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 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.

Fuel cell system with interconnect

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

Goettler, Richard; Liu, Zhien

The present invention includes a fuel cell system having a plurality of adjacent electrochemical cells formed of an anode layer, a cathode layer spaced apart from the anode layer, and an electrolyte layer disposed between the anode layer and the cathode layer. The fuel cell system also includes at least one interconnect, the interconnect being structured to conduct free electrons between adjacent electrochemical cells. Each interconnect includes a primary conductor embedded within the electrolyte layer and structured to conduct the free electrons.

Fuel cell system with interconnect

DOEpatents

Goettler, Richard; Liu, Zhien

2015-08-11

The present invention includes a fuel cell system having a plurality of adjacent electrochemical cells formed of an anode layer, a cathode layer spaced apart from the anode layer, and an electrolyte layer disposed between the anode layer and the cathode layer. The fuel cell system also includes at least one interconnect, the interconnect being structured to conduct free electrons between adjacent electrochemical cells. Each interconnect includes a primary conductor embedded within the electrolyte layer and structured to conduct the free electrons.

Fuel cell system with interconnect

DOEpatents

Goettler, Richard; Liu, Zhien

2015-03-10

The present invention includes a fuel cell system having a plurality of adjacent electrochemical cells formed of an anode layer, a cathode layer spaced apart from the anode layer, and an electrolyte layer disposed between the anode layer and the cathode layer. The fuel cell system also includes at least one interconnect, the interconnect being structured to conduct free electrons between adjacent electrochemical cells. Each interconnect includes a primary conductor embedded within the electrolyte layer and structured to conduct the free electrons.

Fuel cell system with interconnect

DOEpatents

Liu, Zhien; Goettler, Richard

2015-09-29

The present invention includes a fuel cell system having a plurality of adjacent electrochemical cells formed of an anode layer, a cathode layer spaced apart from the anode layer, and an electrolyte layer disposed between the anode layer and the cathode layer. The fuel cell system also includes at least one interconnect, the interconnect being structured to conduct free electrons between adjacent electrochemical cells. Each interconnect includes a primary conductor embedded within the electrolyte layer and structured to conduct the free electrons.

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.

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.

Corrosion resistant solar mirror

DOEpatents

Medwick, Paul A.; Abbott, Edward E.

2016-07-19

A reflective article includes a transparent substrate having a first major surface and a second major surface. A base coat is formed over at least a portion of the second major surface. A primary reflective coating having at least one metallic layer is formed over at least a portion of the base coat. A protective coating is formed over at least a portion of the primary reflective coating. The article further includes a solar cell and an anode, with the solar cell connected to the metallic layer and the anode.

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.

Advanced energy storage for space applications: A follow-up

NASA Technical Reports Server (NTRS)

Halpert, Gerald; Surampudi, Subbarao

1994-01-01

Viewgraphs on advanced energy storage for space applications are presented. Topics covered include: categories of space missions using batteries; battery challenges; properties of SOA and advanced primary batteries; lithium primary cell applications; advanced rechargeable battery applications; present limitations of advanced battery technologies; and status of Li-TiS2, Ni-MH, and Na-NiCl2 cell technologies.

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.

The role of the cilium in hereditary tumor predisposition syndromes

PubMed Central

Klasson, Timothy D.; Giles, Rachel H.

2014-01-01

The primary cilium is a highly conserved cell organelle that is closely connected to processes involved in cell patterning and replication. Amongst their many functions, cilia act as “signal towers” through which cell-cell signaling cascades pass. Dysfunction of cilia or the myriad processes that are connected with cilium function can lead to disease. Due to the sheer number of cellular processes that at some point involve the primary cilium, the effects of misregulation are highly heterogeneous between different cell populations. However, because of the importance of primary cilia in the development, growth, patterning and orientation of cells and tissues, a common thread has emerged in which defective cilia can lead to disorganization, which can contribute to the growth of neoplasms, including cancer and pre-cancerous phenotypes. Because cilia are so vital for signaling during cell replication and the cell fate decisions that are important in childhood growth, symptoms often arise early in life. Here we review recent work connecting misregulation of the primary cilium with tumor formation in a variety of tissues in the developing body, with a particular focus on the syndromes in which classic tumor genes are mutated, including von Hippel-Lindau disease (OMIM 193300), adenomatous polyposis coli (OMIM 175100), tuberous sclerosis (OMIM 191100) and Birt-Hogg-Dubé syndrome (OMIM 135150). Timely diagnosis of these syndromes is essential for entry into appropriate screening protocols, which have been shown to effectively prolong life expectancy in these cohorts of patients. PMID:27625869

TGFβ1-induced down-regulation of microRNA-138 contributes to epithelial-mesenchymal transition in primary lung cancer cells.

PubMed

Zhang, Fang; Li, Tiepeng; Han, Lu; Qin, Peng; Wu, Zhao; Xu, Benling; Gao, Quanli; Song, Yongping

2018-02-19

The existence of cancer stem cells within the tumor could lead to cancer therapy resistance. TGFβ1 is considered as one of the most powerful players in the generation of CSCs through induction of epithelial-mesenchymal transition in different types of cancer including lung cancer, however, the detailed mechanisms by which TGFβ1 contribute to EMT induction and CSC maintenance remains unclear. Here, we showed primary lung cancer cells treated by TGFβ1 exhibit mesenchymal features, including morphology and expression of mesenchymal marker in a time-dependent manner. We also observed long-term TGFβ1 exposure leads to an enrichment of a sub-population of CD44 + CD90 + cells which represent CSCs in lung cancer cells. Moreover, the differential expression microRNAs between CSCs and non-CSCs were identified using next-generation sequencing to screen key miRNAs which might contribute to TGFβ1-induced EMT and CSCs generation. Among those differentially expressed miRNAs, the expression of microRNA-138 was time-dependently down-regulated by TGFβ1 treatment. We further demonstrated primary lung cancer cells, in which we knockdown the expression of miR-138, exhibit mesenchymal phenotypes and stem cell properties. Taken together, these findings indicate TGFβ1-induced down-regulation of microRNA-138 contributes to EMT in primary lung cancer cells, and suggest that miR-138 might serve as a potential therapeutic target. Copyright © 2018 Elsevier Inc. All rights reserved.

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

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

Primary Squamous Cell Carcinoma of the Thyroid: A Population-Based Analysis.

PubMed

Au, Joshua K; Alonso, Jose; Kuan, Edward C; Arshi, Armin; St John, Maie A

2017-07-01

Objectives To analyze the epidemiology and describe the prognostic indicators of patients with primary squamous cell carcinoma of the thyroid. Study Design and Setting Retrospective cohort study based on a national database. Methods The US National Cancer Institute's SEER registry (Surveillance, Epidemiology, and End Results) was reviewed for patients with primary squamous cell carcinoma of the thyroid from 1973 to 2012. Study variables included age, sex, race, tumor size, tumor grade, regional and distant metastases, and treatment modality. Survival measures included overall survival (OS) and disease-specific survival (DSS). Results A total of 199 cases of primary squamous cell carcinoma of the thyroid were identified. Mean age at diagnosis was 68.1 years; 58.3% were female; and 79.4% were white. Following diagnosis, 46.3% of patients underwent surgery; 55.7%, radiation therapy; and 45.8%, surgery with radiation therapy. Kaplan-Meier analysis demonstrated OS and DSS of 16% and 21% at 5 years, respectively. Median survival after diagnosis was 9.1 months. Multivariate Cox regression analysis showed that predictors of OS and DSS included age ( P < .001, P < .001, respectively), tumor grade ( P < .001, P = .001), and tumor size ( P < .001, P = .001). Surgical management was a predictor of OS but not DSS. Conclusion Squamous cell carcinoma of the thyroid is a rare malignancy with a very poor prognosis. Surgical resection confers an overall survival benefit. Age, tumor grade, and tumor size are predictors of OS and DSS.

Orbitofacial Metastatic Basal Cell Carcinoma: Report of 10 Cases.

PubMed

Branson, Sara V; McClintic, Elysa; Ozgur, Omar; Esmaeli, Bita; Yeatts, R Patrick

To explore the clinical features, management, and prognosis of metastatic basal cell carcinoma originating in the orbitofacial region. Ten cases of orbitofacial metastatic basal cell carcinoma were identified by searching databases at 2 institutions from 1995 to 2015. A retrospective chart review was performed. Main outcome measures included patient demographics, lesion size, location of metastases, histologic subtype, recurrence rate, time between primary tumor diagnosis and metastasis, perineural invasion, treatment modalities, and survival from time of metastasis. The median tumor size at largest dimension was 3.3 cm (range, 1.9-11.5 cm), and 6 of 10 patients had at least 1 local recurrence before metastasis (range, 0-2 recurrences). The most common sites of metastasis included the ipsilateral parotid gland (n = 6) and cervical lymph nodes (n = 5). Histologic subtypes included infiltrative (n = 5), basosquamous (n = 2), nodular (n = 1), and mixed (n = 1). The median time from primary tumor diagnosis to metastasis was 7.5 years (range, 0-13). The median survival time from diagnosis of metastasis to last documented encounter or death was 5.3 years (range, 7 months-22.8 years). Treatment regimens included surgical excision, radiotherapy, and hedgehog inhibitors. Based on our findings, the following features may be markers of high risk orbitofacial basal cell carcinoma: 1) increasing tumor size, 2) local recurrence of the primary tumor, 3) aggressive histologic subtype, and 4) perineural invasion. Screening should include close observation of the primary site and tissues in the distribution of regional lymphatics, particularly the parotid gland and cervical lymph nodes.

An avian cell line designed for production of highly attenuated viruses.

PubMed

Jordan, Ingo; Vos, Ad; Beilfuss, Stefanie; Neubert, Andreas; Breul, Sabine; Sandig, Volker

2009-01-29

Several viral vaccines, including highly promising vectors such as modified vaccinia Ankara (MVA), are produced on chicken embryo fibroblasts. Dependence on primary cells complicates production especially in large vaccination programs. With primary cells it is also not possible to create packaging lines for replication-deficient vectors that are adapted to proliferation in an avian host. To obviate requirement for primary cells permanent lines from specific tissues of muscovy duck were derived (AGE1.CR, CS, and CA) and further modified: we demonstrate that stable expression of the structural gene pIX from human adenovirus increases titers for unrelated poxvirus in the avian cells. This augmentation appears to be mediated via induction of heat shock and thus provides a novel cellular substrate that may allow further attenuation of vaccine strains.

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

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 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).

Long-term Culture and Cloning of Primary Human Bronchial Basal Cells that Maintain Multipotent Differentiation Capacity and CFTR Channel Function.

PubMed

Peters-Hall, Jennifer Ruth; Coquelin, Melissa L; Torres, Michael J; LaRanger, Ryan; Alabi, Busola Ruth; Sho, Sei; Calva-Moreno, Jose Francisco; Thomas, Philip J; Shay, Jerry William

2018-05-03

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome editing tools. Recently, conditional reprogramming of cells (CRC) with a ROCK inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the lifespan of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial growth medium instead of F-medium and 2% oxygen instead of 21% oxygen, that extend HBEC lifespan while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof of concept, CRISPR/Cas9 genome editing and cloning was used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis.

PubMed

Ferrari, Angelo; Petterino, Claudio; Ratto, Alessandra; Campanella, Chiara; Wurth, Roberto; Thellung, Stefano; Vito, Guendalina; Barbieri, Federica; Florio, Tullio

2012-03-14

Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours. These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.

CXCR4 expression in feline mammary carcinoma cells: evidence of a proliferative role for the SDF-1/CXCR4 axis

PubMed Central

2012-01-01

Background Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation. Results A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours. Conclusions These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis. PMID:22417013

[Cytomegalovirus-associated infectious mononucleosis-like syndrome accompanied by transient monoclonal expansion of CD8+ T-cells].

PubMed

Yonezawa, Akihito; Onaka, Takashi; Imada, Kazunori

2009-08-01

Most cases of infectious mononucleosis (IM) are caused by Epstein-Barr virus (EBV). Other pathogens have been reported to cause heterophile-negative mononucleosis-like syndrome, including cytomegalovirus (CMV) and human immunodeficiency virus type-1 (HIV-1). Primary CMV infection is often asymptomatic in immunocompetent individuals. In this article, we describe a patient with prolonged fever and fatigue, who developed transient monoclonal CD8+ T-cell lymphocytosis after primary CMV infection. Monoclonal gene rearrangement of T-cell receptor (TCR) beta locus was transiently detected in DNA from peripheral lymphocytes. Monoclonal rearrangement and atypical lymphocytosis disappeared after treatment with anti-viral agents. These observations imply that monoclonal expansion of T-cells could be a reactive phenomenon of primary CMV infection and TCR gene rearrangement is not specific for malignancy. Physicians should carefully follow patients with monoclonal expansion of CD8+ T-cells after CMV-IM in order to rule out T cell malignancy.

Pathophysiology of primary open-angle glaucoma from a neuroinflammatory and neurotoxicity perspective: a review of the literature.

PubMed

Evangelho, Karine; Mogilevskaya, Maria; Losada-Barragan, Monica; Vargas-Sanchez, Jeinny Karina

2017-12-30

Glaucoma is the leading cause of blindness in humans, affecting 2% of the population. This disorder can be classified into various types including primary, secondary, glaucoma with angle closure and with open angle. The prevalence of distinct types of glaucoma differs for each particular region of the world. One of the most common types of this disease is primary open-angle glaucoma (POAG), which is a complex inherited disorder characterized by progressive retinal ganglion cell death, optic nerve head excavation and visual field loss. Nowadays, POAG is considered an optic neuropathy, while intraocular pressure is proposed to play a fundamental role in its pathophysiology and especially in optic disk damage. However, the exact mechanism of optic nerve head damage remains a topic of debate. This literature review aims to bring together the information on the pathophysiology of primary open-angle glaucoma, particularly focusing on neuroinflammatory mechanisms leading to the death of the retinal ganglion cell. A literature search was done on PubMed using key words including primary open-angle glaucoma, retinal ganglion cells, Müller cells, glutamate, glial cells, ischemia, hypoxia, exitotoxicity, neuroinflammation, axotomy and neurotrophic factors. The literature was reviewed to collect the information published about the pathophysiologic mechanisms of RGC death in the POAG, from a neuroinflammatory and neurotoxicity perspective. Proposed mechanisms for glaucomatous damage are a result of pressure in RGC followed by ischemia, hypoxia of the ONH, and consequently death due to glutamate-induced excitotoxicity, deprivation of energy and oxygen, increase in levels of inflammatory mediators and alteration of trophic factors flow. These events lead to blockage of anterograde and retrograde axonal transport with ensuing axotomy and eventually blindness. The damage to ganglion cells and eventually glaucomatous injury can occur via various mechanisms including baric trauma, ischemia and impact of metabolic toxins, which triggers an inflammatory process and secondary degeneration in the ONH.

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.

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

Improving Primary Care Residents' Proficiency in the Diagnosis of Skin Cancer

PubMed Central

Gerbert, Barbara; Bronstone, Amy; Wolff, Mimi; Maurer, Toby; Berger, Timothy; Pantilat, Steven; McPhee, Stephen J

1998-01-01

OBJECTIVE To determine whether a brief, multicomponent intervention could improve the skin cancer diagnosis and evaluation planning performance of primary care residents to a level equivalent to that of dermatologists. PARTICIPANTS Fifty-two primary care residents (26 in the control group and 26 in the intervention group) and 13 dermatologists completed a pretest and posttest. DESIGN A randomized, controlled trial with pretest and posttest measurements of residents' ability to diagnose and make evaluation plans for lesions indicative of skin cancer. INTERVENTION The intervention included face-to-face feedback sessions focusing on residents' performance deficiencies; an interactive seminar including slide presentations, case examples, and live demonstrations; and the Melanoma Prevention Kit including a booklet, magnifying tool, measuring tool, and skin color guide. MEASUREMENTS AND MAIN RESULTS We compared the abilities of a control and an intervention group of primary care residents, and a group of dermatologists to diagnose and make evaluation plans for six categories of skin lesions including three types of skin cancer—malignant melanoma, squamous cell carcinoma, and basal cell carcinoma. At posttest, both the intervention and control group demonstrated improved performance, with the intervention group revealing significantly larger gains. The intervention group showed greater improvement than the control group across all six diagnostic categories (a gain of 13 percentage points vs 5, p < .05), and in evaluation planning for malignant melanoma (a gain of 46 percentage points vs 36, p < .05) and squamous cell carcinoma (a gain of 42 percentage points vs 21, p < .01). The intervention group performed as well as the dermatologists on five of the six skin cancer diagnosis and evaluation planning scores with the exception of the diagnosis of basal cell carcinoma. CONCLUSIONS Primary care residents can diagnose and make evaluation plans for cancerous skin lesions, including malignant melanoma, at a level equivalent to that of dermatologists if they receive relevant, targeted education. PMID:9502368

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.

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.

Two Different Cell Populations Is an Important Clue for Diagnosis of Primary Cutaneous Adenoid Cystic Carcinoma: Immunohistochemical Study

PubMed Central

Alkan, Banu Ince; Karadeniz, Müjde; Bozdoğan, Nazan

2017-01-01

Primary cutaneous adenoid cystic carcinoma (PCACC) is a very rare malignancy. The differential diagnosis of PCACCs in pathology practice can be difficult and a group of primary and metastatic lesions, including adenoid basal cell carcinoma of the skin, should be considered in the differential diagnosis. Besides histomorphological clues, immunohistochemistry studies are very helpful in the differential diagnosis of PCACC. We report herein a case of PCACC with extensive immunohistochemical studies and review the literature from an immunohistochemistry perspective. PMID:28243477

A distinct plasmablast and naïve B-cell phenotype in primary immune thrombocytopenia

PubMed Central

Flint, Shaun M.; Gibson, Adele; Lucas, Geoff; Nandigam, Raghava; Taylor, Louise; Provan, Drew; Newland, Adrian C.; Savage, Caroline O.; Henderson, Robert B.

2016-01-01

Primary immune thrombocytopenia is an autoimmune disorder in which platelet destruction is a consequence of both B- and T-cell dysregulation. Flow cytometry was used to further characterize the B- and T-cell compartments in a cross-sectional cohort of 26 immune thrombocytopenia patients including antiplatelet antibody positive (n=14) and negative (n=12) patients exposed to a range of therapies, and a cohort of matched healthy volunteers. Markers for B-cell activating factor and its receptors, relevant B-cell activation markers (CD95 and CD21) and markers for CD4+ T-cell subsets, including circulating T-follicular helper-like cells, were included. Our results indicate that an expanded population of CD95+ naïve B cells correlated with disease activity in immune thrombocytopenia patients regardless of treatment status. A population of CD21-naïve B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in naïve B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia. PMID:26969086

A distinct plasmablast and naïve B-cell phenotype in primary immune thrombocytopenia.

PubMed

Flint, Shaun M; Gibson, Adele; Lucas, Geoff; Nandigam, Raghava; Taylor, Louise; Provan, Drew; Newland, Adrian C; Savage, Caroline O; Henderson, Robert B

2016-06-01

Primary immune thrombocytopenia is an autoimmune disorder in which platelet destruction is a consequence of both B- and T-cell dysregulation. Flow cytometry was used to further characterize the B- and T-cell compartments in a cross-sectional cohort of 26 immune thrombocytopenia patients including antiplatelet antibody positive (n=14) and negative (n=12) patients exposed to a range of therapies, and a cohort of matched healthy volunteers. Markers for B-cell activating factor and its receptors, relevant B-cell activation markers (CD95 and CD21) and markers for CD4(+) T-cell subsets, including circulating T-follicular helper-like cells, were included. Our results indicate that an expanded population of CD95(+) naïve B cells correlated with disease activity in immune thrombocytopenia patients regardless of treatment status. A population of CD21-naïve B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in naïve B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia. Copyright© Ferrata Storti Foundation.

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.

High-rate lithium thionyl chloride cells

NASA Technical Reports Server (NTRS)

Goebel, F.

1982-01-01

A high-rate C cell with disc electrodes was developed to demonstrate current rates which are comparable to other primary systems. The tests performed established the limits of abuse beyond which the cell becomes hazardous. Tests include: impact, shock, and vibration tests; temperature cycling; and salt water immersion of fresh cells.

HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors.

PubMed

Ahmed, Nabil; Salsman, Vita S; Kew, Yvonne; Shaffer, Donald; Powell, Suzanne; Zhang, Yi J; Grossman, Robert G; Heslop, Helen E; Gottschalk, Stephen

2010-01-15

Glioblastoma multiforme (GBM) is the most aggressive human primary brain tumor and is currently incurable. Immunotherapies have the potential to target GBM stem cells, which are resistant to conventional therapies. Human epidermal growth factor receptor 2 (HER2) is a validated immunotherapy target, and we determined if HER2-specific T cells can be generated from GBM patients that will target autologous HER2-positive GBMs and their CD133-positive stem cell compartment. HER2-specific T cells from 10 consecutive GBM patients were generated by transduction with a retroviral vector encoding a HER2-specific chimeric antigen receptor. The effector function of HER2-specific T cells against autologous GBM cells, including CD133-positive stem cells, was evaluated in vitro and in an orthotopic murine xenograft model. Stimulation of HER2-specific T cells with HER2-positive autologous GBM cells resulted in T-cell proliferation and secretion of IFN-gamma and interleukin-2 in a HER2-dependent manner. Patients' HER2-specific T cells killed CD133-positive and CD133-negative cells derived from primary HER2-positive GBMs, whereas HER2-negative tumor cells were not killed. Injection of HER2-specific T cells induced sustained regression of autologous GBM xenografts established in the brain of severe combined immunodeficient mice. Gene transfer allows the reliable generation of HER2-specific T cells from GBM patients, which have potent antitumor activity against autologous HER2-positive tumors including their putative stem cells. Hence, the adoptive transfer of HER2-redirected T cells may be a promising immunotherapeutic approach for GBM.

Combined enzymatic and metabolic analysis of grapevine cell responses to elicitors.

PubMed

Krzyzaniak, Yuko; Negrel, Jonathan; Lemaitre-Guillier, Christelle; Clément, Gilles; Mouille, Grégory; Klinguer, Agnès; Trouvelot, Sophie; Héloir, Marie-Claire; Adrian, Marielle

2018-02-01

Elicitors trigger plant defense responses, including phytoalexin production and cell-wall reinforcement. Primary metabolism plays an important role in these responses as it fuels the associated energetic costs and provides precursors for the synthesis of the numerous secondary metabolites involved in defenses against pathogens. In this context, we aimed to determine whether oligosaccharidic elicitors differing in their capacity to activate defense-associated secondary metabolism in grapevine would differently impact primary metabolism. To answer this question, cell suspensions were treated with two elicitors: an oligogalacturonide, and the β-glucan laminarin. Enzymatic activity assays together with targeted (HPLC) and global (GC-MS) analyses of metabolites were next performed to compare their impact on plant primary or secondary metabolism. The results showed that the oligogalacturonide, which induced the highest level of the phytoalexin resveratrol and the highest activity of stilbene synthase, also induced the highest activity of shikimate hydroxycinnamoyltransferase, a key enzyme involved in the synthesis of lignin. The oligogalacturonide-induced defenses had a significant impact on primary metabolism 24 h following elicitor treatment, with a reduced abundance of pyruvate and 2-oxoglutarate, together with an increase of a set of metabolites including carbohydrates and amino acids. Interestingly, an accumulation of galacturonate and gentiobiose was observed in the oligogalacturonide- and laminarin-treated cells, respectively, suggesting that both elicitors are rapidly hydrolyzed in grapevine cell suspension cultures. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Primary cilia: cellular sensors for the skeleton.

PubMed

Anderson, Charles T; Castillo, Alesha B; Brugmann, Samantha A; Helms, Jill A; Jacobs, Christopher R; Stearns, Tim

2008-09-01

The primary cilium is a solitary, immotile cilium that is present in almost every mammalian cell type. Primary cilia are thought to function as chemosensors, mechanosensors, or both, depending on cell type, and have been linked to several developmental signaling pathways. Primary cilium malfunction has been implicated in several human diseases, the symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the development and homeostasis of the skeleton. In this review, we discuss the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus on skeletal biology. We also raise several unanswered questions regarding the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions.

Primary Cilia: Cellular Sensors for the Skeleton

PubMed Central

Anderson, Charles T.; Castillo, Alesha B.; Brugmann, Samantha A.; Helms, Jill A.; Jacobs, Christopher R.; Stearns, Tim

2010-01-01

The primary cilium is a solitary, immotile cilium that is present in almost every mammalian cell type. Primary cilia are thought to function as chemosensors, mechanosensors, or both, depending on cell type, and have been linked to several developmental signaling pathways. Primary cilium malfunction has been implicated in several human diseases, the symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the development and homeostasis of the skeleton. In this review, we discuss the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus on skeletal biology. We also raise several unanswered questions regarding the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions. PMID:18727074

Host responses in tissue repair and fibrosis.

PubMed

Duffield, Jeremy S; Lupher, Mark; Thannickal, Victor J; Wynn, Thomas A

2013-01-24

Myofibroblasts accumulate in the spaces between organ structures and produce extracellular matrix (ECM) proteins, including collagen I. They are the primary "effector" cells in tissue remodeling and fibrosis. Previously, leukocyte progenitors termed fibrocytes and myofibroblasts generated from epithelial cells through epithelial-to-mesenchymal transition (EMT) were considered the primary sources of ECM-producing myofibroblasts in injured tissues. However, genetic fate mapping experiments suggest that mesenchyme-derived cells, known as resident fibroblasts, and pericytes are the primary precursors of scar-forming myofibroblasts, whereas epithelial cells, endothelial cells, and myeloid leukocytes contribute to fibrogenesis predominantly by producing key fibrogenic cytokines and by promoting cell-to-cell communication. Numerous cytokines derived from T cells, macrophages, and other myeloid cell populations are important drivers of myofibroblast differentiation. Monocyte-derived cell populations are key regulators of the fibrotic process: They act as a brake on the processes driving fibrogenesis, and they dismantle and degrade established fibrosis. We discuss the origins, modes of activation, and fate of myofibroblasts in various important fibrotic diseases and describe how manipulation of macrophage activation could help ameliorate fibrosis.

[Primary culture of human normal epithelial cells].

PubMed

Tang, Yu; Xu, Wenji; Guo, Wanbei; Xie, Ming; Fang, Huilong; Chen, Chen; Zhou, Jun

2017-11-28

The traditional primary culture methods of human normal epithelial cells have disadvantages of low activity of cultured cells, the low cultivated rate and complicated operation. To solve these problems, researchers made many studies on culture process of human normal primary epithelial cell. In this paper, we mainly introduce some methods used in separation and purification of human normal epithelial cells, such as tissue separation method, enzyme digestion separation method, mechanical brushing method, red blood cell lysis method, percoll layered medium density gradient separation method. We also review some methods used in the culture and subculture, including serum-free medium combined with low mass fraction serum culture method, mouse tail collagen coating method, and glass culture bottle combined with plastic culture dish culture method. The biological characteristics of human normal epithelial cells, the methods of immunocytochemical staining, trypan blue exclusion are described. Moreover, the factors affecting the aseptic operation, the conditions of the extracellular environment, the conditions of the extracellular environment during culture, the number of differential adhesion, and the selection and dosage of additives are summarized.

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

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.

Epstein-Barr virus-associated T/natural killer-cell lymphoproliferative disorders.

PubMed

Park, Sanghui; Ko, Young H

2014-01-01

Primary infection with Epstein-Barr virus (EBV) is usually asymptomatic and, in a normal host, EBV remains latent in B cells after primary infection for the remainder of life. Uncommonly, EBV can infect T or natural killer (NK) cells in a person with a defect in innate immunity, and EBV infection can cause unique systemic lymphoproliferative diseases (LPD) of childhood. Primary infection in young children can be complicated by hemophagocytic lymphohistiocytosis or fulminant systemic T-cell LPD of childhood. Uncommonly, patients can develop chronic active EBV (CAEBV) disease-type T/NK LPD, which includes CAEBV infection of the systemic form, hydroa vacciniforme-like T-cell LPD, and mosquito-bite hypersensitivity. The clinical course of CAEBV disease-type T/NK LPD can be smoldering, persistent or progressive, depending on the balance between viral factors and host immunity. Aggressive NK-cell leukemia, hydroa vacciniforme-like T-cell lymphoma, or uncommonly extranodal NK/T-cell lymphoma can develop in children and young adults with CAEBV disease-type T/NK-cell LPD. Extranodal T/NK-cell lymphoma is a disease of adults, and its incidence begins to increase in the third decade and comprises the major subtype of T/NK LPD throughout life. Aggressive NK-cell leukemia and nodal T/NK-cell lymphoma of the elderly are fulminant diseases, and immune senescence may be an important pathogenetic factor. This review describes the current progress in identifying different types of EBV-associated T/NK-cell LPD and includes a brief presentation of data from Korea. © 2014 Japanese Dermatological Association.

BMI1 Is Expressed in Canine Osteosarcoma and Contributes to Cell Growth and Chemotherapy Resistance

PubMed Central

Gandour-Edwards, Regina; Withers, Sita S.; Holt, Roseline; Rebhun, Robert B.

2015-01-01

BMI1, a stem cell factor and member of the polycomb group of genes, has been shown to contribute to growth and chemoresistance of several human malignancies including primary osteosarcoma (OSA). Naturally occurring OSA in the dog represents a large animal model of human OSA, however the potential role of BMI1 in canine primary and metastatic OSA has not been examined. Immunohistochemical staining of canine primary and metastatic OSA tumors revealed strong nuclear expression of BMI1. An identical staining pattern was found in both primary and metastatic human OSA tissues. Canine OSA cell lines (Abrams, Moresco, and D17) expressed high levels of BMI1 compared with canine osteoblasts and knockdown or inhibition of BMI1 by siRNA or by small molecule BMI1-inhibitor PTC-209 demonstrated a role for BMI1 in canine OSA cell growth and resistance to carboplatin and doxorubicin chemotherapy. These findings suggest that inhibition of BMI1 in primary or metastatic OSA may improve response to chemotherapy and that the dog may serve as a large animal model to evaluate such therapy. PMID:26110620

BMI1 is expressed in canine osteosarcoma and contributes to cell growth and chemotherapy resistance.

PubMed

Shahi, Mehdi Hayat; York, Daniel; Gandour-Edwards, Regina; Withers, Sita S; Holt, Roseline; Rebhun, Robert B

2015-01-01

BMI1, a stem cell factor and member of the polycomb group of genes, has been shown to contribute to growth and chemoresistance of several human malignancies including primary osteosarcoma (OSA). Naturally occurring OSA in the dog represents a large animal model of human OSA, however the potential role of BMI1 in canine primary and metastatic OSA has not been examined. Immunohistochemical staining of canine primary and metastatic OSA tumors revealed strong nuclear expression of BMI1. An identical staining pattern was found in both primary and metastatic human OSA tissues. Canine OSA cell lines (Abrams, Moresco, and D17) expressed high levels of BMI1 compared with canine osteoblasts and knockdown or inhibition of BMI1 by siRNA or by small molecule BMI1-inhibitor PTC-209 demonstrated a role for BMI1 in canine OSA cell growth and resistance to carboplatin and doxorubicin chemotherapy. These findings suggest that inhibition of BMI1 in primary or metastatic OSA may improve response to chemotherapy and that the dog may serve as a large animal model to evaluate such therapy.

Primary malignant perivascular epithelioid cell neoplasm (PEComa) of the bone mimicking granular cell tumor in core biopsy: A case report and literature review

PubMed Central

Sadigh, Sam; Shah, Preya; Weber, Kristy; Sebro, Ronnie; Zhang, Paul J.

2018-01-01

The present study investigated the case of a 46-year-old female with primary malignant perivascular epithelioid cell neoplasm (PEComa) of the femur. The patient presented with a 5-month history of right distal thigh pain following trauma. Radiographs of the right distal femur revealed a mixed lytic and sclerotic lesion with subtle areas of cortical destruction and soft tissue extension, consistent with an aggressive tumor. A core biopsy revealed an epithelioid tumor with granular cell features, but a definitive diagnosis could not be made. Due to the aggressive features on radiologic evaluation, the patient underwent a resection of the distal femur and reconstruction with a distal femoral megaprosthesis and hinged knee replacement. The post-resection pathology led to a final diagnosis of primary bone PEComa, with histologic features including epithelioid, granular cell and spindled cell morphologies and biphasic immunoreactivity for melanocytic and smooth muscle markers. The large tumor size (>5 cm), rapid mitotic rate, infiltrative growth pattern, high nuclear grade and cellularity, and the presence of necrosis rendered this a malignant PEComa. The present study discussed the case, including radiographic (radiographs, magnetic resonance imaging and positron emission tomography scans) and histologic appearance and a literature review. PMID:29435023

Malignancy without immortality? Cellular immortalization as a possible late event in melanoma progression

PubMed Central

Soo, Julia K; MacKenzie Ross, Alastair D; Kallenberg, David M; Milagre, Carla; Heung Chong, W; Chow, Jade; Hill, Lucy; Hoare, Stacey; Collinson, Rebecca S; Hossain, Mehnaz; Keith, W Nicol; Marais, Richard; Bennett, Dorothy C

2011-01-01

Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: β-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event. PMID:21418545

Primary brain tumors, neural stem cell, and brain tumor cancer cells: where is the link?

PubMed Central

Germano, Isabelle; Swiss, Victoria; Casaccia, Patrizia

2010-01-01

The discovery of brain tumor-derived cells (BTSC) with the properties of stem cells has led to the formulation of the hypothesis that neural stem cells could be the cell of origin of primary brain tumors (PBT). In this review we present the most common molecular changes in PBT, define the criteria of identification of BTSC and discuss the similarities between the characteristics of these cells and those of the endogenous population of neural stem cells (NPCs) residing in germinal areas of the adult brain. Finally, we propose possible mechanisms of cancer initiation and progression and suggest a model of tumor initiation that includes intrinsic changes of resident NSC and potential changes in the microenvironment defining the niche where the NSC reside. PMID:20045420

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.

Distinctive properties of metastasis-initiating cells

PubMed Central

Celià-Terrassa, Toni; Kang, Yibin

2016-01-01

Primary tumors are known to constantly shed a large number of cancer cells into systemic dissemination, yet only a tiny fraction of these cells is capable of forming overt metastases. The tremendous rate of attrition during the process of metastasis implicates the existence of a rare and unique population of metastasis-initiating cells (MICs). MICs possess advantageous traits that may originate in the primary tumor but continue to evolve during dissemination and colonization, including cellular plasticity, metabolic reprogramming, the ability to enter and exit dormancy, resistance to apoptosis, immune evasion, and co-option of other tumor and stromal cells. Better understanding of the molecular and cellular hallmarks of MICs will facilitate the development and deployment of novel therapeutic strategies. PMID:27083997

Profiles of Brain Metastases: Prioritization of Therapeutic Targets.

PubMed

Ferguson, Sherise D; Zheng, Siyuan; Xiu, Joanne; Zhou, Shouhao; Khasraw, Mustafa; Brastianos, Priscilla K; Kesari, Santosh; Hu, Jethro; Rudnick, Jeremy; Salacz, Michael E; Piccioni, David; Huang, Suyun; Davies, Michael A; Glitza, Isabella C; Heymach, John V; Zhang, Jianjun; Ibrahim, Nuhad K; DeGroot, John F; McCarty, Joseph; O'Brien, Barbara J; Sawaya, Raymond; Verhaak, Roeland G W; Reddy, Sandeep K; Priebe, Waldemar; Gatalica, Zoran; Spetzler, David; Heimberger, Amy B

2018-06-19

We sought to compare the tumor profiles of brain metastases from common cancers with those of primary tumors and extracranial metastases in order to identify potential targets and prioritize rational treatment strategies. Tumor samples were collected from both the primary and metastatic sites of non-small cell lung cancer, breast cancer, and melanoma from patients in locations worldwide, and these were submitted to Caris Life Sciences for tumor multiplatform analysis, including gene sequencing (Sanger and next-generation sequencing with a targeted 47-gene panel), protein expression (assayed by immunohistochemistry), and gene amplification (assayed by in situ hybridization). The data analysis considered differential protein expression, gene amplification, and mutations among brain metastases, extracranial metastases, and primary tumors. The analyzed population included: 16,999 unmatched primary tumor and/or metastasis samples: 8178 non-small cell lung cancers (5098 primaries; 2787 systemic metastases; 293 brain metastases), 7064 breast cancers (3496 primaries; 3469 systemic metastases; 99 brain metastases), and 1757 melanomas (660 primaries; 996 systemic metastases; 101 brain metastases). TOP2A expression was increased in brain metastases from all 3 cancers, and brain metastases overexpressed multiple proteins clustering around functions critical to DNA synthesis and repair and implicated in chemotherapy resistance, including RRM1, TS, ERCC1, and TOPO1. cMET was overexpressed in melanoma brain metastases relative to primary skin specimens. Brain metastasis patients may particularly benefit from therapeutic targeting of enzymes associated with DNA synthesis, replication, and/or repair. This article is protected by copyright. All rights reserved. © 2018 UICC.

Sonic Hedgehog promotes proliferation of Notch-dependent monociliated choroid plexus tumour cells

PubMed Central

Li, Li; Grausam, Katie B.; Wang, Jun; Lun, Melody P.; Ohli, Jasmin; Lidov, Hart G. W.; Calicchio, Monica L.; Zeng, Erliang; Salisbury, Jeffrey L.; Wechsler-Reya, Robert J.; Lehtinen, Maria K.; Schüller, Ulrich; Zhao, Haotian

2016-01-01

Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly pediatric brain neoplasms. We developed animal models of CP tumours by inducing sustained expression of Notch1 that recapitulate properties of human CP tumours with aberrant NOTCH signaling. Whole transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumours, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues. PMID:26999738

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.

The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

PubMed

Munier, C Mee Ling; van Bockel, David; Bailey, Michelle; Ip, Susanna; Xu, Yin; Alcantara, Sheilajen; Liu, Sue Min; Denyer, Gareth; Kaplan, Warren; Suzuki, Kazuo; Croft, Nathan; Purcell, Anthony; Tscharke, David; Cooper, David A; Kent, Stephen J; Zaunders, John J; Kelleher, Anthony D

2016-10-17

Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

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

Chromosome microduplication in somatic cells decreases the genetic stability of human reprogrammed somatic cells and results in pluripotent stem cells.

PubMed

Yu, Yang; Chang, Liang; Zhao, Hongcui; Li, Rong; Fan, Yong; Qiao, Jie

2015-05-12

Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%-53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells.

RNA sequencing analysis reveals quiescent microglia isolation methods from postnatal mouse brains and limitations of BV2 cells.

PubMed

He, Yingbo; Yao, Xiang; Taylor, Natalie; Bai, Yuchen; Lovenberg, Timothy; Bhattacharya, Anindya

2018-05-22

Microglia play key roles in neuron-glia interaction, neuroinflammation, neural repair, and neurotoxicity. Currently, various microglial in vitro models including primary microglia derived from distinct isolation methods and immortalized microglial cell lines are extensively used. However, the diversity of these existing models raises difficulty in parallel comparison across studies since microglia are sensitive to environmental changes, and thus, different models are likely to show widely varied responses to the same stimuli. To better understand the involvement of microglia in pathophysiological situations, it is critical to establish a reliable microglial model system. With postnatal mouse brains, we isolated microglia using three general methods including shaking, mild trypsinization, and CD11b magnetic-associated cell sorting (MACS) and applied RNA sequencing to compare transcriptomes of the isolated cells. Additionally, we generated a genome-wide dataset by RNA sequencing of immortalized BV2 microglial cell line to compare with primary microglia. Furthermore, based on the outcomes of transcriptional analysis, we compared cellular functions between primary microglia and BV2 cells including immune responses to LPS by quantitative RT-PCR and Luminex Multiplex Assay, TGFβ signaling probed by Western blot, and direct migration by chemotaxis assay. We found that although the yield and purity of microglia were comparable among the three isolation methods, mild trypsinization drove microglia in a relatively active state, evidenced by high amount of amoeboid microglia, enhanced expression of microglial activation genes, and suppression of microglial quiescent genes. In contrast, CD11b MACS was the most reliable and consistent method, and microglia isolated by this method maintained a relatively resting state. Transcriptional and functional analyses revealed that as compared to primary microglia, BV2 cells remain most of the immune functions such as responses to LPS but showed limited TGFβ signaling and chemotaxis upon chemoattractant C5a. Collectively, we determined the optimal isolation methods for quiescent microglia and characterized the limitations of BV2 cells as an alternative of primary microglia. Considering transcriptional and functional differences, caution should be taken when extrapolating data from various microglial models. In addition, our RNA sequencing database serves as a valuable resource to provide novel insights for appropriate application of microglia as in vitro models.

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.

A hypothesis on the formation of the primary ossification centers in the membranous neurocranium: a mathematical and computational model.

