluated the kinetics of uptake, germination and proliferation of B. anthracis Sterne spores in association with human primary lung epithelial cells, Calu-3, and A549 cell lines. We also analyzed the influence of various cell culture media formulations related to spore germination. Conclusions: We found negligible spore uptake by epithelial cells, but germination and proliferation of spores in the extracellular environment was evident, and was appreciably higher in A549 and Calu-3 cultures than in primary epithelial cells. Additionally, ourmore » results revealed spores in association with primary cells submerged in cell culture media germinated 1 h« less

Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

PubMed

Zhang, Jinhui; Chen, Songlin; Cai, Jing; Hou, Zhiqiang; Wang, Xiaohan; Kachelmeier, Allan; Shi, Xiaorui

2017-03-01

The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

[Isolation, purification and primary culture of rat pancreatic beta-cells].

PubMed

Liu, Yu-Pu; Lü, Qing-Guo; Tong, Nan-Wei

2009-01-01

To isolate and purify rat pancreatic beta-cells and to explore the best conditions for the primary culture of the pancreatic beta-cells in vitro. The pancreas of Norman Wistar rats were digested by collagenase V. The islets were purified by mesh sieve. The activity of the islets was stimulated by different concentrations of glucose and detected by dithizone dye. The purified islets were put into RPMI-1640 nutritive medium for culture overnight. The cultured islets were digested again with trypsin and DNAase to obtain the suspension containing single pancreatic cells. The beta-cells were separated and purified in a fluorescence-activated cell sorter (FACS) in the medium containing 2.8 mmol/L glucose. The purified beta-cells were identified by immunohistochemistry and glucose stimulating test. Ham's F-10 with different concentrations of glucose and 3-Isobutyl-1-methylxanthine (IBMX) were used as nutritive medium for the primary cell culture for 24 hours. The best conditions for the culture were identified. An average of 550 +/- 90 islets with fine activities were obtained per rat. The purification with FACS obtained about 5688 beta-cells per rat, with a recovery rate of (93.69 +/- 1.26)% and a purity of (85.5 +/- 1.24)%. A concentration of 10.0 mmol/L and 16.0 mmol/L glucose in primary culture for 24 hours produced the highest survival rates of beta-cells, but IBMX did not increase the survival rates of beta-cells. FACS is effective in purifying pancreatic beta-cells from the suspension with a medium containing 2.8 mmol/L glucose. Pancreatic beta-cells maintain relatively high activities in Ham's F-10 medium containing 10.0-16.0 mmol/L glucose in primary culture.

Odf2-deficient mother centrioles lack distal/subdistal appendages and the ability to generate primary cilia.

PubMed

Ishikawa, Hiroaki; Kubo, Akiharu; Tsukita, Shoichiro; Tsukita, Sachiko

2005-05-01

Outer dense fibre 2 (Odf2; also known as cenexin) was initially identified as a main component of the sperm tail cytoskeleton, but was later shown to be a general scaffold protein that is specifically localized at the distal/subdistal appendages of mother centrioles. Here we show that Odf2 expression is suppressed in mouse F9 cells when both alleles of Odf2 genes are deleted. Unexpectedly, the cell cycle of Odf2(-/-) cells does not seem to be affected. Immunofluorescence and ultrathin-section electron microscopy reveals that in Odf2(-/-) cells, distal/subdistal appendages disappear from mother centrioles, making it difficult to distinguish mother from daughter centrioles. In Odf2(-/-) cells, however, the formation of primary cilia is completely suppressed, although approximately 25% of wild-type F9 cells are ciliated under the steady-state cell cycle. The loss of primary cilia in Odf2(-/-) F9 cells can be rescued by exogenous Odf2 expression. These findings indicate that Odf2 is indispensable for the formation of distal/subdistal appendages and the generation of primary cilia, but not for other cell-cycle-related centriolar functions.

In vitro FTIR microspectroscopy analysis of primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil: a new spectroscopic approach for studying the drug-cell interaction.

PubMed

Giorgini, Elisabetta; Sabbatini, Simona; Rocchetti, Romina; Notarstefano, Valentina; Rubini, Corrado; Conti, Carla; Orilisi, Giulia; Mitri, Elisa; Bedolla, Diana E; Vaccari, Lisa

2018-06-22

In the present study, human primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil were analyzed, for the first time, by in vitro FTIR Microspectroscopy (FTIRM), to improve the knowledge on the biochemical pathways activated by these two chemotherapy drugs. To date, most of the studies regarding FTIRM cellular analysis have been executed on fixed cells from immortalized cell lines. FTIRM analysis performed on primary tumor cells under controlled hydrated conditions provides more reliable information on the biochemical processes occurring in in vivo tumor cells. This spectroscopic analysis allows to get on the same sample and at the same time an overview of the composition and structure of the most remarkable cellular components. In vitro FTIRM analysis of primary oral squamous carcinoma cells evidenced a time-dependent drug-specific cellular response, also including apoptosis triggering. Furthermore, the univariate and multivariate analyses of IR data evidenced meaningful spectroscopic differences ascribable to alterations affecting cellular proteins, lipids and nucleic acids. These findings suggest for the two drugs different pathways and extents of cellular damage, not provided by conventional cell-based assays (MTT assay and image-based cytometry).

Primary central nervous system diffuse large B-cell lymphoma in the immunocompetent: Immunophenotypic subtypes and Epstein-Barr virus association.

PubMed

Mahadevan, Anita; Rao, Clementina Rama; Shanmugham, M; Shankar, Susarla Krishna

2015-01-01

Primary central nervous system diffuse large B-cell lymphoma (PCNSL DLBCL) in the immunocompetent is an uncommon tumor that has an activated B-cell immunophenotype resembling germinal center exit B cells. They also differ from primary central nervous diffuse large B-cell lymphomas in the immunocompromised as they show no association with the Epstein-Barr virus. To determine if immunophenotypic subtyping of PCNS DLBCL from Asian subcontinent are also different similar to its systemic counterpart is unclear, as there are only limited studies from Asia, and none from India. The immunohistochemical profile of 24 South Indian patients with primary central nervous system diffuse large B-cell lymphoma was studied using germinal center markers - CD10 and Bcl-6, and activation markers - MUM1 and CD138, which are markers for late/post germinal centre B cells. Insitu hybridization for EBV genome and LMP1 by immunohistochemistry was carried out in all cases to determine association with EBV. Centroblastic morphology and uniform activated B-cell phenotype with positivity for MUM1 was seen in 91.6% of tumors. Co-expression of Bcl-6 and MUM1 was evident in 50%, which is more frequent than in systemic diffuse large B-cell lymphomas. All cases were negative for Epstein-Barr virus using EBER in-situ hybridization and LMP1 immunohistochemistry. Primary diffuse large B-cell lymphoma in the immunocompetent is a distinct clinicopathological entity with centroblastic morphology, a uniform activated B-cell immunophenotype that is not associated with the Epstein-Barr virus regardless of geographic origin.

[Tricostantin A inhibits self-renewal of breast cancer stem cells in vitro].

PubMed

Peng, Li; Li, Fu-Xi; Shao, Wen-Feng; Xiong, Jing-Bo

2013-10-01

To investigate the effect of tricostantin A (TSA) on self-renewal of breast cancer stem cells and explore the mechanisms. Breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 and SKBR3 were cultured in suspension and treated with different concentrations of TSA for 7 days, using 0.1% DMSO as the control. Secondary mammosphere formation efficiency and percentage of CD44(+)/CD24(-) sub-population in the primary mammospheres were used to evaluate the effects of TSA on self-renewal of breast cancer stem cells. The breast cancer stem cell surface marker CD44(+)/CD24(-) and the percentage of apoptosis in the primary mammospheres were assayed using flow cytometry. The mRNA expressions of Nanog, Sox2 and Oct4 in the primary mammospheres were assayed with quantitative PCR. TSA at both 100 and 500 nmol/L, but not at 10 nmol/L, partially inhibited the self-renewal of breast cancer stem cells from the 4 cell lines. TSA at 500 nmol/L induced cell apoptosis in the primary mammospheres. TSA down-regulated the mRNA expression of Nanog and Sox2 in the primary mammospheres. TSA can partially inhibit the self-renewal of breast cancer stem cells through a mechanism involving the down-regulation of Nanog and Sox2 expression, indicating the value of combined treatments with low-dose TSA and other anticancer drugs to achieve maximum inhibition of breast cancer stem cell self-renewal. The core transcriptional factor of embryonic stem cells Nanog and Sox2 can be potential targets of anticancer therapy.

A Primary Xenograft Model of Small Cell Lung Cancer Reveals Irreversible Changes in Gene Expression Imposed by Culture In-Vitro

PubMed Central

Daniel, Vincent C.; Marchionni, Luigi; Hierman, Jared S.; Rhodes, Jonathan T.; Devereux, Wendy L.; Rudin, Charles M.; Yung, Rex; Parmigani, Giovanni; Dorsch, Marion; Peacock, Craig D.; Watkins, D. Neil

2009-01-01

Traditional approaches to the preclinical investigation of cancer therapies rely on the use of established cell lines maintained in serum-based growth media. This is particularly true of small cell lung cancer (SCLC), where surgically resected tissue is rarely available. Recent attention has focused on the need for better models that preserve the integrity of cancer stem cell populations, as well as three-dimensional tumor-stromal interactions. Here we describe a primary xenograft model of SCLC in which endobronchial tumor specimens obtained from chemo-naive patients are serially propagated in vivo in immunodeficient mice. In parallel, cell lines grown in conventional tissue culture conditions were derived from each xenograft line, passaged for 6 months, and then re-implanted to generate secondary xenografts. Using the Affymetrix platform, we analyzed gene expression in primary xenograft, xenograft-derived cell line, and secondary xenograft, and compared these data to similar analyses of unrelated primary SCLC samples and laboratory models. When compared to normal lung, primary tumors, xenografts and cell lines displayed a gene expression signature specific for SCLC. Comparison of gene expression within the xenograft model identified a group of tumor-specific genes expressed in primary SCLC and xenografts that was lost during the transition to tissue culture, and that was not regained when the tumors were re-established as secondary xenografts. Such changes in gene expression may be a common feature of many cancer cell culture systems, with functional implications for the use of such models for preclinical drug development. PMID:19351829

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

PubMed Central

Peixoto, António; Evaristo, César; Munitic, Ivana; Monteiro, Marta; Charbit, Alain; Rocha, Benedita; Veiga-Fernandes, Henrique

2007-01-01

To study in vivo CD8 T cell differentiation, we quantified the coexpression of multiple genes in single cells throughout immune responses. After in vitro activation, CD8 T cells rapidly express effector molecules and cease their expression when the antigen is removed. Gene behavior after in vivo activation, in contrast, was quite heterogeneous. Different mRNAs were induced at very different time points of the response, were transcribed during different time periods, and could decline or persist independently of the antigen load. Consequently, distinct gene coexpression patterns/different cell types were generated at the various phases of the immune responses. During primary stimulation, inflammatory molecules were induced and down-regulated shortly after activation, generating early cells that only mediated inflammation. Cytotoxic T cells were generated at the peak of the primary response, when individual cells simultaneously expressed multiple killer molecules, whereas memory cells lost killer capacity because they no longer coexpressed killer genes. Surprisingly, during secondary responses gene transcription became permanent. Secondary cells recovered after antigen elimination were more efficient killers than cytotoxic T cells present at the peak of the primary response. Thus, primary responses produced two transient effector types. However, after boosting, CD8 T cells differentiate into long-lived killer cells that persist in vivo in the absence of antigen. PMID:17485515

Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells.

PubMed

Farrell, Emma K; Chen, Yuden; Barazanji, Muna; Jeffries, Kristen A; Cameroamortegui, Felipe; Merkler, David J

2012-02-01

Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.

Flavonoids isolated from Tridax procumbens (TPF) inhibit osteoclasts differentiation and bone resorption.

PubMed

Al Mamun, Md Abdullah; Islam, Kamrul; Alam, Md Jahangir; Khatun, Amina; Alam, M Masihul; Al-Bari, Md Abdul Alim; Alam, Md Jahangir

2015-09-12

The Tridax procumbens flavonoids (TPF), are well known for their medicinal properties among local natives. The TPF are traditionally used for dropsy, anaemia, arthritis, gout, asthma, ulcer, piles, and urinary problems. It also used in treating gastric problems, body pain, and rheumatic pains of joints. The TPF have been reported to increase osteogenic functioning in mesenchymal stem cells. However, their effects on osteoclastogenesis remain unclear. The TPF isolated from T. procumbens and investigated the effects of the TPF inhibit on osteoclast differentiation and bone resorption activities using primary osteoclastic cells. Osteoclast formation was assessed by counting the number of tartrate resistant acid phosphatase (TRAP) positive multinucleated cells and by measuring both TRAP activities. The TPF significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in primary osteoclastic cells. The TPF also decreased the expression of mRNAs related to osteoclast differentiation, including Trap, Cathepsin K, Mmp-9, and Mmp-13 in primary osteoclastic cells. The treatment of primary osteoclastic cells with the TPF decreased Cathepsin K, Mmp-9, and Mmp-13 proteins expression in primary osteoclastic cells. These results indicated that TPF inhibit osteoclastogenesis and pits formation activities. Our results suggest that the TPF could be a potential anti-bone resorptic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells1[S

PubMed Central

Farrell, Emma K.; Chen, Yuden; Barazanji, Muna; Jeffries, Kristen A.; Cameroamortegui, Felipe; Merkler, David J.

2012-01-01

Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N18TG2 and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N18TG2 and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N18TG2 and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation. PMID:22095832

Primary cutaneous marginal zone B-cell lymphoma: clinical and histological aspects.

PubMed

Khaled, A; Sassi, S; Fazaa, B; Ben Hassouna, J; Ben Romdhane, K; Kamoun, M R

2009-02-01

According to the WHO-EORTC classification of cutaneous lymphomas, primary cutaneous marginal zone B-cell lymphoma are now well characterized. We report here a case of primary cutaneous marginal zone B-cell lymphoma in a 51 year-old man in which the diagnosis was made using both histology and immunopathology. The patient had no remarkable medical history, no history of either acute inflammation or insect bite, and presented with a 5 cm solitary asymptomatic erythematous firm, multinodular and infiltrated plaque on the back for 12 months. Histological examination and immunohistochemical study of a cutaneous biopsy provided a differential diagnosis between B cell lymphoma and lymphocytoma cutis. Full body work up revealed no signs of extracutaneous dissemination. The patient underwent surgical excision of the nodule. Histological examination showed a histological and immunophenotyping profile typical of primary cutaneous marginal zone B-cell lymphoma. The lesion was completely excised with clear margins and no recurrence occurred after a 12 month-follow-up period. Primary cutaneous marginal zone B-cell lymphoma are low-grade lymphomas that have an indolent course and a high tendency to recur. They should be differentiated from lymphocytoma cutis and from the other types of cutaneous B cell lymphomas that have a different course and prognosis.

Persistence of T-cell immune response induced by two acellular pertussis vaccines in children five years after primary vaccination.