PubMed

Garzón-Alvarado, Diego A

2013-01-21

This article develops a model of the appearance and location of the primary centers of ossification in the calvaria. The model uses a system of reaction-diffusion equations of two molecules (BMP and Noggin) whose behavior is of type activator-substrate and its solution produces Turing patterns, which represents the primary ossification centers. Additionally, the model includes the level of cell maturation as a function of the location of mesenchymal cells. Thus the mature cells can become osteoblasts due to the action of BMP2. Therefore, with this model, we can have two frontal primary centers, two parietal, and one, two or more occipital centers. The location of these centers in the simplified computational model is highly consistent with those centers found at an embryonic level. Copyright © 2012 Elsevier Ltd. All rights reserved.

Allogeneic stem cell transplantation for advanced cutaneous T-cell lymphomas: a study from the French Society of Bone Marrow Transplantation and French Study Group on Cutaneous Lymphomas

PubMed Central

de Masson, Adèle; Beylot-Barry, Marie; Bouaziz, Jean-David; de Latour, Régis Peffault; Aubin, François; Garciaz, Sylvain; d’Incan, Michel; Dereure, Olivier; Dalle, Stéphane; Dompmartin, Anne; Suarez, Felipe; Battistella, Maxime; Vignon-Pennamen, Marie-Dominique; Rivet, Jacqueline; Adamski, Henri; Brice, Pauline; François, Sylvie; Lissandre, Séverine; Turlure, Pascal; Wierzbicka-Hainaut, Ewa; Brissot, Eolia; Dulery, Rémy; Servais, Sophie; Ravinet, Aurélie; Tabrizi, Reza; Ingen-Housz-Oro, Saskia; Joly, Pascal; Socié, Gérard; Bagot, Martine

2014-01-01

The treatment of advanced stage primary cutaneous T-cell lymphomas remains challenging. In particular, large-cell transformation of mycosis fungoides is associated with a median overall survival of two years for all stages taken together. Little is known regarding allogeneic hematopoietic stem cell transplantation in this context. We performed a multicenter retrospective analysis of 37 cases of advanced stage primary cutaneous T-cell lymphomas treated with allogeneic stem cell transplantation, including 20 (54%) transformed mycosis fungoides. Twenty-four patients (65%) had stage IV disease (for mycosis fungoides and Sézary syndrome) or disseminated nodal or visceral involvement (for non-epidermotropic primary cutaneous T-cell lymphomas). After a median follow up of 29 months, 19 patients experienced a relapse, leading to a 2-year cumulative incidence of relapse of 56% (95%CI: 0.38–0.74). Estimated 2-year overall survival was 57% (95%CI: 0.41–0.77) and progression-free survival 31% (95%CI: 0.19–0.53). Six of 19 patients with a post-transplant relapse achieved a subsequent complete remission after salvage therapy, with a median duration of 41 months. A weak residual tumor burden before transplantation was associated with increased progression-free survival (HR=0.3, 95%CI: 0.1–0.8; P=0.01). The use of antithymocyte globulin significantly reduced progression-free survival (HR=2.9, 95%CI: 1.3–6.2; P=0.01) but also transplant-related mortality (HR=10−7, 95%CI: 4.10−8–2.10−7; P<0.001) in univariate analysis. In multivariate analysis, the use of antithymocyte globulin was the only factor significantly associated with decreased progression-free survival (P=0.04). Allogeneic stem cell transplantation should be considered in advanced stage primary cutaneous T-cell lymphomas, including transformed mycosis fungoides. PMID:24213148

Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis

EPA Science Inventory

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary ne...

Radiation Therapy for Primary Cutaneous Anaplastic Large Cell Lymphoma: An International Lymphoma Radiation Oncology Group Multi-institutional Experience

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

Million, Lynn, E-mail: lmillion@stanford.edu; Yi, Esther J.; Wu, Frank

Purpose: To collect response rates of primary cutaneous anaplastic large cell lymphoma, a rare cutaneous T-cell lymphoma, to radiation therapy (RT), and to determine potential prognostic factors predictive of outcome. Methods and Materials: The study was a retrospective analysis of patients with primary cutaneous anaplastic large cell lymphoma who received RT as primary therapy or after surgical excision. Data collected include initial stage of disease, RT modality (electron/photon), total dose, fractionation, response to treatment, and local recurrence. Radiation therapy was delivered at 8 participating International Lymphoma Radiation Oncology Group institutions worldwide. Results: Fifty-six patients met the eligibility criteria, and 63 tumorsmore » were treated: head and neck (27%), trunk (14%), upper extremities (27%), and lower extremities (32%). Median tumor size was 2.25 cm (range, 0.6-12 cm). T classification included T1, 40 patients (71%); T2, 12 patients (21%); and T3, 4 patients (7%). The median radiation dose was 35 Gy (range, 6-45 Gy). Complete clinical response (CCR) was achieved in 60 of 63 tumors (95%) and partial response in 3 tumors (5%). After CCR, 1 tumor recurred locally (1.7%) after 36 Gy and 7 months after RT. This was the only patient to die of disease. Conclusions: Primary cutaneous anaplastic large cell lymphoma is a rare, indolent cutaneous lymphoma with a low death rate. This analysis, which was restricted to patients selected for treatment with radiation, indicates that achieving CCR was independent of radiation dose. Because there were too few failures (<2%) for statistical analysis on dose response, 30 Gy seems to be adequate for local control, and even lower doses may suffice.« less

Primary cilia maintain corneal epithelial homeostasis by regulation of the Notch signaling pathway

PubMed Central

Grisanti, Laura; Revenkova, Ekaterina; Gordon, Ronald E.

2016-01-01

Primary cilia have been linked to signaling pathways involved in cell proliferation, cell motility and cell polarity. Defects in ciliary function result in developmental abnormalities and multiple ciliopathies. Patients affected by severe ciliopathies, such as Meckel syndrome, present several ocular surface disease conditions of unclear pathogenesis. Here, we show that primary cilia are predominantly present on basal cells of the mouse corneal epithelium (CE) throughout development and in the adult. Conditional ablation of cilia in the CE leads to an increase in proliferation and vertical migration of basal corneal epithelial cells (CECs). A consequent increase in cell density of suprabasal layers results in a thicker than normal CE. Surprisingly, in cilia-deficient CE, cilia-mediated signaling pathways, including Hh and Wnt pathways, were not affected but the intensity of Notch signaling was severely diminished. Although Notch1 and Notch2 receptors were expressed normally, nuclear Notch1 intracellular domain (N1ICD) expression was severely reduced. Postnatal development analysis revealed that in cilia-deficient CECs downregulation of the Notch pathway precedes cell proliferation defects. Thus, we have uncovered a function of the primary cilium in maintaining homeostasis of the CE by balancing proliferation and vertical migration of basal CECs through modulation of Notch signaling. PMID:27122169

Development Status of PEM Non-Flow-Through Fuel Cell System Technology for NASA Applications

NASA Technical Reports Server (NTRS)

Hoberecht, Mark A.; Jakupca, Ian J.

2011-01-01

Today s widespread development of proton-exchange-membrane (PEM) fuel cell technology for commercial users owes its existence to NASA, where fuel cell technology saw its first applications. Beginning with the early Gemini and Apollo programs, and continuing to this day with the Shuttle Orbiter program, fuel cells have been a primary source of electrical power for many NASA missions. This is particularly true for manned missions, where astronauts are able to make use of the by-product of the fuel cell reaction, potable water. But fuel cells also offer advantages for unmanned missions, specifically when power requirements exceed several hundred watts and primary batteries are not a viable alternative. In recent years, NASA s Exploration Technology Development Program (ETDP) funded the development of fuel cell technology for applications that provide both primary power and regenerative fuel cell energy storage for planned Exploration missions that involved a return to the moon. Under this program, the Altair Lunar Lander was a mission requiring fuel cell primary power. There were also various Lunar Surface System applications requiring regenerative fuel cell energy storage, in which a fuel cell and electrolyzer combine to form an energy storage system with hydrogen, oxygen, and water as common reactants. Examples of these systems include habitat modules and large rovers. In FY11, the ETDP has been replaced by the Enabling Technology Development and Demonstration Program (ETDDP), with many of the same technology goals and requirements applied against NASA s revised Exploration portfolio.

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.

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.

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

PubMed Central

2011-01-01

Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

EORTC, ISCL, and USCLC consensus recommendations for the treatment of primary cutaneous CD30-positive lymphoproliferative disorders: lymphomatoid papulosis and primary cutaneous anaplastic large-cell lymphoma*

PubMed Central

Pfaltz, Katrin; Vermeer, Maarten H.; Cozzio, Antonio; Ortiz-Romero, Pablo L.; Bagot, Martine; Olsen, Elise; Kim, Youn H.; Dummer, Reinhard; Pimpinelli, Nicola; Whittaker, Sean; Hodak, Emmilia; Cerroni, Lorenzo; Berti, Emilio; Horwitz, Steve; Prince, H. Miles; Guitart, Joan; Estrach, Teresa; Sanches, José A.; Duvic, Madeleine; Ranki, Annamari; Dreno, Brigitte; Ostheeren-Michaelis, Sonja; Knobler, Robert; Wood, Gary; Willemze, Rein

2011-01-01

Primary cutaneous CD30+ lymphoproliferative disorders (CD30+ LPDs) are the second most common form of cutaneous T-cell lymphomas and include lymphomatoid papulosis and primary cutaneous anaplastic large-cell lymphoma. Despite the anaplastic cytomorphology of tumor cells that suggest an aggressive course, CD30+ LPDs are characterized by an excellent prognosis. Although a broad spectrum of therapeutic strategies has been reported, these have been limited mostly to small retrospective cohort series or case reports, and only very few prospective controlled or multicenter studies have been performed, which results in a low level of evidence for most therapies. The response rates to treatment, recurrence rates, and outcome have not been analyzed in a systematic review. Moreover, international guidelines for staging and treatment of CD30+ LPDs have not yet been presented. Based on a literature analysis and discussions, recommendations were elaborated by a multidisciplinary expert panel of the Cutaneous Lymphoma Task Force of the European Organization for Research and Treatment of Cancer, the International Society for Cutaneous Lymphomas, and the United States Cutaneous Lymphoma Consortium. The recommendations represent the state-of-the-art management of CD30+ LPDs and include definitions for clinical endpoints as well as response criteria for future clinical trials in CD30+ LPDs. PMID:21841159

Survival data for 299 patients with primary cutaneous lymphomas: a monocentre study.

PubMed

Hallermann, Christian; Niermann, Christoph; Fischer, Rudolf-Josef; Schulze, Hans-Joachim

2011-09-01

The aim of this study was retrospectively to assess the validity of the 2005 WHO-EORTC classification for primary cutaneous lymphomas (PCL) in a large cohort of patients of a single German skin cancer unit. All patients with PCLs consecutively visiting our hospital between January 1980 and December 2005 were included in a retrospective monocentre study, analysing their histological and clinical data. A total of 312 patients fulfilled the inclusion criteria for PCL. In 299 patients clinical information and paraffin material were sufficient for detailed classification. Of the 299 patients, 63% expressed a T-cell and 37% a B-cell phenotype. Mycosis fungoides was the entity with the highest frequency (30.9%), followed by primary cutaneous follicle centre lymphomas (16.9%) and lymphomatoid papulosis (15.9%). The mean follow-up period was 38.4 months. Five-year disease-specific survival was 80.5% for mycosis fungoides, 92.5% in primary cutaneous anaplastic large cell lymphoma, 100% in lymphomatoid papulosis, 98.1% in primary cutaneous follicle center lymphoma, 100% in primary cutaneous marginal zone lymphoma and 63.2% in diffuse large B-cell lymphoma, leg type. Our data are in line with the data collected by the WHO-EORTC. This is further evidence for the reliability of the WHO-EORTC classification and staging system.

PRIMARY STROKE PREVENTION IN CHILDREN WITH SICKLE CELL ANEMIA LIVING IN AFRICA: THE FALSE CHOICE BETWEEN PATIENT-ORIENTED RESEARCH AND HUMANITARIAN SERVICE

PubMed Central

DEBAUN, MICHAEL R.; GALADANCI, NAJIBAH A.; KASSIM, ADETOLA A.; JORDAN, LORI C.; PHILLIPS, SHARON; ALIYU, MUKTAR H.

2016-01-01

In the United States, primary stroke prevention in children with sickle cell anemia (SCA) is now the standard of care and includes annual transcranial Doppler ultrasound evaluation to detect elevated intracranial velocities; and for those at risk, monthly blood transfusion therapy for at least a year followed by the option of hydroxyurea therapy. This strategy has decreased stroke prevalence in children with SCA from approximately 11% to 1%. In Africa, where 80% of all children with SCA are born, no systematic approach exists for primary stroke prevention. The two main challenges for primary stroke prevention in children with SCA in Africa include: 1) identifying an alternative to blood transfusion therapy, because safe monthly blood transfusion therapy is not feasible; and 2) assembling a health care team to implement and expand this effort. We will emphasize early triumphs and challenges to decreasing the incidence of strokes in African children with SCA. PMID:28066035

GATA3 expression in triple-negative breast cancers.

PubMed

Byrne, David J; Deb, Siddhartha; Takano, Elena A; Fox, Stephen B

2017-07-01

GATA-binding protein 3 (GATA3) is a well-studied transcription factor found to be essential in the development of luminal breast epithelium and has been identified in a variety of tumour types, including breast and urothelial carcinomas, making it a useful immunohistochemistry marker in the diagnosis of both primary and metastatic disease. We investigated GATA3 protein expression in a 106 primary triple-negative breast carcinomas (100 basal-like, six non-basal-like) using Cell Marque mouse monoclonal anti-GATA3 (L50-823). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to quantify mRNA expression in 22 triple-negative breast cancers (TNBCs) (20 primary and two cell lines), four luminal (three primary and one cell line) and five human epidermal growth factor receptor 2 (HER2) (four primary and one cell line) amplified tumours. In 98 TNBCs where IHC was assessable, 47 (48%) had a 1+ or greater staining with 20 (21%) having high GATA3 expression when using a weighted scoring. Our study has demonstrated that GATA3 expression is common in primary triple-negative breast carcinomas. It also suggests that although GATA3 is an oestrogen receptor (ER) regulated gene, it still proves useful in differentiating between primary and metastatic tumours in patients with a history of breast cancer regardless of its molecular subtype. © 2017 John Wiley & Sons Ltd.

Targeting dorsal root ganglia and primary sensory neurons for the treatment of chronic pain

PubMed Central

Berta, Temugin; Qadri, Yawar; Tan, Ping-Heng; Ji, Ru-Rong

2018-01-01

Introduction Currently the treatment of chronic pain is inadequate and compromised by debilitating central nervous system side effects. Here we discuss new therapeutic strategies that target dorsal root ganglia (DRGs) in the peripheral nervous system for a better and safer treatment of chronic pain. Areas covered The DRGs contain the cell bodies of primary sensory neurons including nociceptive neurons. After painful injuries, primary sensory neurons demonstrate maladaptive molecular changes in DRG cell bodies and in their axons. These changes result in hypersensitivity and hyperexcitability of sensory neurons (peripheral sensitization) and are crucial for the onset and maintenance of chronic pain. We discuss the following new strategies to target DRGs and primary sensory neurons as a means of alleviating chronic pain and minimizing side effects: inhibition of sensory neuron-expressing ion channels such as TRPA1, TRPV1, and Nav1.7, selective blockade of C- and Aβ-afferent fibers, gene therapy, and implantation of bone marrow stem cells. Expert opinion These peripheral pharmacological treatments, as well as gene and cell therapies, aimed at DRG tissues and primary sensory neurons can offer better and safer treatments for inflammatory, neuropathic, cancer, and other chronic pain states. PMID:28480765

DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.

PubMed

Houshdaran, Sahar; Hawley, Sarah; Palmer, Chana; Campan, Mihaela; Olsen, Mari N; Ventura, Aviva P; Knudsen, Beatrice S; Drescher, Charles W; Urban, Nicole D; Brown, Patrick O; Laird, Peter W

2010-02-22

Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. We obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited 'subtype-specific' DNA methylation patterns (FDR<1%) among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene. The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of potential tumor progenitor cells, which may help illuminate the etiology and natural history of these cancers.

Breast Cancer with Synchronous Renal Cell Carcinoma: A Rare Presentation.

PubMed

Arjunan, Ravi; Kumar, Durgesh; Kumar, K V Veerendra; Premlatha, C S

2016-10-01

Primary cancer arising from multiple organs is a well known fact. Synchronous tumours have been most commonly associated with kidney cancer. Bladder, prostate, colorectal and lung cancer are the most common synchronous primaries with Renal Cell Carcinoma (RCC) identified till date. We found metachronous tumours of breast with RCC in literature search which included both metastatic tumours as well second primaries. Overall, 25 cases of metastatic breast tumours and eight cases of second primary in previously treated RCC have been reported in the literature. Here, we are reporting a case of synchronous presentation of carcinoma breast with RCC which is very rare because most of the multiple malignancies reported in the literature are metastatic tumours or metachronous breast malignancy with RCC.

Breast Cancer with Synchronous Renal Cell Carcinoma: A Rare Presentation

PubMed Central

Arjunan, Ravi; Kumar, K V Veerendra; Premlatha, C S

2016-01-01

Primary cancer arising from multiple organs is a well known fact. Synchronous tumours have been most commonly associated with kidney cancer. Bladder, prostate, colorectal and lung cancer are the most common synchronous primaries with Renal Cell Carcinoma (RCC) identified till date. We found metachronous tumours of breast with RCC in literature search which included both metastatic tumours as well second primaries. Overall, 25 cases of metastatic breast tumours and eight cases of second primary in previously treated RCC have been reported in the literature. Here, we are reporting a case of synchronous presentation of carcinoma breast with RCC which is very rare because most of the multiple malignancies reported in the literature are metastatic tumours or metachronous breast malignancy with RCC. PMID:27891445

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.

Cellular Phenotype-Dependent and -Independent Effects of Vitamin C on the Renewal and Gene Expression of Mouse Embryonic Fibroblasts

PubMed Central

Kuo, Shiu-Ming; Burl, Lana R.; Hu, Zihua

2012-01-01

Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF) and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10−5 M), but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells, the growth-inhibitory effect of the redox-active form of vitamin C was preferentially observed in immortalized MEF. All effects of vitamin C required its intracellular presence since the transporter-deficient SVCT2−/− MEF did not respond to vitamin C. SVCT2−/− MEF divided and became immortalized readily indicating little dependence on vitamin C for the cell division. Immortalized SVCT2−/− MEF required higher concentration of vitamin C for the growth inhibition compared to the immortalized wildtype MEF suggesting an intracellular vitamin C toxicity. The relevance of our observation in aging and human cancer prevention was discussed. PMID:22427916

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 Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel Cell Development.

PubMed

Xiao, Ying; Thoresen, Daniel T; Miao, Lingling; Williams, Jonathan S; Wang, Chaochen; Atit, Radhika P; Wong, Sunny Y; Brownell, Isaac

2016-07-01

The Sonic hedgehog (Shh) signaling pathway regulates developmental, homeostatic, and repair processes throughout the body. In the skin, touch domes develop in tandem with primary hair follicles and contain sensory Merkel cells. The developmental signaling requirements for touch dome specification are largely unknown. We found dermal Wnt signaling and subsequent epidermal Eda/Edar signaling promoted Merkel cell morphogenesis by inducing Shh expression in early follicles. Lineage-specific gene deletions revealed intraepithelial Shh signaling was necessary for Merkel cell specification. Additionally, a Shh signaling agonist was sufficient to rescue Merkel cell differentiation in Edar-deficient skin. Moreover, Merkel cells formed in Fgf20 mutant skin where primary hair formation was defective but Shh production was preserved. Although developmentally associated with hair follicles, fate mapping demonstrated Merkel cells primarily originated outside the hair follicle lineage. These findings suggest that touch dome development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectoderm, including Eda signaling to primary hair placodes and ultimately Shh signaling from primary follicles to extrafollicular Merkel cell progenitors. Shh signaling often demonstrates pleiotropic effects within a structure over time. In postnatal skin, Shh is known to regulate the self-renewal, but not the differentiation, of touch dome stem cells. Our findings relate the varied effects of Shh in the touch dome to the ligand source, with locally produced Shh acting as a morphogen essential for lineage specification during development and neural Shh regulating postnatal touch dome stem cell maintenance.

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.

Bovine colostrum enhances natural killer cell activity and immune response in a mouse model of influenza infection and mediates intestinal immunity through toll-like receptors 2 and 4.

PubMed

Wong, Eric B; Mallet, Jean-François; Duarte, Jairo; Matar, Chantal; Ritz, Barry W

2014-04-01

Oral administration of bovine colostrum affects intestinal immunity, including an increased percentage of natural killer (NK) cells. However, effects on NK cell cytotoxic activity and resistance to infection as well as a potential mechanism remain unclear. Therefore, we investigated the effects of bovine colostrum (La Belle, Inc, Bellingham, WA) on the NK cytotoxic response to influenza infection and on toll-like receptor (TLR) activity in a primary intestinal epithelial cell culture. We hypothesized that colostrum would increase NK cell activity and that TLR-2 and TLR-4 blocking would reduce interleukin 6 production by epithelial cells in response to contact stimulation with colostrum. Four-month-old female C57BL/6 mice were supplemented with 1 g of colostrum per kilogram of body weight before and after infection with influenza A virus (H1N1). Animals were assessed for weight loss, splenic NK cell activity, and lung virus titers. Colostrum-supplemented mice demonstrated less reduction in body weight after influenza infection, indicating a less severe infection, increased NK cell cytotoxicity, and less virus burden in the lungs compared with controls. Colostrum supplementation enhanced NK cell cytotoxicity and improved the immune response to primary influenza virus infection in mice. To investigate a potential mechanism, a primary culture of small intestine epithelial cells was then stimulated with colostrum. Direct activation of epithelial cells resulted in increased interleukin 6 production, which was inhibited with TLR-2 and TLR-4 blocking antibodies. The interaction between colostrum and immunity may be dependent, in part, on the interaction of colostrum components with innate receptors at the intestinal epithelium, including TLR-2 and TLR-4. Copyright © 2014 Elsevier Inc. All rights reserved.

Awareness of Stem cells & Health Implications of SHED found in Pediatric Dentition among Indian Population

PubMed Central

Goomer, Pallvi; Sidhu, Arshpreet Kaur; Tuli, Preety; Kansal, Shinam; Bansal, Kanishka; Thakre, Gauri R

2014-01-01

Background: Primary teeth may be an ideal source of postnatal stem cells to regenerate tooth structures and bone, and possibly to treat neural tissue injury or degenerative diseases. SHED (stem cells from human exfoliated deciduous teeth) were identified to be a population of highly proliferative, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts. The present study was carried out to assess the knowledge, awareness & attitude of parents visiting various dental clinics in tricity area of india regarding stem cells from primary teeth and their potential health benefits. Materials & Methods: A total of 250 parents of pediatric patients seeking dental treatment at various dental clinics in tricity area were included in the study. Parents were personally interviewed with a questionnaire and their responses were immediately computed. Results: Among 250 parents only 95(62%) had knowledge regarding stem cells. While only 47(18.8) were informed regarding stem cells from baby teeth & their benefits. Maximum subjects were informed through internet 21(44.6%) followed by information through friends(23.4%) and dentist(21.2%). Very few were informed through magazines, newspaper and only one (2.1%) person was informed by television. Conclusion: It is important to create more awareness among the populace of our country about the potential health benefits of stem cells from primary teeth. Dentist should educate parents, caregivers and teachers regarding SHED & its benefits, ensuring good health for every Indian child and hence health of future citizens. How to cite the article: Goomer P, Sidhu AK, Tuli P, Kansal S, Bansal K, Thakre GR. Awareness of Stem cells & Health Implications of SHED found in Pediatric Dentition among Indian Population. J Int Oral Health 2014;6(1):44-7. PMID:24653602

CD8+ T-Cell Deficiency, Epstein-Barr Virus Infection, Vitamin D Deficiency, and Steps to Autoimmunity: A Unifying Hypothesis.

PubMed

Pender, Michael P

2012-01-01

CD8+ T-cell deficiency is a feature of many chronic autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, dermatomyositis, primary biliary cirrhosis, primary sclerosing cholangitis, ulcerative colitis, Crohn's disease, psoriasis, vitiligo, bullous pemphigoid, alopecia areata, idiopathic dilated cardiomyopathy, type 1 diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, IgA nephropathy, membranous nephropathy, and pernicious anaemia. It also occurs in healthy blood relatives of patients with autoimmune diseases, suggesting it is genetically determined. Here it is proposed that this CD8+ T-cell deficiency underlies the development of chronic autoimmune diseases by impairing CD8+ T-cell control of Epstein-Barr virus (EBV) infection, with the result that EBV-infected autoreactive B cells accumulate in the target organ where they produce pathogenic autoantibodies and provide costimulatory survival signals to autoreactive T cells which would otherwise die in the target organ by activation-induced apoptosis. Autoimmunity is postulated to evolve in the following steps: (1) CD8+ T-cell deficiency, (2) primary EBV infection, (3) decreased CD8+ T-cell control of EBV, (4) increased EBV load and increased anti-EBV antibodies, (5) EBV infection in the target organ, (6) clonal expansion of EBV-infected autoreactive B cells in the target organ, (7) infiltration of autoreactive T cells into the target organ, and (8) development of ectopic lymphoid follicles in the target organ. It is also proposed that deprivation of sunlight and vitamin D at higher latitudes facilitates the development of autoimmune diseases by aggravating the CD8+ T-cell deficiency and thereby further impairing control of EBV. The hypothesis makes predictions which can be tested, including the prevention and successful treatment of chronic autoimmune diseases by controlling EBV infection.

CD8+ T-Cell Deficiency, Epstein-Barr Virus Infection, Vitamin D Deficiency, and Steps to Autoimmunity: A Unifying Hypothesis

PubMed Central

Pender, Michael P.

2012-01-01

CD8+ T-cell deficiency is a feature of many chronic autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, dermatomyositis, primary biliary cirrhosis, primary sclerosing cholangitis, ulcerative colitis, Crohn's disease, psoriasis, vitiligo, bullous pemphigoid, alopecia areata, idiopathic dilated cardiomyopathy, type 1 diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, IgA nephropathy, membranous nephropathy, and pernicious anaemia. It also occurs in healthy blood relatives of patients with autoimmune diseases, suggesting it is genetically determined. Here it is proposed that this CD8+ T-cell deficiency underlies the development of chronic autoimmune diseases by impairing CD8+ T-cell control of Epstein-Barr virus (EBV) infection, with the result that EBV-infected autoreactive B cells accumulate in the target organ where they produce pathogenic autoantibodies and provide costimulatory survival signals to autoreactive T cells which would otherwise die in the target organ by activation-induced apoptosis. Autoimmunity is postulated to evolve in the following steps: (1) CD8+ T-cell deficiency, (2) primary EBV infection, (3) decreased CD8+ T-cell control of EBV, (4) increased EBV load and increased anti-EBV antibodies, (5) EBV infection in the target organ, (6) clonal expansion of EBV-infected autoreactive B cells in the target organ, (7) infiltration of autoreactive T cells into the target organ, and (8) development of ectopic lymphoid follicles in the target organ. It is also proposed that deprivation of sunlight and vitamin D at higher latitudes facilitates the development of autoimmune diseases by aggravating the CD8+ T-cell deficiency and thereby further impairing control of EBV. The hypothesis makes predictions which can be tested, including the prevention and successful treatment of chronic autoimmune diseases by controlling EBV infection. PMID:22312480

Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia

NASA Technical Reports Server (NTRS)

Fritzsch, Bernd

2003-01-01

The molecular and cellular origin of the primary neurons of the inner ear, the vestibular and spiral neurons, is reviewed including how they connect to the specific sensory epithelia and what the molecular nature of their survival is. Primary neurons of the ear depend on a single basic Helix-Loop-Helix (bHLH) protein for their formation, neurogenin 1 (ngn1). An immediate downstream gene is the bHLH gene neuronal differentiation (NeuroD). Targeted null mutations of ngn1 results in absence of primary neuron formation; targeted null mutation of NeuroD results in loss of almost all spiral and many vestibular neurons. NeuroD and a later expressed gene, Brn3a, play a role in pathfinding to and within sensory epithelia. The molecular nature of this pathfinding property is unknown. Reduction of hair cells in ngn1 null mutations suggests a clonal relationship with primary neurons. This relationship may play some role in specifying the identity of hair cells and the primary neurons that connect with them. Primary neuron neurites growth to sensory epithelia is initially independent of trophic factors released from developing sensory epithelia, but becomes rapidly dependent on those factors. Null mutations of specific neurotrophic factors lose distinct primary neuron populations which undergo rapid embryonic cell death.

Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport.

PubMed

Veszelka, Szilvia; Tóth, András; Walter, Fruzsina R; Tóth, Andrea E; Gróf, Ilona; Mészáros, Mária; Bocsik, Alexandra; Hellinger, Éva; Vastag, Monika; Rákhely, Gábor; Deli, Mária A

2018-01-01

Cell culture-based blood-brain barrier (BBB) models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC), ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA). As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L), and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1) and influx transporters (GLUT-1, LAT-1) were present in all models at mRNA levels. The transcript of BCRP (ABCG2) was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which are substrates of these transporters. Brain endothelial cell lines GP8, RBE4, D3 and D3L did not form a restrictive paracellular barrier necessary for screening small molecular weight pharmacons. Therefore, among the tested culture models, the primary cell-based EPA model is suitable for the functional analysis of the BBB.

Primary perivascular epithelioid cell tumors of the liver: CT/MRI findings and clinical outcomes.

PubMed

O'Malley, Martin E; Chawla, Tanya P; Lavelle, Lisa P; Cleary, Sean; Fischer, Sandra

2017-06-01

The purpose of our study was to describe the CT and MRI features of primary PEComas of the liver and to document the associated clinical outcomes. Retrospective study included 20 patients with primary hepatic perivascular epithelioid cell tumors (PEComa) with pathology and clinical outcomes for correlation. Study group included 20 patients: 16 women, 4 men; mean age 53 (range 35-77) years. Initial pathology diagnoses were classic angiomyolipoma (AML) (n = 11), epithelioid AML (n = 7), and PEComa not otherwise specified (n = 2). Mean tumor size was 5.1 (range 1.3-15.0) cm. CT/MRI features included well-defined margins 20/20 (100%), arterial enhancement 18/19 (95%), subcapsular location 17/20 (85%), heterogeneous 16/20 (80%), dysmorphic vessels 14/20 (70%), fat 13/20 (65%), hemorrhage 4/20 (20%), cystic components 4/20 (20%), and calcification 1/20 (5%). At the time of discovery, 18 patients were asymptomatic and their tumors were incidentally detected on imaging, and 2 patients were symptomatic. Ultimately, 18 tumors were benign and 2 developed metastases. On CT/MRI, most primary hepatic PEComas were well-defined, arterial enhancing, subcapsular, heterogeneous masses that often had dysmorphic vessels and contained fat. Most tumors were benign but complications included local symptoms, bleeding, and malignant change.

Inflammatory Responses and Barrier Function of Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

PubMed

Halaidych, Oleh V; Freund, Christian; van den Hil, Francijna; Salvatori, Daniela C F; Riminucci, Mara; Mummery, Christine L; Orlova, Valeria V

2018-05-08

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, which may be important in future regenerative medicine applications requiring vascularization. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Primary cutaneous CD8+ cytotoxic T-cell lymphoma involving the epidermis and subcutis in a young child.

PubMed

Wang, Lei; Gao, Tianwen; Wang, Gang

2015-04-01

CD8+ cytotoxic T-cell lymphoma involving the skin represents a heterogeneous group of diseases that include subcutaneous panniculitis-like T-cell lymphoma, primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma, and 'type D' lymphomatoid papulosis. In this report, we describe a case of CD8+ cytotoxic T-cell lymphoma involving both the epidermis and subcutis. The patient was a 6-year-old girl who presented with a 3-year history of multiple plaques on her trunk and legs. The lesions had relapsed twice but responded well to prednisone. Histopathologic examination showed the proliferation of atypical lymphocytes in the epidermis, dermis and subcutaneous tissue. On immunohistochemical analysis, the atypical lymphocytes were positive for βF1, CD3, CD8, perforin, granzyme B and TIA-1, but negative for T-cell receptor (TCR) γ, CD4, CD30 and CD56. It was difficult to classify this tumor in terms of the known types of cutaneous lymphoma, and this case should be differentiated with subcutaneous panniculitis-like T-cell lymphoma and primary cutaneous aggressive epidermotropic CD8+ T-cell lymphoma. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Role of Activin-A and Myostatin and Their Signaling Pathway in Human Myometrial and Leiomyoma Cell Function

PubMed Central

Islam, Md Soriful; Catherino, William H.; Protic, Olga; Janjusevic, Milijana; Gray, Peter Clarke; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Lamanna, Pasquale; Tranquilli, Andrea Luigi; Petraglia, Felice

2014-01-01

Context: Uterine leiomyomas are highly prevalent benign tumors of premenopausal women and the most common indication for hysterectomy. However, the exact etiology of this tumor is not fully understood. Objective: The objective of the study was to evaluate the role of activin-A and myostatin and their signaling pathways in human myometrial and leiomyoma cells. Design: This was a laboratory study. Setting: Myometrial and leiomyoma cells (primary and cell lines) were cultured in vitro. Patients: The study included premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Interventions: Primary myometrial and leiomyoma cells and/or cell lines were treated with activin-A (4 nM) and myostatin (4 nM) for different days of interval (to measure proliferation rate) or 30 minutes (to measure signaling molecules) or 48 hours to measure proliferating markers, extracellular matrix mRNA, and/or protein expression by real-time PCR, Western blot, and/or immunocytochemistry. Results: We found that activin-A and myostatin significantly reduce cell proliferation in primary myometrial cells but not in leiomyoma cells as measured by a CyQUANT cell proliferation assay kit. Reduced expression of proliferating cell nuclear antigen and Ki-67 were also observed in myometrial cells in response to activin-A and myostatin treatment. Activin-A also significantly increased mRNA expression of fibronectin, collagen1A1, and versican in primary leiomyoma cells. Finally, we found that activin-A and myostatin activate Smad-2/3 signaling but do not affect ERK or p38 signaling in both myometrial and leiomyoma cells. Conclusions: This study results suggest that activin-A and myostatin can exert antiproliferative and/or fibrotic effects on these cell types via Smad-2/3 signaling. PMID:24606069

Block Copolymers for Alkaline Fuel Cell Membrane Materials

DTIC Science & Technology

2014-07-30

temperature fuel cells including proton exchange membrane fuel cell ( PEMFC ) and alkaline fuel cell (AFC) with operation temperature usually lower than 120...advantages over proton exchange membrane fuel cells ( PEMFCs ) resulting in the popularity of AFCs in the US space program.[8-11] The primary benefit AFC...offered over PEMFC is better electrochemical kinetics on the anode and cathode under the alkaline environment, which results in the ability to use

Human herpesvirus 8-associated lymphoma mimicking cutaneous anaplastic large T-cell lymphoma in a patient with human immunodeficiency virus infection.

PubMed

Li, Meng-Fang; Hsiao, Cheng-Hsiang; Chen, Yi-Lin; Huang, Wen-Ya; Lee, Yi-Hsuan; Huang, Hsien-Neng; Lien, Huang-Chun

2012-02-01

Primary effusion lymphoma, a human herpesvirus 8 (HHV8)-associated lymphoma, is uncommon, and it is usually seen in human immunodeficiency virus (HIV)-infected patients. It presents as a body cavity-based lymphomatous effusion, but several cases of the so-called solid primary effusion lymphoma presenting as solid tumors without associated lymphomatous effusion have been reported. They have similar clinical, histopathological and immunophenotypical features. Most of them have a B-cell genotype. This suggests the solid variant may represent a clinicopathological spectrum of primary effusion lymphoma. We report a case of HHV8-associated lymphoma histopathologically and immunophenotypically mimicking cutaneous anaplastic large cell lymphoma. The patient was a 31-year-old HIV-seropositive man presenting with skin nodules over his right thigh. Biopsy of the nodules showed anaplastic large cells infiltrating the dermis. These malignant cells strongly expressed CD3, CD30 and CD43. Cutaneous anaplastic large T-cell lymphoma was initially diagnosed, but further tests, including immunoreactivity for HHV8 protein and clonal rearrangements of immunoglobulin genes, confirmed the diagnosis of HHV8-associated B-cell lymphoma with aberrant T-cell marker expression. This case provides an example of solid primary effusion lymphoma mimicking cutaneous anaplastic large T-cell lymphoma and highlights the importance of HHV8 immunohistochemistry and molecular tests in the diagnosis of HHV8-associated lymphoma with a cutaneous presentation. Copyright © 2011 John Wiley & Sons A/S.

Diagnosing and discriminating between primary and secondary aneurysmal bone cysts

PubMed Central

Sasaki, Hiromi; Nagano, Satoshi; Shimada, Hirofumi; Yokouchi, Masahiro; Setoguchi, Takao; Ishidou, Yasuhiro; Kunigou, Osamu; Maehara, Kosuke; Komiya, Setsuro

2017-01-01

Aneurysmal bone cysts (ABCs) are benign bony lesions frequently accompanied by multiple cystic lesions and aggressive bone destruction. They are relatively rare lesions, representing only 1% of bone tumors. The pathogenesis of ABCs has yet to be elucidated. In the present study, a series of 22 cases of primary and secondary ABC from patients treated in Department of Orthopedic Surgery, Kagoshima University Hospital (Kagoshima, Japan) from 2001–2015 were retrospectively analyzed. The average age at the time of diagnosis of primary ABC was 17.9 years. Intralesional curettage and artificial bone grafting were performed in the majority of the patients with primary ABC. The local recurrence rate following curettage for primary ABC was 18%, and the cause of local recurrence was considered to be insufficient curettage. Although no adjuvant therapy was administered during the surgeries, it may assist the prevention of local recurrence in certain cases. The cases of secondary ABC were preceded by benign bone tumors, including fibrous dysplasia, giant cell tumors, chondroblastoma and non-ossifying fibroma. The features of the secondary ABC typically reflected those of the preceding bone tumor. In the majority of cases, distinguishing the primary ABC from the secondary ABC was possible based on characteristic features, including age of the patient at diagnosis and the tumor location. In cases that exhibit ambiguous features, including a soft tissue mass or a thick septal enhancement on the preoperative magnetic resonance images, a biopsy must be obtained in order to exclude other types of aggressive bone tumors, including giant cell tumor, osteosarcoma and telangiectatic osteosarcoma. PMID:28454393

GEOSYNTHETIC DESIGN GUIDANCE FOR HAZARDOUS WASTE LANDFILL CELLS AND SURFACE IMPOUNDMENTS

EPA Science Inventory

The report provides guidance design procedures for the use of geosynthetic materials in hazardous waste land disposal cells. Primary geosynthetic components include flexible membrane liners (FML) used to limit the flow of leachate, and leachate collection and removal systems (LCR...

Stromal p16 Overexpression in Adult Granulosa Cell Tumors of the Ovary.

PubMed

Na, Kiyong; Sung, Ji-Youn; Kim, Hyun-Soo

2017-05-01

Adult granulosa cell tumor of the ovary is usually diagnosed at an early stage. However, most patients with advanced or recurrent disease will die of the disease due to limited treatment options. Data on the stromal p16 expression of ovarian adult granulosa cell tumors are limited. The aim of this study was to analyze the immunohistochemical p16 expression in the peritumoral stroma of primary and recurrent adult granulosa cell tumors and investigate whether there were significant differences in stromal p16 expression among nonpathological ovaries, benign sex cord-stromal tumors, and adult granulosa cell tumors. This study included 13 and 11 cases of primary and recurrent adult granulosa cell tumors, respectively. Non-pathological ovaries and benign sex cord-stromal tumors showed negative or weak positive expression, whereas most of the adult granulosa cell tumors showed diffuse and moderate-to-strong immunostaining. Primary adult granulosa cell tumors had significantly higher stromal p16 expression levels than nonpathological ovaries and benign sex cord-stromal tumors (p<0.001). Moreover, recurrent adult granulosa cell tumors showed significantly elevated levels of stromal p16 expression compared to primary adult granulosa cell tumors (p=0.032). In contrast, the difference in stromal p16 expression between non-pathological ovaries and benign sex cord-stromal tumors was not statistically significant (p=0.522). Our observations suggest that stromal p16 expression may be involved in the development and progression of ovarian adult granulosa cell tumors. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Human brain metastatic stroma attracts breast cancer cells via chemokines CXCL16 and CXCL12.

PubMed

Chung, Brile; Esmaeili, Ali A; Gopalakrishna-Pillai, Sailesh; Murad, John P; Andersen, Emily S; Kumar Reddy, Naveen; Srinivasan, Gayathri; Armstrong, Brian; Chu, Caleb; Kim, Young; Tong, Tommy; Waisman, James; Yim, John H; Badie, Behnam; Lee, Peter P

2017-01-01

The tumor microenvironment is composed of heterogeneous populations of cells, including cancer, immune, and stromal cells. Progression of tumor growth and initiation of metastasis is critically dependent on the reciprocal interactions between cancer cells and stroma. Through RNA-Seq and protein analyses, we found that cancer-associated fibroblasts derived from human breast cancer brain metastasis express significantly higher levels of chemokines CXCL12 and CXCL16 than fibroblasts from primary breast tumors or normal breast. To further understand the interplay between cancer cells and cancer-associated fibroblasts from each site, we developed three-dimensional organoids composed of patient-derived primary or brain metastasis cancer cells with matching cancer-associated fibroblasts. Three-dimensional CAF aggregates generated from brain metastasis promote migration of cancer cells more effectively than cancer-associated fibroblast aggregates derived from primary tumor or normal breast stromal cells. Treatment with a CXCR4 antagonist and/or CXCL16 neutralizing antibody, alone or in combination, significantly inhibited migration of cancer cells to brain metastatic cancer-associated fibroblast aggregates. These results demonstrate that human brain metastasis cancer-associated fibroblasts potently attract breast cancer cells via chemokines CXCL12 and CXCL16, and blocking CXCR6-CXCL16/CXCR4-CXCL12 receptor-ligand interactions may be an effective therapy for preventing breast cancer brain metastasis.

Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

PubMed

Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

2018-03-05

Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fine needle aspiration biopsy of three cases of squamous cell carcinoma presenting as a thyroid mass: cytological findings and differential diagnosis.

PubMed

Rosa, M; Toronczyk, K

2012-02-01

Primary squamous cell carcinomas of the thyroid gland are extremely rare, comprising about 1% of thyroid malignancies. Although squamous cell carcinomas are readily identified as such on aspiration cytology in the majority of cases, the differentiation of primary versus metastatic tumour might not always be easy. Herein, we report three cases of squamous cell carcinomas involving the thyroid gland. Fine needle aspiration cytology (FNAC) was performed in three patients with a thyroid mass using standard guidelines. Smears were stained with Diff-Quik and Papanicolaou stains. Two patients were male and one was female, aged 59, 45 and 35 years, respectively. In all three patients a thyroid mass was present. FNAC smears in all cases showed cytological features of squamous cell carcinoma including keratinization and necrosis. After clinical and cytological correlation, one case appeared to be primary, one case metastatic, and in the third case no additional clinical information or biopsy follow-up was available for further characterization. Because primary squamous cell carcinoma of the thyroid is a rare finding, metastatic squamous cell carcinoma should always be excluded first. Metastatic disease usually presents in the setting of widespread malignancy, therefore a dedicated clinical and radiological investigation is necessary in these cases. In both clinical scenarios the patient's prognosis is poor. © 2010 Blackwell Publishing Ltd.

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells

PubMed Central

Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

2013-01-01

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001 PMID:24252877

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.

PubMed

Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-Ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

2013-11-19

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

A mathematical model of a lithium/thionyl chloride primary cell

NASA Technical Reports Server (NTRS)

Evans, T. I.; Nguyen, T. V.; White, R. E.

1987-01-01

A 1-D mathematical model for the lithium/thionyl chloride primary cell was developed to investigate methods of improving its performance and safety. The model includes many of the components of a typical lithium/thionyl chloride cell such as the porous lithium chloride film which forms on the lithium anode surface. The governing equations are formulated from fundamental conservation laws using porous electrode theory and concentrated solution theory. The model is used to predict 1-D, time dependent profiles of concentration, porosity, current, and potential as well as cell temperature and voltage. When a certain discharge rate is required, the model can be used to determine the design criteria and operating variables which yield high cell capacities. Model predictions can be used to establish operational and design limits within which the thermal runaway problem, inherent in these cells, can be avoided.

Loss of Proliferation and Antigen Presentation Activity following Internalization of Polydispersed Carbon Nanotubes by Primary Lung Epithelial Cells

PubMed Central

Kumari, Mandavi; Sachar, Sumedha; Saxena, Rajiv K.

2012-01-01

Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells. PMID:22384094

Cell sources for in vitro human liver cell culture models.

PubMed

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

2016-09-01

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

Cell sources for in vitro human liver cell culture models

PubMed Central

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

2016-01-01

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

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

PubMed

Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P; Walles, Heike; Braspenning, Joris; Breitkopf-Heinlein, Katja

2015-01-01

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Purification and characterization of the amine dehydrogenase from a facultative methylotroph.

PubMed

Coleman, J P; Perry, J J

1984-01-01

Strain RA-6 is a pink-pigmented organism which can grow on a variety of substrates including methylamine. It can utilize methylamine as sole source of carbon via an isocitrate lyase negative serine pathway. Methylamine grown cells contain an inducible primary amine dehydrogenase [primary amine: (acceptor) oxidoreductase (deaminating)] which is not present in succinate grown cells. The amine dehydrogenase was purified to over 90% homogeneity. It is an acidic protein (isoelectric point of 5.37) with a molecular weight of 118,000 containing subunits with approximate molecular weights of 16,500 and 46,000. It is active on an array of primary terminal amines and is strongly inhibited by carbonyl reagents. Cytochrome c or artificial electron acceptors are required for activity; neither NAD nor NADP can serve as primary electron acceptor.

Patients with primary diffuse large B-cell lymphoma of female genital tract have high risk of central nervous system relapse.

PubMed

Cao, Xin-xin; Li, Jian; Zhang, Wei; Duan, Ming-hui; Shen, Ti; Zhou, Dao-bin

2014-06-01

The objective of this study was to evaluate retrospectively the clinical characteristics, treatments, and outcomes of patients with primary diffuse large B-cell lymphoma (DLBCL) of the female genital tract. The basic characteristics, treatments, and outcomes of six patients diagnosed with primary DLBCL of the female genital tract, including the ovary, uterine cervix, and vagina, treated in our hospital between 2000 and 2012, were analyzed retrospectively. Seven of 323 (2.2 %) newly diagnosed DLBCLs were diagnosed as primary female genital tract DLBCL. Six patients with complete medical data were included in the analysis. The median age at diagnosis was 52.5 years (range 20-65). The presenting symptoms included abnormal vaginal bleeding, increased vaginal discharge, abdominal fullness, and abdominal pain. Two patients had stage IE disease and four patients had stage IIE disease. Treatment included chemotherapy only in five patients, and combined chemotherapy and localized radiation in one patient. After a median follow-up of 58 months, four patients showed relapse in the central nervous system and two had died from progressive disease. The median progression-free survival was 27 months and the median overall survival for this group has not been reached. Patients with primary female genital tract DLBCL may have poor outcomes and a high risk of central nervous system relapse. Central nervous system prophylaxis might be considered in addition to systemic chemotherapy for DLBCL of the female genital tract.

HtrA3 Is Downregulated in Cancer Cell Lines and Significantly Reduced in Primary Serous and Granulosa Cell Ovarian Tumors.

PubMed

Singh, Harmeet; Li, Ying; Fuller, Peter J; Harrison, Craig; Rao, Jyothsna; Stephens, Andrew N; Nie, Guiying

2013-01-01

Objective. The high temperature requirement factor A3 (HtrA3) is a serine protease homologous to bacterial HtrA. Four human HtrAs have been identified. HtrA1 and HtrA3 share a high degree of domain organization and are downregulated in a number of cancers, suggesting a widespread loss of these proteases in cancer. This study examined how extensively the HtrA (HtrA1-3) proteins are downregulated in commonly used cancer cell lines and primary ovarian tumors.Methods. RT-PCR was applied to various cancer cell lines (n=17) derived from the ovary, endometrium, testes, breast, prostate, and colon, and different subtypes of primary ovarian tumors [granulosa cell tumors (n=19), mucinous cystadenocarcinomas (n=6), serous cystadenocarcinomas (n=8)] and normal ovary (n = 9). HtrA3 protein was localized by immunohistochemistry.Results. HtrA3 was extensively downregulated in the cancer cell lines examined including the granulosa cell tumor-derived cell lines. In primary ovarian tumors, the HtrA3 was significantly lower in serous cystadenocarcinoma and granulosa cell tumors. In contrast, HtrA1 and HtrA2 were expressed in all samples with no significant differences between the control and tumors. In normal postmenopausal ovary, HtrA3 protein was localized to lutenizing stromal cells and corpus albicans. In serous cystadenocarcinoma, HtrA3 protein was absent in the papillae but detected in the mesenchymal cyst wall.Conclusion. HtrA3 is more extensively downregulated than HtrA1-2 in cancer cell lines. HtrA3, but not HtrA1 or HtrA2, was decreased in primary ovarian serous cystadenocarcinoma and granulosa cell tumors. This study provides evidence that HtrA3 may be the most relevant HtrA associated with ovarian malignancy.

Head and neck cutaneous squamous cell carcinoma metastatic to cervical sublevel IIb lymph nodes occurred from primary sites involving the auricle and adjacent neck.

PubMed

Maher, Nigel Gordon; Hoffman, Gary Russell

2014-03-01

Neck dissections that include sublevel IIb increase the risk of postoperative shoulder dysfunction. The purpose of this investigation was to document the incidence of level IIb metastatic lymphatic spread in a group of patients undergoing neck dissection as part of the surgical management of cutaneous squamous cell carcinoma of the head and neck. A retrospective review of the pathology records taken from 1 surgeon from June 2006 through June 2013 was carried out. The predictor variable was the primary tumor site. The outcome variable was the metastatic nodal involvement according to neck level and sublevel. Secondary variables included T stage, pathologist, tumor depth, and the presence of perineural, perilymphatic, and perivascular invasion. Data analyses were by descriptive statistics. Thirty-six patients with a total of 40 neck dissections met the inclusion criteria. The average primary site tumor depth was 14.7 mm, and there were 16 cases of poorly differentiated squamous cell carcinoma. Sublevel IIb was involved in 7.5% of cases, all of which occurred from lateralized primary sites of the head and neck. Cutaneous squamous cell carcinoma arising from the auricle and neck sites adjacent to sublevel IIb may have increased risk of metastatic involvement of sublevel IIb nodes. Further studies with larger numbers are required to determine the risk of metastasis to sublevel IIb from midline sites of the face. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness.

PubMed

Lee, Jisoo; Kim, Yoo-Sun; Lee, JaeHwan; Heo, Seung Chul; Lee, Kook Lae; Choi, Sang-Woon; Kim, Yuri

2016-07-21

Walnut has been known for its health benefits, including anti-cardiovascular disease and anti-oxidative properties. However, there is limited evidence elucidating its effects on cancer stem cells (CSCs) which represent a small subset of cancer cells that provide resistance against chemotherapy. This study aimed to evaluate the anti-CSCs potential of walnut phenolic extract (WPE) and its bioactive compounds, including (+)-catechin, chlorogenic acid, ellagic acid, and gallic acid. In the present study, CD133⁺CD44⁺ cells were isolated from HCT116 cells using fluorescence-activated cell sorting (FACS) and then treated with WPE. As a result, survival of the CD133⁺CD44⁺ HCT116 cells was inhibited and cell differentiation was induced by WPE. In addition, WPE down-regulated the CSC markers, CD133, CD44, DLK1, and Notch1, as well as the β-catenin/p-GSK3β signaling pathway. WPE suppressed the self-renewal capacity of CSCs. Furthermore, the WPE exhibited stronger anti-CSC effects than its individual bioactive compounds. Finally, the WPE inhibited specific CSC markers in primary colon cancer cells isolated from primary colon tumor. These results suggest that WPE can suppress colon cancer by regulating the characteristics of colon CSCs.

Walnut Phenolic Extract and Its Bioactive Compounds Suppress Colon Cancer Cell Growth by Regulating Colon Cancer Stemness

PubMed Central

Lee, Jisoo; Kim, Yoo-Sun; Lee, JaeHwan; Heo, Seung Chul; Lee, Kook Lae; Choi, Sang-Woon; Kim, Yuri

2016-01-01

Walnut has been known for its health benefits, including anti-cardiovascular disease and anti-oxidative properties. However, there is limited evidence elucidating its effects on cancer stem cells (CSCs) which represent a small subset of cancer cells that provide resistance against chemotherapy. This study aimed to evaluate the anti-CSCs potential of walnut phenolic extract (WPE) and its bioactive compounds, including (+)-catechin, chlorogenic acid, ellagic acid, and gallic acid. In the present study, CD133+CD44+ cells were isolated from HCT116 cells using fluorescence-activated cell sorting (FACS) and then treated with WPE. As a result, survival of the CD133+CD44+ HCT116 cells was inhibited and cell differentiation was induced by WPE. In addition, WPE down-regulated the CSC markers, CD133, CD44, DLK1, and Notch1, as well as the β-catenin/p-GSK3β signaling pathway. WPE suppressed the self-renewal capacity of CSCs. Furthermore, the WPE exhibited stronger anti-CSC effects than its individual bioactive compounds. Finally, the WPE inhibited specific CSC markers in primary colon cancer cells isolated from primary colon tumor. These results suggest that WPE can suppress colon cancer by regulating the characteristics of colon CSCs. PMID:27455311

Hemophagocytic Lymphohistiocytosis in Children: Pathogenesis and Treatment

PubMed Central

Ishii, Eiichi

2016-01-01

Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder in children that is characterized by persistent fever, splenomegaly with cytopenia, hypertriglyceridemia, and hypofibrinogenemia. Increased levels of various cytokines and soluble interleukin-2 receptor are biological markers of HLH. HLH can be classified into two major forms: primary and secondary. Familial hemophagocytic lymphohistiocytosis (FHL), a type of primary HLH, is an autosomal recessive disorder that typically occurs in infancy and can be classified into five different subtypes (FHL types 1–5). In Japan, >80% of patients with FHL have either PRF1 (FHL type 2) or UNC13D (FHL type 3) defects. FHL is considered to be a disorder of T-cell function because the activity of NK cells or cytotoxic T lymphocytes as target cells is usually impaired. Moreover, Epstein–Barr virus-associated HLH (EBV-HLH) is considered a major subtype of secondary HLH. Any genetic background could have an effect on the pathogenesis of secondary HLH because EBV-HLH is considered to be particularly prevalent in Asian countries. For primary HLH, hematopoietic stem cell transplantation is the only accepted curative therapy, although cord blood transplantation with a reduced-conditioning regimen has been used with superior outcomes. For secondary HLH, including EBV-HLH, immunochemotherapy based on the HLH-2004 protocol has been used. In the near future, the entire mechanism of HLH should be clarified to establish less toxic therapies, including cell therapy and gene targeting therapy. PMID:27242976

HSV-1 infection of human corneal epithelial cells: receptor-mediated entry and trends of re-infection.

PubMed

Shah, Arpeet; Farooq, Asim V; Tiwari, Vaibhav; Kim, Min-Jung; Shukla, Deepak

2010-11-20

The human cornea is a primary target for herpes simplex virus-1 (HSV-1) infection. The goals of the study were to determine the cellular modalities of HSV-1 entry into human corneal epithelial (HCE) cells. Specific features of the study included identifying major entry receptors, assessing pH dependency, and determining trends of re-infection. A recombinant HSV-1 virus expressing beta-galactosidase was used to ascertain HSV-1 entry into HCE cells. Viral replication within cells was confirmed using a time point plaque assay. Lysosomotropic agents were used to test for pH dependency of entry. Flow cytometry and immunocytochemistry were used to determine expression of three cellular receptors--nectin-1, herpesvirus entry mediator (HVEM), and paired immunoglobulin-like 2 receptor alpha (PILR-a). The necessity of these receptors for viral entry was tested using antibody-blocking. Finally, trends of re-infection were investigated using viral entry assay and flow cytometry post-primary infection. Cultured HCE cells showed high susceptibility to HSV-1 entry and replication. Entry was demonstrated to be pH dependent as blocking vesicular acidification decreased entry. Entry receptors expressed on the cell membrane include nectin-1, HVEM, and PILR-α. Receptor-specific antibodies blocked entry receptors, reduced viral entry and indicated nectin-1 as the primary receptor used for entry. Cells re-infected with HSV-1 showed a decrease in entry, which was correlated to decreased levels of nectin-1 as demonstrated by flow cytometry. HSV-1 is capable of developing an infection in HCE cells using a pH dependent entry process that involves primarily nectin-1 but also the HVEM and PILR-α receptors. Re-infected cells show decreased levels of entry, correlated with a decreased level of nectin-1 receptor expression.

Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

PubMed

Labenski, Verena; Suerth, Julia D; Barczak, Elke; Heckl, Dirk; Levy, Camille; Bernadin, Ornellie; Charpentier, Emmanuelle; Williams, David A; Fehse, Boris; Verhoeyen, Els; Schambach, Axel

2016-08-01

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

RNAi Screening with Self-Delivering, Synthetic siRNAs for Identification of Genes That Regulate Primary Human T Cell Migration.

PubMed

Freeley, Michael; Derrick, Emily; Dempsey, Eugene; Hoff, Antje; Davies, Anthony; Leake, Devin; Vermeulen, Annaleen; Kelleher, Dermot; Long, Aideen

2015-09-01

Screening of RNA interference (RNAi) libraries in primary T cells is labor-intensive and technically challenging because these cells are hard to transfect. Chemically modified, self-delivering small interfering RNAs (siRNAs) offer a solution to this problem, because they enter hard-to-transfect cell types without needing a delivery reagent and are available in library format for RNAi screening. In this study, we have screened a library of chemically modified, self-delivering siRNAs targeting the expression of 72 distinct genes in conjunction with an image-based high-content-analysis platform as a proof-of-principle strategy to identify genes involved in lymphocyte function-associated antigen-1 (LFA-1)-mediated migration in primary human T cells. Our library-screening strategy identified the small GTPase RhoA as being crucial for T cell polarization and migration in response to LFA-1 stimulation and other migratory ligands. We also demonstrate that multiple downstream assays can be performed within an individual RNAi screen and have used the remainder of the cells for additional assays, including cell viability and adhesion to ICAM-1 (the physiological ligand for LFA-1) in the absence or presence of the chemokine SDF-1α. This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function. © 2015 Society for Laboratory Automation and Screening.

Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer.

PubMed

Mouradov, Dmitri; Sloggett, Clare; Jorissen, Robert N; Love, Christopher G; Li, Shan; Burgess, Antony W; Arango, Diego; Strausberg, Robert L; Buchanan, Daniel; Wormald, Samuel; O'Connor, Liam; Wilding, Jennifer L; Bicknell, David; Tomlinson, Ian P M; Bodmer, Walter F; Mariadason, John M; Sieber, Oliver M

2014-06-15

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses. ©2014 American Association for Cancer Research.

Translating stem cell research: challenges at the research frontier.

PubMed

Magnus, David

2010-01-01

This paper will address the translation of basic stem cell research into clinical research. While "stem cell" trials are sometimes used to describe established practices of bone marrow transplantation or transplantation of primary cells derived from bone marrow, for the purposes of this paper, I am primarily focusing on stem cell trials which are far less established, including use of hESC derived stem cells. The central ethical challenges in stem cell clinical trials arise in frontier research, not in standard, well-established areas of research.

Jet fuel based high pressure solid oxide fuel cell system

NASA Technical Reports Server (NTRS)

Gummalla, Mallika (Inventor); Yamanis, Jean (Inventor); Olsommer, Benoit (Inventor); Dardas, Zissis (Inventor); Bayt, Robert (Inventor); Srinivasan, Hari (Inventor); Dasgupta, Arindam (Inventor); Hardin, Larry (Inventor)

2013-01-01

A power system for an aircraft includes a solid oxide fuel cell system which generates electric power for the aircraft and an exhaust stream; and a heat exchanger for transferring heat from the exhaust stream of the solid oxide fuel cell to a heat requiring system or component of the aircraft. The heat can be transferred to fuel for the primary engine of the aircraft. Further, the same fuel can be used to power both the primary engine and the SOFC. A heat exchanger is positioned to cool reformate before feeding to the fuel cell. SOFC exhaust is treated and used as inerting gas. Finally, oxidant to the SOFC can be obtained from the aircraft cabin, or exterior, or both.

Jet Fuel Based High Pressure Solid Oxide Fuel Cell System

NASA Technical Reports Server (NTRS)

Srinivasan, Hari (Inventor); Hardin, Larry (Inventor); Gummalla, Mallika (Inventor); Yamanis, Jean (Inventor); Olsommer, Benoit (Inventor); Dardas, Zissis (Inventor); Dasgupta, Arindam (Inventor); Bayt, Robert (Inventor)

2015-01-01

A power system for an aircraft includes a solid oxide fuel cell system which generates electric power for the aircraft and an exhaust stream; and a heat exchanger for transferring heat from the exhaust stream of the solid oxide fuel cell to a heat requiring system or component of the aircraft. The heat can be transferred to fuel for the primary engine of the aircraft. Further, the same fuel can be used to power both the primary engine and the SOFC. A heat exchanger is positioned to cool reformate before feeding to the fuel cell. SOFC exhaust is treated and used as inerting gas. Finally, oxidant to the SOFC can be obtained from the aircraft cabin, or exterior, or both.

T cell lymphoblastic lymphoma/leukemia within an adrenocorticotropic hormone and thyroid stimulating hormone positive pituitary adenoma: A cytohistological correlation emphasizing importance of intra-operative squash smear.

PubMed

Gupta, Rakesh K; Saran, Ravindra K; Srivastava, Arvind K; Jagetia, Anita; Garg, Lalit; Sharma, Mehar C

2017-08-01

We present a rare case of primary pituitary T cell lymphoma/leukemia (T-LBL) in association with adrenocorticotropic hormone (ACTH) and thyroid stimulating hormone (TSH) expressing pituitary adenoma in a 55-year-old woman highlighting the importance of intra-operative squash smears examination. The patient presented with complaints of headache, diminution of vision and recent onset altered sensorium. MRI revealed a mass lesion in the sellar-suprasellar region with non-visualization of pituitary gland separately, extending to involve adjacent structures diagnosed as invasive pituitary macroadenoma. Intra-operative tissue was sent for squash smear examination. The cytology showed a tumor comprising of sheets of immature lymphoid cells intermixed with clusters of pituitary acinar cells with many mitoses and tingible body macrophages. A diagnosis of presence of immature lymphoid cells within the pituitary was offered and differentials of infiltration by lymphoma cells from systemic disease versus primary central nervous lymphoma-like lymphoma arising in the pituitary adenoma were considered. Later paraffin section examination and immunohistochemistry corroborated with the squash findings and a final diagnosis of primary pituitary T cell lymphoma/leukemia in association with ACTH and TSH expressing pituitary adenoma was made. To date, only six cases of primary pituitary T cell lymphomas, including three T-LBL cases, have been reported. This is the seventh case and first one additionally describing cytohistological correlation and importance of intra-operative cytology. © 2017 Japanese Society of Neuropathology.

Live-Cell Imaging of Phagosome Motility in Primary Mouse RPE Cells.

PubMed

Hazim, Roni; Jiang, Mei; Esteve-Rudd, Julian; Diemer, Tanja; Lopes, Vanda S; Williams, David S

2016-01-01

The retinal pigment epithelium (RPE) is a post-mitotic epithelial monolayer situated between the light-sensitive photoreceptors and the choriocapillaris. Given its vital functions for healthy vision, the RPE is a primary target for insults that result in blinding diseases, including age-related macular degeneration (AMD). One such function is the phagocytosis and digestion of shed photoreceptor outer segments. In the present study, we examined the process of trafficking of outer segment disk membranes in live cultures of primary mouse RPE, using high speed spinning disk confocal microscopy. This approach has enabled us to track phagosomes, and determine parameters of their motility, which are important for their efficient degradation.

Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

PubMed

de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

2010-12-15

Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology. Copyright © 2010 Elsevier B.V. All rights reserved.

The Incidence of Other Primary Cancers in Patients with Cutaneous Lymphoma.

PubMed

Kim, Young Jae; Shin, Ho Jeong; Won, Chong Hyun; Chang, Sung Eun; Lee, Mi Woo; Choi, Jee Ho; Lee, Woo Jin

2018-06-01

Skin cancer is the most common other primary cancer in patients with lymphoma. However, an intriguing association between cutaneous lymphoma and other primary cancers has been suggested in a few studies. This study investigated other primary cancers in patients with cutaneous lymphoma to evaluate the risk for occurrence of each type of cancer. We screened for other primary cancers in 428 patients with cutaneous lymphoma. Clinical features were analyzed according to the lineage and origin of the lymphomas. We calculated the standardized incidence ratio with statistical analysis for each group according to age. Among 330 patients with cutaneous T cell lymphoma and 98 with cutaneous B cell lymphoma, a total of 43 cancers in 38 patients were finally included. Other primary cancers were prevalent in patients with cutaneous B cell lymphoma and patients with secondary cutaneous lymphoma. However, those differences were not significant when the age was calibrated by multiple logistic regression. Metachronously higher standardized incidence ratios were observed for primary lung (standardized incidence ratio [SIR], 14.81; 95% confidence interval [CI], 3.05~39.54), skin (SIR, 68.05; 95% CI, 14.03~181.62), and breast (SIR, 12.91; 95% CI, 1.56~41.41) cancers with statistical significance. Other primary cancers more preferentially occurred in patients with cutaneous lymphoma. Clinicians should carefully examine patients with cutaneous lymphoma for other cancers, especially lung, skin, and breast cancers.

The Incidence of Other Primary Cancers in Patients with Cutaneous Lymphoma

PubMed Central

Kim, Young Jae; Shin, Ho Jeong; Won, Chong Hyun; Chang, Sung Eun; Lee, Mi Woo; Choi, Jee Ho

2018-01-01

Background Skin cancer is the most common other primary cancer in patients with lymphoma. However, an intriguing association between cutaneous lymphoma and other primary cancers has been suggested in a few studies. Objective This study investigated other primary cancers in patients with cutaneous lymphoma to evaluate the risk for occurrence of each type of cancer. Methods We screened for other primary cancers in 428 patients with cutaneous lymphoma. Clinical features were analyzed according to the lineage and origin of the lymphomas. We calculated the standardized incidence ratio with statistical analysis for each group according to age. Results Among 330 patients with cutaneous T cell lymphoma and 98 with cutaneous B cell lymphoma, a total of 43 cancers in 38 patients were finally included. Other primary cancers were prevalent in patients with cutaneous B cell lymphoma and patients with secondary cutaneous lymphoma. However, those differences were not significant when the age was calibrated by multiple logistic regression. Metachronously higher standardized incidence ratios were observed for primary lung (standardized incidence ratio [SIR], 14.81; 95% confidence interval [CI], 3.05~39.54), skin (SIR, 68.05; 95% CI, 14.03~181.62), and breast (SIR, 12.91; 95% CI, 1.56~41.41) cancers with statistical significance. Conclusion Other primary cancers more preferentially occurred in patients with cutaneous lymphoma. Clinicians should carefully examine patients with cutaneous lymphoma for other cancers, especially lung, skin, and breast cancers. PMID:29853749

Factors associated with a primary surgical approach for sinonasal squamous cell carcinoma.

PubMed

Cracchiolo, Jennifer R; Patel, Krupa; Migliacci, Jocelyn C; Morris, Luc T; Ganly, Ian; Roman, Benjamin R; McBride, Sean M; Tabar, Viviane S; Cohen, Marc A

2018-03-01

Primary surgery is the preferred treatment of T1-T4a sinonasal squamous cell carcinoma (SNSCC). Patients with SNSCC in the National Cancer Data Base (NCDB) were analyzed. Factors that contributed to selecting primary surgical treatment were examined. Overall survival (OS) in surgical patients was analyzed. Four-thousand seven hundred and seventy patients with SNSCC were included. In T1-T4a tumors, lymph node metastases, maxillary sinus location, and treatment at high-volume centers were associated with selecting primary surgery. When primary surgery was utilized, tumor factors and positive margin guided worse OS. Adjuvant therapy improved OS in positive margin resection and advanced T stage cases. Tumor and non-tumor factors are associated with selecting surgery for the treatment of SNSCC. When surgery is selected, tumor factors drive OS. Negative margin resection should be the goal of a primary surgical approach. When a positive margin resection ensues, adjuvant therapy may improve OS. © 2017 Wiley Periodicals, Inc.

Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line

PubMed Central

Choi, Sung S.; Yoon, Seung-Bin; Lee, Sang-Rae; Kim, Sun-Uk; Cha, Young Joo; Lee, Daniel; Kim, Seung U.; Chang, Kyu-Tae; Lee, Hong J.

2017-01-01

Despite the increasing importance of minipigs in biomedical research, there has been relatively little research concerning minipig-derived adult stem cells as a promising research tool that could be used to develop stem cell-based therapies. We first generated immortalized neural stem cells (iNSCs) from primary minipig olfactory bulb cells (pmpOBCs) and defined the characteristics of the cell line. Primary neural cells were prepared from minipig neonate olfactory bulbs and immortalized by infection with retrovirus carrying the v-myc gene. The minipig iNSCs (mpiNSCs) had normal karyotypes and expressed NSC-specific markers, including nestin, vimentin, Musashi1, and SOX2, suggesting a similarity to human NSCs. On the basis of the global gene expression profiles from the microarray analysis, neurogenesis-associated transcript levels were predominantly altered in mpiNSCs compared with pmpOBCs. These findings increase our understanding of minipig stem cells and contribute to the utility of mpiNSCs as resources for immortalized stem cell experiments. PMID:27524466

Centrosomal Latency of Incoming Foamy Viruses in Resting Cells

PubMed Central

Giron, Marie Lou; Roingeard, Philippe; Clave, Emmanuel; Tobaly-Tapiero, Joelle; Bittoun, Patricia; Toubert, Antoine; de Thé, Hugues; Saïb, Ali

2007-01-01

Completion of early stages of retrovirus infection depends on the cell cycle. While gammaretroviruses require mitosis for proviral integration, lentiviruses are able to replicate in post-mitotic non-dividing cells. Resting cells such as naive resting T lymphocytes from peripheral blood cannot be productively infected by retroviruses, including lentiviruses, but the molecular basis of this restriction remains poorly understood. We demonstrate that in G0 resting cells (primary fibroblasts or peripheral T cells), incoming foamy retroviruses accumulate in close proximity to the centrosome, where they lie as structured and assembled capsids for several weeks. Under these settings, virus uncoating is impaired, but upon cell stimulation, Gag proteolysis and capsid disassembly occur, which allows viral infection to proceed. The data imply that foamy virus uncoating is the rate-limiting step for productive infection of primary G0 cells. Incoming foamy retroviruses can stably persist at the centrosome, awaiting cell stimulation to initiate capsid cleavage, nuclear import, and viral gene expression. PMID:17530924

Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line.

PubMed

Choi, Sung S; Yoon, Seung-Bin; Lee, Sang-Rae; Kim, Sun-Uk; Cha, Young Joo; Lee, Daniel; Kim, Seung U; Chang, Kyu-Tae; Lee, Hong J

2017-02-16

Despite the increasing importance of minipigs in biomedical research, there has been relatively little research concerning minipig-derived adult stem cells as a promising research tool that could be used to develop stem cell-based therapies. We first generated immortalized neural stem cells (iNSCs) from primary minipig olfactory bulb cells (pmpOBCs) and defined the characteristics of the cell line. Primary neural cells were prepared from minipig neonate olfactory bulbs and immortalized by infection with retrovirus carrying the v-myc gene. The minipig iNSCs (mpiNSCs) had normal karyotypes and expressed NSC-specific markers, including nestin, vimentin, Musashi1, and SOX2, suggesting a similarity to human NSCs. On the basis of the global gene expression profiles from the microarray analysis, neurogenesis-associated transcript levels were predominantly altered in mpiNSCs compared with pmpOBCs. These findings increase our understanding of minipig stem cells and contribute to the utility of mpiNSCs as resources for immortalized stem cell experiments.

Development and characterization of a preclinical model of breast cancer lung micrometastatic to macrometastatic progression.

PubMed

Bailey-Downs, Lora C; Thorpe, Jessica E; Disch, Bryan C; Bastian, Anja; Hauser, Paul J; Farasyn, Taleah; Berry, William L; Hurst, Robert E; Ihnat, Michael A

2014-01-01

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500-1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30-60 micrometastases in the lung (with many animals also having 2-30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis.

Development and Characterization of a Preclinical Model of Breast Cancer Lung Micrometastatic to Macrometastatic Progression

PubMed Central

Disch, Bryan C.; Bastian, Anja; Hauser, Paul J.; Farasyn, Taleah; Berry, William L.; Hurst, Robert E.; Ihnat, Michael A.

2014-01-01

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500–1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30–60 micrometastases in the lung (with many animals also having 2–30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis. PMID:24878664

ASSESSMENT OF SYNAPSE FORMATION IN RAT PRIMARY NEURAL CELL CULTURE USING HIGH CONTENT MICROSCOPY.

EPA Science Inventory

Cell-based assays can model neurodevelopmental processes including neurite growth and synaptogenesis, and may be useful for screening and evaluation of large numbers of chemicals for developmental neurotoxicity. This work describes the use of high content screening (HCS) to dete...

SBIR reports on the chemistry of lithium battery technology

NASA Astrophysics Data System (ADS)

Kilroy, W. P.

1989-11-01

The following contents are included: Identification of an Improved Mixed Solvent Electrolyte for a Lithium Secondary Battery; Catalyzed Cathodes for Lithium-Thionyl Chloride Batteries; Improved Lithium/Thionyl Chloride Cells Using New Electrolyte Salts; Development of Calcium Primary Cells With Improved Anode Stability and Energy Density.

RNF17 blocks promiscuous activity of PIWI proteins in mouse testes.

PubMed

Wasik, Kaja A; Tam, Oliver H; Knott, Simon R; Falciatori, Ilaria; Hammell, Molly; Vagin, Vasily V; Hannon, Gregory J

2015-07-01

PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction. © 2015 Wasik et al.; Published by Cold Spring Harbor Laboratory Press.

Development of bioluminescent chick chorioallantoic membrane (CAM) models for primary pancreatic cancer cells: a platform for drug testing

PubMed Central

Rovithi, Maria; Avan, Amir; Funel, Niccola; Leon, Leticia G.; Gomez, Valentina E.; Wurdinger, Thomas; Griffioen, Arjan W.; Verheul, Henk M. W.; Giovannetti, Elisa

2017-01-01

The aim of the present study was to develop chick-embryo chorioallantoic membrane (CAM) bioluminescent tumor models employing low passage cell cultures obtained from primary pancreatic ductal adenocarcinoma (PDAC) cells. Primary PDAC cells transduced with lentivirus expressing Firefly-luciferase (Fluc) were established and inoculated onto the CAM membrane, with >80% engraftment. Fluc signal reliably correlated with tumor growth. Tumor features were evaluated by immunohistochemistry and genetic analyses, including analysis of mutations and mRNA expression of PDAC pivotal genes, as well as microRNA (miRNA) profiling. These studies showed that CAM tumors had histopathological and genetic characteristic comparable to the original tumors. We subsequently tested the modulation of key miRNAs and the activity of gemcitabine and crizotinib on CAM tumors, showing that combination treatment resulted in 63% inhibition of tumor growth as compared to control (p < 0.01). These results were associated with reduced expression of miR-21 and increased expression of miR-155. Our study provides the first evidence that transduced primary PDAC cells can form tumors on the CAM, retaining several histopathological and (epi)genetic characteristics of original tumors. Moreover, our results support the use of these models for drug testing, providing insights on molecular mechanisms underlying antitumor activity of new drugs/combinations. PMID:28304379

RNF17 blocks promiscuous activity of PIWI proteins in mouse testes

PubMed Central

Wasik, Kaja A.; Tam, Oliver H.; Knott, Simon R.; Falciatori, Ilaria; Hammell, Molly; Vagin, Vasily V.; Hannon, Gregory J.

2015-01-01

PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAs—primary and secondary—are defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction. PMID:26115953

Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

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

Shitara, Shingo; Kakeda, Minoru; Nagata, Keiko

2008-05-09

Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-{beta}-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 daysmore » after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells.« less

Lower gingival squamous cell carcinoma with brain metastasis during long-term cetuximab treatment: A case report.

PubMed

Naruse, Tomofumi; Tokuhisa, Mitsuko; Yanamoto, Souichi; Sakamoto, Yuki; Okuyama, Kohei; Tsuchihashi, Hiroki; Umeda, Masahiro

2018-05-01

Long-term cetuximab treatment can lead to acquired resistance, and tumor progression and/or new lesions often occur. The present report describes a case of lower gingival squamous cell carcinoma with brain metastasis during long-term cetuximab treatment in a 60-year-old man, including findings of an immunohistochemical study. The resected primary tumors, biopsy of the lung metastasis before administration of cetuximab, and brain metastasis specimens mediated by cetuximab were immunohistochemically examined. Histologically, the metastatic brain lesion showed hyperkeratinizing tumor cells with deeply stained irregular nuclei with necrotizing tumor cells, and a decrease in cell density was exhibited in part of the tumor nest. Moreover, the brain lesion was less malignant compared with the primary tumor and metastatic lung lesions. Immunohistochemically, the metastatic brain lesions showed low expression of epidermal growth factor receptor (EGFR) and high expression of N-cadherin compared with the primary tumor and metastatic lung lesions. These results suggest that acquired resistance to cetuximab may be associated with low EGFR expression and increased epithelial-to-mesenchymal transition potential.

Application of carbon-ion beams or gamma-rays on primary tumors does not change the expression profiles of metastatic tumors in an in vivo murine model.

PubMed

Tamaki, Tomoaki; Iwakawa, Mayumi; Ohno, Tatsuya; Imadome, Kaori; Nakawatari, Miyako; Sakai, Minako; Tsujii, Hirohiko; Nakano, Takashi; Imai, Takashi

2009-05-01

To clarify how carbon-ion radiotherapy (C-ion) on primary tumors affects the characteristics of subsequently arising metastatic tumor cells. Mouse squamous cell carcinomas, NR-S1, in synergic C3H/HeMsNrs mice were irradiated with a single dose of 5-50 Gy of C-ion (290 MeV per nucleon, 6-cm spread-out Bragg peak) or gamma-rays ((137)Cs source) as a reference beam. The volume of the primary tumors and the number of metastatic nodules in lung were studied, and histologic analysis and microarray analysis of laser-microdissected tumor cells were also performed. Including 5 Gy of C-ion and 8 Gy of gamma-rays, which did not inhibit the primary tumor growth, all doses used in this study inhibited lung metastasis significantly. Pathologic findings showed no difference among the metastatic tumor nodules in the nonirradiated, C-ion-irradiated, and gamma-ray-irradiated groups. Clustering analysis of expression profiles among metastatic tumors and primary tumors revealed a single cluster consisting of metastatic tumors different from their original primary tumors, indicating that the expression profiles of the metastatic tumor cells were not affected by the local application of C-ion or gamma-ray radiotherapy. We found no difference in the incidence and histology, and only small differences in expression profile, of distant metastasis between local C-ion and gamma-ray radiotherapy. The application of local radiotherapy per se or the type of radiotherapy applied did not influence the transcriptional changes caused by metastasis in tumor cells.

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma.

PubMed

Sarkar, Saheli; Sabhachandani, Pooja; Ravi, Dashnamoorthy; Potdar, Sayalee; Purvey, Sneha; Beheshti, Afshin; Evens, Andrew M; Konry, Tania

2017-01-01

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level.

SOX4 inhibits GBM cell growth and induces G0/G1 cell cycle arrest through Akt-p53 axis.

PubMed

Zhang, Jing; Jiang, Huawei; Shao, Jiaofang; Mao, Ruifang; Liu, Jie; Ma, Yingying; Fang, Xuefeng; Zhao, Na; Zheng, Shu; Lin, Biaoyang

2014-11-01

SOX4 is a transcription factor required for tissue development and differentiation in vertebrates. Overexpression of SOX4 has been reported in many cancers including glioblastoma multiforme (GBM), however, the underlying mechanism of actions has not been studied. In this study, we investigated the role of SOX4 in GBM. Kaplan-Meier analysis was performed to assess the association between SOX4 expression levels and survival times in primary GBM samples. Cre/lox P system was used to generate gain or loss of SOX4 in GBM cells, and microarray analysis uncovered the regulation network of SOX4 in GBM cells. High SOX4 expression was significantly associated with good prognosis of primary GBMs. SOX4 inhibited the growth of GBM cell line LN229, A172G and U87MG, partly via the activation of p53-p21 signaling and down-regulation of phosphorylated AKT1. Gene expression profiling and subsequent gene ontology analysis showed that SOX4 influenced several key pathways including the Wnt/ beta-catenin and TGF-beta signaling pathways. Our study found that SOX4 acts as a tumor suppressor in GBM cells by induce cell cycle arrest and inhibiting cell growth.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

PubMed

Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

2018-05-04

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

Extensive and systematic rewiring of histone post-translational modifications in cancer model systems

PubMed Central

Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

2018-01-01

Abstract Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations. PMID:29618087

Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioral differences in vitro

PubMed Central

Holmes, Katie E.; Thompson, Victoria; Piskun, Caroline M.; Kohnken, Rebecca A.; Huelsmeyer, Michael K.; Fan, Timothy M.; Stein, Timothy J.