PubMed

Palazzo, Raffaella; Carollo, Maria; Bianco, Manuela; Fedele, Giorgio; Schiavoni, Ilaria; Pandolfi, Elisabetta; Villani, Alberto; Tozzi, Alberto E; Mascart, Françoise; Ausiello, Clara M

2016-01-01

The resurgence of pertussis suggests the need for greater efforts to understand the long-lasting protective responses induced by vaccination. In this paper we dissect the persistence of T memory responses induced by primary vaccination with two different acellular pertussis (aP) vaccines, hexavalent Hexavac® vaccine (Hexavac) (Sanofi Pasteur MSD) and Infanrix hexa® (Infanrix) (Glaxo-SmithKline Biologicals). We evaluated magnitude and duration of T-cell responses to pertussis toxin (PT) by measuring T-cell proliferation, cytokines (IL-2 and IFNγ) production and memory subsets in two groups of children 5 years after primary vaccination. Some of the enrolled children received only primary vaccination, while others had the pre-school boost dose. Positive T-cell responses to PT were detected in 36% of children. Percentage of responsive children, T-cell proliferation and CD4IL-2+ cells were significantly higher in the children primed with Hexavac than in those who received Infanrix vaccine. No major effects of the boost on PT-specific proliferation were observed. Overall, our data documented a persistence of T-cell memory against PT in a minor fraction of children 5 years after primary vaccination. The different responses induced by Hexavac and Infanrix vaccine could rely on differences in PT inactivation process or excipients/adjuvants formulations.

Pomalidomide and Dexamethasone in Treating Patients With Relapsed or Refractory Primary Central Nervous System Lymphoma or Newly Diagnosed or Relapsed or Refractory Intraocular Lymphoma

ClinicalTrials.gov

2017-08-28

B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma; Central Nervous System Lymphoma; Intraocular Lymphoma; Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System; Recurrent Adult Diffuse Large Cell Lymphoma; Retinal Lymphoma

In Vitro Transformation of Rat and Mouse Cells by DNA from Simian Virus 40

PubMed Central

Abrahams, P. J.; van der Eb, A. J.

1975-01-01

Primary rat kidney cells and mouse 3T3 cells can be transformed by DNA of simian virus 40 when use is made of the calcium technique (Graham and van der Eb, 1973). The transformation assay in primary rat cells is reproducible, but the dose response is not linear. PMID:166204

HDAC6 inhibition suppresses chondrosarcoma by restoring the expression of primary cilia.

PubMed

Xiang, Wei; Guo, Fengjing; Cheng, Weiting; Zhang, Jiaming; Huang, Junming; Wang, Rui; Ma, Zhongxi; Xu, Kai

2017-07-01

Chondrosarcoma is a bone tumor characterized by the secretion of a cartilage-like extracellular matrix. It has been proved to lack extracellular sensor primary cilia. This study aimed to illustrate a feasible therapeutic method for chondrosarcoma by regulating primary cilia assembly through inhibiting histone deacetylases 6 (HDAC6) activation. In order to detect the interaction between primary cilia and HDAC6 in human chondrosarcoma, Tubastatin A and small interfering RNA (siRNA) were used to inhibit the endogenous expression of HDAC6. Cell viability test and Transwell assay were applied to evaluate the effects of malignant biological properties. Primary cilia staining and related proteins were detected. The abnormal expression of HDAC6 and cilia intraflagellar transport protein 88 (IFT88) was found in chondrosarcoma tissues. The inhibition of HDAC6 could downregulate the proliferation of chondrosarcoma cells in a concentration- and time-dependent manner and suppress the invasion capacity of tumor cells. Besides, the downregulation of HDAC6 exhibited a negative effect on the proliferation of relevant proteins but a positive effect on the primary cilia-related expression of IFT88 and acetylated α-tubulin. Primary cilia restoration could be observed after HDAC6 siRNA transfection. The Aurora A-HDAC6 cascade was involved in regulating primary cilia resorption by affecting α-tubulin deacetylation and Tubastatin A could inhibit chondrosarcoma cell growth in vivo. These results indicate that restricting HDAC6 can restore primary cilia assembly accompanied with suppressed chondrosarcoma cell proliferation and invasion capacities. Thus, promoting primary cilia restoration by targeting HDAC6 may be a feasible potential therapeutic method for chondro-sarcoma treatment.

Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

PubMed Central

Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

1998-01-01

In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

NASA Astrophysics Data System (ADS)

Salazar-García, Samuel; Silva-Ramírez, Ana Sonia; Ramirez-Lee, Manuel A.; Rosas-Hernandez, Hector; Rangel-López, Edgar; Castillo, Claudia G.; Santamaría, Abel; Martinez-Castañon, Gabriel A.; Gonzalez, Carmen

2015-11-01

The aim of this work was to compare the effects of 24-h exposure of rat primary astrocytes and C6 rat glioma cells to 7.8 nm AgNPs. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and current treatments lead to diverse side-effects; for this reason, it is imperative to investigate new approaches, including those alternatives provided by nanotechnology, like nanomaterials (NMs) such as silver nanoparticles. Herein, we found that C6 rat glioma cells, but no primary astrocytes, decreased cell viability after AgNPs treatment; however, both cell types diminished their proliferation. The decrease of glioma C6 cells proliferation was related with necrosis, while in primary astrocytes, the decreased proliferation was associated with the induction of apoptosis. The ionic control (AgNO3) exerted a different profile than AgNPs; the bulk form did not modify the basal effect in each determination, whereas cisplatin, a well-known antitumoral drug used as a comparative control, promoted cytotoxicity in both cell types at specific concentrations. Our findings prompt the need to determine the fine molecular and cellular mechanisms involved in the differential biological responses to AgNPs in order to develop new tools or alternatives based on nanotechnology that may contribute to the understanding, impact and use of NMs in specific targets, like glioblastoma cells.

Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

PubMed Central

Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-chung; Rosenblum, Norman D.

2013-01-01

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms. PMID:23762375

Lack of centrioles and primary cilia in STIL−/− mouse embryos

PubMed Central

David, Ahuvit; Liu, Fengying; Tibelius, Alexandra; Vulprecht, Julia; Wald, Diana; Rothermel, Ulrike; Ohana, Reut; Seitel, Alexander; Metzger, Jasmin; Ashery-Padan, Ruth; Meinzer, Hans-Peter; Gröne, Hermann-Josef; Izraeli, Shai; Krämer, Alwin

2014-01-01

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL−/− mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL−/− cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL−/− cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development. PMID:25486474

Lack of centrioles and primary cilia in STIL(-/-) mouse embryos.

PubMed

David, Ahuvit; Liu, Fengying; Tibelius, Alexandra; Vulprecht, Julia; Wald, Diana; Rothermel, Ulrike; Ohana, Reut; Seitel, Alexander; Metzger, Jasmin; Ashery-Padan, Ruth; Meinzer, Hans-Peter; Gröne, Hermann-Josef; Izraeli, Shai; Krämer, Alwin

2014-01-01

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL(-/-) mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL(-/-) cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL(-/-) cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.

Increased risk of gastric adenocarcinoma after treatment of primary gastric diffuse large B-cell lymphoma.

PubMed

Inaba, Koji; Kushima, Ryoji; Murakami, Naoya; Kuroda, Yuuki; Harada, Ken; Kitaguchi, Mayuka; Yoshio, Kotaro; Sekii, Shuhei; Takahashi, Kana; Morota, Madoka; Mayahara, Hiroshi; Ito, Yoshinori; Sumi, Minako; Uno, Takashi; Itami, Jun

2013-10-26

There have been sporadic reports about synchronous as well as metachronous gastric adenocarcinoma and primary gastric lymphoma. Many reports have dealt with metachronous gastric adenocarcinoma in mucosa-associated lymphoid tissue lymphoma of stomach. But to our knowledge, there have been no reports that document the increased incidence of metachronous gastric adenocarcinoma in patients with gastric diffuse large B-cell lymphoma. This retrospective study was conducted to estimate the incidence of metachronous gastric adenocarcinoma after primary gastric lymphoma treatment, especially in diffuse large B-cell lymphoma. The retrospective cohort study of 139 primary gastric lymphoma patients treated with radiotherapy at our hospital. Mean observation period was 61.5 months (range: 3.7-124.6 months). Patients profile, characteristics of primary gastric lymphoma and metachronous gastric adenocarcinoma were retrieved from medical records. The risk of metachronous gastric adenocarcinoma was compared with the risk of gastric adenocarcinoma in Japanese population. There were 10 (7.2%) metachronous gastric adenocarcinoma patients after treatment of primary gastric lymphomas. It was quite high risk compared with the risk of gastric carcinoma in Japanese population of 54.7/100,000. Seven patients of 10 were diffuse large B-cell lymphoma and other 3 patients were mixed type of diffuse large B-cell lymphoma and mucosa associated lymphoid tissue lymphoma. Four patients of 10 metachronous gastric adenocarcinomas were signet-ring cell carcinoma and two patients died of gastric adenocarcinoma. Metachronous gastric adenocarcinoma may have a more malignant potential than sporadic gastric adenocarcinoma. Old age, Helicobacter pylori infection and gastric mucosal change of chronic gastritis and intestinal metaplasia were possible risk factors for metachronous gastric adenocarcinoma. There was an increased risk of gastric adenocarcinoma after treatment of primary gastric lymphoma, especially of diffuse large B-cell lymphoma.

Primary central nervous system diffuse large B-cell lymphoma shows an activated B-cell-like phenotype with co-expression of C-MYC, BCL-2, and BCL-6.

PubMed

Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan

2017-06-01

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.

Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3.

PubMed

Dwivedi, Prem P; Anderson, Peter J; Powell, Barry C

2012-08-03

Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.

Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells.

PubMed

Gründling, Angelika; Gonzalez, Mark D; Higgins, Darren E

2003-11-01

In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

Onset of human preterm and term birth is related to unique inflammatory transcriptome profiles at the maternal fetal interface.

PubMed

Bukowski, Radek; Sadovsky, Yoel; Goodarzi, Hani; Zhang, Heping; Biggio, Joseph R; Varner, Michael; Parry, Samuel; Xiao, Feifei; Esplin, Sean M; Andrews, William; Saade, George R; Ilekis, John V; Reddy, Uma M; Baldwin, Donald A

2017-01-01

Preterm birth is a main determinant of neonatal mortality and morbidity and a major contributor to the overall mortality and burden of disease. However, research of the preterm birth is hindered by the imprecise definition of the clinical phenotype and complexity of the molecular phenotype due to multiple pregnancy tissue types and molecular processes that may contribute to the preterm birth. Here we comprehensively evaluate the mRNA transcriptome that characterizes preterm and term labor in tissues comprising the pregnancy using precisely phenotyped samples. The four complementary phenotypes together provide comprehensive insight into preterm and term parturition. Samples of maternal blood, chorion, amnion, placenta, decidua, fetal blood, and myometrium from the uterine fundus and lower segment ( n  = 183) were obtained during cesarean delivery from women with four complementary phenotypes: delivering preterm with (PL) and without labor (PNL), term with (TL) and without labor (TNL). Enrolled were 35 pregnant women with four precisely and prospectively defined phenotypes: PL ( n  = 8), PNL ( n  = 10), TL ( n  = 7) and TNL ( n  = 10). Gene expression data were analyzed using shrunken centroid analysis to identify a minimal set of genes that uniquely characterizes each of the four phenotypes. Expression profiles of 73 genes and non-coding RNA sequences uniquely identified each of the four phenotypes. The shrunken centroid analysis and 10 times 10-fold cross-validation was also used to minimize false positive finings and overfitting. Identified were the pathways and molecular processes associated with and the cis-regulatory elements in gene's 5' promoter or 3'-UTR regions of the set of genes which expression uniquely characterized the four phenotypes. The largest differences in gene expression among the four groups occurred at maternal fetal interface in decidua, chorion and amnion. The gene expression profiles showed suppression of chemokines expression in TNL, withdrawal of this suppression in TL, activation of multiple pathways of inflammation in PL, and an immune rejection profile in PNL. The genes constituting expression signatures showed over-representation of three putative regulatory elements in their 5'and 3' UTR regions. The results suggest that pregnancy is maintained by downregulation of chemokines at the maternal-fetal interface. Withdrawal of this downregulation results in the term birth and its overriding by the activation of multiple pathways of the immune system in the preterm birth. Complications of the pregnancy associated with impairment of placental function, which necessitated premature delivery of the fetus in the absence of labor, show gene expression patterns associated with immune rejection.

Viable placental allograft as a biological dressing in the clinical management of full-thickness thermal occupational burns

PubMed Central

Johnson, Eric L.; Tassis, Elisabet K.; Michael, Georgina M.; Whittinghill, Susan G.

2017-01-01

Abstract Rationale: Occupational burn injuries can be detrimental and difficult to manage. The majority of complex cases are referred and managed at regional burn centers where access to specialized care is available. As an alternative to hospitalization with staged surgical procedures, placental products may be used for outpatient medical management of these common burn injuries, especially if access to a regional burn center is limited or restricted. Fresh amnion has been a treatment of choice in burns for more than 100 years. As a biological covering with a broad scope of potential uses, human placental membranes represent a dressing that is particularly advantageous for burn therapy. Recent advances in tissue-preservation technology have allowed for the commercialization of placental amnion products. Patient concerns: To address several complications associated with burn injuries—contractures, scar formation, and pain—a viable cryopreserved placental membrane (vCPM) (Grafix—PRIME, Osiris Therapeutics, Inc., MD) retaining the anti-inflammatory, anti-fibrotic, and antimicrobial properties of fresh placental tissues was chosen for clinical use in the 2 cases reported, where both patients had restricted access to the regional burn center. Diagnoses: Two cases of work-related extremity burns presented to a local rural hospital for immediate post-injury assessment. The 1st case was of a man who sustained a 55.4 cm2 full-thickness 3rd degree thermal burn with exposed bone and tendon, to the left dorsal forefoot after having an industrial pressure washer caught on his work boot. The 2nd case was of a female who sustained a 4.7 cm2 full-thickness 3rd degree crush burn to the dorsum extensor surface of her dominant hand's index finger after applying 80-pounds per square inch of heated pressure from a hydraulic press. Interventions: Both burn patients elected to continue their care at the outpatient-based wound and hyperbaric center, receiving a combination of weekly ad libitum debridement, applications of vCPM, and occupational therapy. Outcomes: Both burns reached timely wound closure, and patients regained full range of motion of the affected limb, allowing for early return to work. The average number of allograft applications was 7.5, allowing both patients to return to work in an average of 63.5 days without adverse events or post-treatment complications. Lessons: The incorporation of this product in the treatment of these complex burns prevented amputation in one patient, and skin autografting and potential index finger contracture-formation in the second patient. The incorporation of vCPM in burn management may offer a new approach to outpatient burn management and may mitigate several of the complications seen post burn injury, leading to favorable patient outcomes. PMID:29245303

Onset of human preterm and term birth is related to unique inflammatory transcriptome profiles at the maternal fetal interface

PubMed Central

Sadovsky, Yoel; Goodarzi, Hani; Zhang, Heping; Biggio, Joseph R.; Varner, Michael; Parry, Samuel; Xiao, Feifei; Esplin, Sean M.; Andrews, William; Saade, George R.; Ilekis, John V.; Reddy, Uma M.; Baldwin, Donald A.

2017-01-01

Background Preterm birth is a main determinant of neonatal mortality and morbidity and a major contributor to the overall mortality and burden of disease. However, research of the preterm birth is hindered by the imprecise definition of the clinical phenotype and complexity of the molecular phenotype due to multiple pregnancy tissue types and molecular processes that may contribute to the preterm birth. Here we comprehensively evaluate the mRNA transcriptome that characterizes preterm and term labor in tissues comprising the pregnancy using precisely phenotyped samples. The four complementary phenotypes together provide comprehensive insight into preterm and term parturition. Methods Samples of maternal blood, chorion, amnion, placenta, decidua, fetal blood, and myometrium from the uterine fundus and lower segment (n = 183) were obtained during cesarean delivery from women with four complementary phenotypes: delivering preterm with (PL) and without labor (PNL), term with (TL) and without labor (TNL). Enrolled were 35 pregnant women with four precisely and prospectively defined phenotypes: PL (n = 8), PNL (n = 10), TL (n = 7) and TNL (n = 10). Gene expression data were analyzed using shrunken centroid analysis to identify a minimal set of genes that uniquely characterizes each of the four phenotypes. Expression profiles of 73 genes and non-coding RNA sequences uniquely identified each of the four phenotypes. The shrunken centroid analysis and 10 times 10-fold cross-validation was also used to minimize false positive finings and overfitting. Identified were the pathways and molecular processes associated with and the cis-regulatory elements in gene’s 5′ promoter or 3′-UTR regions of the set of genes which expression uniquely characterized the four phenotypes. Results The largest differences in gene expression among the four groups occurred at maternal fetal interface in decidua, chorion and amnion. The gene expression profiles showed suppression of chemokines expression in TNL, withdrawal of this suppression in TL, activation of multiple pathways of inflammation in PL, and an immune rejection profile in PNL. The genes constituting expression signatures showed over-representation of three putative regulatory elements in their 5′and 3′ UTR regions. Conclusions The results suggest that pregnancy is maintained by downregulation of chemokines at the maternal-fetal interface. Withdrawal of this downregulation results in the term birth and its overriding by the activation of multiple pathways of the immune system in the preterm birth. Complications of the pregnancy associated with impairment of placental function, which necessitated premature delivery of the fetus in the absence of labor, show gene expression patterns associated with immune rejection. PMID:28879060

Carboplatin, Paclitaxel and Gemcitabine Hydrochloride With or Without Bevacizumab After Surgery in Treating Patients With Recurrent Ovarian, Epithelial, Primary Peritoneal, or Fallopian Tube Cancer

ClinicalTrials.gov

2018-06-06

Clear Cell Adenocarcinoma; Fallopian Tube Clear Cell Adenocarcinoma; Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Serous Adenocarcinoma; Mucinous Adenocarcinoma; Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinofibroma; Ovarian Endometrioid Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Adenocarcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

PubMed

Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

2016-01-01

The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.

Establishment and characterization of clear cell renal cell carcinoma cell lines with different metastatic potential from Chinese patients.

PubMed

Tan, Xiaojie; He, Songqin; Han, Yifang; Yu, Yongwei; Xiao, Jianru; Xu, Danfeng; Wang, Guoping; Du, Yan; Chang, Wenjun; Yin, Jianhua; Su, Tong; Hou, Jianguo; Cao, Guangwen

2013-02-26

Clear cell renal cell carcinoma (ccRCC) cell lines with distinct metastatic potential are essential to study the mechanism of ccRCC metastasis. However, none of them originated from Chinese. Primary cell cultures were performed using a primary tumor of a 49-year-old male ccRCC patient and a metastatic tumor of a 62-year-old male patient who had received nephrectomy to excise primary ccRCC 10 years ago. Cell growth, microstructure, cytogenetics, cytometry, expression of metastasis-associated molecules, tumorigenesis and metastasis were subsequently characterized. Two successive cell lines named NRCC from the primary ccRCC and MRCC from the metastatic ccRCC were established, respectively. Compared to NRCC, MRCC exhibited stronger anchorage-independent growth and invasion potentials and contained more glycogen granules in the cytoplasm. Gains of chromosomes and some translocations were the major chromosomal aberrations in both cell strains. CD24 expression was more frequent in MRCC than in NRCC and the same was true for CD56. The transcriptional levels of TNFα, IL-6, VEGF, HIF2α, MMP2, and RhoC were significantly higher in MRCC than in NRCC. Cytosolic IκBα protein was more degraded in MRCC than in NRCC following TNFα treatment. Both cell lines had strong tumorigenicity in athymic nude mice. However, MRCC had strong potential in generating metastasis to lung and hemorrhagic ascites than NRCC following orthotopic transplantations. Cancer cells isolated from metastatic ccRCC have more malignant and metastatic potential than those from the primary tumor from the patients who shared the similar race background. Establishment of MRCC and NRCC may provide suitable models with which to investigate molecular mechanisms of ccRCC metastasis.

Cytotoxic effects of denture adhesives on primary human oral keratinocytes, fibroblasts and permanent L929 cell lines.

PubMed

Chen, Fengying; Wu, Tianfu; Cheng, Xiangrong

2014-03-01

To date, there have been very little data on the cytotoxic responses of different cell lines to denture adhesives. To determine the cytotoxicity of three denture adhesives on primary human oral keratinocytes (HOKs), fibroblasts (HOFs) and permanent mouse fibroblasts cell lines (L929). Three commercial denture adhesives (two creams and one powder) were prepared for indirect contact using the agar diffusion test, as well as extracts in MTT assay. The results of the MTT assay were statistically analysed by one-way anova and Tukey's test (p < 0.05). All of the tested denture adhesives showed mild to moderate cytotoxicity to primary HOKs (p < 0.001), whereas none of three was toxic to L929 cells (p > 0.05) in both assays. For primary HOFs cultures, slight cytotoxicity was observed for one of the products from the agar diffusion test and undiluted eluates of all tested adhesives with MTT assay (p < 0.01). Denture adhesives are toxic to the primary HOKs and HOFs cultures, whereas non-toxic to L929 cells. The results suggest that primary human oral mucosal cells may provide more valuable information in toxicity screening of denture adhesives. © 2012 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd.

Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures

PubMed Central

Popova, Daria; Stonier, Adam; Pain, David; Titchener‐Hooker, Nigel J.

2016-01-01

Abstract Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost‐effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale‐down (USD) mimics requiring 25–110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost‐effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario. PMID:27067803

Multi-walled carbon nanotube length as a critical determinant of bioreactivity with primary human pulmonary alveolar cells

PubMed Central

Sweeney, Sinbad; Berhanu, Deborah; Misra, Superb K.; Thorley, Andrew J.; Valsami-Jones, Eugenia; Tetley, Teresa D.

2015-01-01

Multiwalled carbon nanotube (MWCNT) length is suggested to critically determine their pulmonary toxicity. This stems from in vitro and in vivo rodent studies and in vitro human studies using cell lines (typically cancerous). There is little data using primary human lung cells. We addressed this knowledge gap, using highly relevant, primary human alveolar cell models exposed to precisely synthesized and thoroughly characterized MWCNTs. In this work, transformed human alveolar type-I-like epithelial cells (TT1), primary human alveolar type-II epithelial cells (ATII) and alveolar macrophages (AM) were treated with increasing concentrations of MWCNTs before measuring cytotoxicity, inflammatory mediator release and MAP kinase signalling. Strikingly, we observed that short MWCNTs (~0.6 µm in length) induced significantly greater responses from the epithelial cells, whilst AM were particularly susceptible to long MWCNTs (~20 µm). These differences in the pattern of mediator release were associated with alternative profiles of JNK, p38 and ERK1/2 MAP kinase signal transduction within each cell type. This study, using highly relevant target human alveolar cells and well defined and characterized MWCNTs, shows marked cellular responses to the MWCNTs that vary according to the target cell type, as well as the aspect ratio of the MWCNT. PMID:25780270

The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens.

PubMed

Lindsten, T; Yaffe, L J; Thompson, C B; Guelde, G; Berning, A; Scher, I; Kenny, J J

1985-05-01

Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.

β-Arrestin2 mediates progression of murine primary myelofibrosis.

PubMed

Rein, Lindsay Am; Wisler, James W; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Minyong; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia Kl; Premont, Richard T; Lefkowitz, Robert J

2017-12-21

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2-/- cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

Squamous cell carcinoma presenting with trigeminal anesthesia: An uncommon presentation of head & neck cancer with unknown primary.

PubMed

Shah, Ameer T; Dagher, Walid I; O'Leary, Miriam A; Wein, Richard O

The differential diagnosis of facial anesthesia is vast. This may be secondary to trauma, neoplasm, both intracranial and extracranial, infection, and neurologic disease. When evaluating a patient with isolated facial anesthesia, the head and neck surgeon often thinks of adenoid cystic carcinoma, which has a propensity for perineural invasion and spread. When one thinks of head and neck squamous cell carcinoma with or without unknown primary, the typical presentation involves dysphagia, odynophagia, weight loss, hoarseness, or more commonly, a neck mass. Squamous cell carcinoma presenting as facial anesthesia and perineural spread, with no primary site is quite rare. Case presentations and review of the literature. Trigeminal anesthesia is an uncommon presentation of head and neck squamous cell carcinoma with unknown primary. We present two interesting cases of invasive squamous cell carcinoma of the trigeminal nerve, with no primary site identified. We will also review the literature of head and neck malignancies with perineural spread and the management techniques for the two different cases presented. Copyright © 2016 Elsevier Inc. All rights reserved.

Promotion of Tumor-Initiating Cells in Primary and Recurrent Breast Tumors

DTIC Science & Technology

2014-10-01

confer stemness . We hypothesize that inhibition of IKK/NF-κB will reduce or eliminate breast camcer TICs, blocking tumorigenesis. Furthermore, we...Korkaya H, Liu S, Wicha MS. Breast cancer stem cells, cytokine networks, and the tumor microenvironment. J Clin Invest. 2011 Oct;121(10):3804-9. Review...cells and sub- population of cells termed cancer stem cells or tumor-initiating cells (TICs).1 The primary characteristic of TICs is their ability to

Calcium phosphate transfection of primary hippocampal neurons.

PubMed

Sun, Miao; Bernard, Laura P; Dibona, Victoria L; Wu, Qian; Zhang, Huaye

2013-11-12

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Profiling Bioactivity of the ToxCast Chemical Library Using BioMAP Primary Human Cell Systems

EPA Science Inventory

The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, ...

Modulated discharge of Purkinje and stellate cells persists after unilateral loss of vestibular primary afferent mossy fibers in mice

PubMed Central

Yakhnitsa, V.

2013-01-01

Cerebellar Purkinje cells are excited by two afferent pathways: climbing and mossy fibers. Climbing fibers evoke large “complex spikes” (CSs) that discharge at low frequencies. Mossy fibers synapse on granule cells whose parallel fibers excite Purkinje cells and may contribute to the genesis of “simple spikes” (SSs). Both afferent systems convey vestibular information to folia 9c–10. After making a unilateral labyrinthectomy (UL) in mice, we tested how the discharge of CSs and SSs was changed by the loss of primary vestibular afferent mossy fibers during sinusoidal roll tilt. We recorded from cells identified by juxtacellular neurobiotin labeling. The UL preferentially reduced vestibular modulation of CSs and SSs in folia 8–10 contralateral to the UL. The effects of a UL on Purkinje cell discharge were similar in folia 9c–10, to which vestibular primary afferents project, and in folia 8–9a, to which they do not project, suggesting that vestibular primary afferent mossy fibers were not responsible for the UL-induced alteration of SS discharge. UL also induced reduced vestibular modulation of stellate cell discharge contralateral to the UL. We attribute the decreased modulation to reduced vestibular modulation of climbing fibers. In summary, climbing fibers modulate CSs directly and SSs indirectly through activation of stellate cells. Whereas vestibular primary afferent mossy fibers cannot account for the modulated discharge of SSs or stellate cells, the nonspecific excitation of Purkinje cells by parallel fibers may set an operating point about which the discharges of SSs are sculpted by climbing fibers. PMID:23966673

SLC6A4 expression and anti-proliferative responses to serotonin transporter ligands chlomipramine and fluoxetine in primary B-cell malignancies.

PubMed

Chamba, Anita; Holder, Michelle J; Jarrett, Ruth F; Shield, Lesley; Toellner, Kai M; Drayson, Mark T; Barnes, Nicholas M; Gordon, John

2010-08-01

B-cell lines of diverse neoplastic origin express the serotonin transporter (SERT/SLC6A4) and growth arrest in response to SERT-ligands, including the antidepressants chlomipramine and fluoxetine. Here we detail SLC6A4 transcript (Q-PCR) and protein (FACS) expression in primary cells from patients with: chronic lymphocytic leukaemia; mantle cell lymphoma; follicular lymphoma; Burkitt's lymphoma; and diffuse large B-cell lymphoma. The ability of the SERT-binding antidepressants to impact the growth of these cells when sustained on CD154-transfected fibroblasts was also determined. The results reveal a broad spectrum of primary B-cell malignancies expressing SLC6A4 with a proportion additionally displaying growth arrest on SERT-ligand exposure. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Epigallocatechin gallate (EGCG) prevents H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells.

PubMed

Cia, David; Vergnaud-Gauduchon, Juliette; Jacquemot, Nathalie; Doly, Michel

2014-09-01

To determine whether the green tea polyphenol epigallocatechin gallate (EGCG) could prevent H(2)O(2)-induced oxidative stress in primary rat retinal pigment epithelial cells. Primary cultures of retinal pigment epithelium (RPE) cells were established from Long-Evans newborn rats. RPE cells were pretreated with various concentrations of EGCG for 24 h before being exposed to hydrogen peroxide (H(2)O(2)) for 2 h to induce oxidative stress. Cell metabolic activity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was quantified by flow cytometry using propidium iodide (PI). Treatment of RPE cells with EGCG alone does not affect the cell viability up to 50 µM. Exposure of RPE cells to 600 µM H(2)O(2) caused a significant decrease in cell viability; whereas pretreatment with 10, 25, and 50 µM EGCG significantly reduced this decrease in a dose-dependent manner. The proportion of PI-positive cells increased significantly in cultures treated with H(2)O(2) alone; whereas pretreatment of RPE cells with 50 µM EGCG significantly reduced H(2)O(2)-induced RPE cell death. Our study shows that EGCG pretreatment can protect primary rat RPE cells from H(2)O(2)-induced death. This suggests potential effect of EGCG in the prevention of retinal diseases associated with H(2)O(2)-induced oxidative stress.

Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

PubMed

Fukuda, Tomokazu; Kurita, Jun; Saito, Tomomi; Yuasa, Kei; Kurita, Masanobu; Donai, Kenichiro; Nitto, Hiroshi; Soichi, Makoto; Nishimori, Katsuhiko; Uchida, Takafumi; Isogai, Emiko; Onuma, Manabu; Sone, Hideko; Oseko, Norihisa; Inoue-Murayama, Miho

2012-12-01

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

Activated T-cell Therapy, Low-Dose Aldesleukin, and Sargramostim in Treating Patients With Ovarian, Fallopian Tube, or Primary Peritoneal Cancer That is Stage III-IV, Refractory, or Recurrent

ClinicalTrials.gov

2016-02-15

Malignant Ovarian Clear Cell Tumor; Malignant Ovarian Serous Tumor; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer

Posttransplantation primary cutaneous CD30 (Ki-1)-positive large-cell lymphoma.

PubMed

Seçkin, D; Demirhan, B; Oğuz Güleç, T; Arikan, U; Haberal, M

2001-12-01

We describe the case of a 51-year-old female renal transplant recipient with primary cutaneous CD30-positive large-cell lymphoma of T-cell origin. Cutaneous T-cell lymphomas are rarely reported in organ transplant recipients, and we believe they should be considered in the differential diagnosis of cutaneous neoplastic and infectious diseases affecting this patient group.

Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite.

PubMed

Ricciardi, M R; Licchetta, R; Mirabilii, S; Scarpari, M; Parroni, A; Fabbri, A A; Cescutti, P; Reverberi, M; Fanelli, C; Tafuri, A

2017-01-01

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor , on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite

PubMed Central

Scarpari, M.; Parroni, A.; Fabbri, A. A.; Cescutti, P.; Reverberi, M.; Fanelli, C.