2013-01-01

Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumor size, presence of metastatic disease, and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behavior of osteosarcoma cells differ based on serum ALP concentration. Here we report on the generation of six canine osteosarcoma cell lines from osteosarcoma-bearing dogs with differences in serum ALP concentration. To determine whether in vitro behavior differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP assays were performed to evaluate proliferation, migration, invasion, and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion, or chemosensitivity between cell lines associated normal or increased serum ALP concentration. PMID:23489774

Dihydromyricetin promotes hepatocellular carcinoma regression via a p53 activation-dependent mechanism

NASA Astrophysics Data System (ADS)

Zhang, Qingyu; Liu, Jie; Liu, Bin; Xia, Juan; Chen, Nianping; Chen, Xiaofeng; Cao, Yi; Zhang, Chen; Lu, Caijie; Li, Mingyi; Zhu, Runzhi

2014-04-01

The development of antitumor chemotherapy drugs remains a key goal for oncologists, and natural products provide a vast resource for anti-cancer drug discovery. In the current study, we found that the flavonoid dihydromyricetin (DHM) exhibited antitumor activity against liver cancer cells, including primary cells obtained from hepatocellular carcinoma (HCC) patients. In contrast, DHM was not cytotoxic to immortalized normal liver cells. Furthermore, DHM treatment resulted in the growth inhibition and remission of xenotransplanted tumors in nude mice. Our results further demonstrated that this antitumor activity was caused by the activation of the p53-dependent apoptosis pathway via p53 phosphorylation at serine (15Ser). Moreover, our results showed that DHM plays a dual role in the induction of cell death when administered in combination with cisplatin, a common clinical drug that kills primary hepatoma cells but not normal liver cells.

Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioural differences in vitro.

PubMed

Holmes, K E; Thompson, V; Piskun, C M; Kohnken, R A; Huelsmeyer, M K; Fan, T M; Stein, T J

2015-09-01

Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumour size, presence of metastatic disease and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behaviour of osteosarcoma cells differ based on serum ALP concentration. Here, we report on the generation of six canine osteosarcoma cell lines from osteosarcoma-bearing dogs with differences in serum ALP concentration. To determine whether in vitro behaviour differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP, assays were performed to evaluate proliferation, migration, invasion and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion or chemosensitivity between cell lines associated with normal or increased serum ALP concentration. © 2013 Blackwell Publishing Ltd.

Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

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

Cuneo, Kyle C.; Mito, Jeffrey K.; Javid, Melodi P.

2013-05-01

Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activatedmore » fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.« less

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements1[OPEN

PubMed Central

Morisaki, Keiko; Sawada, Yuji; Sano, Ryosuke; Yamamoto, Atsushi; Kurata, Tetsuya; Suzuki, Shiro; Matsuda, Mami; Hasunuma, Tomohisa; Hirai, Masami Yokota

2016-01-01

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell’s metabolic activity for the biosynthesis of secondary wall polymers. PMID:27600813

Primary pulmonary plasmacytoma with diffuse alveolar consolidation: a case report.

PubMed

Mohammad Taheri, Zohreh; Mohammadi, Forouzan; Karbasi, Mehrdad; Seyfollahi, Leila; Kahkoei, Shahram; Ghadiany, Mojtaba; Fayazi, Nader; Mansouri, Davood

2010-06-13

Solitary extramedullary plasmacytomas are plasma cell tumors that tend to develop in mucosa-associated lymphoid tissues including the sinonasal or nasopharyngeal regions. Primary plasmacytoma of the lung is exceedingly rare and often presents as a solitary mass or nodule in mid-lung or hilar areas and diagnosed after resection. Herein, we report a case of primary pulmonary plasmacytoma that presented with diffuse alveolar consolidation and diagnosed by transbronchial lung biopsy.

Primary Pulmonary Plasmacytoma with Diffuse Alveolar Consolidation: A Case Report

PubMed Central

Mohammad Taheri, Zohreh; Mohammadi, Forouzan; Karbasi, Mehrdad; Seyfollahi, Leila; Kahkoei, Shahram; Ghadiany, Mojtaba; Fayazi, Nader; Mansouri, Davood

2010-01-01

Solitary extramedullary plasmacytomas are plasma cell tumors that tend to develop in mucosa-associated lymphoid tissues including the sinonasal or nasopharyngeal regions. Primary plasmacytoma of the lung is exceedingly rare and often presents as a solitary mass or nodule in mid-lung or hilar areas and diagnosed after resection. Herein, we report a case of primary pulmonary plasmacytoma that presented with diffuse alveolar consolidation and diagnosed by transbronchial lung biopsy. PMID:21151727

Cross talk between primary human renal tubular cells and endothelial cells in cocultures.

PubMed

Tasnim, Farah; Zink, Daniele

2012-04-15

Interactions between renal tubular epithelial cells and adjacent endothelial cells are essential for normal renal functions but also play important roles in renal disease and repair. Here, we investigated cocultures of human primary renal proximal tubular cells (HPTC) and human primary endothelial cells to address the cross talk between these cell types. HPTC showed improved proliferation, marker gene expression, and enzyme activity in cocultures. Also, the long-term maintenance of epithelia formed by HPTC was improved, which was due to the secretion of transforming growth factor-β1 and its antagonist α2-macroglobulin. HPTC induced endothelial cells to secrete increased amounts of these factors, which balanced each other functionally and only displayed in combination the observed positive effects. In addition, in the presence of HPTC endothelial cells expressed increased amounts of hepatocyte growth factor and vascular endothelial growth factor, which have well-characterized effects on renal tubular epithelial cells as well as on endothelial cells. Together, the results showed that HPTC stimulated endothelial cells to express a functionally balanced combination of various factors, which in turn improved the performance of HPTC. The results give new insights into the cross talk between renal epithelial and endothelial cells and suggest that cocultures could be also useful models for the analysis of cellular communication in renal disease and repair. Furthermore, the characterization of defined microenvironments, which positively affect HPTC, will be helpful for improving the performance of this cell type in in vitro applications including in vitro toxicology and kidney tissue engineering.

Progranulin Is a Chemoattractant for Microglia and Stimulates Their Endocytic Activity

PubMed Central

Pickford, Fiona; Marcus, Jacob; Camargo, Luiz Miguel; Xiao, Qiurong; Graham, Danielle; Mo, Jan-Rung; Burkhardt, Matthew; Kulkarni, Vinayak; Crispino, Jamie; Hering, Heike; Hutton, Michael

2011-01-01

Mutations resulting in progranulin haploinsufficiency cause disease in patients with a subset of frontotemporal lobar degeneration; however, the biological functions of progranulin in the brain remain unknown. To address this subject, the present study initially assessed changes in gene expression and cytokine secretion in rat primary cortical neurons treated with progranulin. Molecular pathways enriched in the progranulin gene set included cell adhesion and cell motility pathways and pathways involved in growth and development. Secretion of cytokines and several chemokines linked to chemoattraction but not inflammation were also increased from progranulin-treated primary neurons. Therefore, whether progranulin is involved in recruitment of immune cells in the brain was investigated. Localized lentiviral expression of progranulin in C57BL/6 mice resulted in an increase of Iba1-positive microglia around the injection site. Moreover, progranulin alone was sufficient to promote migration of primary mouse microglia in vitro. Primary microglia and C4B8 cells demonstrated more endocytosis of amyloid β1-42 when treated with progranulin. These data demonstrate that progranulin acts as a chemoattractant in the brain to recruit or activate microglia and can increase endocytosis of extracellular peptides such as amyloid β. PMID:21224065

Human papilloma virus status and chromosomal imbalances in primary cervical carcinomas and tumour cell lines.

PubMed

Hidalgo, A; Schewe, C; Petersen, S; Salcedo, M; Gariglio, P; Schlüns, K; Dietel, M; Petersen, I

2000-03-01

Human papilloma virus (HPV) infection is the crucial step in the initiation of cervical carcinomas. In addition, HPV18 has been implicated in tumour progression and adverse clinical outcome. We determined the HPV types in 12 primary cervical carcinomas and 12 cell lines and compared the findings with the comparative genetic hybridisation (CGH) pattern of chromosomal alterations. The most frequent alteration was the deletion at 3p14 followed by the loss of 2q34-q36 along with 3q gain. High risk HPV types were detected in all samples except one primary tumour. In contrast to the normal distribution, HPV18 was present in 75% of cases including all cell lines. The cell lines carried a higher number of genetic alterations and a different CGH pattern for several chromosomes than the primary tumours, despite microdissection. Purely HPV18 positive cases indicated a high incidence of imbalances at specific loci with peaks of the histogram coinciding with known HPV integration sites. The study suggests that HPV infection is associated with a recurrent pattern of chromosomal changes in cervical carcinomas and that the development and progression of these alterations is triggered by integration into the host genome.

Establishment and Characterization of Novel Human Primary and Metastatic Anaplastic Thyroid Cancer Cell Lines and Their Genomic Evolution Over a Year as a Primagraft

PubMed Central

Okamoto, Ryoko; Nagata, Yasunobu; Kanojia, Deepika; Venkatesan, Subhashree; M. T., Anand; Braunstein, Glenn D.; Said, Jonathan W.; Doan, Ngan B.; Ho, Quoc; Akagi, Tadayuki; Gery, Sigal; Liu, Li-zhen; Tan, Kar Tong; Chng, Wee Joo; Yang, Henry; Ogawa, Seishi; Koeffler, H. Phillip

2015-01-01

Context: Anaplastic thyroid cancer (ATC) has no effective treatment, resulting in a high rate of mortality. We established cell lines from a primary ATC and its lymph node metastasis, and investigated the molecular factors and genomic changes associated with tumor growth. Objective: The aim of the study was to understand the molecular and genomic changes of highly aggressive ATC and its clonal evolution to develop rational therapies. Design: We established unique cell lines from primary (OGK-P) and metastatic (OGK-M) ATC specimen, as well as primagraft from the metastatic ATC, which was serially xeno-transplanted for more than 1 year in NOD scid gamma mice were established. These cell lines and primagraft were used as tools to examine gene expression, copy number changes, and somatic mutations using RNA array, SNP Chip, and whole exome sequencing. Results: Mice carrying sc (OGK-P and OGK-M) tumors developed splenomegaly and neutrophilia with high expression of cytokines including CSF1, CSF2, CSF3, IL-1β, and IL-6. Levels of HIF-1α and its targeted genes were also elevated in these tumors. The treatment of tumor carrying mice with Bevacizumab effectively decreased tumor growth, macrophage infiltration, and peripheral WBCs. SNP chip analysis showed homozygous deletion of exons 3–22 of the PARD3 gene in the cells. Forced expression of PARD3 decreased cell proliferation, motility, and invasiveness, restores cell-cell contacts and enhanced cell adhesion. Next generation exome sequencing identified the somatic changes present in the primary, metastatic, and primagraft tumors demonstrating evolution of the mutational signature over the year of passage in vivo. Conclusion: To our knowledge, we established the first paired human primary and metastatic ATC cell lines offering unique possibilities for comparative functional investigations in vitro and in vivo. Our exome sequencing also identified novel mutations, as well as clonal evolution in both the metastasis and primagraft. PMID:25365311

Primary T-cell Lymphoma of the Colon

PubMed Central

Son, Hee Jung; Rhee, Poong Lyul; Kim, Jae-Jun; Koh, Kwang Choel; Paik, Seong Woon; Rhee, Jong Chul; Koh, Young Hae

1997-01-01

A 40-year-old woman had been diagnosed with Crohns disease in September 1994, but later examinations revealed a primary T-cell lymphoma of the colon. Colonoscopic and histological examination showed ulcerative lesions simulating Crohns disease involving the entire colon and the terminal ileum, and she was first diagnosed as having Crohns disease. Differential therapeutic strategies, including corticosteroid, had improved the symptoms which were dominated by abdominal pain. When she visited our institute in April 1995, she presented with bloody stool twice a day, 7kg weight loss in a period of six months and a slightly painful abdomen. Colonoscopic finding showed geographic ulceration on the entire colon, especially rectum and terminal ileum. The histologic examination of specimens from colonoscopic biopsy showed primary peripheral T-cell lymphoma of the colon. Any dense lymphocyte infiltrates seen in the biopsy specimens obtained from lesions simulating ulcerative colitis or Crohns disease should be assessed to exclude intestinal lymphoma PMID:9439161

A study of the mechanism of in vitro cytotoxicity of metal oxide nanoparticles using catfish primary hepatocytes and human HepG2 cells

PubMed Central

Wang, Yonggang; Aker, Winfred G.; Hwang, Huey-min; Yedjou, Clement G.; Yu, Hongtao; Tchounwou, Paul B.

2011-01-01

Nanoparticles (NPs), including nano metal oxides, are being used in diverse applications such as medicine, clothing, cosmetics and food. In order to promote the safe development of nanotechnology, it is essential to assess the potential adverse health consequences associated with human exposure. The liver is a target site for NP toxicity, due to NP accumulation within it after ingestion, inhalation or absorption. The toxicity of nano-ZnO, TiO2, CuO and Co3O4 was investigated using a primary culture of channel catfish hepatocytes and human HepG2 cells as in vitro model systems for assessing the impact of metal oxide NPs on human and environmental health. Some mechanisms of nanotoxicity were determined by using phase contrast inverted microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, reactive oxygen species (ROS) assays, and flow cytometric assays. Nano-CuO and ZnO showed significant toxicity in both HepG2 cells and catfish primary hepatocytes. The results demonstrate that HepG2 cells are more sensitive than catfish primary hepatocytes to the toxicity of metal oxide NPs. The overall ranking of the toxicity of metal oxides to the test cells is as follows: TiO2 < Co3O4< ZnO < CuO. The toxicity is due not only to ROS-induced cell death, but also damages to cell and mitochondrial membranes. PMID:21851965

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants1[OPEN

PubMed Central

Li, Xingxing; Huang, Shixin; Van de Meene, Allison M.L.; Tran, Mai L.; Killeavy, Erin; Mercure, Danielle; Burton, Rachel A.

2017-01-01

The secondary cell walls of tracheary elements and fibers are rich in cellulose microfibrils that are helically oriented and laterally aggregated. Support cells within the leaf midribs of mosses deposit cellulose-rich secondary cell walls, but their biosynthesis and microfibril organization have not been examined. Although the Cellulose Synthase (CESA) gene families of mosses and seed plants diversified independently, CESA knockout analysis in the moss Physcomitrella patens revealed parallels with Arabidopsis (Arabidopsis thaliana) in CESA functional specialization, with roles for both subfunctionalization and neofunctionalization. The similarities include regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls, a requirement for two or more functionally distinct CESA isoforms for secondary cell wall synthesis, interchangeability of some primary and secondary CESAs, and some CESA redundancy. The cellulose-deficient midribs of ppcesa3/8 knockouts provided negative controls for the structural characterization of stereid secondary cell walls in wild type P. patens. Sum frequency generation spectra collected from midribs were consistent with cellulose microfibril aggregation, and polarization microscopy revealed helical microfibril orientation only in wild type leaves. Thus, stereid secondary walls are structurally distinct from primary cell walls, and they share structural characteristics with the secondary walls of tracheary elements and fibers. We propose a mechanism for the convergent evolution of secondary walls in which the deposition of aggregated and helically oriented microfibrils is coupled to rapid and highly localized cellulose synthesis enabled by regulatory uncoupling from primary wall synthesis. PMID:28768816

Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype

PubMed Central

Wu, Li-an; Feng, Junsheng; Wang, Lynn; Mu, Yan-dong; Baker, Andrew; Donly, Kevin J.; Harris, Stephen E.; MacDougall, Mary; Chen, Shuo

2011-01-01

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways. PMID:21271257

Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis

EPA Science Inventory

During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary...

Reviewing primary Sjögren's syndrome: beyond the dryness - From pathophysiology to diagnosis and treatment.

PubMed

Both, Tim; Dalm, Virgil A S H; van Hagen, P Martin; van Daele, Paul L A

2017-01-01

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease, characterized by lymphocytic infiltration of the secretory glands. This process leads to sicca syndrome, which is the combination of dryness of the eyes, oral cavity, pharynx, larynx and/or vagina. Extraglandular manifestations may also be prevalent in patients with pSS, including cutaneous, musculoskeletal, pulmonary, renal, hematological and neurological involvement. The pathogenesis of pSS is currently not well understood, but increased activation of B cells followed by immune complex formation and autoantibody production are thought to play important roles. pSS is diagnosed using the American-European consensus group (AECG) classification criteria which include subjective symptoms and objective tests such as histopathology and serology. The treatment of pSS warrants an organ based approach, for which local treatment (teardrops, moistures) and systemic therapy (including non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (DMARDS) and biologicals) can be considered. Biologicals used in the treatment of pSS mainly affect the total numbers of B cells (B cell depletion (Rituximab)) or target proteins required for B cell proliferation and/or activation (e.g. B cell activating factor (BAFF)) resulting in decreased B cell activity. The aim of this review is to provide physicians a general overview concerning the pathogenesis, diagnosis and management of pSS patients.

The Cables Gene on Chromosome 18q Is Silenced by Promoter Hypermethylation and Allelic Loss in Human Colorectal Cancer

PubMed Central

Park, Do Youn; Sakamoto, Hideo; Kirley, Sandra D.; Ogino, Shuji; Kawasaki, Takako; Kwon, Eunjeong; Mino-Kenudson, Mari; Lauwers, Gregory Y.; Chung, Daniel C.; Rueda, Bo R.; Zukerberg, Lawrence R.

2007-01-01

Cables is a cyclin-dependent kinase-binding nuclear protein that maps to chromosome 18q11-12. Here, we assessed Cables expression in 160 colorectal cancers (CRCs), its role in colon cancer cell growth, and the potential mechanisms of Cables inactivation. Expression levels, promoter methylation, and mutational status of Cables were investigated in colon cancer cell lines and primary colon tumors. Chromosome 18q loss of heterozygosity (LOH) was evaluated with multiple polymorphic markers. Cables inhibited cellular proliferation and colony formation in colon cancer cell lines. Cables expression was reduced in 65% of primary CRCs. No mutations were detected in 10 exons of Cables in 20 primary colon tumors. Cables promoter was methylated in cell lines with decreased Cables expression and vice versa. 5-Aza-2′-deoxycytidine resulted in increased Cables expression in methylated cell lines. There was a significant correlation between promoter methylation and Cables gene expression in primary colon tumors. Sixty-five percent of primary colon tumors demonstrated chromosome 18q LOH. LOH involving the Cables region was observed in 35% of cases, including those in which more distal portions of chromosome 18q were retained, and Cables expression was decreased in all such cases. Loss of Cables expression in 65% of CRCs suggests that it is a common event in colonic carcinogenesis, with promoter methylation and LOH appearing to be important mechanisms of Cables gene inactivation. PMID:17982127

Cell Fate and Differentiation of the Developing Ocular Lens

PubMed Central

Greiling, Teri M. S.; Aose, Masamoto

2010-01-01

Purpose. Even though zebrafish development does not include the formation of a lens vesicle, the authors' hypothesis is that the processes of cell differentiation are similar in zebrafish and mammals and determine cell fates in the lens. Methods. Two-photon live embryo imaging was used to follow individual fluorescently labeled cells in real-time from the placode stage at 16 hours postfertilization (hpf) until obvious morphologic differentiation into epithelium or fiber cells had occurred at approximately 28 hpf. Immunohistochemistry was used to label proliferating, differentiating, and apoptotic cells. Results. Similar to the mammal, cells in the teleost peripheral lens placode migrated to the anterior lens mass and differentiated into an anterior epithelium. Cells in the central lens placode migrated to the posterior lens mass and differentiated into primary fiber cells. Anterior and posterior polarization in the zebrafish lens mass was similar to mammalian lens vesicle polarization. Primary fiber cell differentiation was apparent at approximately 21 hpf, before separation of the lens from the surface ectoderm, as evidenced by cell elongation, exit from the cell cycle, and expression of Zl-1, a marker for fiber differentiation. TUNEL labeling demonstrated that apoptosis was not a primary mechanism for lens separation from the surface ectoderm. Conclusions. Despite the absence of a lens vesicle in the zebrafish embryo, lens organogenesis appears to be well conserved among vertebrates. Results using three-dimensional live embryo imaging of zebrafish development showed minimal differences and strong similarities in the fate of cells in the zebrafish and mammalian lens placode. PMID:19834024

Initiation of primary cell culture from amphioxus Branchiostoma belcheri tsingtauense

NASA Astrophysics Data System (ADS)

Wang, Changliu; Zhang, Shicui; Su, Feng; Wang, Lei; Li, Hongyan

2009-02-01

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C.

Orpk mouse model of polycystic kidney disease reveals essential role of primary cilia in pancreatic tissue organization.

PubMed

Cano, David A; Murcia, Noel S; Pazour, Gregory J; Hebrok, Matthias

2004-07-01

Polycystic kidney disease (PKD) includes a group of disorders that are characterized by the presence of cysts in the kidney and other organs, including the pancreas. Here we show that in orpk mice, a model system for PKD that harbors a mutation in the gene that encodes the polaris protein, pancreatic defects start to occur at the end of gestation, with an initial expansion of the developing pancreatic ducts. Ductal dilation continues rapidly after birth and results in the formation of large, interconnected cysts. Expansion of pancreatic ducts is accompanied by apoptosis of neighboring acinar cells, whereas endocrine cell differentiation and islet formation appears to be unaffected. Polaris has been shown to co-localize with primary cilia, and these structures have been implicated in the formation of renal cysts. In the orpk pancreas, cilia numbers are reduced and cilia length is decreased. Expression of polycystin-2, a protein involved in PKD, is mislocalized in orpk mice. Furthermore, the cellular localization of beta-catenin, a protein involved in cell adhesion and Wnt signaling, is altered. Thus, polaris and primary cilia function are required for the maturation and maintenance of proper tissue organization in the pancreas.

A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

PubMed

Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

2015-08-01

At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

Nonimaging light concentrator with uniform irradiance

DOEpatents

Winston, Roland; Gee, Randy C.

2003-04-01

A nonimaging light concentrator system including a primary collector of light, an optical mixer disposed near the focal zone for collecting light from the primary collector, the optical mixer having a transparent entrance aperture, an internally reflective housing for substantially total internal reflection of light, a transparent exit aperture and an array of photovoltaic cells disposed near the transparent exit aperture.

The IL-6 receptor super-antagonist Sant7 enhances antiproliferative and apoptotic effects induced by dexamethasone and zoledronic acid on multiple myeloma cells.

PubMed

Tassone, Pierfrancesco; Galea, Eulalia; Forciniti, Samantha; Tagliaferri, Pierosandro; Venuta, Salvatore

2002-10-01

Interleukin-6 (IL-6) is the major growth and survival factor for multiple myeloma (MM), and has been shown to protect MM cells from apoptosis induced by a variety of agents. IL-6 receptor antagonists, which prevent the assembly of functional IL-6 receptor complexes, inhibit cell proliferation and induce apoptosis in MM cells. We have investigated whether the IL-6 receptor super-antagonist Sant7 might enhance the antiproliferative and apoptotic effects induced by the combination of dexamethasone (Dex) and zoledronic acid (Zln) on human MM cell lines and primary cells from MM patients. Here we show that each of these compounds individually induced detectable antiproliferative effects on MM cells. Sant7 significantly enhanced growth inhibition and apoptosis induced by Dex and Zln on both MM cell lines and primary MM cells. These results indicate that overcoming IL-6 mediated cell resistance by Sant7 potentiates the effect of glucocorticoides and bisphosphonates on MM cell growth and survival, providing a rationale for therapies including IL-6 antagonists in MM.

West Valley feasibility study

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

Pirro, J.

This report presents the results of a technical assessment of decontamination alternative prepared for the Western New York Nuclear Service Center (WNYNSC). The purpose of the assessment is to determine the recommended method for decontamination of cell surfaces and decontamination and removal of fuel reprocessing cell equipment to permit manual entry into the cells for the installation of waste solidification equipment. The primary cells of interest are the PMC, GPC, and CPC because they offer the largest usable volume for the solidification program. The secondary cells include XC-1, XC-2, XC-3 and the PPC which may be needed to support themore » solidification program. Five decontamination assessments were evaluated (A-E). The assessments included the estimated cost, occupational exposure, duration, manpower, waste volume generated, and final cell radiation levels achieved with the alternative decontamination methods. The methods varied from thorough destructive decontamination to equipment removal without decontamination followed by cell wall and floor decontamination. The recommended method for the primary cells is to utilize the remote manipulators and cranes to the maximum extent possible to decontaminate equipment and cell surfaces remotely, and to remove the equipment for temporary on-site storage. The recommended method for secondary cell decontamination is to remotely decontaminate the cells to the maximum extent possible prior to manned entry for contact-removal of the fuel reprocessing equipment (Assessment D). Assessment A is expected to cost $8,713,500 in 1980 dollars (including a 25% contingency) and will result in an occupational exposure of 180.3 manRem. Assessment D is expected to cost $11,039,800 and will result in an occupational exposure of 259 manRems.« less

Growth of human breast tissues from patient cells in 3D hydrogel scaffolds.

PubMed

Sokol, Ethan S; Miller, Daniel H; Breggia, Anne; Spencer, Kevin C; Arendt, Lisa M; Gupta, Piyush B

2016-03-01

Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix. These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.

Rapid Selection of Mesenchymal Stem and Progenitor Cells in Primary Prostate Stromal Cultures

PubMed Central

Brennen, W. Nathaniel; Kisteman, L. Nelleke; Isaacs, John T.

2016-01-01

BACKGROUND Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (>25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. RESULTS MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 μm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping properties. The lack of adipogenesis in stromal cultures derived from normal prostates suggests they have a lineage-restricted progenitor phenotype. The retention of adipogenic differentiation in cultures from a subset of prostate cancer patients suggests the active recruitment of less committed progenitors or MSCs from the bone marrow as a function of disease progression. This recruitment can potentially be exploited for prognostic purposes or a cell-based platform for the systemic delivery of cytotoxic agents to sites of prostate cancer. PMID:26732992

Rapid selection of mesenchymal stem and progenitor cells in primary prostate stromal cultures.

PubMed

Brennen, W Nathaniel; Kisteman, L Nelleke; Isaacs, John T

2016-05-01

Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (<25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 µm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping properties. The lack of adipogenesis in stromal cultures derived from normal prostates suggests they have a lineage-restricted progenitor phenotype. The retention of adipogenic differentiation in cultures from a subset of prostate cancer patients suggests the active recruitment of less committed progenitors or MSCs from the bone marrow as a function of disease progression. This recruitment can potentially be exploited for prognostic purposes or a cell-based platform for the systemic delivery of cytotoxic agents to sites of prostate cancer. © 2016 Wiley Periodicals, Inc.

S1PR1 drives a feedforward signalling loop to regulate BATF3 and the transcriptional programme of Hodgkin lymphoma cells

PubMed Central

Vrzalikova, K; Ibrahim, M; Vockerodt, M; Perry, T; Margielewska, S; Lupino, L; Nagy, E; Soilleux, E; Liebelt, D; Hollows, R; Last, A; Reynolds, G; Abdullah, M; Curley, H; Care, M; Krappmann, D; Tooze, R; Allegood, J; Spiegel, S; Wei, W; Woodman, C B J; Murray, P G

2018-01-01

The Hodgkin/Reed–Sternberg cells of classical Hodgkin lymphoma (HL) are characterised by the aberrant activation of multiple signalling pathways. Here we show that a subset of HL displays altered expression of sphingosine-1-phosphate (S1P) receptors (S1PR)s. S1P activates phosphatidylinositide 3-kinase (PI3-K) in these cells that is mediated by the increased expression of S1PR1 and the decreased expression of S1PR2. We also showed that genes regulated by the PI3-K signalling pathway in HL cell lines significantly overlap with the transcriptional programme of primary HRS cells. Genes upregulated by the PI3-K pathway included the basic leucine zipper transcription factor, ATF-like 3 (BATF3), which is normally associated with the development of dendritic cells. Immunohistochemistry confirmed that BATF3 was expressed in HRS cells of most HL cases. In contrast, in normal lymphoid tissues, BATF3 expression was confined to a small fraction of CD30-positive immunoblasts. Knockdown of BATF3 in HL cell lines revealed that BATF3 contributed to the transcriptional programme of primary HRS cells, including the upregulation of S1PR1. Our data suggest that disruption of this potentially oncogenic feedforward S1P signalling loop could provide novel therapeutic opportunities for patients with HL. PMID:28878352

Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

PubMed

Takayama, Kazuo; Mizuguchi, Hiroyuki

2017-02-01

Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Targeting monoamine oxidase A in advanced prostate cancer.

PubMed

Flamand, Vincent; Zhao, Hongjuan; Peehl, Donna M

2010-11-01

Inhibitors of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades neurotransmitters including serotonin and norepinephrine, are commonly used to treat neurological conditions including depression. Recently, we and others identified high expression of MAOA in normal basal prostatic epithelium and high-grade primary prostate cancer (PCa). In contrast, MAOA is low in normal secretory prostatic epithelium and low-grade PCa. An irreversible inhibitor of MAOA, clorgyline, induced secretory differentiation in primary cultures of normal basal epithelial cells and high-grade PCa. Furthermore, clorgyline inhibited several oncogenic pathways in PCa cells, suggesting clinical value of MAOA inhibitors as a pro-differentiation and anti-oncogenic therapy for high-risk PCa. Here, we extended our studies to a model of advanced PCa, VCaP cells, which were derived from castration-resistant metastatic PCa and express a high level of MAOA. Growth of VCaP cells in the presence or absence of clorgyline was evaluated in vitro and in vivo. Gene expression changes in response to clorgyline were determined by microarray and validated by quantitative real-time polymerase chain reaction. Treatment with clorgyline in vitro inhibited growth and altered the transcriptional pattern of VCaP cells in a manner consistent with the pro-differentiation and anti-oncogenic effects seen in treated primary PCa cells. Src, beta-catenin, and MAPK oncogenic pathways, implicated in androgen-independent growth and metastasis, were significantly downregulated. Clorgyline treatment of mice bearing VCaP xenografts slowed tumor growth and induced transcriptome changes similar to those noted in vitro. Our results support the possibility that anti-depressant drugs that target MAOA might find a new application in treating PCa.

Comparison of Neurovascular Characteristics of Facial Skin in Patients After Primary and Revision Rhytidectomies.

PubMed

Ardeshirpour, Farhad; Hurliman, Elisabeth; Wendelschafer-Crabb, Gwen; McAdams, Brian; Hilger, Peter A; Kennedy, William R; Lassig, Amy Anne D; Brenner, Michael J

2017-09-01

Wound healing influences both the cosmetic and functional outcomes of facial surgery. Study of cutaneous innervation may afford insight into patients' preoperative wound healing potential and aid in their selection of appropriate surgical procedures. To present the quantitative and qualitative differences of epidermal nerve fibers (ENFs), neurotransmitters, vasculature, and mast cells in facial skin among patients after primary and revision rhytidectomies. This pilot study collected cutaneous specimens from 8 female patients aged 42 to 66 years who underwent primary rhytidectomy (n = 5) and revision rhytidectomy (n = 3) at Centennial Lakes Surgery Center, Edina, Minnesota, from July 2010 to March 2014. Tissue was processed for confocal/epifluorescence microscopy and indirect immunofluorescent localization of several neural and tissue antigens as well as basement membrane and mast cell markers. Primary rhytidectomy vs revision rhytidectomy with selection of a small area of redundant, otherwise disposed of tissue anterior to the tragus for ENF study. Demographic characteristics included smoking status; 10-point rating scales for facial sensation, pain, and paresthesias; and confocal/epifluorescence microscopy to quantify ENFs, neurotransmitters, vasculature, and mast cells. Patients in the primary rhytidectomy group had a mean (SD) of 54.4 (31.6) ENFs/mm (range, 14.2-99.2 ENFs/mm), and those in the revision rhytidectomy group had a mean (SD) of 18.6 (5.8) ENFs/mm (range, 13.8-25.0 ENFs/mm). A patient in the primary rhytidectomy group was a 25-pack-year smoker and had 14.2 ENFs/mm, the lowest in both groups. In addition to these structural neural changes, functional neural changes in revision rhytidectomy samples included qualitative changes in normal neural antigen prevalence (substance P, calcitonin gene-related peptide, and vasoactive intestinal peptide). Capillary loops appeared less robust and were less common in dermal papilla among samples from both the primary and revision groups, and mast cells were more degranulated. No differences were found in subjective, self-reported postoperative facial sensation. Previous skin elevation was associated with decreased epidermal nerve fiber density and qualitative changes in dermal nerves, capillaries, and mast cells in a clinical sample of patients undergoing rhytidectomy. Future research is needed to determine whether histological findings predict wound healing and to better understand the effects of surgery on regenerative capacity of epidermal nerve fibers. NA.

Study of Silymarin and Vitamin E Protective Effects on Silver Nanoparticle Toxicity on Mice Liver Primary Cell Culture.

PubMed

Faedmaleki, Firouz; Shirazi, Farshad H; Ejtemaeimehr, Shahram; Anjarani, Soghra; Salarian, Amir-Ahmad; Ahmadi Ashtiani, Hamidreza; Rastegar, Hossein

2016-02-01

Nanotechnology is a most promising field for generating new applications in medicine, although, only few nano products are currently in use for medical purposes. A most prominent nanoproduct is nanosilver. Nano-silver has biological properties which are significant for consumer products, food technology, textiles, and medical applications (e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging). For their antibacterial activity, silver nanoparticles (Ag NPs) are largely used in various commercially available products. The use of nano-silver is becoming more and more widespread in medicine and related applications, and due to its increasing exposure, toxicological and environmental issues need to be raised. Cytotoxicity induced by silver nanoparticles (AgNPs) and the role that oxidative stress plays in this process were demonstrated in human hepatoma cells AgNPs agglomerated in the cytoplasm and nuclei of treated cells, and they induced intracellular oxidative stress. AgNP reduced ATP content of the cell and caused damage to mitochondria and increased production of reactive oxygen species (ROS) in a dose-dependent manner. Silymarin was known as a hepatoprotective agent that is used in the treatment of hepatic diseases including viral hepatitis, alcoholic liver diseases, Amanita mushroom poisoning, liver cirrhosis, toxic and drug-induced liver diseases. It promotes protein synthesis, helps in regenerating liver tissue, controls inflammation, enhances glucuronidation, and protects against glutathione depletion. Vitamin E is a well-known antioxidant and has hepatoprotective effect in liver diseases. In this study, we investigated the cytotoxic effects of Ag NPs on primary liver cells of mice. Cell viability (cytotoxicity) was examined with MTT assay after primary liver cells of mice exposure to AgNPs at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration- dependent decrease of cell viability (IC50 value = 121.7 ppm or µg/ml). Then the hepatoprotective effect of silymarin and vitamin E were experimented on silver nanoparticle toxicity on mice liver primary cell culture. The results showed that silymarin at 600 µg/ml and vitamin E at 2500 µmol/l have protective effects on silver nanoparticle toxicity on mice liver primary cell culture. Viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles at 121.7 ppm and co-treatment of silymarin, and vitamin E is more than viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles and silymarin or silver nanoparticles and vitamin E.

Fuel cell system combustor

DOEpatents

Pettit, William Henry

2001-01-01

A fuel cell system including a fuel reformer heated by a catalytic combustor fired by anode and cathode effluents. The combustor includes a turbulator section at its input end for intimately mixing the anode and cathode effluents before they contact the combustors primary catalyst bed. The turbulator comprises at least one porous bed of mixing media that provides a tortuous path therethrough for creating turbulent flow and intimate mixing of the anode and cathode effluents therein.

Thymidylate synthase (TS) gene expression in primary lung cancer patients: a large-scale study in Japanese population.

PubMed

Tanaka, F; Wada, H; Fukui, Y; Fukushima, M

2011-08-01

Previous small-sized studies showed lower thymidylate synthase (TS) expression in adenocarcinoma of the lung, which may explain higher antitumor activity of TS-inhibiting agents such as pemetrexed. To quantitatively measure TS gene expression in a large-scale Japanese population (n = 2621) with primary lung cancer, laser-captured microdissected sections were cut from primary tumors, surrounding normal lung tissues and involved nodes. TS gene expression level in primary tumor was significantly higher than that in normal lung tissue (mean TS/β-actin, 3.4 and 1.0, respectively; P < 0.01), and TS gene expression level was further higher in involved node (mean TS/β-actin, 7.7; P < 0.01). Analyses of TS gene expression levels in primary tumor according to histologic cell type revealed that small-cell carcinoma showed highest TS expression (mean TS/β-actin, 13.8) and that squamous cell carcinoma showed higher TS expression as compared with adenocarcinoma (mean TS/β-actin, 4.3 and 2.3, respectively; P < 0.01); TS gene expression was significantly increased along with a decrease in the grade of tumor cell differentiation. There was no significant difference in TS gene expression according to any other patient characteristics including tumor progression. Lower TS expression in adenocarcinoma of the lung was confirmed in a large-scale study.

Clinicopathological findings of primary esophageal malignant melanoma: report of six cases and review of literature.

PubMed

Zheng, Jinfeng; Mo, Haiying; Ma, Shufang; Wang, Zhenzheng

2014-01-01

We studied images and histopathological features of primary esophageal malignant melanoma to explore the clinical pathological features, diagnosis, differential diagnoses, and treatment. Immunolabelling was conducted on six cases of esophageal malignant melanoma using histological and immunohistochemical techniques. Combined with the related literature, the clinical manifestations, imaging, histopathological and immunohistochemical features, treatment, and prognosis of primary esophageal malignant melanoma were observed and analyzed. The six patients with primary esophageal malignant melanoma were all male with an average age of 63.4 years. Poor food intake was observed in all patients, and the symptoms showed progressive aggravation. Endoscopic feed tube revealed dark brown and black nodular and polypoid lesions, 1/4-1/2 loop cavity. Tumor histopathology revealed the following characteristics: tumor cells arranged in nests, sheets and cords, round or polygonal, abundant and red-stained cytoplasm, melanin granules in the cytoplasm, heterogeneous nucleus sizes, centered or deviated nuclei, clearly identifiable nucleoli, and apparent pathological mitosis. The immune phenotype was as follows: tumor cells had diffuse expression of HMB45, Melan A, and S100. The cells were CK negative, and the Ki67-positive cell number was 40%-45%. Primary esophageal malignant melanoma is rare with high malignancy and poor prognosis. Immunohistochemical staining is helpful for diagnosing this tumor. The differential diagnosis includes low differentiated carcinoma, primitive neuroectodermal tumor, esophageal sarcomatoid carcinoma, esophageal lymphoma, and other tumors.

Comprehensive Method for Culturing Embryonic Dorsal Root Ganglion Neurons for Seahorse Extracellular Flux XF24 Analysis

PubMed Central

Lange, Miranda; Zeng, Yan; Knight, Andrew; Windebank, Anthony; Trushina, Eugenia

2012-01-01

Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG) neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15) rat DRG neurons suitable for analysis with the Seahorse XF24 platform. PMID:23248613

Comprehensive Method for Culturing Embryonic Dorsal Root Ganglion Neurons for Seahorse Extracellular Flux XF24 Analysis.

PubMed

Lange, Miranda; Zeng, Yan; Knight, Andrew; Windebank, Anthony; Trushina, Eugenia

2012-01-01

Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG) neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15) rat DRG neurons suitable for analysis with the Seahorse XF24 platform.

Collision tumor of primary laryngeal mucosal melanoma and invasive squamous cell carcinoma with IL-17A and CD70 gene over-expression.

PubMed

Sirikanjanapong, Sasis; Lanson, Biana; Amin, Milan; Martiniuk, Frank; Kamino, Hideko; Wang, Beverly Y

2010-12-01

The most common primary malignancy of the larynx is the squamous cell carcinoma (SCC). The primary malignant melanoma is quite rare in this location. Less than 60 cases of laryngeal melanomas have been reported to date. To our knowledge, collision primary malignant melanoma and invasive squamous cell carcinoma in the vocal cords has not been reported. We report a 53-year-old male patient who was diagnosed with a collision tumor of laryngeal melanoma and invasive SCC. Multiple Th17 pathway related genes including CTLA-4, IL-17A-F, PLZF, FoxP3, RorγT, CD27, and CD70 were analyzed by reverse transcriptase-polymerase chain reaction (Rt-PCR) in this case. Both IL-17A and CD70 genes were detected in this case of collision tumor. The results may define useful biomarkers for early diagnosis of mucosal melanoma and open an immunotherapeutic field for clinical management with the potential benefit from the immunomodulators that enhance both genes.

Performance characteristics of lithium primary cells after controlled storage. [on-orbit for energy power supply

NASA Technical Reports Server (NTRS)

Deligiannis, F.; Shen, D. H.; Halpert, G.; Ang, V.; Donley, S.

1991-01-01

A program was initiated to investigate the effects of storage on the performance of lithium primary cells. Two types of liquid cathode cells were chosen to investigate these effects. The cell types included Li-SOCl2/BCX cells, Li-SO2 cells from two different manufacturers, and a small sample size of 8-year-old Li-SO2 cells. The following measurements are performed at each test interval: open circuit voltage, resistance and weight, microcalorimetry, ac impedance, capacity, and voltage delay. The authors examine the performance characteristics of these cells after one year of controlled storage at two temperatures (10 and 30 C). The Li-SO2 cells experienced little to no voltage and capacity degradation after one year storage. The Li-SOCl2/BCX cells exhibited significant voltage and capacity degradation after 30 C storage. Predischarging shortly prior to use appears to be an effective method of reducing the initial voltage drop. Studies are in progress to correlate ac impedance and microcalorimetry measurements with capacity losses and voltage delay.