2017-01-01

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity. PMID:29270245

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1

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

Rosa Borges, Andrew; Wieczorek, Lindsay; Johnson, Benitra

2010-12-05

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC{sub 50}s ranging from 0.1 to 7.4 {mu}g/ml. Inhibition of Env-mediated membrane fusion by MVC wasmore » also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions. -- Research Highlights: {yields}Multivalent carbohydrates (MVCs) inhibited infection of PBMCs by HIV-1. {yields}MVCs inhibited infection by T cell line-adapted viruses. {yields}MVCs inhibited infection by primary isolates of HIV-1. {yields}MVCs inhibited Env-mediated membrane fusion.« less

Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

PubMed Central

Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

2014-01-01

ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

Primary cilia proteins: ciliary and extraciliary sites and functions.

PubMed

Hua, Kiet; Ferland, Russell J

2018-05-01

Primary cilia are immotile organelles known for their roles in development and cell signaling. Defects in primary cilia result in a range of disorders named ciliopathies. Because this organelle can be found singularly on almost all cell types, its importance extends to most organ systems. As such, elucidating the importance of the primary cilium has attracted researchers from all biological disciplines. As the primary cilia field expands, caution is warranted in attributing biological defects solely to the function of this organelle, since many of these "ciliary" proteins are found at other sites in cells and likely have non-ciliary functions. Indeed, many, if not all, cilia proteins have locations and functions outside the primary cilium. Extraciliary functions are known to include cell cycle regulation, cytoskeletal regulation, and trafficking. Cilia proteins have been observed in the nucleus, at the Golgi apparatus, and even in immune synapses of T cells (interestingly, a non-ciliated cell). Given the abundance of extraciliary sites and functions, it can be difficult to definitively attribute an observed phenotype solely to defective cilia rather than to some defective extraciliary function or a combination of both. Thus, extraciliary sites and functions of cilia proteins need to be considered, as well as experimentally determined. Through such consideration, we will understand the true role of the primary cilium in disease as compared to other cellular processes' influences in mediating disease (or through a combination of both). Here, we review a compilation of known extraciliary sites and functions of "cilia" proteins as a means to demonstrate the potential non-ciliary roles for these proteins.

Primary mucinous carcinoma with rhabdoid cells of the thyroid gland: a case report.

PubMed

Matsuo, Mioko; Tuneyoshi, Masazumi; Mine, Mari

2016-06-10

Primary mucinous carcinoma of the thyroid gland is a rare disease; only 6 cases of primary mucinous carcinoma of the thyroid have been previously reported. Primary mucinous carcinoma of the thyroid gland with incomplete tumor resection tends to be associated with a poor prognosis, resulting in death within a few months. An early and appropriate diagnosis may contribute to improvement in patient prognosis; however, it is extremely difficult to diagnose primary mucinous carcinoma of the thyroid. We present the seventh reported case of primary mucinous carcinoma in the thyroid gland; moreover, rhabdoid cells were detected, which, to our knowledge, is a novel finding. An 81-year-old Japanese woman was initially diagnosed with a poorly differentiated thyroid carcinoma, and she underwent a hemithyroidectomy. Pathological examination revealed the presence of abundant mucus and agglomeration of large atypical cells. Rhabdoid cells were also seen scattered among the tumor cells. Immunostaining was performed for various markers, and on the basis of these results, we diagnosed the lesion as primary mucinous carcinoma with rhabdoid cells in the thyroid gland. Ten months after surgery, recurrence was noted in the paratracheal lymph nodes; therefore, total resection of the residual thyroid gland and paratracheal lymphadenectomy with thyroid-stimulating hormone suppression were performed. The patient is currently alive and disease-free. The current case is of interest not only because of the rare histological findings, but also because the patient achieved long-term survival following diagnosis of a mucinous carcinoma. We believe this report will be helpful for diagnosing future cases of mucinous carcinoma of the thyroid.

Discontinuing Pneumocystis jirovecii Pneumonia Prophylaxis in HIV-Infected Patients With a CD4 Cell Count <200 cells/mm3.

PubMed

Sidhu, Vaninder K; Foisy, Michelle M; Hughes, Christine A

2015-12-01

To review the evidence for discontinuing primary and secondary Pneumocystis jirovecii pneumonia (PJP) prophylaxis in HIV-infected patients with a CD4 count <200 cells/mm(3). We conducted a literature search in MEDLINE, EMBASE, Cochrane Library, Google Scholar, and the International Aids Society Library (up to August 2015) using the following key search terms: Pneumocystis jirovecii, pneumonia, human immunodeficiency virus, primary prophylaxis, secondary prophylaxis, and discontinuation. All English-language studies that evaluated discontinuation of primary and/or secondary PJP prophylaxis in HIV-infected patients with CD4 count <200 cells/mm(3) were included. Five studies were identified, which varied in design, sample size, outcomes, and duration of follow-up. Three studies examined discontinuation of primary and secondary PJP prophylaxis; 1 study evaluated discontinuing primary PJP prophylaxis; and 1 study evaluated stopping secondary PJP prophylaxis. Two out of the 5 studies pooled data for all opportunistic infections. Overall, there was a low incidence of PJP among HIV-infected patients who discontinued primary PJP prophylaxis and were well controlled on antiretroviral therapy (ART). Discontinuation of primary PJP prophylaxis appears to be safe in patients on combination ART with a suppressed HIV viral load and a CD4 count >100 cells/mm(3). Additional data are needed to support the safety of discontinuing secondary PJP prophylaxis. Decisions to discontinue PJP prophylaxis in patients with a CD4 count <200 cells/mm(3) should be done on an individual patient basis, taking into consideration clinical factors, including ongoing adherence to ART. © The Author(s) 2015.

Molluscan cells in culture: primary cell cultures and cell lines

PubMed Central

Yoshino, T. P.; Bickham, U.; Bayne, C. J.

2013-01-01

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

COMPARISON OF PM-INDUCED GENE EXPRESSION PROFILES BETWEEN BRONCHIAL EPITHELIAL CELLS AND NASAL EPITHELIAL CELLS IN HUMAN

EPA Science Inventory

Epidemiologic studies have linked exposures to particulate matter (PM) and increased pulmonary mortality and morbidity. Bronchial epithelial cells (BEC) are the primary target of PM. PM exposure induces a wide array of biological responses in BEC. Primary human BEC, however, need...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

PubMed Central

DiBona, Victoria L.; Wu, Qian; Zhang, Huaye

2013-01-01

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells. PMID:24300106

Cranberry Products Inhibit Adherence of P-Fimbriated Escherichia Coli to Primary Cultured Bladder and Vaginal Epithelial Cells

PubMed Central

Gupta, K.; Chou, M. Y.; Howell, A.; Wobbe, C.; Grady, R.; Stapleton, A. E.

2011-01-01

Purpose Cranberry proanthocyanidins have been identified as possible inhibitors of Escherichia coli adherence to uroepithelial cells. However, little is known about the dose range of this effect. Furthermore, it has not been studied directly in the urogenital system. To address these issues we tested the effect of a cranberry powder and proanthocyanidin extract on adherence of a P-fimbriated uropathogenic E. coli isolate to 2 new urogenital model systems, namely primary cultured bladder epithelial cells and vaginal epithelial cells. Materials and Methods E. coli IA2 was pre-incubated with a commercially available cranberry powder (9 mg proanthocyanidin per gm) or with increasing concentrations of proanthocyanidin extract. Adherence of E. coli IA2 to primary cultured bladder epithelial cells or vaginal epithelial cells was measured before and after exposure to these products. Results Cranberry powder decreased mean adherence of E. coli IA2 to vaginal epithelial cells from 18.6 to 1.8 bacteria per cell (p <0.001). Mean adherence of E. coli to primary cultured bladder epithelial cells was decreased by exposure to 50 μg/ml proanthocyanidin extract from 6.9 to 1.6 bacteria per cell (p <0.001). Inhibition of adherence of E. coli by proanthocyanidin extract occurred in linear, dose dependent fashion over a proanthocyanidin concentration range of 75 to 5 μg/ml. Conclusions Cranberry products can inhibit E. coli adherence to biologically relevant model systems of primary cultured bladder and vaginal epithelial cells. This effect occurs in a dose dependent relationship. These findings provide further mechanistic evidence and biological plausibility for the role of cranberry products for preventing urinary tract infection. PMID:17509358

The Neuroprotective Potential of Cyanidin-3-glucoside Fraction Extracted from Mulberry Following Oxygen-glucose Deprivation.

PubMed

Bhuiyan, Mohammad Iqbal Hossain; Kim, Hyun-Bok; Kim, Seong Yun; Cho, Kyung-Ok

2011-12-01

In this study, cyanidin-3-glucoside (C3G) fraction extracted from the mulberry fruit (Morus alba L.) was investigated for its neuroprotective effects against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Cell membrane damage and mitochondrial function were assessed by LDH release and MTT reduction assays, respectively. A time-course study of OGD-induced cell death of primary cortical neurons at 7 days in vitro (DIV) indicated that neuronal death was OGD duration-dependent. It was also demonstrated that OGD for 3.5 h resulted in approximately 50% cell death, as determined by the LDH release assay. Treatments with mulberry C3G fraction prevented membrane damage and preserved the mitochondrial function of the primary cortical neurons exposed to OGD for 3.5 h in a concentration-dependent manner. Glutamate-induced cell death was more pronounced in DIV-9 and DIV-11 cells than that in DIV-7 neurons, and an application of 50µM glutamate was shown to induce approximately 40% cell death in DIV-9 neurons. Interestingly, treatment with mulberry C3G fraction did not provide a protective effect against glutamate-induced cell death in primary cortical neurons. On the other hand, treatment with mulberry C3G fraction maintained the mitochondrial membrane potential (MMP) in primary cortical neurons exposed to OGD as assessed by the intensity of rhodamine-123 fluorescence. These results therefore suggest that the neuroprotective effects of mulberry C3G fraction are mediated by the maintenance of the MMP and mitochondrial function but not by attenuating glutamate-induced excitotoxicity in rat primary cortical neurons.

Uniformity under in vitro conditions: Changes in the phenotype of cancer cell lines derived from different medulloblastoma subgroups.

PubMed

Chlapek, Petr; Zitterbart, Karel; Kren, Leos; Filipova, Lenka; Sterba, Jaroslav; Veselska, Renata

2017-01-01

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

Establishment and characterization of a telomerase immortalized porcine luteal cells.

PubMed

Zhang, Liang; Huang, Yong; Wang, Zhenyu; Luo, Xiaomao; Zhang, Hongling; Du, Qian; Chang, Lingling; Zhao, Xiaomin; Tong, Dewen

2017-05-01

Luteal cells play a crucial role in pregnancy through secreting progesterone to maintain pregnancy and support of fetus. However, low cellular yields and inability to passage primary porcine luteal cells (PLCs) in vitro limit the luteal cell study. Therefore, developing an immortalized porcine luteal cell line is necessary for studying luteal cells activity and function in different diseases. In this study, primary PLCs were obtained from gilts at day 30 to day 50 of gestation and immortalized by human telomerase reverse transcriptase (hTERT). The porcine corpus luteal cell line (hTERT-PLCs) expressed hTERT gene steady, maintained high hTERT activity and normal karyotype. The phase contrast microscope and transmission electron microscope observation showed primary PLCs and hTERT-PLCs were polygonal and exhibited abundant mitochondria, smooth endoplasmic reticulum and lipid droplets. 3β hydroxysteroid dehydrogenase (3βHSD) and Oil-Red-O staining showed that hTERT-PLCs at passage 30 and 50 were similar to primary PLCs. The hTERT-PLCs expressed steroidogenesis-related proteins, enzymes and receptors, such as steroidogenic acute regulatory protein, P450 cholesterol side-chain cleavage, 3βHSD, 20αHSD, luteinizing hormone receptor, progesterone receptor, prolactin receptor, estrogen receptorα/β, as well as primary PLCs. Consequently, hTERT-PLCs could secret progesterone and exhibited similar responses to luteinizing hormone and prostaglandin F2α as primary PLCs. In addition, the hTERT-PLCs did not show neoplastic transformation or anchorage independent growth. In summary, we developed an immortalized porcine luteal cell line which maintained its originally morphological, biological and functional characteristics. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential expression of c-Met between primary and metastatic sites in clear-cell renal cell carcinoma and its association with PD-L1 expression.

PubMed

Lalani, Aly-Khan A; Gray, Kathryn P; Albiges, Laurence; Callea, Marcella; Pignon, Jean-Christophe; Pal, Soumitro; Gupta, Mamta; Bhatt, Rupal S; McDermott, David F; Atkins, Michael B; Woude, G F Vande; Harshman, Lauren C; Choueiri, Toni K; Signoretti, Sabina

2017-11-28

In preclinical models, c-Met promotes survival of renal cancer cells through the regulation of programmed death-ligand 1 (PD-L1). However, this relationship in human clear cell renal cell carcinoma (ccRCC) is not well characterized. We evaluated c-Met expression in ccRCC patients using paired primary and metastatic samples and assessed the association with PD-L1 expression and other clinical features. Areas with predominant and highest Fuhrman nuclear grade (FNG) were selected. c-Met expression was evaluated by IHC using an anti-Met monoclonal antibody (MET4 Ab) and calculated by a combined score (CS, 0-300): intensity of c-Met staining (0-3) x % of positive cells (0-100). PD-L1 expression in tumor cells was previously assessed by IHC and PD-L1+ was defined as PD-L1 > 0% positive cells. Our cohort consisted of 45 pairs of primary and metastatic ccRCC samples. Overall, c-Met expression was higher in metastatic sites compared to primary sites (average c-Met CS: 55 vs. 28, p = 0.0003). Higher c-Met expression was associated with higher FNG (4 vs. 3) in primary tumors (average c-Met CS: 52 vs. 20, p = 0.04). c-Met expression was numerically greater in PD-L1+ vs. PD-L1- tumors. Higher c-Met expression in metastatic sites compared to primary tumors suggests that testing for biomarkers of response to c-Met inhibitors should be conducted in metastases. While higher c-Met expression in PD-L1+ tumors requires further investigation, it supports exploring these targets in combination clinical trials.

Primary diffuse large B cell lymphoma arising from a leiomyoma of the uterine corpus.

PubMed

Zhao, Lianhua; Ma, Qiang; Wang, Qiushi; Zeng, Ying; Luo, Qingya; Xiao, Hualiang

2016-01-20

Primary diffuse large B cell lymphoma (DLBCL) of the uterus is rare, and primary DLBCL arising from a uterine leiomyoma (collision tumor) has not been reported in the literature. We describe the clinical, histological, immunohistochemical, and molecular features of primary DLBCL arising from a leiomyoma in the uterine corpus. A 73-year-old female patient had a uterine mass for 23 years. An ultrasound scan revealed marked enlargement of the uterus, measuring 18.2 × 13 × 16.3 cm, with a 17.6 × 10.9 × 11.6 cm hypoechoic mass in the uterine corpus. The tumors consisted of medium- to large-sized cells exhibiting a diffuse pattern of growth with a well-circumscribed leiomyoma. The neoplastic cells strongly expressed CD79α, CD20 and PAX5. Molecular analyses indicated clonal B-cell receptor gene rearrangement. To the best of our knowledge, no previous cases of primary DLBCL arising from a leiomyoma have been reported. It is necessary to differentiate a diagnosis of primary DLBCL arising from a leiomyoma from that of leiomyoma with florid reactive lymphocytic infiltration (lymphoma-like lesion). Careful analysis of clinical, histological, immunophenotypic, and genetic features is required to establish the correct diagnosis.

Induced Pluripotent Stem Cells: A novel frontier in the study of human primary immunodeficiencies

PubMed Central

Pessach, Itai M.; Ordovas-Montanes, Jose; Zhang, Shen-Ying; Casanova, Jean-Laurent; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Manos, Philip D.; Schlaeger, Thorsten M.; Park, In-Hyun; Rucci, Francesca; Agarwal, Suneet; Mostoslavsky, Gustavo; Daley, George Q.; Notarangelo, Luigi D.