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

PubMed Central

2011-01-01

Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. Conclusion The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450. PMID:21961524

MHC class-I associated phosphopeptides are the targets of memory-like immunity in leukemia

PubMed Central

Cobbold, Mark; De La Peña, Hugo; Norris, Andrew; Polefrone, Joy; Qian, Jie; English, A. Michelle; Cummings, Kara; Penny, Sarah; Turner, James E.; Cottine, Jennifer; Abelin, Jennifer G; Malaker, Stacy A; Zarling, Angela L; Huang, Hsing-Wen; Goodyear, Oliver; Freeman, Sylvie; Shabanowitz, Jeffrey; Pratt, Guy; Craddock, Charles; Williams, Michael E; Hunt, Donald F; Engelhard, Victor H

2014-01-01

Deregulation of signaling pathways involving phosphorylation is a hallmark of malignant transformation. Degradation of phosphoproteins generates cancer-specific phosphopeptides that are associated with MHC-I and II molecules and recognized by T-cells. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8 T-cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and HLA-matched primary leukemia cells ex vivo. Healthy individuals showed surprisingly high levels of CD8 T-cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients, which correlated with clinical outcome, and was restored following allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T-cell adoptive transfer immunotherapies.. PMID:24048523

Fuel cells

NASA Astrophysics Data System (ADS)

Hooie, D. T.; Harrington, B. C., III; Mayfield, M. J.; Parsons, E. L.

1992-07-01

The primary objective of DOE's Fossil Energy Fuel Cell program is to fund the development of key fuel cell technologies in a manner that maximizes private sector participation and in a way that will give contractors the opportunity for a competitive posture, early market entry, and long-term market growth. This summary includes an overview of the Fuel Cell program, an elementary explanation of how fuel cells operate, and a synopsis of the three major fuel cell technologies sponsored by the DOE/Fossil Energy Phosphoric Acid Fuel Cell program, the Molten Carbonate Fuel Cell program, and the Solid Oxide Fuel Cell program.

High-content adhesion assay to address limited cell samples†

PubMed Central

Warrick, Jay W.; Young, Edmond W. K.; Schmuck, Eric G.; Saupe, Kurt W.

2013-01-01

Cell adhesion is a broad topic in cell biology that involves physical interactions between cells and other cells or the surrounding extracellular matrix, and is implicated in major research areas including cancer, development, tissue engineering, and regenerative medicine. While current methods have contributed significantly to our understanding of cell adhesion, these methods are unsuitable for tackling many biological questions requiring intermediate numbers of cells (102–105), including small animal biopsies, clinical samples, and rare cell isolates. To overcome this fundamental limitation, we developed a new assay to quantify the adhesion of ~102–103 cells at a time on engineered substrates, and examined the adhesion strength and population heterogeneity via distribution-based modeling. We validated the platform by testing adhesion strength of cancer cells from three different cancer types (breast, prostate, and multiple myeloma) on both IL-1β activated and non-activated endothelial monolayers, and observed significantly increased adhesion for each cancer cell type upon endothelial activation, while identifying and quantifying distinct subpopulations of cell-substrate interactions. We then applied the assay to characterize adhesion of primary bone marrow stromal cells to different cardiac fibroblast-derived matrix substrates to demonstrate the ability to study limited cell populations in the context of cardiac cell-based therapies. Overall, these results demonstrate the sensitivity and robustness of the assay as well as its ability to enable extraction of high content, functional data from limited and potentially rare primary samples. We anticipate this method will enable a new class of biological studies with potential impact in basic and translational research. PMID:23426645

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

Wu, Li-An; Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an; Yuan, Guohua

Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show thatmore » transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.« less

The Mitotic and Metabolic Effects of Phosphatidic Acid in the Primary Muscle Cells of Turbot (Scophthalmus maximus).

PubMed

Wang, Tingting; Wang, Xuan; Zhou, Huihui; Jiang, Haowen; Mai, Kangsen; He, Gen

2018-01-01

Searching for nutraceuticals and understanding the underlying mechanism that promote fish growth is at high demand for aquaculture industry. In this study, the modulatory effects of soy phosphatidic acids (PA) on cell proliferation, nutrient sensing, and metabolic pathways were systematically examined in primary muscle cells of turbot ( Scophthalmus maximus ). PA was found to stimulate cell proliferation and promote G1/S phase transition through activation of target of rapamycin signaling pathway. The expression of myogenic regulatory factors, including myoD and follistatin , was upregulated, while that of myogenin and myostatin was downregulated by PA. Furthermore, PA increased intracellular free amino acid levels and enhanced protein synthesis, lipogenesis, and glycolysis, while suppressed amino acid degradation and lipolysis. PA also was found to increased cellular energy production through stimulated tricarboxylic acid cycle and oxidative phosphorylation. Our results identified PA as a potential nutraceutical that stimulates muscle cell proliferation and anabolism in fish.

Preparation of herpes simplex virus-infected primary neurons for transmission electron microscopy.

PubMed

Miranda-Saksena, Monica; Boadle, Ross; Cunningham, Anthony L

2014-01-01

Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus-host cell interaction. Whilst there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus-infected primary neurons, grown on plastic cover slips, to allow sectioning of neurons and axons in their growth plane. This technique allows TEM examination of cell bodies, axons, growth cones, and varicosities, providing powerful insights into virus-cell interaction.

Induction of the IL-9 gene by HTLV-I Tax stimulates the spontaneous proliferation of primary adult T-cell leukemia cells by a paracrine mechanism

PubMed Central

Chen, Jing; Petrus, Mike; Bryant, Bonita R.; Phuc Nguyen, Vinh; Stamer, Mindy; Goldman, Carolyn K.; Bamford, Richard; Morris, John C.; Janik, John E.

2008-01-01

The etiologic agent of adult T-cell leukemia (ATL) is human T cell lymphotropic virus type I (HTLV-I). The HTLV-I protein Tax alters gene expression, including those of cytokines and their receptors, which plays an important role in early stages of ATL. Here we demonstrate that expression of interleukin-9 (IL-9) is activated by Tax via an NF-κB motif in its proximal promoter, whereas IL-9 receptor-α (IL-9Rα) expression is not induced by Tax. However, supporting a role for IL-9/IL-9Rα in ATL, a neutralizing monoclonal antibody directed toward IL-9Rα inhibited ex vivo spontaneous proliferation of primary ATL cells from several patients. Fluorescence-activated cell sorter analysis of freshly isolated peripheral blood mononuclear cells from these patients revealed high level expression of IL-9Rα on their CD14-expressing monocytes. Furthermore, purified T cells or monocytes alone from these patients did not proliferate ex vivo, whereas mixtures of these cell types manifested significant proliferation through a contact-dependent manner. Taken together, our data suggest that primary ATL cells, via IL-9, support the action of IL-9Rα/CD14-expressing monocytes, which subsequently support the ex vivo spontaneous proliferation of malignant T cells. In summary, these data support a role for IL-9 and its receptor in ATL by a paracrine mechanism. PMID:18339896

Antagonism of SET using OP449 enhances the efficacy of tyrosine kinase inhibitors and overcome drug resistance in myeloid leukemia

PubMed Central

Agarwal, Anupriya; MacKenzie, Ryan J.; Pippa, Raffaella; Eide, Christopher A.; Oddo, Jessica; Tyner, Jeffrey W.; Sears, Rosalie; Vitek, Michael P.; Odero, María D.; Christensen, Dale; Druker, Brian J.

2014-01-01

Purpose The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A. Experimental Design In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis and colonogenic assays. Efficacy of target inhibition by OP449 is evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model. Results We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34+ CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1-mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells. Conclusions We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML. PMID:24436473

Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

PubMed

Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

2012-01-01

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples. Copyright © 2011 Elsevier Inc. All rights reserved.

Testing of SNS-032 in a Panel of Human Neuroblastoma Cell Lines with Acquired Resistance to a Broad Range of Drugs.

PubMed

Löschmann, Nadine; Michaelis, Martin; Rothweiler, Florian; Zehner, Richard; Cinatl, Jaroslav; Voges, Yvonne; Sharifi, Mohsen; Riecken, Kristoffer; Meyer, Jochen; von Deimling, Andreas; Fichtner, Iduna; Ghafourian, Taravat; Westermann, Frank; Cinatl, Jindrich

2013-12-01

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3(r)CDDP(1000) in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.

Testing of SNS-032 in a Panel of Human Neuroblastoma Cell Lines with Acquired Resistance to a Broad Range of Drugs12

PubMed Central

Löschmann, Nadine; Michaelis, Martin; Rothweiler, Florian; Zehner, Richard; Cinatl, Jaroslav; Voges, Yvonne; Sharifi, Mohsen; Riecken, Kristoffer; Meyer, Jochen; von Deimling, Andreas; Fichtner, Iduna; Ghafourian, Taravat; Westermann, Frank; Cinatl, Jindrich

2013-01-01

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3rCDDP1000 in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases. PMID:24466371

Variation in pestivirus growth in testicle primary cell culture is more dependent on the individual cell donor than cattle breed

USDA-ARS?s Scientific Manuscript database

Bovine respiratory disease complex (BRDC) affects cattle from all breeds living in all regions. However, different breeds of cattle appear to have different susceptibilities to developing BRDC. The causes of BRDC are multifactorial and include infection with both viral and bacterial pathogens. Infec...

A combination HIV reporter virus system for measuring post-entry event efficiency and viral outcome in primary CD4+ T cell subsets.

PubMed

Tilton, Carisa A; Tabler, Caroline O; Lucera, Mark B; Marek, Samantha L; Haqqani, Aiman A; Tilton, John C

2014-01-01

Fusion between the viral membrane of human immunodeficiency virus (HIV) and the host cell marks the end of the HIV entry process and the beginning of a series of post-entry events including uncoating, reverse transcription, integration, and viral gene expression. The efficiency of post-entry events can be modulated by cellular factors including viral restriction factors and can lead to several distinct outcomes: productive, latent, or abortive infection. Understanding host and viral proteins impacting post-entry event efficiency and viral outcome is critical for strategies to reduce HIV infectivity and to optimize transduction of HIV-based gene therapy vectors. Here, we report a combination reporter virus system measuring both membrane fusion and viral promoter-driven gene expression. This system enables precise determination of unstimulated primary CD4+ T cell subsets targeted by HIV, the efficiency of post-entry viral events, and viral outcome and is compatible with high-throughput screening and cell-sorting methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Dielectrophoretic Capture and Genetic Analysis of Single Neuroblastoma Tumor Cells

PubMed Central

Carpenter, Erica L.; Rader, JulieAnn; Ruden, Jacob; Rappaport, Eric F.; Hunter, Kristen N.; Hallberg, Paul L.; Krytska, Kate; O’Dwyer, Peter J.; Mosse, Yael P.

2014-01-01

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients. PMID:25133137

Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma.

PubMed

Kremer, Veronika; Ligtenberg, Maarten A; Zendehdel, Rosa; Seitz, Christina; Duivenvoorden, Annet; Wennerberg, Erik; Colón, Eugenia; Scherman-Plogell, Ann-Helén; Lundqvist, Andreas

2017-09-19

Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer. Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays. We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells. To increase the success of NK cell-based therapies of solid tumors, it is of great importance to promote their homing to the tumor site. In this study, we show that stable engineering of human primary NK cells to express a chemokine receptor thereby enhancing their migration is a promising strategy to improve anti-tumor responses following adoptive transfer of NK cells.

Myeloid-derived suppressor cells

PubMed Central

Chandra, Dinesh; Gravekamp, Claudia

2013-01-01

While conventional anticancer therapies, including surgical resection, radiotherapy, and/or chemotherapy, are relatively efficient at eliminating primary tumors, these treatment modalities are largely ineffective against metastases. At least in part, this reflects the rather inefficient delivery of conventional anticancer agents to metastatic lesions. We have recently demonstrated that myeloid-derived suppressor cells (MDSCs) can be used as cellular missiles to selectively deliver a radioisotope-coupled attenuated variant of Listeria monocytogenes to both primary and metastatic neoplastic lesions in mice with pancreatic cancer. This novel immunotherapeutic intervention robustly inhibited tumor growth while promoting a dramatic decrease in the number of metastases. PMID:24427545

Myeloid-derived suppressor cells: Cellular missiles to target tumors.

PubMed

Chandra, Dinesh; Gravekamp, Claudia

2013-11-01

While conventional anticancer therapies, including surgical resection, radiotherapy, and/or chemotherapy, are relatively efficient at eliminating primary tumors, these treatment modalities are largely ineffective against metastases. At least in part, this reflects the rather inefficient delivery of conventional anticancer agents to metastatic lesions. We have recently demonstrated that myeloid-derived suppressor cells (MDSCs) can be used as cellular missiles to selectively deliver a radioisotope-coupled attenuated variant of Listeria monocytogenes to both primary and metastatic neoplastic lesions in mice with pancreatic cancer. This novel immunotherapeutic intervention robustly inhibited tumor growth while promoting a dramatic decrease in the number of metastases.

Multi-Cellular 3D Human Primary Liver Cell Cultures Elevate Metabolic Activity Under Fluidic Flow

PubMed Central

Esch, Mandy B.; Prot, Jean-Matthieu; Wang, Ying I.; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A.; Applegate, Dawn R.

2015-01-01

Predicting drug-induced liver injury with in vitro cell culture models more accurately would be of significant value to the pharmaceutical industry. To this end we have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop and bidirectional fluid flow (average flow rate of 650 μL/min, and a maximum shear stress of 0.64 dyne/cm2). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures derived from human tissues increase their metabolic activity in response to bidirectional fluidic flow. Since bidirectional flow drastically changes the behavior of other cells types that are shear sensitive, the finding that bidirectional flow increases the metabolic activity of primary liver cells also supports the theory that this increase in metabolic activity is likely caused by increased levels of gas and metabolite exchange or by the accumulation of soluble growth factors rather than by shear sensing. Our results indicate that device operation with bi-directional gravity-driven medium flow supports the 14-day culture of a mix of primary human liver cells with the benefits of enhanced metabolic activity. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs. PMID:25857666

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

Status of commercial fuel cell powerplant system development

NASA Technical Reports Server (NTRS)

Warshay, Marvin

1987-01-01

The primary focus is on the development of commercial Phosphoric Acid Fuel Cell (PAFC) powerplant systems because the PAFC, which has undergone extensive development, is currently the closest fuel cell system to commercialization. Shorter discussions are included on the high temperature fuel cell systems which are not as mature in their development, such as the Molten Carbonate Fuel Cell (MCFC) and the Solid Oxide Fuel Cell (SOFC). The alkaline and the Solid Polymer Electrolyte (SPE) fuel cell systems, are also included, but their discussions are limited to their prospects for commercial development. Currently, although the alkaline fuel cell continues to be used for important space applications there are no commercial development programs of significant size in the USA and only small efforts outside. The market place for fuel cells and the status of fuel cell programs in the USA receive extensive treatment. The fuel cell efforts outside the USA, especially the large Japanese programs, are also discussed.

Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine.

PubMed

Lee, Charlotte A; Sinha, Siddharth; Fitzpatrick, Emer; Dhawan, Anil

2018-06-01

Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases.

Primary distal femur T-cell lymphoma after allogeneic haematopoietic stem cell transplantation for chronic myeloid leukaemia: a rare case report and literature review.

PubMed

Han, Qiaoyan; Sun, Miao; Wu, Lingyu; Chen, Jing; Wang, Wei; Liu, Chunhua; Chen, Haoyue; Du, Guibin

2014-04-01

Post-transplant lymphoproliferative disorders originating from T lymphocytes are a rare complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) that are not usually associated with Epstein-Barr virus infection. A male patient diagnosed at the age of 15 years with chronic myeloid leukaemia (in the chronic phase) was initially treated with oral hydroxyurea. The disease entered an accelerated phase when the patient was 22 years old. Complete remission was achieved after one course of homoharringtonine and cytarabine. The patient then underwent human leucocyte antigen-matched sibling donor allo-HSCT. Just over 6.5 years after the allo-HSCT, a second primary tumour was located in the distal femur and diagnosed as T-cell non-Hodgkin's lymphoma (stage IV, group B). This was treated with various chemotherapy and radiotherapy regimens, but the outcomes were poor and the disease progressed. The T-cell lymphoma invaded many sites, including the skeleton, spleen and skin, and the patient died within 8 months of the diagnosis. This current case report highlights the need for the early detection and prevention of subsequent primary malignancies after allo-HSCT.

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.

Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation

PubMed Central

Blake, Sophia M; Stricker, Stefan H; Halavach, Hanna; Poetsch, Anna R; Cresswell, George; Kelly, Gavin; Kanu, Nnennaya; Marino, Silvia; Luscombe, Nicholas M; Pollard, Steven M; Behrens, Axel

2016-01-01

Glioblastoma multiforme (GBM) is the most aggressive human primary brain cancer. Using a Trp53-deficient mouse model of GBM, we show that genetic inactivation of the Atm cofactor Atmin, which is dispensable for embryonic and adult neural development, strongly suppresses GBM formation. Mechanistically, expression of several GBM-associated genes, including Pdgfra, was normalized by Atmin deletion in the Trp53-null background. Pharmacological ATM inhibition also reduced Pdgfra expression, and reduced the proliferation of Trp53-deficient primary glioma cells from murine and human tumors, while normal neural stem cells were unaffected. Analysis of GBM datasets showed that PDGFRA expression is also significantly increased in human TP53-mutant compared with TP53-wild-type tumors. Moreover, combined treatment with ATM and PDGFRA inhibitors efficiently killed TP53-mutant primary human GBM cells, but not untransformed neural stem cells. These results reveal a new requirement for ATMIN-dependent ATM signaling in TP53-deficient GBM, indicating a pro-tumorigenic role for ATM in the context of these tumors. DOI: http://dx.doi.org/10.7554/eLife.08711.001 PMID:26984279

Golgi-Mediated Synthesis and Secretion of Matrix Polysaccharides of the Primary Cell Wall of Higher Plants

PubMed Central

Driouich, Azeddine; Follet-Gueye, Marie-Laure; Bernard, Sophie; Kousar, Sumaira; Chevalier, Laurence; Vicré-Gibouin, Maïté; Lerouxel, Olivier

2012-01-01

The Golgi apparatus of eukaryotic cells is known for its central role in the processing, sorting, and transport of proteins to intra- and extra-cellular compartments. In plants, it has the additional task of assembling and exporting the non-cellulosic polysaccharides of the cell wall matrix including pectin and hemicelluloses, which are important for plant development and protection. In this review, we focus on the biosynthesis of complex polysaccharides of the primary cell wall of eudicotyledonous plants. We present and discuss the compartmental organization of the Golgi stacks with regards to complex polysaccharide assembly and secretion using immuno-electron microscopy and specific antibodies recognizing various sugar epitopes. We also discuss the significance of the recently identified Golgi-localized glycosyltransferases responsible for the biosynthesis of xyloglucan (XyG) and pectin. PMID:22639665

Golgi-mediated synthesis and secretion of matrix polysaccharides of the primary cell wall of higher plants.

PubMed

Driouich, Azeddine; Follet-Gueye, Marie-Laure; Bernard, Sophie; Kousar, Sumaira; Chevalier, Laurence; Vicré-Gibouin, Maïté; Lerouxel, Olivier

2012-01-01

The Golgi apparatus of eukaryotic cells is known for its central role in the processing, sorting, and transport of proteins to intra- and extra-cellular compartments. In plants, it has the additional task of assembling and exporting the non-cellulosic polysaccharides of the cell wall matrix including pectin and hemicelluloses, which are important for plant development and protection. In this review, we focus on the biosynthesis of complex polysaccharides of the primary cell wall of eudicotyledonous plants. We present and discuss the compartmental organization of the Golgi stacks with regards to complex polysaccharide assembly and secretion using immuno-electron microscopy and specific antibodies recognizing various sugar epitopes. We also discuss the significance of the recently identified Golgi-localized glycosyltransferases responsible for the biosynthesis of xyloglucan (XyG) and pectin.

A case of oculo-cerebral B-cell lymphoma in a cat.

PubMed

Giordano, Cristina; Giudice, Chiara; Bellino, Claudio; Borrelli, Antonio; D'Angelo, Antonio; Gianella, Paola

2013-01-01

To describe a case of a cat with primary B-cell lymphoma affecting the eye and brain and which shared features similar to oculo-cerebral lymphoma in humans. A 13-year-old castrated male Persian cat presented with clinical signs of anterior uveitis and increased intraocular pressure (IOP) in the left eye (OS). A complete diagnostic work-up was declined, and left-eye enucleation was performed. The globe was submitted for histopathology. One week after surgery, the cat became inappetent, hypothermic, and aggressive. Euthanasia was requested by the owner, and a necropsy was permitted.   Histopathology of the enucleated globe revealed an extensive neoplastic infiltration consistent with large-cell lymphoma, affecting the anterior uvea, neuroretina and optic nerve. At necropsy, all organs were unremarkable except for the brain, where there was a neoplastic cell population consistent with that described in the left eye, infiltrated and expanded meninges, and perivascular spaces. Immunohistochemically, the neoplastic cells were positive for B-cell marker (CD20) and negative for T-cell marker (CD3). Histology and immunophenotyping suggested a diagnosis of primary central nervous system and ocular large B-cell lymphoma. The lymphoma in this cat resembled oculo-cerebral lymphoma in humans, sharing similar clinical features and histopathological findings, including the perivascular pattern of neoplastic cell infiltration. To the best of the authors' knowledge, this is the first description of a primary oculo-cerebral B-cell lymphoma in a cat. © 2012 American College of Veterinary Ophthalmologists.

Characterization and risk estimate of cancer in patients with primary Sjögren syndrome.

PubMed

Brito-Zerón, Pilar; Kostov, Belchin; Fraile, Guadalupe; Caravia-Durán, Daniel; Maure, Brenda; Rascón, Francisco-Javier; Zamora, Mónica; Casanovas, Arnau; Lopez-Dupla, Miguel; Ripoll, Mar; Pinilla, Blanca; Fonseca, Eva; Akasbi, Miriam; de la Red, Gloria; Duarte-Millán, Miguel-Angel; Fanlo, Patricia; Guisado-Vasco, Pablo; Pérez-Alvarez, Roberto; Chamorro, Antonio J; Morcillo, César; Jiménez-Heredia, Iratxe; Sánchez-Berná, Isabel; López-Guillermo, Armando; Ramos-Casals, Manuel

2017-04-17

The purpose of this study is to characterize the risk of cancer in a large cohort of patients with primary Sjögren syndrome (SjS). We had analyzed the development of cancer in 1300 consecutive patients fulfilling the 2002 SjS classification criteria. The baseline clinical and immunological characteristics and systemic activity (ESSDAI scores) were assessed at diagnosis as predictors of cancer using Cox proportional hazards regression analysis adjusted for age at diagnosis and gender. The sex-and age-specific standardized incidence ratios (SIR) of cancer were estimated from 2012 Spanish mortality data. After a mean follow-up of 91 months, 127 (9.8%) patients developed 133 cancers. The most frequent type of cancer was B-cell lymphoma (including 27 MALT and 19 non-MALT B-cell lymphomas). Systemic activity at diagnosis of primary SjS correlated with the risk of hematological neoplasia and cryoglobulins with a high risk of either B-cell or non-B-cell lymphoma subtypes. Patients with cytopenias had a high risk of non-MALT B-cell and non-B-cell cancer, while those with low C3 levels had a high risk of MALT lymphomas and those with monoclonal gammopathy and low C4 levels had a high risk of non-MALT lymphomas. The estimated SIR for solid cancer was 1.13 and 11.02 for hematological cancer. SIRs for specific cancers were 36.17 for multiple myeloma and immunoproliferative diseases, 19.41 for Hodgkin lymphoma, 6.04 for other non-Hodgkin lymphomas, 5.17 for thyroid cancer, 4.81 for cancers of the lip and oral cavity, and 2.53 for stomach cancer. One third of cancers developed by patients with primary SjS are B-cell lymphomas. The prognostic factors identified at SjS diagnosis differed according to the subtype of B-cell lymphoma developed. Primary SjS is also associated with the development of some non-hematological cancers (thyroid, oral cavity, and stomach).

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

PubMed

Pamies, David; Bal-Price, Anna; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Gstraunthaler, Gerhard; Hartung, Thomas; Jennings, Paul; Leist, Marcel; Martin, Ulrich; Passier, Robert; Schwamborn, Jens C; Stacey, Glyn N; Ellinger-Ziegelbauer, Heidrun; Daneshian, Mardas

2018-04-13

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

Cell-based optical assay for amyloid β-induced neuronal cell dysfunction using femtosecond-pulsed laser

NASA Astrophysics Data System (ADS)

Lee, Seunghee; Yoon, Jonghee; Choi, Chulhee

2015-03-01

Amyloid β-protein (Aβ) is known as a key molecule related to the pathogenesis of Alzheimer's disease (AD). Over time, the amyloid cascade disrupts essential function of mitochondria including Ca2+ homeostasis and reactive oxygen species (ROS) regulation, and eventually leads to neuronal cell death. However, there have been no methods that analyze and measure neuronal dysfuction in pathologic conditions quantitatively. Here, we suggest a cell-based optical assay to investigate neuronal function in AD using femtosecond-pulsed laser stimulation. We observed that laser stimulation on primary rat hippocampal neurons for a few microseconds induced intracellular Ca2+ level increases or produced intracellular ROS which was a primary cause of neuronal cell death depending on delivered energy. Although Aβ treatment alone had little effect on the neuronal morphologies and networks in a few hours, Aβ-treated neurons showed delayed Ca2+ increasing pattern and were more vulnerable to laser-induced cell death compared to normal neurons. Our results collectively indicate that femtosecond laser stimulation can be a useful tool to study neuronal dysfuction related to AD pathologies. We anticipate this optical method to enable studies in the early progression of neuronal impairments and the quantitative evaluation of drug effects on neurons in neurodegenerative diseases, including AD and Parkinson's disease in a preclinical study.

HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion

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

Cavrois, Marielle; Neidleman, Jason; Yonemoto, Wes

2004-10-15

We recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells. This assay involves the use of virions containing {beta}-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of {beta}-lactamase. Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr. Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating. We also show that wildtype andmore » Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells. Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry. Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane.« less

Medicare claims data reliably identify treatments for basal cell carcinoma and squamous cell carcinoma: a prospective cohort study.

PubMed

Thompson, Bridie S; Olsen, Catherine M; Subramaniam, Padmini; Neale, Rachel E; Whiteman, David C

2016-04-01

To investigate the accuracy of Medical Benefit Schedule (MBS) item numbers to identify treatments for basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). We linked records from QSkin Study participants (n=37,103) to Medicare. We measured the proportion of Medicare claims for primary excision of BCC/SCC that had corresponding claims for histopathology services. In subsets of participants, we estimated the sensitivity and external concordance of MBS item numbers for identifying BCC/SCC diagnoses by comparing against 'gold-standard' histopathology reports. A total of 2,821 (7.6%) participants had 4,830 separate Medicare claims for BCC/SCC excision; almost all (97%) had contemporaneous Medicare claims for histopathology services. Among participants with BCC/SCC confirmed by histology reports, 76% had a corresponding Medicare claim for primary surgical excision of BCC/SCC. External concordance for Medicare claims for primary BCC/SCC excision was 68%, increasing to 97% when diagnoses for intra-epidermal carcinomas and keratoacanthomas were included. MBS item numbers for primary excision of BCC/SCC are reasonably reliable for determining incident cases of keratinocyte skin cancers, but may underestimate incidence by up to 24%. Medicare claims data may have utility in monitoring trends in conditions for which there is no mandatory reporting. © 2015 Public Health Association of Australia.

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

Clinical features and management of primary sclerosing cholangitis

PubMed Central

Silveira, Marina G; Lindor, Keith D

2008-01-01

Primary sclerosing cholangitis is a chronic cholestatic liver disease characterized by inflammation and fibrosis of the bile ducts, resulting in cirrhosis and need for liver transplantation and reduced life expectancy. The majority of cases occur in young and middle-aged men, often in association with inflammatory bowel disease. The etiology of primary sclerosing cholangitis includes immune-mediated components and elements of undefined nature. No effective medical therapy has been identified. The multiple complications of primary sclerosing cholangitis include metabolic bone disease, dominant strictures, bacterial cholangitis, and malignancy, particularly cholangiocarcinoma, which is the most lethal complication of primary sclerosing cholangitis. Liver transplantation is currently the only life-extending therapeutic alternative for patients with end-stage disease, although recurrence in the allografted liver has been described. A PSC-like variant attracting attention is cholangitis marked by raised levels of the immunoglobulin G4 subclass, prominence of plasma cells within the lesions, and steroid responsiveness. PMID:18528931

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

Ultrastructural characterization of primary cilia in pathologically characterized human glioblastoma multiforme (GBM) tumors.

PubMed

Moser, Joanna J; Fritzler, Marvin J; Rattner, Jerome B

2014-01-01

Primary cilia are non-motile sensory cytoplasmic organelles that are involved in cell cycle progression. Ultrastructurally, the primary cilium region is complex, with normal ciliogenesis progressing through five distinct morphological stages in human astrocytes. Defects in early stages of ciliogenesis are key features of astrocytoma/glioblastoma cell lines and provided the impetus for the current study which describes the morphology of primary cilia in molecularly characterized human glioblastoma multiforme (GBM) tumors. Seven surgically resected human GBM tissue samples were molecularly characterized according to IDH1/2 mutation status, EGFR amplification status and MGMT promoter methylation status and were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. We report for the first time that primary cilia are disrupted in the early stages of ciliogenesis in human GBM tumors. We confirm that immature primary cilia and basal bodies/centrioles have aberrant ciliogenesis characteristics including absent paired vesicles, misshaped/swollen vesicular hats, abnormal configuration of distal appendages, and discontinuity of centriole microtubular blades. Additionally, the transition zone plate is able to form in the absence of paired vesicles on the distal end of the basal body and when a cilium progresses beyond the early stages of ciliogenesis, it has electron dense material clumped along the transition zone and a darkening of the microtubules at the proximal end of the cilium. Primary cilia play a role in a variety of human cancers. Previously primary cilia structure was perturbed in cultured cell lines derived from astrocytomas/glioblastomas; however there was always some question as to whether these findings were a cell culture phenomena. In this study we confirm that disruptions in ciliogenesis at early stages do occur in GBM tumors and that these ultrastructural findings bear resemblance to those previously observed in cell cultures. This is the first study to demonstrate that defects in cilia expression and function are a true hallmark of GBM tumors and correlate with their unrestrained growth. A review of the current ultrastructural profiles in the literature provides suggestions as to the best possible candidate protein that underlies defects in the early stages of ciliogenesis within GBM tumors.

Acute or chronic life-threatening diseases associated with Epstein-Barr virus infection.

PubMed

Okano, Motohiko; Gross, Thomas G

2012-06-01

Infectious mononucleosis (IM) is one of the representative, usually benign, acute diseases associated with primary Epstein-Barr virus (EBV) infection. IM is generally self-limiting and is characterized mostly by transient fever, lymphadenopathy and hepatosplenomegaly. However, very rarely primary EBV infection results in severe or fatal conditions such as hemophagocytic lymphohistiocytosis together with fulminant hepatitis designated as severe or fatal IM or EBV-associated hemophagocytic lymphohistiocytosis alone. In addition, chronic EBV-associated diseases include Burkitt's lymphoma, undifferentiated nasopharyngeal carcinoma, Hodgkin lymphoma, T-cell lymphoproliferative disorder (LPD)/lymphoma, natural killer-cell LPD including leukemia or lymphoma, gastric carcinoma, pyothorax-associated lymphoma and senile B-cell LPD as well as chronic active EBV infection and LPD/lymphoma in patients with immunodeficiency. The number of chronic life-threatening diseases linked to the EBV infection is increasingly reported and many of these diseases have a poor prognosis. This review will focus on the historical, pathogenetic, diagnostic, therapeutic and prophylactic issues of EBV-associated life-threatening diseases.

Isolation, Characterization, and Functional Analysis of Ferret Lymphatic Endothelial Cells

PubMed Central

Berendam, Stella J.; Fallert-Junecko, Beth A.; Murphy-Corb, Michael A.; Fuller, Deborah H.; Reinhart, Todd A.

2014-01-01

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases -- the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-1 ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles of LECs might play in health and disease in animal models. PMID:25540877

Isolation, characterization, and functional analysis of ferret lymphatic endothelial cells.

PubMed

Berendam, Stella J; Fallert Junecko, Beth A; Murphey-Corb, Michael A; Fuller, Deborah H; Reinhart, Todd A

2015-02-15

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases - the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-I ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles LECs might play in health and disease in animal models. Copyright © 2014 Elsevier B.V. All rights reserved.

MRI with DWI for the Detection of Posttreatment Head and Neck Squamous Cell Carcinoma: Why Morphologic MRI Criteria Matter.

PubMed

Ailianou, A; Mundada, P; De Perrot, T; Pusztaszieri, M; Poletti, P-A; Becker, M

2018-04-01

Although diffusion-weighted imaging combined with morphologic MRI (DWIMRI) is used to detect posttreatment recurrent and second primary head and neck squamous cell carcinoma, the diagnostic criteria used so far have not been clarified. We hypothesized that precise MRI criteria based on signal intensity patterns on T2 and contrast-enhanced T1 complement DWI and therefore improve the diagnostic performance of DWIMRI. We analyzed 1.5T MRI examinations of 100 consecutive patients treated with radiation therapy with or without additional surgery for head and neck squamous cell carcinoma. MRI examinations included morphologic sequences and DWI ( b =0 and b =1000 s/mm 2 ). Histology and follow-up served as the standard of reference. Two experienced readers, blinded to clinical/histologic/follow-up data, evaluated images according to clearly defined criteria for the diagnosis of recurrent head and neck squamous cell carcinoma/second primary head and neck squamous cell carcinoma occurring after treatment, post-radiation therapy inflammatory edema, and late fibrosis. DWI analysis included qualitative (visual) and quantitative evaluation with an ADC threshold. Recurrent head and neck squamous cell carcinoma/second primary head and neck squamous cell carcinoma occurring after treatment was present in 36 patients, whereas 64 patients had post-radiation therapy lesions only. The Cohen κ for differentiating tumor from post-radiation therapy lesions with MRI and qualitative DWIMRI was 0.822 and 0.881, respectively. Mean ADCmean in recurrent head and neck squamous cell carcinoma/second primary head and neck squamous cell carcinoma occurring after treatment (1.097 ± 0.295 × 10 -3 mm 2 /s) was significantly lower ( P < .05) than in post-radiation therapy inflammatory edema (1.754 ± 0.343 × 10 -3 mm 2 /s); however, it was similar to that in late fibrosis (0.987 ± 0.264 × 10 -3 mm 2 /s, P > .05). Although ADCs were similar in tumors and late fibrosis, morphologic MRI criteria facilitated distinction between the 2 conditions. The sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios (95% CI) of DWIMRI with ADCmean < 1.22 × 10 -3 mm 2 /s and precise MRI criteria were 92.1% (83.5-100.0), 95.4% (90.3-100.0), 92.1% (83.5-100.0), 95.4% (90.2-100.0), 19.9 (6.58-60.5), and 0.08 (0.03-0.24), respectively, indicating a good diagnostic performance to rule in and rule out disease. Adding precise morphologic MRI criteria to quantitative DWI enables reproducible and accurate detection of recurrent head and neck squamous cell carcinoma/second primary head and neck squamous cell carcinoma occurring after treatment. © 2018 by American Journal of Neuroradiology.

*NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.

PubMed

Blasig, I E; Giese, H; Schroeter, M L; Sporbert, A; Utepbergenov, D I; Buchwalow, I B; Neubert, K; Schönfelder, G; Freyer, D; Schimke, I; Siems, W E; Paul, M; Haseloff, R F; Blasig, R

2001-09-01

To investigate the relevance of *NO and oxyradicals in the blood-brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (gamma-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced gamma-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn-, Cu/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 microM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in *NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature. Copyright 2001 Academic Press.

Genome-wide methylation sequencing of paired primary and metastatic cell lines identifies common DNA methylation changes and a role for EBF3 as a candidate epigenetic driver of melanoma metastasis

PubMed Central

Chatterjee, Aniruddha; Stockwell, Peter A; Ahn, Antonio; Rodger, Euan J; Leichter, Anna L; Eccles, Michael R

2017-01-01

Epigenetic alterations are increasingly implicated in metastasis, whereas very few genetic mutations have been identified as authentic drivers of cancer metastasis. Yet, to date, few studies have identified metastasis-related epigenetic drivers, in part because a framework for identifying driver epigenetic changes in metastasis has not been established. Using reduced representation bisulfite sequencing (RRBS), we mapped genome-wide DNA methylation patterns in three cutaneous primary and metastatic melanoma cell line pairs to identify metastasis-related epigenetic drivers. Globally, metastatic melanoma cell lines were hypomethylated compared to the matched primary melanoma cell lines. Using whole genome RRBS we identified 75 shared (10 hyper- and 65 hypomethylated) differentially methylated fragments (DMFs), which were associated with 68 genes showing significant methylation differences. One gene, Early B Cell Factor 3 (EBF3), exhibited promoter hypermethylation in metastatic cell lines, and was validated with bisulfite sequencing and in two publicly available independent melanoma cohorts (n = 40 and 458 melanomas, respectively). We found that hypermethylation of the EBF3 promoter was associated with increased EBF3 mRNA levels in metastatic melanomas and subsequent inhibition of DNA methylation reduced EBF3 expression. RNAi-mediated knockdown of EBF3 mRNA levels decreased proliferation, migration and invasion in primary and metastatic melanoma cell lines. Overall, we have identified numerous epigenetic changes characterising metastatic melanoma cell lines, including EBF3-induced aggressive phenotypic behaviour with elevated EBF3 expression in metastatic melanoma, suggesting that EBF3 promoter hypermethylation may be a candidate epigenetic driver of metastasis. PMID:28030832

Thermo-responsive cell culture carrier: Effects on macrophage functionality and detachment efficiency.

PubMed

Rennert, Knut; Nitschke, Mirko; Wallert, Maria; Keune, Natalie; Raasch, Martin; Lorkowski, Stefan; Mosig, Alexander S

2017-01-01

Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyte-derived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyte-derived macrophages. In summary, we observed similar functionality and viability of primary monocyte-derived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications.

Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin.

PubMed

Wiegering, Armin; Matthes, Niels; Mühling, Bettina; Koospal, Monika; Quenzer, Anne; Peter, Stephanie; Germer, Christoph-Thomas; Linnebacher, Michael; Otto, Christoph

2017-04-01

Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n=9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n=5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC 50 <3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Patients with primary breast and primary female genital tract diffuse large B cell lymphoma have a high frequency of MYD88 and CD79B mutations.

PubMed

Cao, Xin-Xin; Li, Jian; Cai, Hao; Zhang, Wei; Duan, Ming-Hui; Zhou, Dao-Bin

2017-11-01

This study is to retrospectively evaluate the prevalence of MYD88 and CD79B mutations and the clinicopathologic characteristics of patients with primary diffuse large B cell lymphoma (DLBCL) of the female genital tract and breast. The characteristics, treatments, and outcomes of 19 patients diagnosed with primary DLBCL of the female genital tract and breast, who had formalin-fixed and paraffin-embedded tissues obtained from diagnostic samples diagnosed between January 2004 and June 2016, were analyzed retrospectively. Nineteen female patients (7 with primary breast and 12 with primary female genital tract DLBCL) were included in this retrospective study. Eleven patients (57.9%) carried a MYD88 mutation, including 10 with MYD8 L265P and 1 with the MYD88 L265S mutation. Seven patients (36.8%) harbored a CD79B mutation, which included two cases with CD79B Y196H, two cases with CD79B Y196N, one case with CD79B Y196D, one case with CD79B Y196F, and one case with CD79B Y196X. Four cases had both MYD88 and CD79B mutations. The clinicopathologic parameters, progression-free survival (PFS), and overall survival (OS) of the MYD88 mutation-carrying group were not significantly different from those of the MYD88 wild-type group except for higher LDH levels. Six patients received cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP), while 13 patients received rituximab plus CHOP, and 13 patients received central nervous system prophylaxis. The median OS and PFS were 73 and 56 months, respectively. Patients with primary breast and primary female genital tract DLBCL have a high frequency of MYD88 and CD79B mutations. The presence of these mutations does not affect survival but may offer additional therapeutic options.

HIV infection-induced transcriptional program in renal tubular epithelial cells activates a CXCR2-driven CD4+ T-cell chemotactic response.

PubMed

Chen, Ping; Yi, Zhengzi; Zhang, Weijia; Klotman, Mary E; Chen, Benjamin K

2016-07-31

Viral replication and interstitial inflammation play important roles in the pathogenesis of HIV-associated nephropathy. Cell-cell interactions between renal tubule epithelial cells (RTECs) and HIV-infected T cells can trigger efficient virus internalization and viral gene expression by RTEC. To understand how HIV replication initiates HIV-associated nephropathy, we studied the cellular response of RTECs to HIV, examining the transcriptional profiles of primary RTECs exposed to cell-free HIV or HIV-infected T cells. HIV-induced gene expression in hRTECs was examined in vitro by Illumina RNA deep sequencing and revealed an innate response to HIV, which was subclassified by gene ontology biological process terms. Chemokine responses were examined by CD4 T-cell chemotaxis assays. As compared with cell-free virus infection, exposure to HIV-infected T cells elicited a stronger upregulation of inflammatory and immune response genes. A major category of upregulated genes are chemokine/cytokine families involved in inflammation and immune response, including inflammatory cytokines CCL20, IL6 and IL8-related chemokines: IL8, CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6. Supernatants from virus-exposed RTECs contained strong chemoattractant activity on primary CD4 T cells, which was potently blocked by a CXCR2 antagonist that antagonizes IL8-related chemokines. We observed a preferential migration of CXCR2-expressing, central memory CD4 T cells in response to HIV infection of RTECs. Interactions between primary RTECs and HIV-infected T cells result in potent induction of inflammatory response genes and release of cytokines/chemokines from RTECs that can attract additional T cells. Activation of these genes reflects an innate response to HIV by nonimmune cells.

Establishment of segment polarity in the ectoderm of the leech Helobdella

NASA Technical Reports Server (NTRS)

Seaver, E. C.; Shankland, M.

2001-01-01

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. We have examined the mechanism by which the leech primary blast cell clones acquire segment polarity - i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the O and P teloblast lineages, the earliest divisions of the primary blast cell segregate anterior and posterior cell fates along the anteroposterior axis. Using a laser microbeam, we ablated single cells from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which we specifically ablated those blast cell progeny that are known to express the engrailed gene, or their lineal precursors. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and our findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms.

Introduction to cell culture.

PubMed

Philippeos, Christina; Hughes, Robin D; Dhawan, Anil; Mitry, Ragai R

2012-01-01

The basics of cell culture as applied to human cells are discussed. Biosafety when working with human tissue, which is often pathogenic, is important. The requirements for a tissue culture laboratory are described, particularly the range of equipment needed to carry out cell isolation, purification, and culture. Steps must be taken to maintain aseptic conditions to prevent contamination of cultures with micro-organisms. Basic cell-handling techniques are discussed, including choice of media, primary culture, and cryopreservation of cells so they can be stored for future use. Common assays which are used to determine cell viability and activity are considered.

Role of non-coding RNAs in maintaining primary airway smooth muscle cells

PubMed Central

2014-01-01

Background The airway smooth muscle (ASM) cell maintains its own proliferative rate and contributes to the inflammatory response in the airways, effects that are inhibited by corticosteroids, used in the treatment of airways diseases. Objective We determined the differential expression of mRNAs, microRNAs (miRNAs) and long noncoding RNA species (lncRNAs) in primary ASM cells following treatment with a corticosteroid, dexamethasone, and fetal calf serum (FCS). Methods mRNA, miRNA and lncRNA expression was measured by microarray and quantitative real-time PCR. Results A small number of miRNAs (including miR-150, −371-5p, −718, −940, −1181, −1207-5p, −1915, and −3663-3p) were decreased following exposure to dexamethasone and FCS. The mRNA targets of these miRNAs were increased in expression. The changes in mRNA expression were associated with regulation of ASM actin cytoskeleton. We also observed changes in expression of lncRNAs, including natural antisense, pseudogenes, intronic lncRNAs, and intergenic lncRNAs following dexamethasone and FCS. We confirmed the change in expression of three of these, LINC00882, LINC00883, PVT1, and its transcriptional activator, c-MYC. We propose that four of these lincRNAs (RP11-46A10.4, LINC00883, BCYRN1, and LINC00882) act as miRNA ‘sponges’ for 4 miRNAs (miR-150, −371-5p, −940, −1207-5p). Conclusion This in-vitro model of primary ASM cell phenotype was associated with the regulation of several ncRNAs. Their identification allows for in-vitro functional experimentation to establish causality with the primary ASM phenotype, and in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). PMID:24886442

Argininosuccinate synthetase (ASS) deficiency in high-grade pulmonary neuroendocrine carcinoma: an opportunity for personalized targeted therapy.

PubMed

Walts, Ann E; Bomalaski, John S; Ines, Delma; Orsulic, Sandra

2015-08-01

Cells deficient in argininosuccinate synthetase (ASS) must absorb the arginine they need for growth from circulating blood. Treatment with pegylated arginine deiminase (ADI-PEG 20) selectively eliminates arginine from the circulation and has shown some efficacy against ASS-deficient tumors including small cell lung cancer (SCLC). We sought to assess ASS expression in a cohort of high-grade pulmonary neuroendocrine carcinomas (PNEC) which include SCLC and large cell neuroendocrine carcinoma (LCNEC). Sixty-nine PNEC (49 SCLC and 20 LCNEC) were retrieved from our pathology archives. Formalin-fixed paraffin-embedded sections of the 54 primary tumors, 15 metastases and appropriate positive and negative controls were immunostained using an ASS-specific monoclonal antibody. Positive staining in <30 % of the tumor was scored as weak; staining in ≥30 % of the tumor was scored as strong. The absence of staining in the tumor was recorded as ASS negative. 58 % of the PNEC including 61.2 % of the SCLC and 50 % of the LCNEC were ASS negative. These ASS-negative tumors included 63 % of the primary and 40 % of the metastatic lesions tested. More than 50 % of the high-grade PNEC tested lack immunohistochemically detectable ASS, suggesting that they are auxotrophic for arginine and potential candidates for arginine deprivation therapy. PNEC comprise about 25 % of primary lung cancers and have a 5-year overall survival of only 5-10 %, underscoring the need for new and more effective therapies. Immunostaining for ASS has potential to improve the selection of patients with PNEC for arginine deprivation therapy with ADI-PEG 20.

The pathogenesis of pseudoachalasia: a clinicopathologic study of 13 cases of a rare entity.

PubMed

Liu, Wendy; Fackler, William; Rice, Thomas W; Richter, Joel E; Achkar, Edgar; Goldblum, John R

2002-06-01

Pseudoachalasia is an esophageal motor disorder usually associated with malignancy that has clinical, radiographic, and manometric findings that are often indistinguishable from primary achalasia. There are few reports examining the histologic features of the associated neoplasms and their relationship with the esophageal myenteric plexus. We studied the clinical and pathologic features of 13 cases of pseudoachalasia seen at our institution between 1979 and 1999. Detailed clinical and radiographic data were obtained from medical records. In all cases available histologic material was reviewed to confirm the presence and type of associated neoplasm. When possible, the relationship of the neoplasm to the esophageal myenteric plexus was examined. In selected cases immunohistochemical stains were performed to further evaluate this relationship. All patients had clinical, radiographic, and manometric features similar to primary achalasia. The cohort included seven men and six women, age range 24-79 years (median 61 years). Associated neoplasms included esophageal adenocarcinoma arising in Barrett's esophagus (n = 1), adenocarcinoma of the esophagogastric junction (n = 7), metastatic renal cell carcinoma to the esophagogastric junction (n = 1), breast adenocarcinoma (n = 1), pulmonary small cell carcinoma (n = 1), pleural malignant mesothelioma (n = 1), and mediastinal fibrosis (n = 1). The mechanism of pseudoachalasia was consistent with neoplastic infiltration of the esophageal myenteric plexus in 11 cases. Neoplastic cells surrounded myenteric ganglion cells, which appeared normal in number and morphology. In the patient with pulmonary small cell carcinoma, there was no evidence of neoplastic infiltration of the esophagogastric junction, and anti-ANNA-1 antibody was detected, suggesting a paraneoplastic syndrome. Tissue obtained at the time of esophagomyotomy revealed lymphocytic myenteric inflammation and marked depletion of ganglion cells identical to that seen in primary achalasia. The mechanism pseudoachalasia in the patient with breast adenocarcinoma is uncertain, as there was no evidence of direct involvement of the esophagogastric junction. In summary, we describe 13 cases of pseudoachalasia resulting in a clinical syndrome indistinguishable from primary achalasia. The most common mechanism is direct involvement of the esophageal myenteric plexus by neoplastic cells. Rarely, a distant neoplasm may cause this syndrome as a paraneoplastic process.

Lithium Iron Phosphate Cell Performance Evaluations for Lunar Extravehicular Activities

NASA Technical Reports Server (NTRS)

Reid, Concha

2007-01-01

Lithium-ion battery cells are being evaluated for their ability to provide primary power and energy storage for NASA s future Exploration missions. These missions include the Orion Crew Exploration Vehicle, the Ares Crew Launch Vehicle Upper Stage, Extravehicular Activities (EVA, the advanced space suit), the Lunar Surface Ascent Module (LSAM), and the Lunar Precursor and Robotic Program (LPRP), among others. Each of these missions will have different battery requirements. Some missions may require high specific energy and high energy density, while others may require high specific power, wide operating temperature ranges, or a combination of several of these attributes. EVA is one type of mission that presents particular challenges for today s existing power sources. The Portable Life Support System (PLSS) for the advanced Lunar surface suit will be carried on an astronaut s back during eight hour long sorties, requiring a lightweight power source. Lunar sorties are also expected to occur during varying environmental conditions, requiring a power source that can operate over a wide range of temperatures. Concepts for Lunar EVAs include a primary power source for the PLSS that can recharge rapidly. A power source that can charge quickly could enable a lighter weight system that can be recharged while an astronaut is taking a short break. Preliminary results of Al23 Ml 26650 lithium iron phosphate cell performance evaluations for an advanced Lunar surface space suit application are discussed in this paper. These cells exhibit excellent recharge rate capability, however, their specific energy and energy density is lower than typical lithium-ion cell chemistries. The cells were evaluated for their ability to provide primary power in a lightweight battery system while operating at multiple temperatures.

Adult multipotent stromal cell cryopreservation: Pluses and pitfalls

PubMed Central

Duan, Wei; Hicok, Kevin

2017-01-01

Abstract Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive improvements in veterinary regenerative medicine. Inherent limitations to long‐term culture preclude use for storage. Until cell line creation from primary isolates becomes routine, MSC stasis at cryogenic temperatures is required for this purpose. Many protocols and reagents, including cryoprotectants, used for veterinary MSCs are derived from those for human and rodent cells. Dissimilarities in cryopreservation strategies play a role in variable MSC behaviors. Familiarity with contemporary cryopreservation reagents and processes is essential to an appreciation of their impact on MSC survival and post‐cryopreservation behavior. In addition to these points, this review includes a brief history and description of current veterinary stem cell regulation. PMID:29023790

Compound heterozygous TYK2 mutations underlie primary immunodeficiency with T-cell lymphopenia.

PubMed

Nemoto, Michiko; Hattori, Hiroyoshi; Maeda, Naoko; Akita, Nobuhiro; Muramatsu, Hideki; Moritani, Suzuko; Kawasaki, Tomonori; Maejima, Masami; Ode, Hirotaka; Hachiya, Atsuko; Sugiura, Wataru; Yokomaku, Yoshiyuki; Horibe, Keizo; Iwatani, Yasumasa

2018-05-03

Complete tyrosine kinase 2 (TYK2) deficiency has been previously described in patients with primary immunodeficiency diseases. The patients were infected with various pathogens, including mycobacteria and/or viruses, and one of the patients developed hyper-IgE syndrome. A detailed immunological investigation of these patients revealed impaired responses to type I IFN, IL-10, IL-12 and IL-23, which are associated with increased susceptibility to mycobacterial and/or viral infections. Herein, we report a recessive partial TYK2 deficiency in two siblings who presented with T-cell lymphopenia characterized by low naïve CD4 + T-cell counts and who developed Epstein-Barr virus (EBV)-associated B-cell lymphoma. Targeted exome-sequencing of the siblings' genomes demonstrated that both patients carried novel compound heterozygous mutations (c.209_212delGCTT/c.691C > T, p.Cys70Serfs*21/p.Arg231Trp) in the TYK2. The TYK2 protein levels were reduced by 35% in the T cells of the patient. Unlike the response under complete TYK2 deficiency, the patient's T cells responded normally to type I IFN, IL-6, IL-10 and IL-12, whereas the cells displayed an impaired response to IL-23. Furthermore, the level of STAT1 was low in the cells of the patient. These studies reveal a new clinical entity of a primary immunodeficiency with T-cell lymphopenia that is associated with compound heterozygous TYK2 mutations in the patients.

Mechanistic insights into the antileukemic activity of hyperforin.

PubMed

Billard, C; Merhi, F; Bauvois, B

2013-01-01

Hyperforin is a prenylated phloroglucinol present in the medicinal plant St John's wort (Hypericum perforatum). The compound has many biological properties, including antidepressant, anti-inflammatory, antibacterial and antitumor activities. This review focuses on the in vitro antileukemic effects of purified hyperforin and related mechanisms in chronic lymphoid leukemia (CLL) and acute myeloid leukemia (AML) - conditions that are known for their resistance to chemotherapy. Hyperforin induces apoptosis in both CLL and AML cells. In AML cell lines and primary AML cells, hyperforin directly inhibits the kinase activity of the serine/threonine protein kinase B/AKT1, leading to activation of the pro-apoptotic Bcl-2 family protein Bad through its non-phosphorylation by AKT1. In primary CLL cells, hyperforin acts by stimulating the expression of the pro-apoptotic Bcl-2 family member Noxa (possibly through the inhibition of proteasome activity). Other hyperforin targets include matrix metalloproteinase-2 in AML cells and vascular endothelial growth factor and matrix metalloproteinase-9 in CLL cells - two mediators of cell migration and angiogenesis. In summary, hyperforin targets molecules involved in signaling pathways that control leukemic cell proliferation, survival, apoptosis, migration and angiogenesis. Hyperforin also downregulates the expression of P-glycoprotein, a protein that is involved in the resistance of leukemia cells to chemotherapeutic agents. Lastly, native hyperforin and its stable derivatives show interesting in vivo properties in animal models. In view of their low toxicity, hyperforin and its derivatives are promising antileukemic agents and deserve further investigation in vivo.

Providing cell phone numbers and email addresses to Patients: the physician's perspective.

PubMed

Peleg, Roni; Avdalimov, Angelika; Freud, Tamar

2011-03-23

The provision of cell phone numbers and email addresses enhances the accessibility of medical consultations, but can add to the burden of physicians' routine clinical practice and affect their free time. The objective was to assess the attitudes of physicians to providing their telephone number or email address to patients. Primary care physicians in the southern region of Israel completed a structured questionnaire that related to the study objective. The study population included 120 primary care physicians with a mean age of 41.2 ± 8.5, 88 of them women (73.3%). Physicians preferred to provide their cell phone number rather than their email address (P = 0.0007). They preferred to answer their cell phones only during the daytime and at predetermined times, but would answer email most hours of the day, including weekends and holidays (P = 0.001). More physicians (79.7%) would have preferred allotted time for email communication than allotted time for cell phone communication (50%). However, they felt that email communication was more likely to lead to miscommunication than telephone calls (P = 0.0001). There were no differences between male and female physicians on the provision of cell phone numbers or email addresses to patients. Older physicians were more prepared to provide cell phone numbers that younger ones (P = 0.039). The attitude of participating physicians was to provide their cell phone number or email address to some of their patients, but most of them preferred to give out their cell phone number.

Disordered follicle development

PubMed Central

Chang, R. Jeffrey; Cook-Andersen, Heidi

2013-01-01

Alterations of ovarian follicle morphology and function have been well documented in women with PCOS. These include increased numbers of growing preantral follicles, failure of follicle growth beyond the mid-antral stage, evidence of granulosa call degeneration, and theca cell hyperplasia. Functional abnormalities include paradoxical granulosa cell hyperresponsiveness to FSH which is clinically linked to ovarian hyperstimulation during ovulation induction. In addition, there is likely a primary theca cell defect that accounts for the majority of excess androgen production in this disorder. The precise mechanisms responsible for altered follicle function are not completely clear. However, several factors appear to influence normal advancement of follicle development as well as impair ovarian steroidogenesis. These include intra- as well as extraovarian influences that distort normal ovarian growth and disrupt steroid production by follicle cells. PMID:22874072

Systematic chemical and molecular profiling of MLL-rearranged infant acute lymphoblastic leukemia reveals efficacy of romidepsin

PubMed Central

Cruickshank, M N; Ford, J; Cheung, L C; Heng, J; Singh, S; Wells, J; Failes, T W; Arndt, G M; Smithers, N; Prinjha, R K; Anderson, D; Carter, K W; Gout, A M; Lassmann, T; O'Reilly, J; Cole, C H; Kotecha, R S; Kees, U R

2017-01-01

To address the poor prognosis of mixed lineage leukemia (MLL)-rearranged infant acute lymphoblastic leukemia (iALL), we generated a panel of cell lines from primary patient samples and investigated cytotoxic responses to contemporary and novel Food and Drug Administration-approved chemotherapeutics. To characterize representation of primary disease within cell lines, molecular features were compared using RNA-sequencing and cytogenetics. High-throughput screening revealed variable efficacy of currently used drugs, however identified consistent efficacy of three novel drug classes: proteasome inhibitors, histone deacetylase inhibitors and cyclin-dependent kinase inhibitors. Gene expression of drug targets was highly reproducible comparing iALL cell lines to matched primary specimens. Histone deacetylase inhibitors, including romidepsin (ROM), enhanced the activity of a key component of iALL therapy, cytarabine (ARAC) in vitro and combined administration of ROM and ARAC to xenografted mice further reduced leukemia burden. Molecular studies showed that ROM reduces expression of cytidine deaminase, an enzyme involved in ARAC deactivation, and enhances the DNA damage–response to ARAC. In conclusion, we present a valuable resource for drug discovery, including the first systematic analysis of transcriptome reproducibility in vitro, and have identified ROM as a promising therapeutic for MLL-rearranged iALL. PMID:27443263

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

PubMed Central

EL Andaloussi, Samir; Lehto, Taavi; Mäger, Imre; Rosenthal-Aizman, Katri; Oprea, Iulian I.; Simonson, Oscar E.; Sork, Helena; Ezzat, Kariem; Copolovici, Dana M.; Kurrikoff, Kaido; Viola, Joana R.; Zaghloul, Eman M.; Sillard, Rannar; Johansson, Henrik J.; Said Hassane, Fatouma; Guterstam, Peter; Suhorutšenko, Julia; Moreno, Pedro M. D.; Oskolkov, Nikita; Hälldin, Jonas; Tedebark, Ulf; Metspalu, Andres; Lebleu, Bernard; Lehtiö, Janne; Smith, C. I. Edvard; Langel, Ülo

2011-01-01

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential. PMID:21245043

Mucin profiles in signet-ring cell carcinoma.

PubMed

Nguyen, Minh D; Plasil, Brian; Wen, Ping; Frankel, Wendy L

2006-06-01

Signet-ring cell carcinoma (SRCC) is a poorly differentiated mucin-producing adenocarcinoma that may arise from many different organs, but all SRCCs share identical morphology. It is not possible to differentiate sites of origin for metastatic SRCC based on morphology alone. Mucins are high-molecular-weight glycoproteins differentially expressed in glandular epithelia and in adenocarcinomas. To identify mucin profiles of primary and metastatic SRCCs using immunohistochemistry to determine whether mucin staining could help distinguish sites of origin. Forty-seven SRCCs, including 38 primary (21 stomach, 11 colorectum, and 6 breast) and 9 metastases from these primary sites were retrieved from archival files. Consecutive tissue sections were immunostained with monoclonal antibodies against MUC1, MUC2, MUC4, MUC5AC (MUC5), and MUC6 on separate slides. Cytoplasmic staining was scored based on proportion of positive tumor cells as follows: 0+ (<5%), 1+ (5%-25%), 2+ (26%-50%), and 3+ (>50%). Mucin profiles were recorded as MUC+, MUCv, and MUC- for consistent, variable, and negative expression, respectively. The mucin profiles for gastric, colorectum, and breast SRCCs are MUC1.2.4.5.6v, MUC2.4+/MUC5v/ MUC1.6-, and MUC1+/MUC2.5.6v/MUC4-, respectively. Mucin profiles of metastatic cases shared profiles with their respective primaries. Signet-ring cell carcinomas of the stomach, colorectum, and breast have distinct mucin expression patterns that are maintained in metastases. Mucin profiling may be useful to identify the origin of a metastatic SRCC of unknown primary.

Characterizing emergency admissions of patients with sickle cell crisis in NHS brent: observational study

PubMed Central

Green, Stuart A; AlJuburi, Ghida; Majeed, Azeem; Okoye, Ogo; Amobi, Carole; Banarsee, Ricky; Phekoo, Karen J

2012-01-01

Objectives To characterize emergency admissions for patients with sickle cell crisis in NHS Brent and to determine which patients and practices may benefit most from primary care intervention. Design Observational study Setting Emergency departments attended by residents of the London borough of Brent Participants Patients with sickle cell disease registered with a general practitioner (GP) in the borough of Brent Main outcome measures Analysis of admissions between January 2008 and July 2010 that included length of stay (average and <2 days versus ≥2 days) by age group and registered GP practice. Results Thirty six percent of sickle cell disease admission spells resulted in a length of stay of less than two days. Seventy four percent of total bed days are associated with patients with more than one admission during the period of analysis, i.e. multiple admissions. Two general practices in Brent were identified as having the highest number of patients admitted to the emergency department for sickle cell crisis and may benefit most from primary care intervention. Discussion Patients with short length of stay and multiple admissions may be potentially amenable to primary care intervention. The practices which have the highest numbers of sickle cell disease patients who frequently seek emergency care will be earmarked for an education intervention designed to help further engage general practitioners in the care and management of their sickle cell patients. PMID:22768371

Characterizing emergency admissions of patients with sickle cell crisis in NHS brent: observational study.

PubMed

Green, Stuart A; Aljuburi, Ghida; Majeed, Azeem; Okoye, Ogo; Amobi, Carole; Banarsee, Ricky; Phekoo, Karen J

2012-06-01

To characterize emergency admissions for patients with sickle cell crisis in NHS Brent and to determine which patients and practices may benefit most from primary care intervention. Observational study Emergency departments attended by residents of the London borough of Brent Patients with sickle cell disease registered with a general practitioner (GP) in the borough of Brent Analysis of admissions between January 2008 and July 2010 that included length of stay (average and <2 days versus ≥2 days) by age group and registered GP practice. Thirty six percent of sickle cell disease admission spells resulted in a length of stay of less than two days. Seventy four percent of total bed days are associated with patients with more than one admission during the period of analysis, i.e. multiple admissions. Two general practices in Brent were identified as having the highest number of patients admitted to the emergency department for sickle cell crisis and may benefit most from primary care intervention. Patients with short length of stay and multiple admissions may be potentially amenable to primary care intervention. The practices which have the highest numbers of sickle cell disease patients who frequently seek emergency care will be earmarked for an education intervention designed to help further engage general practitioners in the care and management of their sickle cell patients.

Clinical presentation, diagnosis, and pharmacotherapy of patients with primary brain tumors.

PubMed

Newton, H B; Turowski, R C; Stroup, T J; McCoy, L K

1999-01-01

To briefly review the clinical presentation and diagnosis of patients with primary brain tumors, followed by an in-depth survey of the pertinent pharmacotherapy. A detailed search of the neurologic, neurosurgical, and oncologic literature for basic science research, clinical studies, and review articles related to chemotherapy and pharmacotherapy of primary brain tumors. Relevant studies on tissue culture systems, animals, and humans examining the mechanisms of action, pharmacokinetics, clinical pharmacology, and treatment results of chemotherapeutic agents for primary brain tumors. In addition, studies of pharmacologic agents administered for supportive care and symptom control are reviewed. Primary brain tumors derive from cells within the intracranial cavity and generally present with headache, seizure activity, cognitive changes, and weakness. They are diagnosed most efficiently with magnetic resonance imaging. After diagnosis, the most common supportive medications include corticosteroids, gastric acid inhibitors, and anticonvulsants. Chemotherapy is adjunctive treatment for patients with malignant tumors and selected recurrent or progressive benign neoplasms. In general, the most effective chemotherapeutic drugs are alkylating agents such as the nitrosoureas, procarbazine, cisplatin, and carboplatin. Other agents used include cyclophosphamide, methotrexate, vincristine, and etoposide. Angiogenesis inhibitors and gene therapy comprise some of the novel therapeutic strategies under investigation. The efficacy of chemotherapy for primary brain tumors remains modest. Novel agents must be discovered that are more specific and attack tumor cells at the molecular level of tumorigenesis. Furthermore, strategies must be developed to counteract the pervasive problem of brain tumor chemoresistance.

The relationship between components of tumour inflammatory cell infiltrate and clinicopathological factors and survival in patients with primary operable invasive ductal breast cancer

PubMed Central

Mohammed, Z MA; Going, J J; Edwards, J; Elsberger, B; Doughty, J C; McMillan, D C

2012-01-01

Background: The importance of the components of host local inflammatory response in determining outcome in primary operable ductal invasive breast cancer is not clear. The aim of this study was to examine the relationship between components of the tumour inflammatory cell infiltrate and standard clinicopathological factors including hormone status (oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER)-2), Ki-67 and survival in patients with primary operable invasive ductal breast cancer. Methods: Tumour inflammatory cell infiltrate, hormone status (ER, PR and HER-2), Ki-67 and standard clinicopathological factors were determined using routine pathological and immuno-histochemical techniques in 468 patients. Results: The large majority (94%) of ductal tumours had evidence of inflammatory cell infiltrate. The general inflammatory cell infiltrate was positively associated with high grade (P<0.001), the absence of ER (P<0.001), the absence of PR (P<0.01), the presence of vascular invasion (P<0.05) and high lymphocytic infiltrate, plasma cell infiltrate, other inflammatory cell infiltrate and macrophage infiltrate (all P<0.001). The median follow-up of the survivors was 165 months. During this period, 93 patients died of their cancer. On univariate analysis, stratified for ER status, tumour size (P<0.01), lymph node involvement (P<0.001), tumour plasma cell infiltrate (P<0.001), other inflammatory cell infiltrate (P<0.05) and treatment (P<0.05) were associated with poorer cancer-specific survival whereas lymphocyte infiltrate (P<0.001) was associated with improved cancer-specific survival. On multivariate analysis, stratified for ER status, lymph node involvement (P<0.05) was independently associated with poorer cancer-specific survival whereas increased tumour lymphocyte infiltrate (P<0.001) was independently associated with improved cancer-specific survival. Conclusion: The results of this study show that, using routine histology, the general inflammatory cell infiltrate was a common feature and was positively associated with high grade, the absence of ER, the absence of PR, the presence of vascular invasion and high-grade infiltration of lymphocytes, plasma cells, other inflammatory cells and macrophages. Also, that within a mature cohort of patients, a high lymphocytic infiltrate was associated with improved survival, independent of clinicopathological characteristics including ER status, in primary operable ductal invasive breast cancer. These results rationalise previous work and provide a sound basis for future studies in this important area of breast cancer research. PMID:22878371

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

Compositions and methods for adoptive and active immunotherapy

DOEpatents

Fahmy, Tarek; Steenblock, Erin

2014-01-14

Modular aAPCs and methods of their manufacture and use are provided. The modular aAPCs are constructed from polymeric microparticles. The aAPCs include encapsulated cytokines and coupling agents which modularly couple functional elements including T cell receptor activators, co-stimulatory molecules and adhesion molecules to the particle. The ability of these aAPCs to release cytokines in a controlled manner, coupled with their modular nature and ease of ligand attachment, results in an ideal, tunable APC capable of stimulating and expanding primary T cells.

The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

PubMed Central

Liu, Xiu-Huai; Xu, Wenqin; Russ, Jill; Eiden, Lee E.; Eiden, Maribeth V.

2011-01-01

Background Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. Methodology/Principal Findings Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. Conclusions/Significance These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease. PMID:21464894

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

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

Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression.more » Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.« less

Mechanisms of Transendothelial Migration of Primary Human Invasive Ductal Carcinoma Cells from ER+, Her2+, and Triple-Negative Disease

DTIC Science & Technology

2015-09-01

Intravital imaging in animal models has revealed many aspects of meta- stasis (3–6), including the essential roles that macrophages play in the...micro- environments inwhichmammary tumor cells invade,migrate, and intravasate (5, 7). In particular, intravital imaging of rodent mammary tumors shows...cell intravasation, called TMEM (tumor micro- environment of metastasis) sites (22, 23). These sites, initially observed by intravital imaging of

Intramyocardial autologous CD34+ cell therapy for refractory angina: A meta-analysis of randomized controlled trials.

PubMed

Velagapudi, Poonam; Turagam, Mohit; Kolte, Dhaval; Khera, Sahil; Hyder, Omar; Gordon, Paul; Aronow, Herbert D; Leopold, Jane; Abbott, J Dawn

2018-06-05

Previous studies have demonstrated that intramyocardial human CD34+ cells may relieve symptoms and improve clinical outcomes in chronic refractory angina unresponsive to optimal medical therapy or not amenable to revascularization. We performed a meta-analysis of randomized controlled trials (RCTs) to evaluate the impact of human CD34+ cells compared with placebo in chronic refractory angina. Primary efficacy outcomes in our analysis were angina frequency and exercise time. Primary safety outcomes included major adverse cardiovascular events such as myocardial infarction (MI), stroke and death. Three eligible randomized trials including 269 patients (placebo = 90, CD34+ = 179) were included. Dose of auto-CD34+ cells ranged from 5 × 10 4 to 5 × 10 5  cells/kg. Follow-up ranged from 6 to 24 months. In a pooled analysis, administration of CD34+ cells decreased the risk of all-cause mortality [OR 0.24, 95% CI (0.08-0.73), p = 0.01], reduced angina frequency [mean difference -2.91, 95% CI (-4.57 to -1.25), p = 0.0006] and improved exercise time [mean difference 58.62 s, 95% CI (21.19 to 96.06), p = 0.02] compared with control group. However, there was no significant difference in the risk of myocardial infarction (MI) and stroke between groups. In a meta-analysis, intra-myocardial CD34+ cell therapy was superior to placebo in improving risk of all - cause mortality, angina frequency with an increase in exercise time, without a significant increase in adverse events. This analysis supports further trials of CD34+ cell therapy for ischemic heart disease. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Recent progress in Si thin film technology for solar cells

NASA Astrophysics Data System (ADS)

Kuwano, Yukinori; Nakano, Shoichi; Tsuda, Shinya

1991-11-01

Progress in Si thin film technology 'specifically amorphous Si (a-Si) and polycrystalline Si (poly-Si) thin film' for solar cells is summarized here from fabrication method, material, and structural viewpoints. In addition to a-Si, primary results on poly-Si thin film research are discussed. Various applications for a-Si solar cells are mentioned, and consumer applications and a-Si solar cell photovoltaic systems are introduced. New product developments include see-through solar cells, solar cell roofing tiles, and ultra-light flexible solar cells. As for new systems, air conditioning equipment powered by solar cells is described. Looking to the future, the proposed GENESIS project is discussed.

Discovery of a proteinaceous cellular receptor for a norovirus

PubMed Central

Orchard, Robert C.; Wilen, Craig B.; Doench, John G.; Baldridge, Megan T.; McCune, Broc T.; Lee, Ying-Chiang J.; Lee, Sanghyun; Pruett-Miller, Shondra M.; Nelson, Christopher A.; Fremont, Daved H.; Virgin, Herbert W.

2017-01-01

Human noroviruses (NoV) are a leading cause of gastroenteritis globally, yet host factors required for NoV infection are poorly understood. We identified host molecules essential for murine NoV (MNoV) induced cell death including CD300lf as a proteinaceous receptor. CD300lf is essential for MNoV binding and replication in cell lines and primary cells. Additionally, Cd300lf−/− mice are resistant to MNoV infection. Expression of CD300lf in human cells breaks the species barrier restricting MNoV replication. The crystal structure of the CD300lf ectodomain revealed a potential ligand binding cleft composed of residues critical for MNoV infection. Therefore, the presence of a proteinaceous receptor is the primary determinant of MNoV species tropism while other components of cellular machinery required for NoV replication are conserved between humans and mice. PMID:27540007

Navigation to the graveyard-induction of various pathways of necrosis and their classification by flow cytometry.

PubMed

Janko, Christina; Munoz, Luis; Chaurio, Ricardo; Maueröder, Christian; Berens, Christian; Lauber, Kirsten; Herrmann, Martin

2013-01-01

Apoptosis and necrosis reflect the program of cell death employed by a dying cell and the final stage of death, respectively. Whereas apoptosis is defined as a physiological, highly organized cell death process, necrosis is commonly considered to be accidental and uncontrolled. Physiological and weak pathological death stimuli preferentially induce apoptosis, while harsh non-physiological insults often immediately instigate (primary) necrosis. If an apoptosing cell transits into a phase of plasma membrane disintegration, this stage of death is referred to as secondary or post-apoptotic necrosis.Here, we present several conditions that stimulate primary and/or secondary necrosis and show that necrosis displays considerably different time courses. For subclassification of necrotic phenotypes we employed a flow cytometric single-tube 4-color staining technique including annexin A5-FITC, propidium iodide, DiIC1(5), and Hoechst 33342.

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.

Splanchnic vein thrombosis as a first manifestation of Primary myelofibrosis

PubMed

Campos-Cabrera, Gregorio; Campos-Cabrera, Virginia; Campos-Cabrera, Salvador; Campos-Villagómez, José-Luis; Romero-González, Alejandra

2017-01-01

Myeloproliferative neoplasms are chronic disorders of clonal hematopoietic stem cells, characterized by an overproduction of functional granulocytes, red blood cells and / or platelets, and one of the major complications is the occurrence of venous and arterial thrombotic problems caused by increased platelet aggregation and thrombin generation. In this study 11 cases of primary myelofibrosis (PM) were evaluated and 2 debuted with splanchnic venous thrombosis (SVT); so after seeing the results of this study and of world literature, it is suggested that in patients with SVT, diagnostic methods for PM like the JAK2V617F mutation should be included. Copyright: © 2017 SecretarÍa de Salud

Gene expression networks underlying ovarian development in wild largemouth bass (Micropterus salmoides).

PubMed

Martyniuk, Christopher J; Prucha, Melinda S; Doperalski, Nicholas J; Antczak, Philipp; Kroll, Kevin J; Falciani, Francesco; Barber, David S; Denslow, Nancy D

2013-01-01

Oocyte maturation in fish involves numerous cell signaling cascades that are activated or inhibited during specific stages of oocyte development. The objectives of this study were to characterize molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in wild female largemouth bass to improve understanding of the molecular sequence of events underlying oocyte maturation. Transcriptomic analysis was performed on eight morphologically diverse stages of the ovary, including primary and secondary stages of oocyte growth, ovulation, and atresia. Ovary histology, plasma vitellogenin, 17β-estradiol, and testosterone were also measured to correlate with gene networks. Global expression patterns revealed dramatic differences across ovarian development, with 552 and 2070 genes being differentially expressed during both ovulation and atresia respectively. Gene set enrichment analysis (GSEA) revealed that early primary stages of oocyte growth involved increases in expression of genes involved in pathways of B-cell and T-cell receptor-mediated signaling cascades and fibronectin regulation. These pathways as well as pathways that included adrenergic receptor signaling, sphingolipid metabolism and natural killer cell activation were down-regulated at ovulation. At atresia, down-regulated pathways included gap junction and actin cytoskeleton regulation, gonadotrope and mast cell activation, and vasopressin receptor signaling and up-regulated pathways included oxidative phosphorylation and reactive oxygen species metabolism. Expression targets for luteinizing hormone signaling were low during vitellogenesis but increased 150% at ovulation. Other networks found to play a significant role in oocyte maturation included those with genes regulated by members of the TGF-beta superfamily (activins, inhibins, bone morphogenic protein 7 and growth differentiation factor 9), neuregulin 1, retinoid X receptor, and nerve growth factor family. This study offers novel insight into the gene networks underlying vitellogenesis, ovulation and atresia and generates new hypotheses about the cellular pathways regulating oocyte maturation.

Absence of Genomic Ikaros/IKZF1 Deletions in Pediatric B-Precursor Acute Lymphoblastic Leukemia

PubMed Central

Qazi, Sanjive; Ma, Hong; Uckun, Fatih M

2013-01-01

Here we report the results of gene expression analyses using multiple probesets aimed at determining the incidence of Ikaros/IKZF1 deletions in pediatric B-precursor acute lymphoblastic leukemia (BPL). Primary leukemia cells from 122 Philadelphia chromosome (Ph)+ BPL patients and 237 Ph− BPL patients as well as normal hematopoietic cells from 74 normal non-leukemic bone marrow specimens were organized according to expression levels of IKZF1 transcripts utilizing two-way hierarchical clustering technique to identify specimens with low IKZF1 expression for the 10 probesets interrogating Exons 1 through 4 and Exon 8. Our analysis demonstrated no changes in expression that would be expected from homozygous or heterozygous deletions of IKZF1 in primary leukemic cells. Similar results were obtained in gene expression analysis of primary leukemic cells from 20 Ph+ positive and 155 Ph− BPL patients in a validation dataset. Taken together, our gene expression analyses in 534 pediatric BPL cases, including 142 cases with Ph+ BPL, contradict previous reports that were based on SNP array data and suggested that Ph+ pediatric BPL is characterized by a high frequency of homozygous or heterozygous IKZF1 deletions. Further, exon-specific genomic PCR analysis of primary leukemia cells from 21 high-risk pediatric BPL patients and 11 standard-risk pediatric BPL patients, and 8 patients with infant BPL did not show any evidence for homozygous IKZF1 locus deletions. Nor was there any evidence for homozygous or heterozygous intragenic IKZF1 deletions. PMID:24478816

The human secretome atlas initiative: Implications in health and disease conditions

PubMed Central

Brown, Kristy J; Seol, Haeri; Pillai, Dinesh K; Sankoorikal, Binu-John; Formolo, Catherine A; Mac, Jenny; Edwards, Nathan J.; Rose, Mary C; Hathout, Yetrib

2013-01-01

Proteomic analysis of human body fluids is highly challenging, therefore many researchers are redirecting efforts towards secretome profiling. The goal is to define potential biomarkers and therapeutic targets in the secretome that can be traced back in accessible human body fluids. However, currently there is a lack of secretome profiles of normal human primary cells making it difficult to assess the biological meaning of current findings. In this study we sought to establish secretome profiles of human primary cells obtained from healthy donors with the goal of building a human secretome atlas. Such an atlas can be used as a reference for discovery of potential disease associated biomarkers and eventually novel therapeutic targets. As a preliminary study, secretome profiles were established for six different types of human primary cell cultures and checked for overlaps with the three major human body fluids including plasma, cerebrospinal fluid and urine. About 67% of the 1054 identified proteins in the secretome of these primary cells occurred in at least one body fluid. Furthermore, comparison of the secretome profiles of two human glioblastoma cell lines to this new human secretome atlas enabled unambiguous identification of potential brain tumor biomarkers. These biomarkers can be easily monitored in different body fluids using stable isotope labeled standard proteins. The long term goal of this study is to establish a comprehensive online human secretome atlas for future use as a reference for any disease related secretome study. PMID:23603790

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

Expression of p27Kip1 and E-cadherin in Head and Neck Squamous Cell Carcinoma of Indonesian Patients.

PubMed

E I, Auerkari; V, Joewono; D R, Handjari; A T, Sarwono; A W, Suhartono; K, Eto; M A, Ikeda

2014-01-01

Cancer cells exhibit characteristic damage of DNA and its expression. The expression of the tumor suppressors E-cadherin and p27(Kip1) has been tested on 57 head and neck squamous cell carcinomas (HNSCC) of Indonesian subjects. HNSCC tumor samples including both primary and (unrelated) nodal cases were obtained from the archives of Indonesian hospitals, in accordance with acknowledged ethical requirements. Only modest correlation was found between reduced expression of E-cadherin or p27(Kip1) with increased malignancy of primary and nodal growth. The observed strong correlation regardless of malignancy between the expressed levels of E-cadherin and p27(Kip1) suggests that also in combination these would not help to better predict the outcome of HNSCC.

Primary cutaneous T-cell lymphoma: experience from the Peruvian National Cancer Institute*

PubMed Central

Ruiz, Rosana; Morante, Zaida; Mantilla, Raul; Mas, Luis; Casanova, Luis; Gomez, Henry L.

2017-01-01

Background Primary cutaneous T-cell lymphomas constitute a heterogeneous and rare group of diseases with regional particularities in Latin America. Objective To determine the clinicopathological features, relative frequency and survival among patients from a Peruvian institution. Methods Primary cutaneous T-cell lymphomas were defined based on the absence of extracutaneous disease at diagnosis. Classification was performed following the 2008 World Health Organization Classification of Neoplasms of the Hematopoietic and Lymphoid tissues. Risk groups were established according to the 2005 World Health Organization-EORTC classification for cutaneous lymphomas. Data of patients admitted between January 2008 and December 2012 were analyzed. Results 74 patients were included. Mean age was 49.5 years. In order of frequency, diagnoses were: mycosis fungoides (40.5%), peripheral T-cell lymphoma not otherwise specified (22.95%), adult T-cell lymphoma/leukemia (18.9%), CD30+ lymphoproliferative disorders (6.8%), hydroa vacciniforme-like lymphoma (5.4%), extranodal NK/T-cell lymphoma (4.1%) and Sézary syndrome (1.4%). Predominant clinical patterns were observed across different entities. Mycosis fungoides appeared mainly as plaques (93%). Peripheral T-cell lymphoma not otherwise specified and adult T-cell lymphoma/leukemia presentation was polymorphic. All patients with hydroa vacciniforme-like lymphoma presented with facial edema. All cases of extranodal NK/T-cell lymphoma appeared as ulcerated nodules/tumors. Disseminated cutaneous involvement was found in 71.6% cases. Forty-six percent of patients were alive at 5 years. Five-year overall survival was 76.4% and 19.2%, for indolent and high-risk lymphomas, respectively (p<0.05). High risk group (HR: 4.6 [2.08-10.18]) and increased DHL level (HR: 3.2 [1.57-6.46]) emerged as prognostic factors for survival. Study limitations Small series. Conclusion Primary cutaneous T-cell lymphomas other than mycosis fungoides or CD30+ lymphoproliferative disorders are aggressive entities with a poor prognosis. PMID:29166501

Use of email, cell phone and text message between patients and primary-care physicians: cross-sectional study in a French-speaking part of Switzerland.