2010-01-01

Background The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells through the exogenous expression of transcription promises to revolutionize the study of human diseases. Objective Here we report on the generation of 25 induced pluripotent stem cell lines from 6 patients with various forms of Primary Immunodeficiencies, affecting adaptive and/or innate immunity. Methods Patients’ dermal fibroblasts were reprogrammed by expression of four transcription factors, OCT4, SOX2, KLF4, and c-MYC using a single excisable polycistronic lentiviral vector. Results Induced pluripotent stem cells derived from patients with primary immunodeficiencies show a stemness profile that is comparable to that observed in human embryonic stem cells. Following in vitro differentiation into embryoid bodies, pluripotency of the patient-derived indiced pluripotent stem cells lines was demonstrated by expression of genes characteristic of each of the three embryonic layers. We have confirmed the patient-specific origin of the induced pluripotent stem cell lines, and ascertained maintenance of karyotypic integrity. Conclusion By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of induced pluripotent stem cell lines from patients with primary immunodeficiencies represents a unique resource to investigate the pathophysiology of hematopoietic and extra-hematopoietic manifestations of these diseases, and may assist in the development of novel therapeutic approaches based on gene correction. PMID:21185069

Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells.

PubMed

Nylund, Reetta; Kuster, Niels; Leszczynski, Dariusz

2010-10-18

Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in expression of numerous proteins. However, exposure of EA.hy926 cells to 1800 MHz GSM signal had only very small effect on cell proteome, as compared with 900 MHz GSM exposure. In the present study, using as model human primary endothelial cells, we have examined whether exposure to 1800 MHz GSM mobile phone radiation can affect cell proteome. Primary human umbilical vein endothelial cells and primary human brain microvascular endothelial cells were exposed for 1 hour to 1800 MHz GSM mobile phone radiation at an average specific absorption rate of 2.0 W/kg. The cells were harvested immediately after the exposure and the protein expression patterns of the sham-exposed and radiation-exposed cells were examined using two dimensional difference gel electrophoresis-based proteomics (2DE-DIGE). There were observed numerous differences between the proteomes of human umbilical vein endothelial cells and human brain microvascular endothelial cells (both sham-exposed). These differences are most likely representing physiological differences between endothelia in different vascular beds. However, the exposure of both types of primary endothelial cells to mobile phone radiation did not cause any statistically significant changes in protein expression. Exposure of primary human endothelial cells to the mobile phone radiation, 1800 MHz GSM signal for 1 hour at an average specific absorption rate of 2.0 W/kg, does not affect protein expression, when the proteomes were examined immediately after the end of the exposure and when the false discovery rate correction was applied to analysis. This observation agrees with our earlier study showing that the 1800 MHz GSM radiation exposure had only very limited effect on the proteome of human endothelial cell line EA.hy926, as compared with the effect of 900 MHz GSM radiation.

Pan-Genotype Hepatitis E Virus Replication in Stem Cell-Derived Hepatocellular Systems.

PubMed

Wu, Xianfang; Dao Thi, Viet Loan; Liu, Peng; Takacs, Constantin N; Xiang, Kuanhui; Andrus, Linda; Gouttenoire, Jérôme; Moradpour, Darius; Rice, Charles M

2018-02-01

The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Intracerebral transplants of primary muscle cells: a potential 'platform' for transgene expression in the brain

NASA Technical Reports Server (NTRS)

Jiao, S.; Schultz, E.; Wolff, J. A.

1992-01-01

After the transplantation of rat primary muscle cells into the caudate or cortex of recipient rats, the muscle cells were able to persist for at least 6 months. Muscle cells transfected with expression plasmids prior to transplantation were able to express reporter genes in the brains for at least 2 months. These results suggest that muscle cells might be a useful 'platform' for transgene expression in the brain.

Paclitaxel and Carboplatin With or Without Bevacizumab in Treating Patients With Stage II, Stage III, or Stage IV Ovarian Epithelial Cancer, Primary Peritoneal Cancer, or Fallopian Tube Cancer

ClinicalTrials.gov

2017-12-28

Fallopian Tube Endometrioid Adenocarcinoma; Fallopian Tube Mucinous Adenocarcinoma; Fallopian Tube Transitional Cell Carcinoma; Malignant Ovarian Mixed Epithelial Tumor; Ovarian Brenner Tumor; Ovarian Clear Cell Adenocarcinofibroma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mucinous Adenocarcinoma; Ovarian Serous Adenocarcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Stage IIA Fallopian Tube Cancer; Stage IIA Ovarian Cancer; Stage IIB Fallopian Tube Cancer; Stage IIB Ovarian Cancer; Stage IIC Fallopian Tube Cancer; Stage IIC Ovarian Cancer; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Undifferentiated Ovarian Carcinoma

Genetics of Primary Intraocular Tumors

PubMed Central

Nagarkatti-Gude, Nisha; Wang, Yujuan; Ali, Mohammad Javed; Honavar, Santosh G.; Jager, Martine J.; Chan, Chi-Chao

2012-01-01

Primary intraocular neoplasms are tumors that originate within the eye. The most common malignant primary intraocular tumor in adults is uveal melanoma and the second is primary intraocular lymphoma or vitreoretinal (intraocular) lymphoma. The most common malignant intraocular tumor in children is retinoblastoma. Genetics plays a vital role in the diagnosis and detection of ocular tumors. In uveal melanoma, monosomy 3 is the most common genetic alteration and somatic mutations of BAP1, a tumor suppressor gene, have been reported in nearly 50% of primary uveal melanomas. The retinoblastoma gene RB1 is the prototype tumor suppressor gene—mutations in RB1 alleles lead to inactivated RB protein and the development of retinoblastoma. Immunoglobulin heavy chain (IgH) or T-cell receptor (TCR) gene rearrangement is observed in B-cell or T-cell primary vitreoretinal lymphoma, respectively. Other factors related to the genetics of these three common malignancies in the eye are discussed and reviewed. PMID:22834783

T-Cell Tropism of Simian Varicella Virus during Primary Infection

PubMed Central

Ouwendijk, Werner J. D.; Mahalingam, Ravi; de Swart, Rik L.; Haagmans, Bart L.; van Amerongen, Geert; Getu, Sarah; Gilden, Don; Osterhaus, Albert D. M. E.; Verjans, Georges M. G. M.

2013-01-01

Varicella-zoster virus (VZV) causes varicella, establishes a life-long latent infection of ganglia and reactivates to cause herpes zoster. The cell types that transport VZV from the respiratory tract to skin and ganglia during primary infection are unknown. Clinical, pathological, virological and immunological features of simian varicella virus (SVV) infection of non-human primates parallel those of primary VZV infection in humans. To identify the host cell types involved in virus dissemination and pathology, we infected African green monkeys intratracheally with recombinant SVV expressing enhanced green fluorescent protein (SVV-EGFP) and with wild-type SVV (SVV-wt) as a control. The SVV-infected cell types and virus kinetics were determined by flow cytometry and immunohistochemistry, and virus culture and SVV-specific real-time PCR, respectively. All monkeys developed fever and skin rash. Except for pneumonitis, pathology produced by SVV-EGFP was less compared to SVV-wt. In lungs, SVV infected alveolar myeloid cells and T-cells. During viremia the virus preferentially infected memory T-cells, initially central memory T-cells and subsequently effector memory T-cells. In early non-vesicular stages of varicella, SVV was seen mainly in perivascular skin infiltrates composed of macrophages, dendritic cells, dendrocytes and memory T-cells, implicating hematogenous spread. In ganglia, SVV was found primarily in neurons and occasionally in memory T-cells adjacent to neurons. In conclusion, the data suggest the role of memory T-cells in disseminating SVV to its target organs during primary infection of its natural and immunocompetent host. PMID:23675304

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

PubMed Central

Ulm, Ashley; Mayhew, Christopher N.; Debley, Jason; Khurana Hershey, Gurjit K.; Ji, Hong

2016-01-01

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research. PMID:27022951

Self-targeting of TNF-releasing cancer cells in preclinical models of primary and metastatic tumors.

PubMed

Dondossola, Eleonora; Dobroff, Andrey S; Marchiò, Serena; Cardó-Vila, Marina; Hosoya, Hitomi; Libutti, Steven K; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2016-02-23

Circulating cancer cells can putatively colonize distant organs to form metastases or to reinfiltrate primary tumors themselves through a process termed "tumor self-seeding." Here we exploit this biological attribute to deliver tumor necrosis factor alpha (TNF), a potent antitumor cytokine, directly to primary and metastatic tumors in a mechanism that we have defined as "tumor self-targeting." For this purpose, we genetically engineered mouse mammary adenocarcinoma (TSA), melanoma (B16-F10), and Lewis lung carcinoma cells to produce and release murine TNF. In a series of intervention trials, systemic administration of TNF-expressing tumor cells was associated with reduced growth of both primary tumors and metastatic colonies in immunocompetent mice. We show that these malignant cells home to tumors, locally release TNF, damage neovascular endothelium, and induce massive cancer cell apoptosis. We also demonstrate that such tumor-cell-mediated delivery avoids or minimizes common side effects often associated with TNF-based therapy, such as acute inflammation and weight loss. Our study provides proof of concept that genetically modified circulating tumor cells may serve as targeted vectors to deliver anticancer agents. In a clinical context, this unique paradigm represents a personalized approach to be translated into applications potentially using patient-derived circulating tumor cells as self-targeted vectors for drug delivery.

Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

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

Gu Yongpeng; Li Hongzhen; Miki, Jun

2006-04-01

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferativemore » capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.« less

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

PubMed

Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

2016-03-10

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

Treatment of Primary Cutaneous CD4 Small/Medium T cell Lymphoproliferative Disorder with Intralesional Triamcinolone Acetonide.

DTIC Science & Technology

2018-02-15

12. REPORT TYPE 02/15/2018 Poster 4. TITLE AND SUBTITLE Treatment of Primary Cutaneous CD4+ Small/Medium T- cell Lymphoproliferative Disorder with...cutaneous CD4+ small/medium T- cell lymphoproliferative disorder (LPD) is a generally indolent cutaneous T- cell proliferation. Most cases follow a benign...lmmunohistochemistry showed diffuse CD3+ CD4+ T- cells without CD30, TIA1 or CD10. A subset of medium to large cells expressed BCL-6. Small subsets of B- cells and CDB

Veliparib, Cisplatin, and Gemcitabine Hydrochloride in Treating Patients With Advanced Biliary, Pancreatic, Urothelial, or Non-Small Cell Lung Cancer

ClinicalTrials.gov

2013-07-01

Advanced Adult Primary Liver Cancer; Localized Unresectable Adult Primary Liver Cancer; Metastatic Transitional Cell Cancer of the Renal Pelvis and Ureter; Regional Transitional Cell Cancer of the Renal Pelvis and Ureter; Stage III Bladder Cancer; Stage III Pancreatic Cancer; Stage IIIA Non-small Cell Lung Cancer; Stage IIIB Non-small Cell Lung Cancer; Stage IV Bladder Cancer; Stage IV Non-small Cell Lung Cancer; Stage IV Pancreatic Cancer; Transitional Cell Carcinoma of the Bladder; Unresectable Extrahepatic Bile Duct Cancer; Unresectable Gallbladder Cancer

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours.

PubMed

Bate-Eya, Laurel T; Ebus, Marli E; Koster, Jan; den Hartog, Ilona J M; Zwijnenburg, Danny A; Schild, Linda; van der Ploeg, Ida; Dolman, M Emmy M; Caron, Huib N; Versteeg, Rogier; Molenaar, Jan J

2014-02-01

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models. Copyright © 2013 Elsevier Ltd. All rights reserved.

B cell lymphoma with lung involvement: what is it about?

PubMed

Mian, Michael; Wasle, Ines; Gritsch, Stefan; Willenbacher, Wolfgang; Fiegl, Michael

2015-01-01

Primary lymphoma of the lung or pleural is a very rare condition. Due to the outdated literature data, the approximate occurrence of primary and secondary lung and/or pleural involvement according to the most common B cell lymphoma entities is unknown. To answer this open question in Austria, we screened the Tyrolean registry for B cell non-Hodgkin's lymphomas regarding primary and secondary lung involvement. Of 854 patients affected by B cell lymphoma, 7.5% had lung/pleural disease. This organ was the primary site in only 0.7%, while a secondary involvement was registered in 6.8%. Most of them were affected by diffuse large B cell lymphoma (DLBCL; 29/368, 8%) followed by follicular lymphoma (7/188, 4%), mantle cell lymphoma (7/57, 12%), mucosa-associated tissue lymphoma (10/37, 27%), posttransplant lymphoproliferative disease (6/24, 25%), Burkitt lymphoma (3/19, 16%), other lymphomas (1/32, 3%) and Richter transformation (1/11, 9%). Moreover, primary lung/pleural lymphoma is one of the rarest neoplasias affecting the lung, accounting for only 0.4% of cases. Lung/pleural involvement is a very rare condition among B cell lymphomas since it mainly occurs in the setting of a generalized disease. A large majority of patients with secondary organ involvement are affected by DLBCL and have similar clinical features at diagnosis to others with advanced-stage disease. © 2014 S. Karger AG, Basel.

Is a Preoperative Gastrointestinal Endoscopy for Second Primary Cancer Detection in Head and Neck Cancer Necessary? Ten-year Registry Data.

PubMed

Heo, Gyeong Mi; Kim, Mi Hee; Kim, Jin Hwan; Rho, Young Soo; Shin, Woon Geon

2016-07-25

In head and neck squamous cell carcinoma, second primary gastrointestinal tumors are not uncommon. However, it is unclear whether a screening endoscopy is needed for detecting gastrointestinal neoplasm in patients with head and neck cancer. Therefore, we analyzed the prevalence and independent risk factors for second primary gastrointestinal neoplasm in head and neck squamous cell carcinoma. A consecutive series of 328 patients with primary head and neck squamous cell carcinoma that underwent esophagogastroduodenoscopy or colonoscopy were included using our registry. An age- and sex-matched group of 328 control subjects was enrolled. We assessed risk factors of synchronous gastrointestinal cancer. The prevalence of esophageal cancer with head and neck squamous cell carcinoma was significantly higher than that of the control group (1.5% vs. 0.0%, p=0.011). An age of 54 years or more (OR, 1.033; 95% CI, 1.008-1.059; p=0.009) and male gender (OR, 4.974; 95% CI, 1.648-15.013; p=0.004) were risk factors for concomitant colorectal cancer or adenomas in the head and neck squamous cell carcinoma patients. Preoperative colonoscopy can be recommended for detecting synchronous second primary colorectal lesions in head and neck squamous cell carcinoma patients with male sex regardless of age, and esophagogastroduodenoscopy is necessary in all head and neck squamous cell carcinoma patients for detecting esophageal cancer.

Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway

PubMed Central

Dai, Jie-Min; Yu, Mu-Xue; Shen, Zhen-Yu; Guo, Chu-Yi; Zhuang, Si-Qi; Qiu, Xiao-Shan

2015-01-01

Signaling through the mammalian target of rapamycin (mTOR) in response to leucine modulates many cellular and developmental processes. However, in the context of satellite cell proliferation and differentiation, the role of leucine and mTORC1 is less known. This study investigates the role of leucine in the process of proliferation and differentiation of primary preterm rat satellite cells, and the relationship with mammalian target of rapamycin complex 1 (mTORC1) activation. Dissociation of primary satellite cells occurred with type I collagenase and trypsin, and purification, via different speed adherence methods. Satellite cells with positive expression of Desmin were treated with leucine and rapamycin. We observed that leucine promoted proliferation and differentiation of primary satellite cells and increased the phosphorylation of mTOR. Rapamycin inhibited proliferation and differentiation, as well as decreased the phosphorylation level of mTOR. Furthermore, leucine increased the expression of MyoD and myogenin while the protein level of MyoD decreased due to rapamycin. However, myogenin expressed no affect by rapamycin. In conclusion, leucine may up-regulate the activation of mTORC1 to promote proliferation and differentiation of primary preterm rat satellite cells. We have shown that leucine promoted the differentiation of myotubes in part through the mTORC1-MyoD signal pathway. PMID:26007333

EF5 and Motexafin Lutetium in Detecting Tumor Cells in Patients With Abdominal or Non-Small Cell Lung Cancer

ClinicalTrials.gov

2013-01-15

Advanced Adult Primary Liver Cancer; Carcinoma of the Appendix; Fallopian Tube Cancer; Gastrointestinal Stromal Tumor; Localized Extrahepatic Bile Duct Cancer; Localized Gallbladder Cancer; Localized Gastrointestinal Carcinoid Tumor; Localized Resectable Adult Primary Liver Cancer; Localized Unresectable Adult Primary Liver Cancer; Metastatic Gastrointestinal Carcinoid Tumor; Ovarian Sarcoma; Ovarian Stromal Cancer; Primary Peritoneal Cavity Cancer; Recurrent Adult Primary Liver Cancer; Recurrent Adult Soft Tissue Sarcoma; Recurrent Colon Cancer; Recurrent Extrahepatic Bile Duct Cancer; Recurrent Gallbladder Cancer; Recurrent Gastric Cancer; Recurrent Gastrointestinal Carcinoid Tumor; Recurrent Non-small Cell Lung Cancer; Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Recurrent Pancreatic Cancer; Recurrent Rectal Cancer; Recurrent Small Intestine Cancer; Recurrent Uterine Sarcoma; Regional Gastrointestinal Carcinoid Tumor; Small Intestine Adenocarcinoma; Small Intestine Leiomyosarcoma; Small Intestine Lymphoma; Stage 0 Non-small Cell Lung Cancer; Stage I Adult Soft Tissue Sarcoma; Stage I Colon Cancer; Stage I Gastric Cancer; Stage I Non-small Cell Lung Cancer; Stage I Ovarian Epithelial Cancer; Stage I Ovarian Germ Cell Tumor; Stage I Pancreatic Cancer; Stage I Rectal Cancer; Stage I Uterine Sarcoma; Stage II Adult Soft Tissue Sarcoma; Stage II Colon Cancer; Stage II Gastric Cancer; Stage II Non-small Cell Lung Cancer; Stage II Ovarian Epithelial Cancer; Stage II Ovarian Germ Cell Tumor; Stage II Pancreatic Cancer; Stage II Rectal Cancer; Stage II Uterine Sarcoma; Stage III Adult Soft Tissue Sarcoma; Stage III Colon Cancer; Stage III Gastric Cancer; Stage III Ovarian Epithelial Cancer; Stage III Ovarian Germ Cell Tumor; Stage III Pancreatic Cancer; Stage III Rectal Cancer; Stage III Uterine Sarcoma; Stage IIIA Non-small Cell Lung Cancer; Stage IIIB Non-small Cell Lung Cancer; Stage IV Adult Soft Tissue Sarcoma; Stage IV Colon Cancer; Stage IV Gastric Cancer; Stage IV Non-small Cell Lung Cancer; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Pancreatic Cancer; Stage IV Rectal Cancer; Stage IV Uterine Sarcoma; Unresectable Extrahepatic Bile Duct Cancer; Unresectable Gallbladder Cancer

Evidence against Resveratrol as a viable therapy for the rescue of defective ΔF508 CFTR

PubMed Central

Jai, Ying; Shah, Kalpit; Bridges, Robert J.; Bradbury, Neil A.

2015-01-01

BACKGROUND Resveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations. METHODS Cells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers. RESULTS Consistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol CONCLUSIONS High concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents. PMID:26342647

Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells

PubMed Central

McMurray, R. J.; Wann, A. K. T.; Thompson, C. L.; Connelly, J. T.; Knight, M. M.

2013-01-01

The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation. PMID:24346024

In vitro and in vivo transfection of primary phagocytes via microbubble-mediated intraphagosomal sonoporation.

PubMed

Lemmon, Jason C M; McFarland, Ryan J; Rybicka, Joanna M; Balce, Dale R; McKeown, Kyle R; Krohn, Regina M; Matsunaga, Terry O; Yates, Robin M

2011-08-31

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies. Copyright © 2011 Elsevier B.V. All rights reserved.

The isolation and characterization of a telomerase immortalized goat trophoblast cell line.

PubMed

Dong, F; Huang, Y; Li, W; Zhao, X; Zhang, W; Du, Q; Zhang, H; Song, X; Tong, D

2013-12-01

Trophoblast cells play vital roles in the processes of embryonic implantation and placentation. Many toxicological compounds can induce the malfunction of trophoblast cells, resulting in implantation failure or early embryonic loss. The finite lifespan of primary trophoblast cells limits investigation of the long-term effects of some toxicological compounds on trophoblast cells in vitro. In this study, primary goat trophoblast cells were purified by density gradient centrifugation and specific immuno-affinity purification. Then, the purified cells were immortalized through transfection of a plasmid containing the human telomerase reverse transcriptase (hTERT) gene. hTERT-transfected goat trophoblast cells (hTERT-GTCs) could steadily express hTERT gene and exhibit higher telomerase activity, and persistently proliferate without any signs of senescence up to 50 passages. The immortalized goat trophoblast cells still possessed the basic and key properties of normal primary goat trophoblast cells to express the specific intracellular marker cytokeratin 7 (CK-7) and secrete chorionic gonadotrophin β-subunit (CG-β) and placental lactogen (PL). Further studies showed that the immortalized goat trophoblast cells expressed vimentin and non-classical MHC class I antigen and exhibited invasive phenotype, suggesting that the immortalized goat trophoblasts resembled human extravillous trophoblasts. In addition, this cell line did not show neoplastic transformation either in vivo or in vitro. We concluded the immortalized goat trophoblast cells by hTERT transfection retained the basic and key characteristics of primary trophoblast cells and may provide a useful model to study the effects of some toxicological compounds on trophoblast cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Flavopiridol sensitivity of cancer cells isolated from ascites and pleural fluids.

PubMed

Richard, Christina; Matthews, Donald; Duivenvoorden, Wilhelmina; Yau, Jonathan; Wright, Paul S; Th'ng, John P H

2005-05-01

We examined the efficacy of flavopiridol, a cyclin-dependent kinase inhibitor that is undergoing clinical trials, on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. Metastasized cancer cells were isolated from the pleural fluids (n = 20) or ascites (n = 15) of patients, most of whom were refractory to chemotherapy. These primary cancer cells were used within 2 weeks of isolation without selecting for proliferative capacities. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay was used to characterize the response of these cancer cells to commonly used chemotherapeutic agents, and their response to flavopiridol was compared with rapidly dividing cultured cell lines. The primary cancer cells displayed phenotypes that were different from established cell lines; they had very low replication rates, dividing every 1 to 2 weeks, and underwent replicative senescence within five passages. These primary tumor cells retained their resistance to chemotherapeutic drugs exhibited by the respective patients but did not show cross-resistance to other agents. However, these cancer cells showed sensitivity to flavopiridol with an average LD50 of 50 nmol/L (range, 21.5-69 nmol/L), similar to the LD50 in established cell lines. Because senescent cells also showed similar sensitivity to flavopiridol, it suggests that the mechanism of action is not dependent on the activity of cyclin-dependent kinases that regulate the progression of the cell cycle. Using cancer cells isolated from the ascites or pleural fluids, this study shows the potential of flavopiridol against cancer cells that have developed resistance to conventional chemotherapeutic agents.

Cnidarian Primary Cell Culture as a Tool to Investigate the Effect of Thermal Stress at Cellular Level.

PubMed

Ventura, P; Toullec, G; Fricano, C; Chapron, L; Meunier, V; Röttinger, E; Furla, P; Barnay-Verdier, S

2018-04-01

In the context of global change, symbiotic cnidarians are largely affected by seawater temperature elevation leading to symbiosis breakdown. This process, also called bleaching, is triggered by the dysfunction of the symbiont photosystems causing an oxidative stress and cell death to both symbiont and host cells. In our study, we wanted to elucidate the intrinsic capacity of isolated animal cells to deal with thermal stress in the absence of symbiont. In that aim, we have characterized an animal primary cell culture form regenerating tentacles of the temperate sea anemone Anemonia viridis. We first compared the potential of whole tissue tentacle or separated epidermal or gastrodermal monolayers as tissue sources to settle animal cell cultures. Interestingly, only isolated cells extracted from whole tentacles allowed establishing a viable and proliferative primary cell culture throughout 31 days. The analysis of the expression of tissue-specific and pluripotency markers defined cultivated cells as differentiated cells with gastrodermal origin. The characterization of the animal primary cell culture allowed us to submit the obtained gastrodermal cells to hyperthermal stress (+ 5 and + 8 °C) during 1 and 7 days. Though cell viability was not affected at both hyperthermal stress conditions, cell growth drastically decreased. In addition, only a + 8 °C hyperthermia induced a transient increase of antioxidant defences at 1 day but no ubiquitin or carbonylation protein damages. These results demonstrated an intrinsic resistance of cnidarian gastrodermal cells to hyperthermal stress and then confirmed the role of symbionts in the hyperthermia sensitivity leading to bleaching.

Primary midgut, salivary gland, and ovary cultures from Boophilus microplus.

PubMed

Mosqueda, Juan; Cossío-Bayugar, Raquel; Rodríguez, Elba; Falcón, Alfonso; Ramos, Alberto; Figueroa, Julio V; Alvarez, Antonio

2008-12-01

Primary cell cultures from different tick organs are a valuable tool for host parasite research in the study of the protozoan Babesia sp., which infects different organs of the tick. In this work we describe the generation of midgut, salivary gland, and ovary primary cell cultures from dissections of Boophilus microplus. Midguts, salivary glands, and ovaries were dissected from B. microplus ticks on different days after bovine infestation; different enzymatic disaggregating protocols were tested in the presence of proteolytic enzymes, such as trypsin and collagenase type I and II, for tissue disaggregation and primary cell culture generation. The dissected tick organs obtained 18-20 days after bovine infestation showed a major cellular differentiation and were easier to identify by cellular morphology. The enzymatic disaggregation results showed that each tissue required a different proteolytic enzyme for optimal disaggregation; collagenase type I produced the most complete disaggregation for ovaries but not for midgut or salivary glands. Collagenase type II was effective for salivary glands but performed poorly on ovaries and midgets, and typsin was effective for midguts only. The midgut and ovary primary cell cultures were maintained for 4 weeks in optimal conditions after the cells were no longer viable. The salivary gland cell cultures were viable for 8 months.

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

PubMed

Liverani, Chiara; La Manna, Federico; Groenewoud, Arwin; Mercatali, Laura; Van Der Pluijm, Gabri; Pieri, Federica; Cavaliere, Davide; De Vita, Alessandro; Spadazzi, Chiara; Miserocchi, Giacomo; Bongiovanni, Alberto; Recine, Federica; Riva, Nada; Amadori, Dino; Tasciotti, Ennio; Snaar-Jagalska, Ewa; Ibrahim, Toni

2017-02-15

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays. © 2016. Published by The Company of Biologists Ltd.

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures.

PubMed

Petrenko, Volodymyr; Saini, Camille; Perrin, Laurent; Dibner, Charna

2016-11-11

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Promotion of haematopoietic activity in embryonic stem cells by the aorta-gonad-mesonephros microenvironment

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

Krassowska, Anna; Gordon-Keylock, Sabrina; Samuel, Kay

We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We concludemore » that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance.« less

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

Van der Hauwaert, Cynthia; Savary, Grégoire; Buob, David

Numerous xenobiotics have been shown to be harmful for the kidney. Thus, to improve our knowledge of the cellular processing of these nephrotoxic compounds, we evaluated, by real-time PCR, the mRNA expression level of 377 genes encoding xenobiotic-metabolizing enzymes (XMEs), transporters, as well as nuclear receptors and transcription factors that coordinate their expression in eight normal human renal cortical tissues. Additionally, since several renal in vitro models are commonly used in pharmacological and toxicological studies, we investigated their metabolic capacities and compared them with those of renal tissues. The same set of genes was thus investigated in HEK293 and HK2more » immortalized cell lines in commercial primary cultures of epithelial renal cells and in proximal tubular cell primary cultures. Altogether, our data offers a comprehensive description of kidney ability to process xenobiotics. Moreover, by hierarchical clustering, we observed large variations in gene expression profiles between renal cell lines and renal tissues. Primary cultures of proximal tubular epithelial cells exhibited the highest similarities with renal tissue in terms of transcript profiling. Moreover, compared to other renal cell models, Tacrolimus dose dependent toxic effects were lower in proximal tubular cell primary cultures that display the highest metabolism and disposition capacity. Therefore, primary cultures appear to be the most relevant in vitro model for investigating the metabolism and bioactivation of nephrotoxic compounds and for toxicological and pharmacological studies. - Highlights: • Renal proximal tubular (PT) cells are highly sensitive to xenobiotics. • Expression of genes involved in xenobiotic disposition was measured. • PT cells exhibited the highest similarities with renal tissue.« less

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase.

PubMed

Kim, H; You, S; Kong, B W; Foster, L K; Farris, J; Foster, D N

2001-08-22

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.

Computer simulation of thermal modeling of primary lithium cells

NASA Technical Reports Server (NTRS)

Young, I. Cho; Frank, Harvey; Halpert, Gersid

1987-01-01

The objective was to gain a better understanding of the safety problem of primary Li-SOCl2 and Li-SO2 cells by carrying out detailed thermal modeling work. In particular, the transient heat generation rates during moderate and extermely high discharge rate tests of Li-SOCl2 cells were predicted and compared with those from the electrochemical heating. The difference between the two may be attributed to the lithium corrosion and other chemical reactions. The present program was also tested for charging of Li-SO2. In addition, the present methodology should be applicable to other primary cylindrical cells as well as rechargeable battery analyses with minor modifications.

Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton.

PubMed

Yuan, Xue; Serra, Rosa A; Yang, Shuying

2015-01-01

Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes. © 2014 New York Academy of Sciences.

The role of cytokeratins 20 and 7 and estrogen receptor analysis in separation of metastatic lobular carcinoma of the breast and metastatic signet ring cell carcinoma of the gastrointestinal tract.

PubMed

Tot, T

2000-06-01

Metastatic signet ring cell carcinomas of unknown primary site can represent a clinical problem. Gastrointestinal signet ring cell carcinomas and invasive lobular carcinomas of the breast are the most common sources of these metastases. Immunohistochemical algorithms have been successfully used in the search for the unknown primary adenocarcinomas. In the present study a series of primary invasive lobular breast carcinomas (79 cases) and their metastases and a series of gastrointestinal signet ring cell carcinomas (22 primary and 13 metastases) were stained with monoclonal antibodies for cytokeratin (CK) 20 and CK7 and for estrogen receptors (ER). The staining was evaluated as negative (no staining), focally (less than 10% of the tumor cells stained) or diffusely positive. All the primary and metastatic gastrointestinal signet ring cell carcinomas proved to be CK20 positive, while only 2/79 (3%) of the primary and 1/21 metastatic lobular carcinomas (5%) stained positively for this CK. None of the gastrointestinal carcinomas and the majority of the lobular carcinomas expressed ER. The majority of the tumors were CK7+. Using CK20 alone, 33 of 34 metastases could be properly classified as gastrointestinal (CK20+) or mammary (CK20-). ER identified 31/34 of breast cancer metastases. By combining the results of CK20 and ER staining all the metastases could be properly classified as the CK20+/ER- pattern identified all the gastrointestinal tumors.