PubMed

Dash, Jonathan; Haller, Dagmar M; Sommer, Johanna; Junod Perron, Noelle

2016-10-05

Physicians' daily work is increasingly affected by the use of emails, text messages and cell phone calls with their patients. The aim of this study was to describe their use between primary-care physicians and patients in a French-speaking part of Switzerland. A cross-sectional mail survey was conducted among all primary-care physicians of Geneva canton (n = 636). The questionnaire focused on the frequency of giving access to, type of use, advantages and disadvantages of email, cell phone calls and text messages communication between physicians and patients. Six hundred thirty-six questionnaires were mailed, 412 (65 %) were returned and 372 (58 %) could be analysed (37 refusals and three blanks). Seventy-two percent physicians gave their email-address and 74 % their cell phone number to their patients. Emails were used to respond to patients' questions (82 %) and change appointments (72 %) while cell phone calls and text messages were used to follow patients' health conditions. Sixty-four percent of those who used email communication never discussed the rules for email exchanges, and 54 % did not address confidentiality issues with their patients. Most commonly identified advantages of emails, cell phone calls and text messages were improved relationship with the patient, saving time (for emails) and improving the follow-up (for cell phone and text messages). The main disadvantages included misuse by the patient, interference with private life and lack of reimbursement. These tools are widely used by primary-care physicians with their patients. More attention should be paid to confidentiality, documentation and reimbursement when using email communication in order to optimize its use.

The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia.

PubMed

de Rooij, Martin F M; Kuil, Annemieke; Geest, Christian R; Eldering, Eric; Chang, Betty Y; Buggy, Joseph J; Pals, Steven T; Spaargaren, Marcel

2012-03-15

Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α(4)β(1)-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis.

PubMed

Dong, Shengli; Maziveyi, Mazvita; Alahari, Suresh K

2015-05-20

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2

PubMed Central

Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E.; Chen, Shuang; Bodie, Wesley W.; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A.; Kramer, Lora B.; Dent, Paul

2011-01-01

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138+ cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138+ primary samples, but spared normal CD138− and CD34+ cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G0/G1 arrest and increased apoptosis in all cell-cycle phases, including G0/G1. To determine whether this regimen is active against quiescent G0/G1 MM cells, cells were cultured in low-serum medium to enrich the G0/G1 population. G0/G1–enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G0/G1–enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G0) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst+), low pyronin Y (PY)–staining (2N Hst+/PY−) G0 population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition. PMID:21911831

Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2.

PubMed

Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E; Chen, Shuang; Bodie, Wesley W; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A; Kramer, Lora B; Dent, Paul; Grant, Steven

2011-11-10

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.

The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds.

PubMed

Johnson, James D

2016-10-01

The production of fully functional insulin-secreting cells to treat diabetes is a major goal of regenerative medicine. In this article, I review progress towards this goal over the last 15 years from the perspective of a beta cell biologist. I describe the current state-of-the-art, and speculate on the general approaches that will be required to identify and achieve our ultimate goal of producing functional beta cells. The need for deeper phenotyping of heterogeneous cultures of stem cell derived islet-like cells in parallel with a better understanding of the heterogeneity of the target cell type(s) is emphasised. This deep phenotyping should include high-throughput single-cell analysis, as well as comprehensive 'omics technologies to provide unbiased characterisation of cell products and human beta cells. There are justified calls for more detailed and well-powered studies of primary human pancreatic beta cell physiology, and I propose online databases of standardised human beta cell responses to physiological stimuli, including both functional and metabolomic/proteomic/transcriptomic profiles. With a concerted, community-wide effort, including both basic and applied scientists, beta cell replacement will become a clinical reality for patients with diabetes.

MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

PubMed

Xu, Hui; Yan, Yaping; Williams, Mark S; Carey, Gregory B; Yang, Jingxian; Li, Hongmei; Zhang, Guang-Xian; Rostami, Abdolmohamad

2010-11-01

MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

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.

Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells.

PubMed

Boonkaew, Benjawan; Kempf, Margit; Kimble, Roy; Cuttle, Leila

2014-12-01

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver(®) and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver(®) showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Dissecting Germ Cell Metabolism through Network Modeling.

PubMed

Whitmore, Leanne S; Ye, Ping

2015-01-01

Metabolic pathways are increasingly postulated to be vital in programming cell fate, including stemness, differentiation, proliferation, and apoptosis. The commitment to meiosis is a critical fate decision for mammalian germ cells, and requires a metabolic derivative of vitamin A, retinoic acid (RA). Recent evidence showed that a pulse of RA is generated in the testis of male mice thereby triggering meiotic commitment. However, enzymes and reactions that regulate this RA pulse have yet to be identified. We developed a mouse germ cell-specific metabolic network with a curated vitamin A pathway. Using this network, we implemented flux balance analysis throughout the initial wave of spermatogenesis to elucidate important reactions and enzymes for the generation and degradation of RA. Our results indicate that primary RA sources in the germ cell include RA import from the extracellular region, release of RA from binding proteins, and metabolism of retinal to RA. Further, in silico knockouts of genes and reactions in the vitamin A pathway predict that deletion of Lipe, hormone-sensitive lipase, disrupts the RA pulse thereby causing spermatogenic defects. Examination of other metabolic pathways reveals that the citric acid cycle is the most active pathway. In addition, we discover that fatty acid synthesis/oxidation are the primary energy sources in the germ cell. In summary, this study predicts enzymes, reactions, and pathways important for germ cell commitment to meiosis. These findings enhance our understanding of the metabolic control of germ cell differentiation and will help guide future experiments to improve reproductive health.

The roles of HOXB7 in promoting migration, invasion, and anti-apoptosis in gastric cancer.

PubMed

Joo, Moon Kyung; Park, Jong-Jae; Yoo, Hyo Soon; Lee, Beom Jae; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

2016-10-01

The aim of this study was to compare HOXB7 expression level between gastric cancer and non-cancerous gastric tissues. Additionally, the functional effects of HOXB7, including its pro-migration or invasion and anti-apoptosis roles, were evaluated in gastric cancer cells. Both gene and protein expression levels of HOXB7 were examined in gastric cancer cell lines, and HOXB7 expression was compared between primary or metastatic gastric cancer tissues and chronic gastritis or intestinal metaplasia tissues. Functional studies included a wound healing assay, a Matrigel invasion assay, and an Annexin-V assay were performed, and Akt/PTEN activity was measured by western blotting. Both gene and protein expression levels of HOXB7 could be clearly detected in various gastric cancer cell lines except MKN-28 cell. HOXB7 expression was significantly higher in primary or metastatic gastric cancer tissues than in chronic gastritis or intestinal metaplasia tissues. HOXB7 knockdown led to inhibition of cell invasion and migration, had an apoptotic effect, downregulated phosphor-Akt, and upregulated PTEN in AGS and SNU-638 cells. Reinforced expression of HOXB7 caused the opposite effects in MKN-28 and MKN-45 cells. Our study suggests that HOXB7 has an oncogenic role in gastric cancer, which might be related to the modulation of Akt/PTEN activity to induce cell migration/invasion and anti-apoptotic effects. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

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...

Natural Hemozoin Stimulates Syncytiotrophoblast to Secrete Chemokines and Recruit Peripheral Blood Mononuclear Cells

PubMed Central

Lucchi, Naomi W.; Sarr, Demba; Owino, Simon O.; Mwalimu, Stephen M.; Peterson, David S.; Moore, Julie M.

2011-01-01

Background Placental malaria is associated with local accumulation of parasitized erythrocytes, deposition of the parasite hemoglobin metabolite, hemozoin, and accumulation of mononuclear cells in the intervillous space. Fetal syncytiotrophoblast cells in contact with maternal blood are known to respond immunologically to cytoadherent Plasmodium falciparum-infected erythrocytes, but their responsiveness to hemozoin, a potent pro-inflammatory stimulator of monocytes, macrophages and dendritic cells, is not known. Methods The biochemical and immunological changes induced in primary syncytiotrophoblast by natural hemozoin was assessed. Changes in syncytiotrophoblast mitogen-activated protein kinase activation was assessed by immunoblotting and secreted cytokine and chemokine proteins were assayed by ELISA. Chemotaxis of peripheral blood mononuclear cells was assessed using a two-chamber assay system and flow cytometry was used to assess the activation of primary monocytes by hemozoin-stimulated syncytiotrophoblast conditioned medium. Results Hemozoin stimulation induced ERK1/2 phosphorylation. Treated cells secreted CXCL8, CCL3, CCL4, and tumor necrosis factor and released soluble intercellular adhesion molecule-1. Furthermore, the dependence of the hemozoin responses on ERK1/2 stimulation was confirmed by inhibition of chemokine release in syncytiotrophoblast treated with an ERK pathway inhibitor. Hemozoin-stimulated cells elicited the specific migration of PBMCs, and conditioned medium from the cells induced the upregulation of intercellular adhesion molecule-1 on primary monocytes. Conclusions These findings confirm an immunostimulatory role for hemozoin and expand the cell types known to be responsive to hemozoin to include fetal syncytiotrophoblast. The results provide further evidence that syncytiotrophoblast cells can influence the local maternal immune response to placental malaria. PMID:21632106

Effects of inhibition of ubiquitin-proteasome pathway on human primary leukemic cells.

PubMed

Lan, Yu; Zhang, Xuemin; Yang, Pingdi; Hu, Meiru; Yu, Ming; Yang, Yi; Shen, Beifen

2002-12-01

Though there were a lot of reports about the totally different responses to the inhibition of ubiquitin-proteasome pathway in different kinds of cell lines, much less has been known about the responses in primary human leukemic cells. In this study, the effects of inhibition of ubiquitin-proteasome pathway on human bone marrow (BM) mononuclear cells (MNCs) obtained from 10 normal persons and 8 leukemia patients were examined. The results showed that the responses obviously varied individually. Among them, BM MNCs in 3 cases of leukemic patients were extremely sensitive, demonstrated by that > 90% cells were induced to undergo apoptosis within 24 h, but MNCs in 10 cases of normal persons showed resistance to the inhibition and no apoptosis was observed. Furthermore, Western blots revealed that the Bcl-2 expression was relatively high in the sensitive primary leukemia cells, and especially the cleavage of 26 ku Bcl-2 into a 22 ku fragment occurred during the induction of apoptosis. In contrast, the Bcl-2 expression was either undetectable or detectable but no cleavage of that above was observed in the cells insensitive to the inhibition of the pathway (including BM MNCs in normal persons). Together with the observations on the leukemic cell lines, these findings suggested the correlation of the specific cleavage of Bcl-2 into a shortened fragment with the sensitivity of cells to the inhibition of ubiquitin-proteasome pathway, which provides clues to the further understanding of the mechanisms of that dramatically different responses existing in different kinds of cells to the inhibition of ubiquitin-proteasome pathway.

[Primary care follow-up of newborns with sickle cell disease detected in neonatal screening in the Community of Madrid].

PubMed

Rodríguez-Moldes, B; Carbajo, A J; Sánchez, B; Fernández, M; Garí, M; Fernández, M C; Álvarez, J; García, A; Cela, E

2015-04-01

The main aim of the study was to assess the effects of the recommended preventive program in the population affected with Sickle Cell Disease in Primary Care. The program included, antibiotic prophylaxis, immunizations and health education, following the introduction of universal neonatal screening program for Sickle Cell Disease in the Community of Madrid. A cross-sectional observational study was performed with retrospective data collected from a cohort of newborns with Sickle Cell Disease diagnosed by neonatal screening test in the Community of Madrid. From the data obtained from a sample of 20 patients, it was found that 95% had been diagnosed by the newborn screening test performed between 5 and 13 days of life. The mean age was 39 months when the study was conducted. During follow-up, from Primary Care Paediatric clinic, it was observed that the compliance for antibiotic prophylaxis was 90%, and the coverage for the official vaccination schedule was 85%. Specific vaccine coverage as a risk population was highly variable (85% for pneumococcal 23V, 50% for influenza, and 15% for hepatitis A). Health education only reached one in every four families. Acceptable compliance with antibiotic prophylaxis was observed during the follow-up of patients with sickle cell disease in Primary Care, but a low coverage of routine immunization, as well as specific immunizations. Coverage of health education was very low. Improving these parameters would require greater coordination and involvement of Primary Care Professionals so that these patients were followed up appropriately, and could be translated into a reduction of disease complications and an improvement in the quality of life of these patients. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier España, S.L.U. All rights reserved.

Mouse Cytomegalovirus Infection in BALB/c Mice Resembles Virus-Associated Secondary Hemophagocytic Lymphohistiocytosis and Shows a Pathogenesis Distinct from Primary Hemophagocytic Lymphohistiocytosis.

PubMed

Brisse, Ellen; Imbrechts, Maya; Put, Karen; Avau, Anneleen; Mitera, Tania; Berghmans, Nele; Rutgeerts, Omer; Waer, Mark; Ninivaggi, Marisa; Kelchtermans, Hilde; Boon, Louis; Snoeck, Robert; Wouters, Carine H; Andrei, Graciela; Matthys, Patrick

2016-04-01

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunological disorder that is characterized by systemic inflammation, widespread organ damage, and hypercytokinemia. Primary HLH is caused by mutations in granule-mediated cytotoxicity, whereas secondary HLH occurs, without a known genetic background, in a context of infections, malignancies, or autoimmune and autoinflammatory disorders. Clinical manifestations of both HLH subtypes are often precipitated by a viral infection, predominantly with Herpesviridae. Exploiting this knowledge, we established an animal model of virus-associated secondary HLH by infecting immunocompetent wild-type mice with the β-herpesvirus murine CMV. C57BL/6 mice developed a mild inflammatory phenotype, whereas BALB/c mice displayed the clinicopathologic features of HLH, as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia, and elevated serum levels of soluble CD25. BALB/c mice also developed lymphadenopathy, liver dysfunction, and decreased NK cell numbers. Lymphoid and myeloid cells were in a hyperactivated state. Nonetheless, depletion of CD8(+) T cells could not inhibit or cure the HLH-like syndrome, highlighting a first dissimilarity from mouse models of primary HLH. Immune cell hyperactivation in BALB/c mice was accompanied by a cytokine storm. Notably, plasma levels of IFN-γ, a key pathogenic cytokine in models of primary HLH, were the highest. Nevertheless, murine CMV-infected IFN-γ-deficient mice still developed the aforementioned HLH-like symptoms. In fact, IFN-γ-deficient mice displayed a more complete spectrum of HLH, including splenomegaly, coagulopathy, and decreased NK cell cytotoxicity, indicating a regulatory role for IFN-γ in the pathogenesis of virus-associated secondary HLH as opposed to its central pathogenic role in primary HLH. Copyright © 2016 by The American Association of Immunologists, Inc.

Insight into the epidemiology of cutaneous squamous cell carcinoma with perineural spread.

PubMed

Warren, Timothy A; Whiteman, David C; Porceddu, Sandro V; Panizza, Benedict J

2016-09-01

Perineural spread (PNS) of cutaneous squamous cell carcinoma of the head and neck (SCCHN) can be associated with poor outcomes. Disease understanding and awareness is limited leading to delayed diagnosis and treatment. The purpose of this study was to identify epidemiological features of patients with PNS of cutaneous SCCHN. Tumor characteristics and demographics of patients with PNS of cutaneous SCCHN managed through a single institution were collected between 1998 and 2013. One hundred twenty patients were included in this study. The majority had a history of skin cancer (85.8%). The median time from primary tumor treatment to PNS symptom onset was 16 months (range, 1-86 months). A total of 34.2% had no perineural invasion (PNI) detected in the primary, and 22.5% had no known primary tumor. Only 5.8% of the patients had nodal involvement at presentation. Patients can present with PNS from cutaneous SCCHN with no known primary tumor or with primary tumors without PNI. The majority of patients presented without regional nodal involvement. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1416-1420, 2016. © 2016 Wiley Periodicals, Inc.

Primary Cilia Negatively Regulate Melanogenesis in Melanocytes and Pigmentation in a Human Skin Model.

PubMed

Choi, Hyunjung; Shin, Ji Hyun; Kim, Eun Sung; Park, So Jung; Bae, Il-Hong; Jo, Yoon Kyung; Jeong, In Young; Kim, Hyoung-June; Lee, Youngjin; Park, Hea Chul; Jeon, Hong Bae; Kim, Ki Woo; Lee, Tae Ryong; Cho, Dong-Hyung

2016-01-01

The primary cilium is an organelle protruding from the cell body that senses external stimuli including chemical, mechanical, light, osmotic, fluid flow, and gravitational signals. Skin is always exposed to the external environment and responds to external stimuli. Therefore, it is possible that primary cilia have an important role in skin. Ciliogenesis was reported to be involved in developmental processes in skin, such as keratinocyte differentiation and hair formation. However, the relation between skin pigmentation and primary cilia is largely unknown. Here, we observed that increased melanogenesis in melanocytes treated with a melanogenic inducer was inhibited by a ciliogenesis inducer, cytochalasin D, and serum-free culture. However, these inhibitory effects disappeared in GLI2 knockdown cells. In addition, activation of sonic hedgehog (SHH)-smoothened (Smo) signaling pathway by a Smo agonist, SAG inhibited melanin synthesis in melanocytes and pigmentation in a human skin model. On the contrary, an inhibitor of primary cilium formation, ciliobrevin A1, activated melanogenesis in melanocytes. These results suggest that skin pigmentation may be regulated partly by the induction of ciliogenesis through Smo-GLI2 signaling.

Innate cell communication kick-starts pathogen-specific immunity

PubMed Central

Rivera, Amariliz; Siracusa, Mark C.; Yap, George S.; Gause, William C.

2016-01-01

Innate cells are responsible for the rapid recognition of infection and mediate essential mechanisms of pathogen elimination, and also facilitate adaptive immune responses. We review here the numerous intricate interactions among innate cells that initiate protective immunity. The efficient eradication of pathogens depends on the coordinated actions of multiple cells, including innate cells and epithelial cells. Rather than acting as isolated effector cells, innate cells are in constant communication with other responding cells of the immune system, locally and distally. These interactions are critically important for the efficient control of primary infections as well for the development of ‘trained’ innate cells that facilitate the rapid elimination of homologous or heterologous infections. PMID:27002843

Primary Surgery vs Radiotherapy for Early Stage Oral Cavity Cancer.

PubMed

Ellis, Mark A; Graboyes, Evan M; Wahlquist, Amy E; Neskey, David M; Kaczmar, John M; Schopper, Heather K; Sharma, Anand K; Morgan, Patrick F; Nguyen, Shaun A; Day, Terry A

2018-04-01

Objective The goal of this study is to determine the effect of primary surgery vs radiotherapy (RT) on overall survival (OS) in patients with early stage oral cavity squamous cell carcinoma (OCSCC). In addition, this study attempts to identify factors associated with receiving primary RT. Study Design Retrospective cohort study. Setting National Cancer Database (NCDB, 2004-2013). Subjects and Methods Reviewing the NCDB from 2004 to 2013, patients with early stage I to II OCSCC were identified. Kaplan-Meier estimates of survival, Cox regression analysis, and propensity score matching were used to examine differences in OS between primary surgery and primary RT. Multivariable logistic regression analysis was performed to identify factors associated with primary RT. Results Of the 20,779 patients included in the study, 95.4% (19,823 patients) underwent primary surgery and 4.6% (956 patients) underwent primary RT. After adjusting for covariates, primary RT was associated with an increased risk of mortality (adjusted hazard ratio [aHR], 1.97; 99% confidence interval [CI], 1.74-2.22). On multivariable analysis, factors associated with primary RT included age ≥70 years, black race, Medicaid or Medicare insurance, no insurance, oral cavity subsite other than tongue, clinical stage II disease, low-volume treatment facilities, and earlier treatment year. Conclusion Primary RT for early stage OCSCC is associated with increased mortality. Approximately 5% of patients receive primary RT; however, this percentage is decreasing. Patients at highest risk for receiving primary RT include those who are elderly, black, with public insurance, and treated at low-volume facilities.

In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity

PubMed Central

Vargas, Ana Cristina; Keith, Patricia; Reid, Lynne; Wockner, Leesa; Amiri, Marjan Askarian; Sarkar, Debina; Simpson, Peter T.; Clarke, Catherine; Schmidt, Chris W.; Reynolds, Brent A.

2013-01-01

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally ‘enriching’ for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity. PMID:23750209

Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles

PubMed Central

Prel, Anne; Caval, Vincent; Gayon, Régis; Ravassard, Philippe; Duthoit, Christine; Payen, Emmanuel; Maouche-Chretien, Leila; Creneguy, Alison; Nguyen, Tuan Huy; Martin, Nicolas; Piver, Eric; Sevrain, Raphaël; Lamouroux, Lucille; Leboulch, Philippe; Deschaseaux, Frédéric; Bouillé, Pascale; Sensébé, Luc; Pagès, Jean-Christophe

2015-01-01

RNA delivery is an attractive strategy to achieve transient gene expression in research projects and in cell- or gene-based therapies. Despite significant efforts investigating vector-directed RNA transfer, there is still a requirement for better efficiency of delivery to primary cells and in vivo. Retroviral platforms drive RNA delivery, yet retrovirus RNA-packaging constraints limit gene transfer to two genome-molecules per viral particle. To improve retroviral transfer, we designed a dimerization-independent MS2-driven RNA packaging system using MS2-Coat-retrovirus chimeras. The engineered chimeric particles promoted effective packaging of several types of RNAs and enabled efficient transfer of biologically active RNAs in various cell types, including human CD34+ and iPS cells. Systemic injection of high-titer particles led to gene expression in mouse liver and transferring Cre-recombinase mRNA in muscle permitted widespread editing at the ROSA26 locus. We could further show that the VLPs were able to activate an osteoblast differentiation pathway by delivering RUNX2- or DLX5-mRNA into primary human bone-marrow mesenchymal-stem cells. Thus, the novel chimeric MS2-lentiviral particles are a versatile tool for a wide range of applications including cellular-programming or genome-editing. PMID:26528487

S100 chemokines mediate bookmarking of premetastatic niches

PubMed Central

Rafii, Shahin; Lyden, David

2010-01-01

Primary tumours release soluble factors, including VEGF-A, TGFβ and TNFα, which induce expression of the chemokines S100A8 and S100A9 in the myeloid and endothelial cells within the lung before tumour metastasis. These chemokine-activated premetastatic niches support adhesion and invasion of disseminating malignant cells, thereby establishing a fertile habitat for metastatic tumours. PMID:17139281

The Mitotic and Metabolic Effects of Phosphatidic Acid in the Primary Muscle Cells of Turbot (Scophthalmus maximus)

PubMed Central

Wang, Tingting; Wang, Xuan; Zhou, Huihui; Jiang, Haowen; Mai, Kangsen; He, Gen

2018-01-01

Searching for nutraceuticals and understanding the underlying mechanism that promote fish growth is at high demand for aquaculture industry. In this study, the modulatory effects of soy phosphatidic acids (PA) on cell proliferation, nutrient sensing, and metabolic pathways were systematically examined in primary muscle cells of turbot (Scophthalmus maximus). PA was found to stimulate cell proliferation and promote G1/S phase transition through activation of target of rapamycin signaling pathway. The expression of myogenic regulatory factors, including myoD and follistatin, was upregulated, while that of myogenin and myostatin was downregulated by PA. Furthermore, PA increased intracellular free amino acid levels and enhanced protein synthesis, lipogenesis, and glycolysis, while suppressed amino acid degradation and lipolysis. PA also was found to increased cellular energy production through stimulated tricarboxylic acid cycle and oxidative phosphorylation. Our results identified PA as a potential nutraceutical that stimulates muscle cell proliferation and anabolism in fish. PMID:29780359

Persistent hyperplastic primary vitreous with retinal tumor in tuberous sclerosis: report of a case including tumoral immunohistochemistry and cytogenetic analyses.

PubMed

Milot, J; Michaud, J; Lemieux, N; Allaire, G; Gagnon, M M

1999-03-01

The authors describe an ocular lesion combining the characteristics of persistent hyperplastic primary vitreous (PHPV) and a retinal tumor in an infant with tuberous sclerosis complex (TSC). Case report. Immunohistochemistry and cytogenetic studies were performed on TSC cells from an intraocular tumor in a 6-week-old infant. Histopathologic examination showed a thick fibrovascular membrane between the aspect of the lens and the astrocytic component of the mass. Glial fibrillary acidic protein (GFAP) showed a variable intracytoplasmic reaction in the astrocytic proliferation, involving approximately 50% of the cells. Tissue culture studies showed a fairly rapid proliferation of fusiform cells, consistent with bipolar astrocytic cells. Cytogenetic studies showed one abnormal clone consisting of three hyperdiploid cells with a loss of chromosome 9 and a gain of chromosomes 6 and 12. The atypical localization of the retinal tumor could be explained by the fact that it was trapped during its proliferation by the retinal detachment associated with the PHPV.

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.

PubMed

Martinovich, Kelly M; Iosifidis, Thomas; Buckley, Alysia G; Looi, Kevin; Ling, Kak-Ming; Sutanto, Erika N; Kicic-Starcevich, Elizabeth; Garratt, Luke W; Shaw, Nicole C; Montgomery, Samuel; Lannigan, Francis J; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2017-12-21

Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1)

PubMed Central

Udasin, Ronald G.; Wen, Xia; Bircsak, Kristin M.; Aleksunes, Lauren M.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2016-01-01

Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants developed as chemical warfare agents. These electrophilic, bifunctional alkylating agents cause skin injury, including inflammation, edema, and blistering. HN2 covalently modifies macromolecules such as DNA, RNA, and proteins or is scavenged by glutathione, forming adducts that can contribute to toxicity. Multidrug resistance-associated protein 1 (Mrp1/MRP1) is a transmembrane ATPase known to efflux glutathione-conjugated electrophiles. In the present studies, we examined the effects of modulating Mrp1-mediated transport activity on the sensitivity of primary and PAM212 mouse keratinocytes to HN2. Primary keratinocytes, and to a lesser extent, PAM212 cells, express Mrp1 mRNA and protein and possess Mrp1 functional activity, as measured by calcein efflux. Sulforaphane, an activator of Nrf2, increased Mrp1 mRNA, protein, and functional activity in primary keratinocytes and PAM212 cells and decreased their sensitivity to HN2-induced growth inhibition (IC50 = 1.4 and 4.8 µM in primary keratinocytes and 1 and 13 µM in PAM212 cells, in the absence and presence of sulforaphane, respectively). The Mrp1 inhibitor, MK-571, reversed the effects of sulforaphane on HN2-induced growth inhibition in both primary keratinocytes and PAM212 cells. In primary keratinocytes from Nrf2−/− mice, sulforaphane had no impact on Mrp1 expression or activity, or on sensitivity to HN2, demonstrating that its effects depend on Nrf2. These data suggest that Mrp1-mediated efflux is important in regulating HN2-induced keratinocyte growth inhibition. Enhancing HN2 efflux from keratinocytes may represent a novel strategy for mitigating vesicant-induced cytotoxicity. PMID:26454883

Comprehensive Evaluation of Programmed Death-Ligand 1 Expression in Primary and Metastatic Prostate Cancer.

PubMed

Haffner, Michael C; Guner, Gunes; Taheri, Diana; Netto, George J; Palsgrove, Doreen N; Zheng, Qizhi; Guedes, Liana Benevides; Kim, Kunhwa; Tsai, Harrison; Esopi, David M; Lotan, Tamara L; Sharma, Rajni; Meeker, Alan K; Chinnaiyan, Arul M; Nelson, William G; Yegnasubramanian, Srinivasan; Luo, Jun; Mehra, Rohit; Antonarakis, Emmanuel S; Drake, Charles G; De Marzo, Angelo M

2018-06-01

Antibodies targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-L1) interaction have shown clinical activity in multiple cancer types. PD-L1 protein expression is a clinically validated predictive biomarker of response for such therapies. Prior studies evaluating the expression of PD-L1 in primary prostate cancers have reported highly variable rates of PD-L1 positivity. In addition, limited data exist on PD-L1 expression in metastatic castrate-resistant prostate cancer (mCRPC). Here, we determined PD-L1 protein expression by immunohistochemistry using a validated PD-L1-specific antibody (SP263) in a large and representative cohort of primary prostate cancers and prostate cancer metastases. The study included 539 primary prostate cancers comprising 508 acinar adenocarcinomas, 24 prostatic duct adenocarcinomas, 7 small-cell carcinomas, and a total of 57 cases of mCRPC. PD-L1 positivity was low in primary acinar adenocarcinoma, with only 7.7% of cases showing detectable PD-L1 staining. Increased levels of PD-L1 expression were noted in 42.9% of small-cell carcinomas. In mCRPC, 31.6% of cases showed PD-L1-specific immunoreactivity. In conclusion, in this comprehensive evaluation of PD-L1 expression in prostate cancer, PD-L1 expression is rare in primary prostate cancers, but increased rates of PD-L1 positivity were observed in mCRPC. These results will be important for the future clinical development of programmed cell death protein 1/PD-L1-targeting therapies in prostate cancer. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Association between environmental factors including second-hand smoke and primary lung cancer in dogs.

PubMed

Zierenberg-Ripoll, A; Pollard, R E; Stewart, S L; Allstadt, S D; Barrett, L E; Gillem, J M; Skorupski, K A

2018-06-01

To estimate prevalence of exposure to environmental tobacco smoke and other environmental toxins in dogs with primary lung tumours and to analyse association between exposure and lung tumour development. In this case-control study, an owner survey was developed to collect data on patient characteristics, general health care and environmental exposures. Dogs diagnosed with primary lung carcinomas formed the Case group. Dogs diagnosed with mast cell tumours served as Control Group 1 and dogs diagnosed with neurologic disease served as Control Group 2. Associations between diagnosis of primary lung tumour and patient and environmental exposure variables were analysed using bivariate and multivariate statistical methods. A total of 1178 owner surveys were mailed and 470 surveys were returned and included in statistical analysis, including 135 Cases, 169 dogs in Control Group 1 and 166 dogs in Control Group 2. An association between exposure to second-hand smoke and prevalence of primary lung cancer was not identified in this study. Second-hand smoke is associated with primary lung cancer in people but a definitive association has not been found in dogs. The results of this study suggest that tobacco smoke exposure may not be associated with primary lung cancer development in dogs but study limitations may have precluded detection of an association. © 2017 British Small Animal Veterinary Association.

Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture.

PubMed Central

Gebhardt, R; Mecke, D

1983-01-01

The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells. Images Fig. 1. PMID:6138251

Less aggressive disease in patients with primary squamous cell carcinomas of the thyroid gland and coexisting lymphocytic thyroiditis.

PubMed

Asik, Mehmet; Binnetoglu, Emine; Sen, Hacer; Gunes, Fahri; Muratli, Asli; Kankaya, Duygu; Uysal, Fatma; Sahin, Mustafa; Ukinc, Kubilay

2015-01-01

Primary squamous cell carcinoma (SCC) of the thyroid gland is extremely rare. Infrequently, primary SCC of the thyroid gland is accompanied by other thyroid diseases such as Hashimoto's thyroiditis (HT). Recently, studies have demonstrated that differentiated thyroid cancer with coexisting HT has a better prognosis. However, the prognosis of patients with primary SCC of the thyroid gland and coexistent HT has not been clearly identified. We compared the clinical characteristics and disease stages of patients with primary SCC with and without lymphocytic thyroiditis (LT). We reviewed reports of primary SCC of the thyroid gland published in the English literature. We identified 46 papers that included 17 cases of primary SCC of the thyroid gland with LT and 77 cases of primary SCC of the thyroid gland without LT. Lymph node metastasis and local invasion rates did not differ between these two groups. Distant metastases were absent in patients with LT, and were observed in 13 (16.9%) patients without LT. A greater proportion of patients without LT had advanced stage disease (stage IV A-B-C) than patients with LT (p < 0.05). Patients with primary SCC of the thyroid gland and coexisting LT had lower tumour-node-metastasis stage and frequency of distant metastasis than those without LT. Lymphocytic infiltration in patients with SCC appears to limit tumour growth and distant metastases.

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

IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M-CAM expression in human renal cell carcinoma.

PubMed

Ueno, Koji; Hirata, Hiroshi; Majid, Shahana; Tabatabai, Z Laura; Hinoda, Yuji; Dahiya, Rajvir

2011-11-15

The Wnt/β-catenin signaling pathway is inactivated by Wnt antagonists in most cancers and IGFBP-4 is an antagonist of the Wnt/ β-catenin signaling pathway. However, the function of IGFBP-4 is not currently understood in renal cell carcinoma (RCC). We initially found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in primary renal cancer. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and β-catenin expression was increased. Also the β-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell lines (ACHN) was knocked-down using a siRNA technique. The cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. Tcf activity in ACHN cells was also decreased with si-IGFBP-4 transfection. This is a first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis. Copyright © 2011 UICC.

Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90

PubMed Central

2011-01-01

Background Multiple myeloma (MM) is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. Results In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. Conclusions Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma. PMID:21871133

Treatment and prognosis of primary thymic carcinoma.

PubMed

Yano, T; Hara, N; Ichinose, Y; Asoh, H; Yokoyama, H; Ohta, M

1993-04-01

From 1972 to 1990, we treated eight cases of thymic carcinoma (6 squamous cell and 2 small cell carcinomas). According to the classification by Masaoka et al., they consisted of one stage I, four stage III, one stage IVa, and two stage IVb. A complete resection of the primary tumour could be done in only three patients; the others had diagnostic biopsy and then radiation treatment. Four of five patients had a prolonged regression of the primary tumors after irradiation at 40-61.2 Gy. Six patients suffered from extrathoracic metastases. All patients received systemic chemotherapy with different regimens to counter either metastatic or locally recurrent lesions. Only two patients (with a regimen including cyclophosphamide, doxorubicin, and vincristine) obtained a partial response. The median survival of the eight patients was 70 months after surgical operation. The identification of an effective drug combination may thus improve the long-term prognosis of thymic carcinoma since radiotherapy is able to control primary lesions, even in the case of unresectable advanced disease.

Primary Immune Deficiency Treatment Consortium (PIDTC) report.

PubMed

Griffith, Linda M; Cowan, Morton J; Notarangelo, Luigi D; Kohn, Donald B; Puck, Jennifer M; Pai, Sung-Yun; Ballard, Barbara; Bauer, Sarah C; Bleesing, Jack J H; Boyle, Marcia; Brower, Amy; Buckley, Rebecca H; van der Burg, Mirjam; Burroughs, Lauri M; Candotti, Fabio; Cant, Andrew J; Chatila, Talal; Cunningham-Rundles, Charlotte; Dinauer, Mary C; Dvorak, Christopher C; Filipovich, Alexandra H; Fleisher, Thomas A; Bobby Gaspar, Hubert; Gungor, Tayfun; Haddad, Elie; Hovermale, Emily; Huang, Faith; Hurley, Alan; Hurley, Mary; Iyengar, Sumathi; Kang, Elizabeth M; Logan, Brent R; Long-Boyle, Janel R; Malech, Harry L; McGhee, Sean A; Modell, Fred; Modell, Vicki; Ochs, Hans D; O'Reilly, Richard J; Parkman, Robertson; Rawlings, David J; Routes, John M; Shearer, William T; Small, Trudy N; Smith, Heather; Sullivan, Kathleen E; Szabolcs, Paul; Thrasher, Adrian; Torgerson, Troy R; Veys, Paul; Weinberg, Kenneth; Zuniga-Pflucker, Juan Carlos

2014-02-01

The Primary Immune Deficiency Treatment Consortium (PIDTC) is a network of 33 centers in North America that study the treatment of rare and severe primary immunodeficiency diseases. Current protocols address the natural history of patients treated for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, and chronic granulomatous disease through retrospective, prospective, and cross-sectional studies. The PIDTC additionally seeks to encourage training of junior investigators, establish partnerships with European and other International colleagues, work with patient advocacy groups to promote community awareness, and conduct pilot demonstration projects. Future goals include the conduct of prospective treatment studies to determine optimal therapies for primary immunodeficiency diseases. To date, the PIDTC has funded 2 pilot projects: newborn screening for SCID in Navajo Native Americans and B-cell reconstitution in patients with SCID after hematopoietic stem cell transplantation. Ten junior investigators have received grant awards. The PIDTC Annual Scientific Workshop has brought together consortium members, outside speakers, patient advocacy groups, and young investigators and trainees to report progress of the protocols and discuss common interests and goals, including new scientific developments and future directions of clinical research. Here we report the progress of the PIDTC to date, highlights of the first 2 PIDTC workshops, and consideration of future consortium objectives. Published by Mosby, Inc.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

PubMed

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

PubMed Central

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening. PMID:26657314

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.

A Novel Role of Peripheral Corticotropin-Releasing Hormone (CRH) on Dermal Fibroblasts

PubMed Central

Rassouli, Olga; Liapakis, George; Lazaridis, Iakovos; Sakellaris, George; Gkountelias, Kostas; Gravanis, Achille; Margioris, Andrew N.

2011-01-01

Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh−/−) had higher proliferation and migration rates and compromised production of IL-6 and TGF-β1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF1 antagonist antalarmin recapitulated the findings in the Crh−/− cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis. PMID:21765902

Human primary myoblast cell cultures from non-diabetic insulin resistant subjects retain defects in insulin action.

PubMed Central

Thompson, D B; Pratley, R; Ossowski, V

1996-01-01

Insulin resistance is a predictor of the development of noninsulin-dependent diabetes mellitus (NIDDM) in humans. It is unclear whether insulin resistance is a primary defect leading to NIDDM or the result of hyperinsulinemia and hyperglycemia. To determine if insulin resistance is the result of extrinsic factors such as hyperinsulinemia primary skeletal muscle cell cultures were established from muscle biopsies from Pima Indians with differing in vivo insulin sensitivities. These cell cultures expressed a variety of muscle-specific phenotypes including the proteins alpha-actinin and myosin, muscle-specific creatine kinase activity, and RNA encoding GLUT4, MYF5, MYOD1, and MYOGENIN. Labeled glucose was used to measure the insulin-stimulated conversion of glucose to glycogen in these cultures. The in vivo rates of insulin-stimulated glycogen production (insulin resistance) were correlated with in vitro measures of glycogen production (P = 0.007, r = 0.58). This defect in insulin action is stable in a uniform culture environment and is retained over time. The retention of insulin resistance in myoblast derived cell cultures is consistent with the expression of an underlying biochemical defect in insulin resistant skeletal muscle. PMID:8941652

Proliferative human cell sources applied as biocomponent in bioartificial livers: a review.

PubMed

Nibourg, Geert A A; Chamuleau, Robert A F M; van Gulik, Thomas M; Hoekstra, Ruurdtje

2012-07-01

Bioartificial livers (BALs) are urgently needed to bridge severe liver failure patients to liver transplantation or liver regeneration. When based on primary hepatocytes, their efficacy has been shown in animal experiments and their safety was confirmed in clinical trials. However, a proliferative human cell source with therapeutic functionality is needed to secure availability and move BAL application forward. This review compares the performance of BALs based on proliferative human biocomponents and primary hepatocytes. This review evaluates relevant studies identified by searching the MEDLINE database until July 2011 and some of our own unpublished data. All the discussed hepatocyte-like biocomponents show deficiencies in their hepatic functionality compared with primary hepatocytes, particularly functions occurring late in liver development. Nonetheless, the HepaRG, HepG2-GS-CYP3A4, and mesenchymal stem cells show efficacy in a statistically well-powered animal model of acute liver failure, when applied in a BAL device. Various methods to gain higher functionality of BALs, including genetic modification, the usage of combinatory cell sources, and improvement of culture methods, have scarcely been applied, but may further pave the path for BAL application. Clinical implementation of a BAL based on a human proliferative biocomponent is still several years away.

Strategic Therapeutic Targeting to Overcome Venetoclax Resistance in Aggressive B-cell Lymphomas.