Dengue Virus Activates Polyreactive, Natural IgG B Cells after Primary and Secondary Infection

PubMed Central

Toh, Ying Xiu; Flamand, Marie; Devi, Shamala; Koh, Mickey B.; Hibberd, Martin L.; Ooi, Eng Eong; Low, Jenny G.; Leo, Yee Sin; Gu, Feng; Fink, Katja

2011-01-01

Background Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. Methodology/Principal Findings We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4–7 days after fever onset was more than 50% even after primary infection. Conclusions/Significance Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and “innate specificities” seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development. PMID:22216280

Hypermethylation of the TSLC1 Gene Promoter in Primary Gastric Cancers and Gastric Cancer Cell Lines

PubMed Central

Honda, Teiichiro; Waki, Takayoshi; Jin, Zhe; Sato, Kiyoshi; Motoyama, Teiichi; Kawata, Sumio; Kimura, Wataru; Nishizuka, Satoshi; Murakami, Yoshinori

2002-01-01

The TSLC1 (tumor suppressor in lung cancer–1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non‐small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non‐cancerous gastric tissues, by bisulfite‐SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO‐III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non‐cancerous gastric tissues, suggesting that this hypermethylation is a cancer‐specific alteration. KATO‐III and ECC10 cells retained two alleles of TSLC1, both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi‐allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers. PMID:12716461

Optimization of methods for the genetic modification of human T cells.

PubMed

Bilal, Mahmood Y; Vacaflores, Aldo; Houtman, Jon Cd

2015-11-01

CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells.

β-Arrestin2 mediates progression of murine primary myelofibrosis

PubMed Central

Rein, Lindsay A.M.; Wisler, James W.; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia K.L.; Premont, Richard T.; Lefkowitz, Robert J.

2017-01-01

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2–/–) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2–/– cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2–/– cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease. PMID:29263312

A Phase 1-2 Multi-Center Study Evaluating Axicabtagene Ciloleucel in Subjects With Refractory Aggressive Non-Hodgkin Lymphoma (ZUMA-1)

ClinicalTrials.gov

2018-06-18

Refractory Diffuse Large B Cell Lymphoma; Refractory Primary Mediastinal B Cell Lymphoma; Refractory Transformed Follicular Lymphoma; Relapsed/Refractory Transplant Ineligible Diffuse Large B Cell Lymphoma; Relapsed/Refractory Transplant Ineligible Primary Mediastinal B Cell Lymphoma; Relapsed/Refractory Transplant Ineligible Transformed Follicular Lymphoma; Relapsed/Refractory Large B Cell Lymphoma Including DLBCL, PMBCL, TFL and HGBCL After Two Systemic Lines of Therapy" in Phase 2 Expanded Cohorts

Microenvironmental Regulation of Mammary Carcinogenesis

DTIC Science & Technology

2009-06-01

cells, leukocytes 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON...metastatic spread to sentinel LNs and increased primary tumor size13. Perhaps more significant, the ratio of CD4+ to CD8+ T cells or TH2 to TH1 cells...present in primary tumors, where CD4+ or TH2 cells are more frequent than CD8+ or TH1 cells, correlates with LN metastasis and reduced overall patient

Patient-derived osteosarcoma cells are resistant to methotrexate.

PubMed

Dos Santos Cavalcanti, Amanda; Meohas, Walter; Ribeiro, Gabriele de Oliveira; de Sá Lopes, Ana Cristina; Gholamin, Sharareh; Razavi, Mostafa; Hanae Kasai Brunswick, Taís; Avan, Amir; Matheus Guimarães, João Antonio; Leite Duarte, Maria Eugenia; Kahn, Suzana Assad

2017-01-01

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas.

Patient-derived osteosarcoma cells are resistant to methotrexate

PubMed Central

dos Santos Cavalcanti, Amanda; Meohas, Walter; Ribeiro, Gabriele de Oliveira; de Sá Lopes, Ana Cristina; Gholamin, Sharareh; Razavi, Mostafa; Hanae Kasai Brunswick, Taís; Avan, Amir; Matheus Guimarães, João Antonio; Leite Duarte, Maria Eugenia

2017-01-01

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas. PMID:28934267

Mussel mimetic tissue adhesive for fetal membrane repair: initial in vivo investigation in rabbits.

PubMed

Kivelio, A; Dekoninck, P; Perrini, M; Brubaker, C E; Messersmith, P B; Mazza, E; Deprest, J; Zimmermann, R; Ehrbar, M; Ochsenbein-Koelble, N

2013-12-01

Iatrogenic preterm prelabour rupture of fetal membranes (iPPROM) remains the main complication after invasive interventions into the intrauterine cavity. The aim of this study was to evaluate the sealing capability and tissue interaction of mussel-mimetic tissue adhesive (mussel glue) in comparison to fibrin glue on punctured fetal membranes in vivo. A mid-gestational rabbit model was used for testing the materials. The fetal sacs of pregnant rabbits at day 23 were randomly assigned into experimental groups: unoperated (negative control), unclosed puncture (positive control), commercially available fibrin glue (FG) with decellularized amnion scaffold (DAM), mussel glue (MG) with DAM, or mussel glue alone. Evaluation was done at term (30 days' gestation) assessing fetal survival, fetal membrane integrity and histology of the membranes. Fetal survival was not significantly lower in any of the treatment groups compared to the negative control. All plugging materials could be found at the end of the pregnancy and no adverse effects on the fetus or the pregnant does could be observed. Sac integrity was higher in all treatment groups compared to the positive control group but significant only in the FG+DAM group. Cellular infiltration could be seen in fibrin glue and DAM in contrast to mussel glue which was only tightly adhering to the surrounding tissue. These cells were mostly of mesenchymal phenotype staining positive for vimentin. CD68 positive macrophages were found clustered around all the plugging materials, but their numbers were only significantly increased for the mussel glue alone group compared to negative controls. Mussel glues performance in sealing fetal membranes in the rabbit model was comparable to that of fibrin glue. Taking into account its other favorable properties, it is a noteworthy candidate for a clinically applicable fetal membrane sealant. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

NOS2 expression in glioma cell lines and glioma primary cell cultures: correlation with neurosphere generation and SOX-2 expression.

PubMed

Palumbo, Paola; Miconi, Gianfranca; Cinque, Benedetta; Lombardi, Francesca; La Torre, Cristina; Dehcordi, Soheila Raysi; Galzio, Renato; Cimini, Annamaria; Giordano, Antonio; Cifone, Maria Grazia

2017-04-11

Nitric oxide has been implicated in biology and progression of glioblastoma (GBM) being able to influence the cellular signal depending on the concentration and duration of cell exposure. NOS2 (inducible nitric oxide synthase) have been proposed as a component of molecular profile of several tumors, including glioma, one of the most aggressive primary brain tumor featuring local cancer stem cells responsible for enhanced resistance to therapies and for tumor recurrence. Here, we investigated the NOS2 mRNA expression by reverse transcription-PCR in human glioma primary cultures at several grade of malignancy and glioma stem cell (GSC) derived neurospheres. Glioma cell lines were used as positive controls both in terms of stemness marker expression that of capacity of generating neurospheres. NOS2 expression was detected at basal levels in cell lines and primary cultures and appeared significantly up-regulated in cultures kept in the specific medium for neurospheres. The immunofluorescence analysis of all cell cultures to evaluate the levels of SOX-2, a stemness marker aberrantly up-regulated in GBM, was also performed. The potential correlation between NOS2 expression and ability to generate neurospheres and between NOS2 and SOX-2 levels was also verified. The results show that the higher NOS2 expression is detected in all primary cultures able to arise neurosphere. A high and significant correlation between NOS2 expression and SOX-2 positive cells (%) in all cell cultures maintained in standard conditions has been observed. The results shed light on the potential relevance of NOS2 as a prognostic factor for glioma malignancy and recurrence.

Primary Airway Epithelial Cell Gene Editing Using CRISPR-Cas9.

PubMed

Everman, Jamie L; Rios, Cydney; Seibold, Max A

2018-01-01

The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction, allowing the CRISPR-Cas9 system to be integrated into the genome of the cell, resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel, advancements have been made in the culture, expansion, selection, and differentiation of AECs, which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods, we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery, (2) efficiently transduce AECs, (3) culture and select for a bulk edited AEC population, (4) molecularly screen AECs for Cas9 cutting and specific sequence edits, and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease.

MV-NIS or Investigator's Choice Chemotherapy in Treating Patients With Ovarian, Fallopian, or Peritoneal Cancer

ClinicalTrials.gov

2018-04-27

Fallopian Tube Transitional Cell Carcinoma; Malignant Ovarian Clear Cell Tumor; Malignant Ovarian Endometrioid Tumor; Malignant Ovarian Serous Tumor; Ovarian Seromucinous Carcinoma; Ovarian Transitional Cell Carcinoma; Primary Peritoneal Serous Adenocarcinoma; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Primary Peritoneal Carcinoma; Undifferentiated Fallopian Tube Carcinoma; Undifferentiated Ovarian Carcinoma

Immune response in melanoma: an in-depth analysis of the primary tumor and corresponding sentinel lymph node

PubMed Central

Ma, Michelle W.; Medicherla, Ratna C.; Qian, Meng; de Miera, Eleazar Vega-Saenz; Friedman, Erica B.; Berman, Russell S.; Shapiro, Richard L.; Pavlick, Anna C.; Ott, Patrick A.; Bhardwaj, Nina; Shao, Yongzhao; Osman, Iman; Darvishian, Farbod

2013-01-01

The sentinel lymph node is the initial site of metastasis. Down-regulation of anti-tumor immunity plays a role in nodal progression. Our objective was to investigate the relationship between immune modulation and sentinel lymph node positivity, correlating it with outcome in melanoma patients. Lymph node/primary tissues from melanoma patients prospectively accrued and followed at New York University Medical Center were evaluated for the presence of regulatory T-cells (Foxp3+) and dendritic cells (conventional: CD11c+, mature: CD86+) using immunohistochemistry. Primary melanoma immune cell profiles from sentinel lymph node-positive/-negative patients were compared. Logistic regression models inclusive of standard-of-care/immunologic primary tumor characteristics were constructed to predict the risk of sentinel lymph node positivity. Immunological responses in the positive sentinel lymph node were also compared to those in the negative non-sentinel node from the same nodal basin and matched negative sentinel lymph node. Decreased immune response was defined as increased regulatory T-cells or decreased dendritic cells. Associations between the expression of these immune modulators, clinicopathologic variables, and clinical outcome were evaluated using univariate/multivariate analyses. Primary tumor conventional dendritic cells and regression were protective against sentinel lymph node metastasis (odds ratio=0.714, 0.067; P=0.0099, 0.0816, respectively). Anti-tumor immunity was down-regulated in the positive sentinel lymph node with an increase in regulatory T-cells compared to the negative non-sentinel node from the same nodal basin (P=0.0005) and matched negative sentinel lymph node (P=0.0002). The positive sentinel lymph node also had decreased numbers of conventional dendritic cells compared to the negative sentinel lymph node (P<0.0001). Adding sentinel lymph node regulatory T-cell expression improved the discriminative power of a recurrence risk assessment model using clinical stage. Primary tumor regression was associated with prolonged disease-free (P=0.025) and melanoma-specific (P=0.014) survival. Our results support an assessment of local immune profiles in both the primary tumor and sentinel lymph node to help guide therapeutic decisions. PMID:22425909

CD79B limits response of diffuse large B cell lymphoma to ibrutinib.

PubMed

Kim, Joo Hyun; Kim, Won Seog; Ryu, Kyungju; Kim, Seok Jin; Park, Chaehwa

2016-01-01

Blockage of B cell receptor signaling with ibrutinib presents a promising clinical approach for treatment of B-cell malignancies. However, many patients show primary resistance to the drug or develop secondary resistance. In the current study, cDNA microarray and Western blot analyses revealed CD79B upregulation in the activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) that display differential resistance to ibrutinib. CD79B overexpression was sufficient to induce resistance to ibrutinib and enhanced AKT and MAPK activation, indicative of an alternative mechanism underlying resistance. Conversely, depletion of CD79B sensitized primary refractory cells to ibrutinib and led to reduced phosphorylation of AKT or MAPK. Combination of the AKT inhibitor or the MAPK inhibitor with ibrutinib resulted in circumvention of both primary and acquired resistance in ABC-DLBCL. Our data collectively indicate that CD79B overexpression leading to activation of AKT/MAPK is a potential mechanism underlying primary ibrutinib resistance in ABC-DLBCL, and support its utility as an effective biomarker to predict therapeutic response to ibrutinib.

cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane Fusion Sites.

PubMed

Xu, Shuting; Ducroux, Aurélie; Ponnurangam, Aparna; Vieyres, Gabrielle; Franz, Sergej; Müsken, Mathias; Zillinger, Thomas; Malassa, Angelina; Ewald, Ellen; Hornung, Veit; Barchet, Winfried; Häussler, Susanne; Pietschmann, Thomas; Goffinet, Christine

2016-10-12

Upon sensing cytoplasmic retroviral DNA in infected cells, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide cGAMP, which activates STING to trigger a type I interferon (IFN) response. We find that membrane fusion-inducing contact between donor cells expressing the HIV envelope (Env) and primary macrophages endogenously expressing the HIV receptor CD4 and coreceptor enable intercellular transfer of cGAMP. This cGAMP exchange results in STING-dependent antiviral IFN responses in target macrophages and protection from HIV infection. Furthermore, under conditions allowing cell-to-cell transmission of HIV-1, infected primary T cells, but not cell-free virions, deliver cGAMP to autologous macrophages through HIV-1 Env and CD4/coreceptor-mediated membrane fusion sites and induce a STING-dependent, but cGAS-independent, IFN response in target cells. Collectively, these findings identify an infection-specific mode of horizontal transfer of cGAMP between primary immune cells that may boost antiviral responses, particularly in infected tissues in which cell-to-cell transmission of virions exceeds cell-free infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential Activation of CD8+ Tumor-Specific Tc1 and Tc2 Cells by an IL-10-Producing Murine Plasmacytoma

PubMed Central

Pauels, Hans-Gerd; Becker, Christian; Kölsch, Eckehart

1998-01-01

The involvement of counteractive CD8+ T-cell subsets during tumor-specific immune responses was analyzed in a syngeneic murine plasmacytoma model. CD8+ Tc cells against the immunogenic IL-10-producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced by immunization of BALB/c mice with X-irradiated ADJ-PC-5 tumor cells in vivo and in vitro. However, the failure of recipient mice to mount a protective Tc response against the tumor during early stages of a real or simulated tumor growth is not due to immunological ignorance, but depends on the induction of tumor-specific tolerance, involving a population of tumorinduced CD8+ T cells that are able to inhibit the generation of tumor-specific Tc cells in a primary ADJ-PC-5-specific MLTC, using IFN-γ as a suppressive factor. Whereas most longterm cultivated CD8+ ADJ-PC-5-specific Tc lines produce type-1 cytokines on stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum. Furthermore, the primary in vitro Tc response against ADJ-PC-5 cells shows characteristics of a Tc2 response. The Tc response is strictly depending on tumor-derived IL-10. CD8+ Tc cells that are induced in a primary MLTC do not produce IFN-γ, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-γ or IL-12. In contrast, ADJ-PC- 5-specific CD8+ Tc cells from immunized mice are IFN-γ producing Tc1 cells. Since the primary in vitro Tc response against the tumor is suppressed even by the smallest numbers of irradiated ADJ-PC-5-specific Tc1 cells via IFN-γ these Tc1 cells behave similar to the suppressive CD8+ T cells that are induced during early stages of ADJ-PC-5 tumorigenesis. PMID:9814607

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

PubMed

Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

PubMed Central

Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

PubMed

Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

2009-11-12

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

The retinoblastoma gene is frequently altered leading to loss of expression in primary breast tumours.

PubMed

Varley, J M; Armour, J; Swallow, J E; Jeffreys, A J; Ponder, B A; T'Ang, A; Fung, Y K; Brammar, W J; Walker, R A

1989-06-01

We have analysed the organisation of the retinoblastoma (RB1) gene in 77 primary breast carcinomas, in metastatic tissue derived from 16 of those primary tumours, and in a variety of benign breast lesions. Expression of RB1 was also assessed in most samples by immunohistochemical detection of the RB1 protein in tissue sections. Structural abnormalities to RB1 were detected in DNA from 15/77 (19%) of primary breast carcinomas examined. Where DNA was available from metastatic tissue derived from such primary tumours, the same aberration could be detected. No alterations were seen in benign breast lesions. 16/56 (29%) of tumours examined for expression by immunohistochemical methods showed a proportion of tumour cells to be completely negative for the RB1 protein. All tumours in which a structural alteration to RB1 was detected had a proportion of negative cells, except for one case where all cells were positive. Several primary tumour samples were identified where there was no detectable structural change to the gene, but there was loss of expression in some tumour cells. The data presented here demonstrate that changes to the RB1 gene leading to loss of expression of both alleles are frequent in primary human breast tumours.

Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision

PubMed Central

Phua, Siew Cheng; Chiba, Shuhei; Suzuki, Masako; Su, Emily; Roberson, Elle C.; Pusapati, Ganesh V.; Setou, Mitsutoshi; Rohatgi, Rajat; Reiter, Jeremy F.; Ikegami, Koji; Inoue, Takanari

2017-01-01

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate to distal cilia. This triggers otherwise forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. Whilst cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer. PMID:28086093

Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells

PubMed Central

Lee, Jonghyeob; Snyder, Emily R.; Liu, Yinghua; Gu, Xueying; Wang, Jing; Flowers, Brittany M.; Kim, Yoo Jung; Park, Sangbin; Szot, Gregory L.; Hruban, Ralph H.; Longacre, Teri A.; Kim, Seung K.

2017-01-01

Development of systems that reconstitute hallmark features of human pancreatic intraepithelial neoplasia (PanINs), the precursor to pancreatic ductal adenocarcinoma, could generate new strategies for early diagnosis and intervention. However, human cell-based PanIN models with defined mutations are unavailable. Here, we report that genetic modification of primary human pancreatic cells leads to development of lesions resembling native human PanINs. Primary human pancreas duct cells harbouring oncogenic KRAS and induced mutations in CDKN2A, SMAD4 and TP53 expand in vitro as epithelial spheres. After pancreatic transplantation, mutant clones form lesions histologically similar to native PanINs, including prominent stromal responses. Gene expression profiling reveals molecular similarities of mutant clones with native PanINs, and identifies potential PanIN biomarker candidates including Neuromedin U, a circulating peptide hormone. Prospective reconstitution of human PanIN development from primary cells provides experimental opportunities to investigate pancreas cancer development, progression and early-stage detection. PMID:28272465

Establishment and characterization of Xenopus oviduct cells in primary culture

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

Marsh, J.; Tata, J.R.

1987-11-01

Based on previously established procedure of Xenopus hepatocytes, the authors describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response, from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the samemore » characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes. The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfill the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.« less

Characteristics of microRNAs enriched in specific cell types and primary tissue types in solid organs.

PubMed

Kriegel, Alison J; Liu, Yong; Liu, Pengyuan; Baker, Maria Angeles; Hodges, Matthew R; Hua, Xing; Liang, Mingyu

2013-12-01

Knowledge of miRNA expression and function in specific cell types in solid organs is limited because of difficulty in obtaining appropriate specimens. We used laser capture microdissection to obtain nine tissue regions from rats, including the nucleus of the solitary tract, hypoglossal motor nucleus, ventral respiratory column/pre-Bötzinger complex, and midline raphe nucleus from the brain stem, myocardium and coronary artery from the heart, and glomerulus, proximal convoluted tubule, and medullary thick ascending limb from the kidney. Each tissue region consists of or is enriched for a specific cell type. Differential patterns of miRNA expression obtained by deep sequencing of minute amounts of laser-captured cells were highly consistent with data obtained from real-time PCR analysis. miRNA expression patterns correctly clustered the specimens by tissue regions and then by primary tissue types (neural, muscular, or epithelial). The aggregate difference in miRNA profiles between tissue regions that contained the same primary tissue type was as large as one-half of the aggregate difference between primary tissue types. miRNAs differentially expressed between primary tissue types are more likely to be abundant miRNAs, while miRNAs differentially expressed between tissue regions containing the same primary tissue type were distributed evenly across the abundance spectrum. The tissue type-enriched miRNAs were more likely to target genes enriched for specific functional categories compared with either cell type-enriched miRNAs or randomly selected miRNAs. These data indicate that the role of miRNAs in determining characteristics of primary tissue types may be different than their role in regulating cell type-specific functions in solid organs.

Breast abscess as the initial manifestation of primary pure squamous cell carcinoma: a rare presentation and literature review.

PubMed

Salemis, Nikolaos S

2011-01-01

Primary squamous cell carcinoma of the breast is a very rare tumor accounting for less than 0.4% of all breast cancers. Fewer than 100 cases have been reported in the literature so far. The diagnosis requires strict pathologic criteria to be fulfilled. Due to the rarity of this tumor the optimal treatment and prognosis are both unclear. Breast abscess as the initial presentation of a primary squamous cell breast carcinoma is an extremely rare clinical entity. In this study, we describe a case of a 61-year-old postmenopausal woman who presented with typical manifestations of a breast abscess and was diagnosed with a pure primary squamous cell breast carcinoma. Diagnostic evaluation and management of the patient are discussed along with a review of the literature. Despite its rarity, the possibility of a primary pure squamous cell breast carcinoma should always be considered in the differential diagnosis in postmenopausal patients presenting with manifestations of a breast abscess, especially in those who respond poorly to the initial treatment. Physicians should be aware of this rare malignancy in order to avoid delays in diagnosis and treatment.

Temsirolimus and Bevacizumab in Treating Patients With Advanced Endometrial, Ovarian, Liver, Carcinoid, or Islet Cell Cancer

ClinicalTrials.gov

2017-07-10

Adult Hepatocellular Carcinoma; Advanced Adult Hepatocellular Carcinoma; Endometrial Serous Adenocarcinoma; Localized Non-Resectable Adult Liver Carcinoma; Lung Carcinoid Tumor; Malignant Pancreatic Gastrinoma; Malignant Pancreatic Glucagonoma; Malignant Pancreatic Insulinoma; Malignant Pancreatic Somatostatinoma; Metastatic Digestive System Neuroendocrine Tumor G1; Ovarian Carcinosarcoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Surface Papillary Adenocarcinoma; Pancreatic Alpha Cell Adenoma; Pancreatic Beta Cell Adenoma; Pancreatic Delta Cell Adenoma; Pancreatic G-Cell Adenoma; Pancreatic Polypeptide Tumor; Recurrent Adult Liver Carcinoma; Recurrent Digestive System Neuroendocrine Tumor G1; Recurrent Fallopian Tube Carcinoma; Recurrent Ovarian Carcinoma; Recurrent Pancreatic Neuroendocrine Carcinoma; Recurrent Primary Peritoneal Carcinoma; Recurrent Uterine Corpus Carcinoma; Regional Digestive System Neuroendocrine Tumor G1; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Primary Peritoneal Cancer; Stage IIIA Uterine Corpus Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Primary Peritoneal Cancer; Stage IIIB Uterine Corpus Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Primary Peritoneal Cancer; Stage IIIC Uterine Corpus Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Primary Peritoneal Cancer; Stage IVA Uterine Corpus Cancer; Stage IVB Uterine Corpus Cancer; Uterine Carcinosarcoma

Silibinin negatively contributes to primary cilia length via autophagy regulated by histone deacetylase 6 in confluent mouse embryo fibroblast 3T3-L1 cells.

PubMed

Xu, Qian; Liu, Wei; Liu, Xiaoling; Liu, Weiwei; Wang, Hongju; Yao, Guodong; Zang, Linghe; Hayashi, Toshihiko; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2016-09-01

Primary cilium is a cellular antenna, signalling as a sensory organelle. Numerous pathological manifestation is associated with change of its length. Although the interaction between autophagy and primary cilia has been suggested, the role of autophagy in primary cilia length is largely unknown. In this study the primary cilia were immunostained and observed by using confocal fluorescence microscopy, and we found that silibinin, a natural flavonoid, shortened the length of primary cilia, meanwhile it also induced autophagy in 3T3-L1 cells. This study was designed to investigate the significance of silibinin-induced autophagy in primary ciliary structure in confluent mouse embryo fibroblast 3T3-L1 cells. Either blocking the autophagic flux with pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), or transfection of siRNA targeting LC3 inhibited the reduction of cilia length caused by silibinin exposure. Autophagy induced by silibinin decreased expressions of the cilia-associated proteins, such as IFT88, KIF3a and Ac-tubulin, while 3-MA restored it, indicating that autophagy induced by silibinin led to a reduction of primary cilia length. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy, was up-regulated by silibinin in a time-dependent manner. In addition, 3T3-L1 cells treated with siRNA against HDAC6 had a reduced autophagic level and were protected from silibinin-induced cilia shortening. Taken together, we conclude that the HDAC6-mediated autophagy negatively regulates primary cilia length during silibinin treatment and has the potential to serve as a therapeutic target for primary cilia-associated ciliopathies. These findings thus provide new information about the potential link between autophagy and primary cilia.

Products of cells from gliomas: VIII. Multiple-well immunoperoxidase assay of immunoreactivity of primary hybridoma supernatants with human glioma and brain tissue and cultured glioma cells.

PubMed

McKeever, P E; Wahl, R L; Shakui, P; Jackson, G A; Letica, L H; Liebert, M; Taren, J A; Beierwaltes, W H; Hoff, J T

1990-06-01

To test the feasibility of primary screening of hybridoma supernatants against human glioma tissue, over 5000 combinations of hybridoma supernatants with glioma tissue, cultured glioma cells, and normal central neural tissue were screened with a new multiple-well (M-well) screening system. This is an immunoperoxidase assay system with visual endpoints for screening 20-30 hybridoma supernatants per single microscope slide. There were extensive differences between specificities to tissue and to cultured glioma cells when both were screened with M-wells and when cultured cells were screened with standard semi-automated fluorescence. Primary M-well screening with glioma tissue detected seven hybridoma supernatants that specifically identified parenchymal cells of glioma tissue and that were not detected with cultured cells. Immunoreactivities of individual supernatants for vascular components (nine supernatants), necrosis (five supernatants), and nuclei (three supernatants) were detected. Other supernatants bound multiple sites on glioma tissue and/or subpopulations of neurons and glia of normal tissue. The results show that primary screening with glioma tissue detects a number of different specificities of hybridoma supernatants to gliomas not detected by conventional screening with cultured cells. These are potentially applicable to diagnosis and therapy.

The potential use of primary human upper urinary tract urothelial cell carcinoma (UUT-UCC) cultured cells for prognostic indicators and chemosensitivity test.

PubMed

Hsieh, Teng-Fu; Chen, Chi-Cheng; Chang, Chao-Hsiang; Yu, Ai-Lin; Ma, Wen-Lung; Shyr, Chih-Rong

2013-07-01

Upper urinary tract urothelial cell carcinoma (UUT-UCC) is a rare yet aggressive urologic tumor with a high rate of recurrence and metastasis, resulting in high mortality. Chemotherapy has been used to prevent recurrence and treat metastatic UUT-UCCs. Although UUT-UCC is sensitive to chemotherapy, the patients' responses to therapy vary and the therapy outcome is unpredictable. Therefore, the identification of patients who are at high risk of failure in chemotherapy is important for accurate prognostication, patient counseling, and better therapy. We have obtained cells from UUT-UCC tumor tissues after surgery and established individual primary cultured cell lines, which were used to evaluate E-cadherin and Ki-67 proliferation marker expression and their chemosensitivity to chemotherapeutic drugs. Differential Ki-67 expression and chemosensitivity were observed in these primary cultured cell lines, suggesting these types of UUT-UCC cell lines could provide a platform for determining prognostic makers and evaluating the efficacy of chemotherapy. In conclusion, primary cultured cell lines from individual patients will be a great tool for evaluating and determining each individual's personalized chemotherapy course and for testing and screening new chemotherapeutic agents against UUT-UCCs. Copyright © 2012 Elsevier GmbH. All rights reserved.

Immune response to functionalized mesoporous silica nanoparticles for targeted drug delivery

NASA Astrophysics Data System (ADS)

Heidegger, Simon; Gößl, Dorothée; Schmidt, Alexandra; Niedermayer, Stefan; Argyo, Christian; Endres, Stefan; Bein, Thomas; Bourquin, Carole

2015-12-01

Multifunctional mesoporous silica nanoparticles (MSN) have attracted substantial attention with regard to their high potential for targeted drug delivery. For future clinical applications it is crucial to address safety concerns and understand the potential immunotoxicity of these nanoparticles. In this study, we assess the biocompatibility and functionality of multifunctional MSN in freshly isolated, primary murine immune cells. We show that the functionalized silica nanoparticles are rapidly and efficiently taken up into the endosomal compartment by specialized antigen-presenting cells such as dendritic cells. The silica nanoparticles showed a favorable toxicity profile and did not affect the viability of primary immune cells from the spleen in relevant concentrations. Cargo-free MSN induced only very low immune responses in primary cells as determined by surface expression of activation markers and release of pro-inflammatory cytokines such as Interleukin-6, -12 and -1β. In contrast, when surface-functionalized MSN with a pH-responsive polymer capping were loaded with an immune-activating drug, the synthetic Toll-like receptor 7 agonist R848, a strong immune response was provoked. We thus demonstrate that MSN represent an efficient drug delivery vehicle to primary immune cells that is both non-toxic and non-inflammagenic, which is a prerequisite for the use of these particles in biomedical applications.Multifunctional mesoporous silica nanoparticles (MSN) have attracted substantial attention with regard to their high potential for targeted drug delivery. For future clinical applications it is crucial to address safety concerns and understand the potential immunotoxicity of these nanoparticles. In this study, we assess the biocompatibility and functionality of multifunctional MSN in freshly isolated, primary murine immune cells. We show that the functionalized silica nanoparticles are rapidly and efficiently taken up into the endosomal compartment by specialized antigen-presenting cells such as dendritic cells. The silica nanoparticles showed a favorable toxicity profile and did not affect the viability of primary immune cells from the spleen in relevant concentrations. Cargo-free MSN induced only very low immune responses in primary cells as determined by surface expression of activation markers and release of pro-inflammatory cytokines such as Interleukin-6, -12 and -1β. In contrast, when surface-functionalized MSN with a pH-responsive polymer capping were loaded with an immune-activating drug, the synthetic Toll-like receptor 7 agonist R848, a strong immune response was provoked. We thus demonstrate that MSN represent an efficient drug delivery vehicle to primary immune cells that is both non-toxic and non-inflammagenic, which is a prerequisite for the use of these particles in biomedical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06122a