PubMed

Pham, Lan V; Huang, Shengjian; Zhang, Hui; Zhang, Jun; Bell, Taylor; Zhou, Shouhao; Pogue, Elizabeth; Ding, Zhiyong; Lam, Laura; Westin, Jason; Davis, R Eric; Young, Ken H; Medeiros, L Jeffrey; Ford, Richard J; Nomie, Krystle; Zhang, Leo; Wang, Michael

2018-04-17

Purpose: B-cell lymphoma-2 (BCL-2), an antiapoptotic protein often dysregulated in B-cell lymphomas, promotes cell survival and provides protection from stress. A recent phase I first-in-human study of the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma showed an overall response rate of 44%. These promising clinical results prompted our examination of the biological effects and mechanism of action underlying venetoclax activity in aggressive B-cell lymphoma, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Experimental Design: MCL and DLBCL cell lines, primary patient samples, and in vivo patient-derived xenograft (PDX) models were utilized to examine venetoclax efficacy. Furthermore, the mechanisms underlying venetoclax response and the development of venetoclax resistance were evaluated using proteomics analysis and Western blotting. Results: Potential biomarkers linked to venetoclax activity and targeted combination therapies that can augment venetoclax response were identified. We demonstrate that DLBCL and MCL cell lines, primary patient samples, and PDX mouse models expressing high BCL-2 levels are extremely sensitive to venetoclax treatment. Proteomics studies showed that venetoclax substantially alters the expression levels and phosphorylation status of key proteins involved in cellular processes, including the DNA damage response, cell metabolism, cell growth/survival, and apoptosis. Short- and long-term exposure to venetoclax inhibited PTEN expression, leading to enhanced AKT pathway activation and concomitant susceptibility to PI3K/AKT inhibition. Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K/AKT inhibition. Conclusions: These findings demonstrate the on-target effect of venetoclax and offer potential mechanisms to overcome acquired and intrinsic venetoclax resistance through PI3K/AKT inhibition. Clin Cancer Res; 1-14. ©2018 AACR. ©2018 American Association for Cancer Research.

Primary cutaneous perivascular epithelioid cell tumor (PEComa): Five new cases and review of the literature.

PubMed

Stuart, Lauren N; Tipton, Russell G; DeWall, Michael R; Parker, Douglas C; Stelton, Christina D; Morrison, Annie O; Coleman, Landon W; Fosko, Scott W; Vidal, Claudia I; Yadira Hurley, Maria; Deeken, Amy H; Gardner, Jerad M

2017-08-01

PEComas represent a family of uncommon mesenchymal tumors composed of "perivascular epithelioid cells" with a distinct immunophenotype that typically shows both myogenic and melanocytic differentiation. The PEComa family includes angiomyolipoma (AML), clear cell "sugar" tumor of the lung and extra pulmonary sites, lymphangioleiomyomatosis and clear cell myomelanocytic tumor of the falciform ligament/ligamentum teres. Very rarely, PEComas may arise in the skin. Primary cutaneous PEComas typically display a dermal proliferation of epithelioid cells with pale, clear, or granular pink cytoplasm arranged in nests and trabecula with an intervening arborizing network of delicate capillaries. Primary cutaneous PEComas have a lower frequency of myogenic marker expression than their deep soft tissue and visceral counterparts. They also often express strong diffuse CD10, leading to potential confusion with metastatic renal cell carcinoma. Most cases behave indolently. We report 5 additional cases of this rare entity. All showed classic histologic features and expression of either HMB-45 and/or Melan-A/MART-1. Four cases were tested for myogenic markers (2 were positive & 2 were negative). Three cases were tested for CD10 (all 3 were positive). All of our cases with clinical follow-up behaved indolently. Table 1 provides a summary of findings for all 5 cases in our series. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Adult Mouse Cortical Cell Taxonomy by Single Cell Transcriptomics

PubMed Central

Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T.; Sorensen, Staci A.; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M.; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

2016-01-01

Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. Here, we construct a cellular taxonomy of one cortical region, primary visual cortex, in adult mice based on single cell RNA-sequencing. We identify 49 transcriptomic cell types including 23 GABAergic, 19 glutamatergic and seven non-neuronal types. We also analyze cell-type specific mRNA processing and characterize genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we show that some of our transcriptomic cell types display specific and differential electrophysiological and axon projection properties, thereby confirming that the single cell transcriptomic signatures can be associated with specific cellular properties. PMID:26727548

ErbB receptors in the biology and pathology of the aerodigestive tract

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

Morgan, Sarah; Suite 500, Pittsburgh, PA 15213; Grandis, Jennifer R.

2009-02-15

The most common sites of malignancies in the aerodigestive tract include the lung, head and neck and the esophagus. Esophageal adenocarcinomas (EA), esophageal squamous cell carcinomas (ESCC), and squamous cell carcinomas of the head and neck (SCCHN) are the primary focus of this review. Traditional treatment for aerodigestive tract cancers includes primary chemoradiotherapy (CRT) or surgical resection followed by radiation (or CRT). Recent developments in treatment have focused increasingly on molecular targeting strategies including cetuximab (a monoclonal antibody against epidermal growth factor receptor (EGFR)). Cetuximab was FDA approved in 2006 for treatment of SCCHN, underscoring the importance of understanding themore » biology of these malignancies. EGFR is a member of the ErbB family of growth factor receptor tyrosine kinases. The major pathways activated by ErbB receptors include Ras/Raf/MAPK; PI3K/AKT; PLC{gamma} and STATs, all of which lead to the transcription of target genes that may contribute to aerodigestive tumor progression. This review explores the expression of ErbB receptors in EA, ESCC and SCCHN and the signaling pathways of EGFR in SCCHN.« less

Medical comorbidities and perioperative allogeneic red blood cell transfusion are risk factors for surgical site infection after shoulder arthroplasty.

PubMed

Everhart, Joshua S; Bishop, Julie Y; Barlow, Jonathan D

2017-11-01

Multiple perioperative factors have been implicated in infection risk after shoulder arthroplasty. The purpose of this study was to determine surgical site infection (SSI) risk due to medical comorbidities or blood transfusion after primary or revision shoulder arthroplasty. Comprehensive data on medical comorbidities, surgical indication, perioperative transfusion, and SSI were obtained for 707 patients who underwent primary or revision hemiarthroplasty or total shoulder arthroplasty in a single hospital system. Multivariate Poisson regression was used to determine the independent association between allogeneic red blood cell transfusion, medical comorbidities, and SSI after controlling for procedure. The SSI rate was 1.9% for primary hemiarthroplasties and 1.3% for primary total shoulder arthroplasties. Among patients without prior shoulder infection, revision arthroplasty or prior open reduction and internal fixation had higher SSI risk than primary arthroplasties (incidence risk ratio [IRR], 11.4; 95% confidence interval [CI], 3.84-34.0; P < .001); among primary arthroplasties, SSI risk factors included male gender (IRR, 60.0; CI, 4.39-819; P = .002), rheumatoid arthritis (IRR, 8.63; CI, 1.84-40.4; P = .006), and long-term corticosteroid use (IRR, 37.4; CI, 5.79-242; P < .001). Perioperative allogeneic red blood cell transfusion significantly increased SSI risk and was dose dependent (IRR, 1.68 per unit packed red blood cell; CI, 1.21-2.35; P = .002). Gender, rheumatoid arthritis, and long-term (>1 year) corticosteroid use affect SSI risk after shoulder arthroplasty. Revision surgery, particularly in the setting of prior infection, increased risk of future infection. Finally, allogeneic red blood cell transfusion increases SSI risk after shoulder arthroplasty in a dose-dependent manner. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

Evolution and diversity of plant cell walls: from algae to flowering plants.

PubMed

Popper, Zoë A; Michel, Gurvan; Hervé, Cécile; Domozych, David S; Willats, William G T; Tuohy, Maria G; Kloareg, Bernard; Stengel, Dagmar B

2011-01-01

All photosynthetic multicellular Eukaryotes, including land plants and algae, have cells that are surrounded by a dynamic, complex, carbohydrate-rich cell wall. The cell wall exerts considerable biological and biomechanical control over individual cells and organisms, thus playing a key role in their environmental interactions. This has resulted in compositional variation that is dependent on developmental stage, cell type, and season. Further variation is evident that has a phylogenetic basis. Plants and algae have a complex phylogenetic history, including acquisition of genes responsible for carbohydrate synthesis and modification through a series of primary (leading to red algae, green algae, and land plants) and secondary (generating brown algae, diatoms, and dinoflagellates) endosymbiotic events. Therefore, organisms that have the shared features of photosynthesis and possession of a cell wall do not form a monophyletic group. Yet they contain some common wall components that can be explained increasingly by genetic and biochemical evidence.

Four-point bending protocols to study the effects of dynamic strain in osteoblastic cells in vitro.

PubMed

Galea, Gabriel L; Price, Joanna S

2015-01-01

Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to those in primary cultures of osteoblast-like cells derived from explants of mouse long bones. These cells show a range of well-characterized responses to physiological levels of strain, including increased proliferation, which in vivo is a feature of the osteogenic response.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

PubMed

Shahi, Payam; Kim, Samuel C; Haliburton, John R; Gartner, Zev J; Abate, Adam R

2017-03-14

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

NASA Astrophysics Data System (ADS)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

PubMed Central

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing. PMID:28290550

Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration.

PubMed

Blum, Walter; Pecze, László; Felley-Bosco, Emanuela; Schwaller, Beat

2015-12-22

The Ca(2+)-binding protein calretinin is currently used as a positive marker for identifying epithelioid malignant mesothelioma (MM) and reactive mesothelium, but calretinin's likely role in mesotheliomagenesis remains unclear. Calretinin protects immortalized mesothelial cells in vitro from asbestos-induced cytotoxicity and thus might be implicated in mesothelioma formation. To further investigate calretinin's putative role in the early steps of MM generation, primary mesothelial cells from calretinin knockout (CR-/-) and wildtype (WT) mice were compared. Primary mouse mesothelial cells from WT and CR-/- mice were investigated with respect to morphology, marker proteins, proliferation, cell cycle parameters and mobility in vitro. Overexpression of calretinin or a nuclear-targeted variant was achieved by a lentiviral expression system. CR-/- mice have a normal mesothelium and no striking morphological abnormalities compared to WT animals were noted. Primary mouse mesothelial cells from both genotypes show a typical "cobblestone-like" morphology and express mesothelial markers including mesothelin. In cells from CR-/- mice in vitro, we observed more giant cells and a significantly decreased proliferation rate. Up-regulation of calretinin in mesothelial cells of both genotypes increases the proliferation rate and induces a cobblestone-like epithelial morphology. The length of the S/G2/M phase is unchanged, however the G1 phase is clearly prolonged in CR-/- cells. They are also much slower to close a scratch in a confluent cell layer (2D-wound assay). In addition to a change in cell morphology, an increase in proliferation and mobility is observed, if calretinin overexpression is targeted to the nucleus. Thus, both calretinin and nuclear-targeted calretinin increase mesothelial cell proliferation and consequently, speed up the scratch-closure time. The increased rate of scratch closure in WT cells is the result of two processes: an increased proliferation rate and augmented cell mobility of the border cells migrating towards the empty space. We hypothesize that the differences in proliferation and mobility between WT and CR-/- mesothelial cells are the likely result from differences in their developmental trajectories. The mechanistic understanding of the function of calretinin and its putative implication in signaling pathways in normal mesothelial cells may help understanding its role during the processes that lead to mesothelioma formation and could possibly open new avenues for mesothelioma therapy, either by directly targeting calretinin expression or indirectly by targeting calretinin-mediated downstream signaling.

Effect of doxycycline on epithelial-mesenchymal transition via the p38/Smad pathway in respiratory epithelial cells.

PubMed

Shin, Jae-Min; Kang, Ju-Hyung; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2017-03-01

Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells. A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability. Doxycycline (0-10 μg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells. Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.

Isolation and Characterization of Current Human Coronavirus Strains in Primary Human Epithelial Cell Cultures Reveal Differences in Target Cell Tropism

PubMed Central

Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker

2013-01-01

The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains. PMID:23427150

Strigolactone analogues induce apoptosis through activation of p38 and the stress response pathway in cancer cell lines and in conditionally reprogrammed primary prostate cancer cells.

PubMed

Pollock, Claire B; McDonough, Sara; Wang, Victor S; Lee, Hyojung; Ringer, Lymor; Li, Xin; Prandi, Cristina; Lee, Richard J; Feldman, Adam S; Koltai, Hinanit; Kapulnik, Yoram; Rodriguez, Olga C; Schlegel, Richard; Albanese, Christopher; Yarden, Ronit I

2014-03-30

Strigolactones are a novel class of plant hormones produced in roots and regulate shoot and root development. We have previously shown that synthetic strigolactone analogues potently inhibit growth of breast cancer cells and breast cancer stem cells. Here we show that strigolactone analogues inhibit the growth and survival of an array of cancer-derived cell lines representing solid and non-solid cancer cells including: prostate, colon, lung, melanoma, osteosarcoma and leukemic cell lines, while normal cells were minimally affected. Treatment of cancer cells with strigolactone analogues was hallmarked by activation of the stress-related MAPKs: p38 and JNK and induction of stress-related genes; cell cycle arrest and apoptosis evident by increased percentages of cells in the sub-G1 fraction and Annexin V staining. In addition, we tested the response of patient-matched conditionally reprogrammed primary prostate normal and cancer cells. The tumor cells exhibited significantly higher sensitivity to the two most potent SL analogues with increased apoptosis confirmed by PARP1 cleavage compared to their normal counterpart cells. Thus, Strigolactone analogues are promising candidates for anticancer therapy by their ability to specifically induce cell cycle arrest, cellular stress and apoptosis in tumor cells with minimal effects on growth and survival of normal cells.

Presumed primary immune-mediated neutropenia in 35 dogs: a retrospective study.

PubMed

Devine, L; Armstrong, P J; Whittemore, J C; Sharkey, L; Bailiff, N; Huang, A; Rishniw, M

2017-06-01

To describe, in a cohort of dogs with presumed primary immune-mediated neutropenia, the presenting clinical characteristics, haematology results, bone marrow characteristics, therapies used (drugs and doses), clinical response to treatment, relapse and outcome at six months and one year. Multi-institutional recruited retrospective descriptive case series with voluntary submissions. Presumed immune-mediated neutropenia was diagnosed based on a neutrophil concentration <1·5×10 9 cells/L on a minimum of two complete blood counts, exclusion of other causes of neutropenia based on a diagnostic bone marrow aspirate or biopsy, and exclusion of secondary immune-mediated neutropenia. Dogs meeting these diagnostic criteria between 2006 and 2013, and that had a haematocrit of ≥29% and minimum of two complete blood clounts performed after initiation of therapy, were included. Information on 35 dogs was included. Neutropenia was less than 0·5×10 9 cells/L in most cases (21 dogs), 0·5 to ·99×10 9 cells/L in 11, and 1.0 to 1·49×10 9 cells/L in three. Eight dogs had thrombocytopenia, which was severe (<49·9×10 9 cells/L) in three. [Correction added on 23 May 2017, after first online publication: the cell numbers were incorrect due to errors in the conversion of cell measurements to international units. The numbers have been corrected throughout the article and Table 2.] Twenty-three dogs had myeloid hyperplasia, 10 dogs had myeloid hypoplasia and two dogs had normal myelopoiesis. Neutropenia resolved in 32 of 33 dogs within two weeks of starting corticosteroid therapy and in all dogs within one month. Relapse of neutropenia occurred in 12 cases within one year. Initial response of presumed primary immune-mediated neutropenia cases to corticosteroid therapy can be excellent. Long-term monitoring for relapse is warranted because 34% of cases relapsed during or after taper of immunosuppressive medications. © 2017 British Small Animal Veterinary Association.

Challenges for fuel cells as stationary power resource in the evolving energy enterprise

NASA Astrophysics Data System (ADS)

Rastler, Dan

The primary market challenges for fuel cells as stationary power resources in evolving energy markets are reviewed. Fuel cell power systems have significant barriers to overcome in their anticipated role as decentralized energy power systems. Market segments for fuel cells include combined heat and power; low-cost energy, premium power; peak shaving; and load management and grid support. Understanding the role and fit of fuel cell systems in evolving energy markets and the highest value applications are a major challenge for developers and government funding organizations. The most likely adopters of fuel cell systems and the challenges facing each adopter in the target market segment are reviewed. Adopters include generation companies, utility distribution companies, retail energy service providers and end-users. Key challenges include: overcoming technology risk; achieving retail competitiveness; understanding high value markets and end-user needs; distribution and service channels; regulatory policy issues; and the integration of these decentralized resources within the electrical distribution system.

The BioDyn facility on ISS: Advancing biomaterial production in microgravity for commercial applications

NASA Astrophysics Data System (ADS)

Myers, Niki; Wessling, Francis; Deuser, Mark; Anderson, C. D.; Lewis, Marian

1999-01-01

The primary goals of the BioDyn program are to foster use of the microgravity environment for commercial production of bio-materials from cells, and to develop services and processes for obtaining these materials through space processing. The scope of products includes commercial bio-molecules such as cytokines, other cell growth regulatory proteins, hormones, monoclonal antibodies and enzymes; transplantable cells or tissues which can be improved by low-G processes, or which cannot be obtained through standard processes in earth gravity; agriculture biotechnology products from plant cells; microencapsulation for diabetes treatment; and factors regulating cellular aging. To facilitate BioDyn's commercial science driven goals, hardware designed for ISS incorporates the flexibility for interchange between the different ISS facilities including the glovebox, various thermal units and centrifuges. By providing a permanent research facility, ISS is the critical space-based platform required by scientists for carrying out the long-term experiments necessary for developing bio-molecules and tissues using several cell culture modalities including suspension and anchorage-dependent cell types.

Providing cell phone numbers and email addresses to Patients: the physician's perspective

PubMed Central

2011-01-01

Background The provision of cell phone numbers and email addresses enhances the accessibility of medical consultations, but can add to the burden of physicians' routine clinical practice and affect their free time. The objective was to assess the attitudes of physicians to providing their telephone number or email address to patients. Methods Primary care physicians in the southern region of Israel completed a structured questionnaire that related to the study objective. Results The study population included 120 primary care physicians with a mean age of 41.2 ± 8.5, 88 of them women (73.3%). Physicians preferred to provide their cell phone number rather than their email address (P = 0.0007). They preferred to answer their cell phones only during the daytime and at predetermined times, but would answer email most hours of the day, including weekends and holidays (P = 0.001). More physicians (79.7%) would have preferred allotted time for email communication than allotted time for cell phone communication (50%). However, they felt that email communication was more likely to lead to miscommunication than telephone calls (P = 0.0001). There were no differences between male and female physicians on the provision of cell phone numbers or email addresses to patients. Older physicians were more prepared to provide cell phone numbers that younger ones (P = 0.039). Conclusions The attitude of participating physicians was to provide their cell phone number or email address to some of their patients, but most of them preferred to give out their cell phone number. PMID:21426591

Business Case for Fuel Cells 2016

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

Curtin, Sandra; Gangi, Jennifer; Benjamin, Thomas G.

The report provides an overview of recent private sector fuel cell installations at U.S. businesses as of December 31, 2016. This list is by no means exhaustive. Over the past few decades, hundreds of thousands of fuel cells have been installed around the world, for primary or backup power, as well as in various other applications including portable and emergency backup power. Fuel cells have also been deployed in other applications such as heat and electricity for homes and apartments, material handling, passenger vehicles, buses, and remote, off-grid sites.

Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO).

PubMed

Winkler, Dirk; Beconi, Maria; Toledo-Sherman, Leticia M; Prime, Michael; Ebneth, Andreas; Dominguez, Celia; Muñoz-Sanjuan, Ignacio

2013-09-01

Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells.

Advances in clinical immunology in 2015.

PubMed

Chinen, Javier; Notarangelo, Luigi D; Shearer, William T

2016-12-01

Advances in clinical immunology in the past year included the report of practice parameters for the diagnosis and management of primary immunodeficiencies to guide the clinician in the approach to these relatively uncommon disorders. We have learned of new gene defects causing immunodeficiency and of new phenotypes expanding the spectrum of conditions caused by genetic mutations such as a specific regulator of telomere elongation (RTEL1) mutation causing isolated natural killer cell deficiency and mutations in ras-associated RAB (RAB27) resulting in immunodeficiency without albinism. Advances in diagnosis included the increasing use of whole-exome sequencing to identify gene defects and the measurement of serum free light chains to identify secondary hypogammaglobulinemias. For several primary immunodeficiencies, improved outcomes have been reported after definitive therapy with hematopoietic stem cell transplantation and gene therapy. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Mass Cytometry Analysis Reveals the Landscape and Dynamics of CD32a+ CD4+ T Cells From Early HIV Infection to Effective cART.

PubMed

Coindre, Sixtine; Tchitchek, Nicolas; Alaoui, Lamine; Vaslin, Bruno; Bourgeois, Christine; Goujard, Cecile; Avettand-Fenoel, Veronique; Lecuroux, Camille; Bruhns, Pierre; Le Grand, Roger; Beignon, Anne-Sophie; Lambotte, Olivier; Favier, Benoit

2018-01-01

CD32a has been proposed as a specific marker of latently HIV-infected CD4 + T cells. However, CD32a was recently found to be expressed on CD4 + T cells of healthy donors, leading to controversy on the relevance of this marker in HIV persistence. Here, we used mass cytometry to characterize the landscape and variation in the abundance of CD32a + CD4 + T cells during HIV infection. To this end, we analyzed CD32a + CD4 + T cells in primary HIV infection before and after effective combination antiretroviral therapy (cART) and in healthy donors. We found that CD32a + CD4 + T cells include heterogeneous subsets that are differentially affected by HIV infection. Our analysis revealed that naive ( N ), central memory ( CM ), and effector/memory ( Eff/Mem ) CD32a + CD4 + T-cell clusters that co-express LILRA2- and CD64-activating receptors were more abundant in primary HIV infection and cART stages. Conversely, LILRA2 - CD32a + CD4 + T-cell clusters of either the T N , T CM , or T Eff/Mem phenotype were more abundant in healthy individuals. Finally, an activated CD32a + CD4 + T Eff/Mem cell cluster co-expressing LILRA2, CD57, and NKG2C was more abundant in all HIV stages, particularly during primary HIV infection. Overall, our data show that multiple abundance modifications of CD32a + CD4 + T-cell subsets occur in the early phase of HIV infection, and some of which are conserved after effective cART. Our study brings a better comprehension of the relationship between CD32a expression and CD4 + T cells during HIV infection.

Tropism and Infectivity of Influenza Virus, Including Highly Pathogenic Avian H5N1 Virus, in Ferret Tracheal Differentiated Primary Epithelial Cell Cultures

PubMed Central

Zeng, Hui; Goldsmith, Cynthia S.; Maines, Taronna R.; Belser, Jessica A.; Gustin, Kortney M.; Pekosz, Andrew; Zaki, Sherif R.; Katz, Jacqueline M.

2013-01-01

Tropism and adaptation of influenza viruses to new hosts is partly dependent on the distribution of the sialic acid (SA) receptors to which the viral hemagglutinin (HA) binds. Ferrets have been established as a valuable in vivo model of influenza virus pathogenesis and transmission because of similarities to humans in the distribution of HA receptors and in clinical signs of infection. In this study, we developed a ferret tracheal differentiated primary epithelial cell culture model that consisted of a layered epithelium structure with ciliated and nonciliated cells on its apical surface. We found that human-like (α2,6-linked) receptors predominated on ciliated cells, whereas avian-like (α2,3-linked) receptors, which were less abundant, were presented on nonciliated cells. When we compared the tropism and infectivity of three human (H1 and H3) and two avian (H1 and H5) influenza viruses, we observed that the human influenza viruses primarily infected ciliated cells and replicated efficiently, whereas a highly pathogenic avian H5N1 virus (A/Vietnam/1203/2004) replicated efficiently within nonciliated cells despite a low initial infection rate. Furthermore, compared to other influenza viruses tested, VN/1203 virus replicated more efficiently in cells isolated from the lower trachea and at a higher temperature (37°C) compared to a lower temperature (33°C). VN/1203 virus infection also induced higher levels of immune mediator genes and cell death, and virus was recovered from the basolateral side of the cell monolayer. This ferret tracheal differentiated primary epithelial cell culture system provides a valuable in vitro model for studying cellular tropism, infectivity, and the pathogenesis of influenza viruses. PMID:23255802

SL-401 and SL-501, Targeted Therapeutics Directed at the Interleukin-3 Receptor, Inhibit the Growth of Leukaemic Cells and Stem Cells in Advanced Phase Chronic Myeloid Leukaemia

PubMed Central

Frolova, Olga; Benito, Juliana; Brooks, Chris; Wang, Rui-Yu; Korchin, Borys; Rowinsky, Eric K.; Cortes, Jorge; Kantarjian, Hagop; Andreeff, Michael; Frankel, Arthur E.; Konopleva, Marina

2014-01-01

SUMMARY While imatinib and other tyrosine kinase inhibitors (TKIs) are highly efficacious in the treatment of chronic myeloid leukaemia (CML), some patients become refractory to these therapies. After confirming that interleukin-3 receptor (IL3R, CD123) is highly expressed on CD34+/CD38− BCR-ABL1+ CML stem cells, we investigated whether targeting IL3R with diphtheria toxin (DT)-IL3 fusion proteins SL-401 (DT388-IL3) and SL-501 (DT388-IL3[K116W]) could eradicate these stem cells. SL-401 and SL-501 inhibited cell growth and induced apoptosis in the KBM5 cell line and its TKI-resistant KBM5-STI subline. Combinations of imatinib with these agents increased apoptosis in KBM5 and in primary CML cells. In six primary CML samples, including CML cells harbouring the ABL1 T315I mutation, SL-401 and SL-501 decreased the absolute numbers of viable CD34+/CD38−/CD123+ CML progenitor cells by inducing apoptosis. IL3-targeting agents reduced clonogenic growth and diminished the fraction of primitive long-term culture-initiating cells in samples from patients with advanced phase CML that were resistant to TKIs or harboured an ABL1 mutation. Survival was also extended in a mouse model of primary TKI-resistant CML blast crisis. These data suggest that the DT-IL3 fusion proteins, SL-401 and SL-501, deplete CML stem cells and may increase the effectiveness of current CML treatment, which principally targets tumour bulk. PMID:24942980

SL-401 and SL-501, targeted therapeutics directed at the interleukin-3 receptor, inhibit the growth of leukaemic cells and stem cells in advanced phase chronic myeloid leukaemia.

PubMed

Frolova, Olga; Benito, Juliana; Brooks, Chris; Wang, Rui-Yu; Korchin, Borys; Rowinsky, Eric K; Cortes, Jorge; Kantarjian, Hagop; Andreeff, Michael; Frankel, Arthur E; Konopleva, Marina

2014-09-01

While imatinib and other tyrosine kinase inhibitors (TKIs) are highly efficacious in the treatment of chronic myeloid leukaemia (CML), some patients become refractory to these therapies. After confirming that interleukin-3 receptor (IL3R, CD123) is highly expressed on CD34(+) /CD38(-) BCR-ABL1(+) CML stem cells, we investigated whether targeting IL3R with diphtheria toxin (DT)-IL3 fusion proteins SL-401 (DT388 -IL3) and SL-501 (DT388 -IL3[K116W]) could eradicate these stem cells. SL-401 and SL-501 inhibited cell growth and induced apoptosis in the KBM5 cell line and its TKI-resistant KBM5-STI subline. Combinations of imatinib with these agents increased apoptosis in KBM5 and in primary CML cells. In six primary CML samples, including CML cells harbouring the ABL1 T315I mutation, SL-401 and SL-501 decreased the absolute numbers of viable CD34(+) /CD38(-) /CD123(+) CML progenitor cells by inducing apoptosis. IL3-targeting agents reduced clonogenic growth and diminished the fraction of primitive long-term culture-initiating cells in samples from patients with advanced phase CML that were resistant to TKIs or harboured an ABL1 mutation. Survival was also extended in a mouse model of primary TKI-resistant CML blast crisis. These data suggest that the DT-IL3 fusion proteins, SL-401 and SL-501, deplete CML stem cells and may increase the effectiveness of current CML treatment, which principally targets tumour bulk. © 2014 John Wiley & Sons Ltd.

Tropism and infectivity of influenza virus, including highly pathogenic avian H5N1 virus, in ferret tracheal differentiated primary epithelial cell cultures.

PubMed

Zeng, Hui; Goldsmith, Cynthia S; Maines, Taronna R; Belser, Jessica A; Gustin, Kortney M; Pekosz, Andrew; Zaki, Sherif R; Katz, Jacqueline M; Tumpey, Terrence M

2013-03-01

Tropism and adaptation of influenza viruses to new hosts is partly dependent on the distribution of the sialic acid (SA) receptors to which the viral hemagglutinin (HA) binds. Ferrets have been established as a valuable in vivo model of influenza virus pathogenesis and transmission because of similarities to humans in the distribution of HA receptors and in clinical signs of infection. In this study, we developed a ferret tracheal differentiated primary epithelial cell culture model that consisted of a layered epithelium structure with ciliated and nonciliated cells on its apical surface. We found that human-like (α2,6-linked) receptors predominated on ciliated cells, whereas avian-like (α2,3-linked) receptors, which were less abundant, were presented on nonciliated cells. When we compared the tropism and infectivity of three human (H1 and H3) and two avian (H1 and H5) influenza viruses, we observed that the human influenza viruses primarily infected ciliated cells and replicated efficiently, whereas a highly pathogenic avian H5N1 virus (A/Vietnam/1203/2004) replicated efficiently within nonciliated cells despite a low initial infection rate. Furthermore, compared to other influenza viruses tested, VN/1203 virus replicated more efficiently in cells isolated from the lower trachea and at a higher temperature (37°C) compared to a lower temperature (33°C). VN/1203 virus infection also induced higher levels of immune mediator genes and cell death, and virus was recovered from the basolateral side of the cell monolayer. This ferret tracheal differentiated primary epithelial cell culture system provides a valuable in vitro model for studying cellular tropism, infectivity, and the pathogenesis of influenza viruses.

Multiple Primary Merkel Cell Carcinomas Presenting as Pruritic, Painful Lower Leg Tumors

PubMed Central

Blumenthal, Laura; VandenBoom, Timothy; Melian, Edward; Peterson, Anthony; Hutchens, Kelli A.

2015-01-01

Merkel cell carcinoma (MCC) is a rare and highly aggressive neuroendocrine tumor of the skin which almost exclusively presents as a solitary tumor. It is most often seen on sun-exposed regions, historically almost exclusively on the head and neck, with only rare case reports on the extremities. Although recent studies have shown increased incidence with up to 20% on the extremities, here we present one of these rare emerging presentations, with the addition of a unique treatment option. Our patient is an 80-year-old male with a 3-month history of multiple raised, rapidly enlarging tumors on the right ankle. Two separate biopsies were performed and demonstrated sheets and clusters of small blue cells filling the dermis with scant cytoplasm, dusty chromatin, and nuclear molding. Subsequent immunohistochemical stains confirmed the diagnosis of multiple primary MCC. Despite the characteristic immunohistochemical profile of primary MCC, the possibility of a metastatic neuroendocrine carcinoma from an alternate primary site was entertained, given his unusual clinical presentation. A complete clinical workup including CT scans of the chest, abdomen, and pelvis showed no evidence of disease elsewhere. Instead of amputation, the patient opted for nonsurgical treatment with radiation therapy alone, resulting in a rapid and complete response. This case represents an unusual presentation of primary MCC and demonstrates further evidence that radiation as monotherapy is an effective local treatment option for inoperable MCC. PMID:26594171

Sex Reporting in Preclinical Microbiological and Immunological Research.

PubMed

Potluri, Tanvi; Engle, Kyrra; Fink, Ashley L; Vom Steeg, Landon G; Klein, Sabra L

2017-11-14

Both sex (i.e., biological construct of male and female) and gender (i.e., social construct of masculine and feminine) impact the pathogenesis of diseases, including those caused by microbial infections. Following the 2015 NIH policy for consideration of sex as a biological variable in preclinical research, in 2018, authors of papers published in primary-research American Society for Microbiology (ASM) journals will be asked to report the sex of the research subjects and animals and of materials derived directly from them. To address the need for sex reporting in ASM journals, we systematically reviewed 2,928 primary-research articles published in six primary-research ASM journals ( Antimicrobial Agents and Chemotherapy , Clinical and Vaccine Immunology , Infection and Immunity , Journal of Bacteriology , Journal of Virology , and mBio ) in 2016. Approximately 37% of animal studies and 9% of primary cell culture papers published in 2016 would have been affected by the new sex-reporting policy. For animal studies (i.e., studies with any nonhuman vertebrate hosts), most published papers either did not report the sex of the animals or used only female animals, and a minority used only males or both sexes. For published studies using primary cells from diverse animal species (i.e., humans and nonhuman vertebrates), almost all studies failed to report the sex of donors from which the cells were isolated. We believe that reporting the sex of animals and even of the donors of derived cells could improve the rigor and reproducibility of research conducted in microbiology and immunology and published in ASM journals. Copyright © 2017 Potluri et al.

MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes

PubMed Central

Polioudakis, Damon; Abell, Nathan S.; Iyer, Vishwanath R.

2015-01-01

miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191’s regulation of primary human fibroblast proliferation. PMID:25992613

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

PubMed

Kaneko, Ai; Sankai, Yoshiyuki

2014-01-01

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

Efficient generation of biliary epithelial cells from rabbit intrahepatic bile duct by Y-27632 and Matrigel.

PubMed

Jin, Lifang; Ji, Shaohui; Sun, Aijing

2013-06-01

Efficient culture of primary biliary epithelial cells (BECs) from adult liver is useful for both experimental studies and clinical applications of tissue engineering. However, an effective culture system for long-term proliferation of adult BECs is still unachieved. Laboratory rabbit has been used in a large number of studies; however, there are no reports of BECs from normal adult rabbit. As little as 5 g of normal rabbit liver tissue were minced, digested, and then clonally cultured in medium containing FBS and ITS. Cells were characterized by cell morphology, immunoassaying, and growth rate assay. Different combination of growth factors and substrates, including Y-27632 and Matrigel, were employed to assess their effect on cell proliferation. In the primary culture, the BECs cellular sheets consisting of cuboidal cells, as well as fibroblast-like cells and other hepatic cells, emerged with time of culture. The BECs cellular sheets were then manually split into cells clumps for further characterization. The subcultured cells had typical cell morphology of cholangiocytes, expressed the specific markers of BECs, including GGT, cytokeratin (CK18), and CK19, and possessed the capacity to form duct-like structure in three-dimensional Matrigel. Y-27632 and Matrigel-treated BECs had a steady growth rate as well as colony-formation capacity. The BECs were maintained in Y-27632 and Matrigel culture system for more than 3 mo. This is the first example, to our knowledge, of the successful culture of BECs from normal adult rabbit liver. Furthermore, our results indicate that treatment of BECs with Y-27632 and Matrigel is a simple method for efficient output of BECs.

Mitotic Dysfunction Associated with Aging Hallmarks.

PubMed

Macedo, Joana Catarina; Vaz, Sara; Logarinho, Elsa

2017-01-01

Aging is a biological process characterized by the progressive deterioration of physiological functions known to be the main risk factor for chronic diseases and declining health. There has been an emerging connection between aging and aneuploidy, an aberrant number of chromosomes, even though the molecular mechanisms behind age-associated aneuploidy remain largely unknown. In recent years, several genetic pathways and biochemical processes controlling the rate of aging have been identified and proposed as aging hallmarks. Primary hallmarks that cause the accumulation of cellular damage include genomic instability, telomere attrition, epigenetic alterations and loss of proteostasis (López-Otín et al., Cell 153:1194-1217, 2013). Here we review the provocative link between these aging hallmarks and the loss of chromosome segregation fidelity during cell division, which could support the correlation between aging and aneuploidy seen over the past decades. Secondly, we review the systemic impacts of aneuploidy in cell physiology and emphasize how these include some of the primary hallmarks of aging. Based on the evidence, we propose a mutual causality between aging and aneuploidy, and suggest modulation of mitotic fidelity as a potential means to ameliorate healthy lifespan.

Expression of monoacylglycerol lipase as a marker of tumour invasion and progression in malignant melanoma.

PubMed

Baba, Yuko; Funakoshi, T; Mori, M; Emoto, K; Masugi, Y; Ekmekcioglu, S; Amagai, M; Tanese, K

2017-12-01

Accumulating evidence suggests that the lipid lytic enzyme monoacylglycerol lipase (MAGL) promotes tumour invasion and metastasis through up-regulation of pro-tumorigenic signalling lipids in several tumour cell lines. However, the expression status of MAGL in clinical melanoma tissues and its clinicopathological significance remain unclear. To correlate the tumour expression status of MAGL with the clinicopathological information of patients with malignant melanoma. Polymerase chain reaction (PCR) array screening was performed, and the results were validated using immunocytochemical analysis of tumour and non-tumour melanocytic cell lines. Immunohistochemical staining for MAGL was performed for 74 melanoma samples, including 48 primary and 26 metastatic tumours, in which the expression of MAGL was determined by evaluating the percentage of MAGL-positive tumour cells and the MAGL staining intensity. Finally, we analysed the association of MAGL expression status with tumour progression, tumour thickness and vascular invasion of the primary lesion. Immunocytochemical analysis revealed that MAGL was expressed in all 12 melanoma cell lines, but not in normal human epidermal melanocytes. In the immunohistochemical analysis, positive staining for MAGL was noted in 32 of 48 (64.5%) primary lesions, 14 of 17 (82.4%) lymph node metastatic lesions and 7 of 9 (77.8%) skin metastatic lesions. Metastatic tumours had a significantly higher staining intensity (P = 0.033 for lymph node, P = 0.010 for skin). In the analysis of primary lesions, higher MAGL expression correlated with greater tumour thickness (P = 0.015) and the presence of vascular invasion (P = 0.017). On further evaluation of MAGL-positive primary lesions, staining intensity of MAGL tended to be higher in deeper areas of the tumour mass. The expression of MAGL in tumour cells reflects the aggressiveness of melanoma cells and may serve as a marker of tumour progression. © 2017 European Academy of Dermatology and Venereology.

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.

Practice makes pretty good: assessment of primary literature reading abilities across multiple large-enrollment biology laboratory courses.

PubMed

Sato, Brian K; Kadandale, Pavan; He, Wenliang; Murata, Paige M N; Latif, Yama; Warschauer, Mark

2014-01-01

Primary literature is essential for scientific communication and is commonly utilized in undergraduate biology education. Despite this, there is often little time spent training our students how to critically analyze a paper. To address this, we introduced a primary literature module in multiple upper-division laboratory courses. In this module, instructors conduct classroom discussions that dissect a paper as researchers do. While previous work has identified classroom interventions that improve primary literature comprehension within a single course, our goal was to determine whether including a scientific paper module in our classes could produce long-term benefits. On the basis of performance in an assessment exam, we found that our module resulted in longitudinal gains, including increased comprehension and critical-thinking abilities in subsequent lab courses. These learning gains were specific to courses utilizing our module, as no longitudinal gains were seen in students who had taken other upper-division labs that lacked extensive primary literature discussion. In addition, we assessed whether performance on our assessment correlated with a variety of factors, including grade point average, course performance, research background, and self-reported confidence in understanding of the article. Furthermore, all of the study conclusions are independent of biology disciplines, as we observe similar trends within each course. © 2014 B. K. Sato et al. CBE—Life Sciences Education © 2014 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Potential Use of Autologous Renal Cells from Diseased Kidneys for the Treatment of Renal Failure.

PubMed

George, Sunil K; Abolbashari, Mehran; Jackson, John D; Aboushwareb, Tamer; Atala, Anthony; Yoo, James J

2016-01-01

Chronic kidney disease (CKD) occurs when certain conditions cause the kidneys to gradually lose function. For patients with CKD, renal transplantation is the only treatment option that restores kidney function. In this study, we evaluated primary renal cells obtained from diseased kidneys to determine whether their normal phenotypic and functional characteristics are retained, and could be used for cell therapy. Primary renal cells isolated from both normal kidneys (NK) and diseased kidneys (CKD) showed similar phenotypic characteristics and growth kinetics. The expression levels of renal tubular cell markers, Aquaporin-1 and E-Cadherin, and podocyte-specific markers, WT-1 and Nephrin, were similar in both NK and CKD kidney derived cells. Using fluorescence- activated cell sorting (FACS), specific renal cell populations were identified and included proximal tubular cells (83.1% from NK and 80.3% from CKD kidneys); distal tubular cells (11.03% from NK and 10.9% from CKD kidneys); and podocytes (1.91% from NK and 1.78% from CKD kidneys). Ultra-structural analysis using scanning electron microscopy (SEM) revealed microvilli on the apical surface of cultured cells from NK and CKD samples. Moreover, transmission electron microscopy (TEM) analysis showed a similar organization of tight junctions, desmosomes, and other intracellular structures. The Na+ uptake characteristics of NK and CKD derived renal cells were also similar (24.4 mmol/L and 25 mmol/L, respectively) and no significant differences were observed in the protein uptake and transport characteristics of these two cell isolates. These results show that primary renal cells derived from diseased kidneys such as CKD have similar structural and functional characteristics to their counterparts from a normal healthy kidney (NK) when grown in vitro. This study suggests that cells derived from diseased kidney may be used as an autologous cell source for renal cell therapy, particularly in patients with CKD or end-stage renal disease (ESRD).

Wide-field Diffuse Amacrine Cells in the Monkey Retina Contain Immunoreactive Cocaine- and Amphetamine-Regulated Transcript (CART)

PubMed Central

Liu, Weiley S.; Davis, Elizabeth P.; Lee, Stephen J.; Tseng, Luke; Chuang, Alice Z.; Whitaker, Christopher M.; Massey, Stephen C.; Sherman, Michael B.; Marshak, David W.

2016-01-01

The goals of this study were to localize the neuropeptide Cocaine- and Amphetamine-Regulated Transcript (CART) in primate retinas and to describe the morphology, neurotransmitter content and synaptic connections of the neurons that contain it. Using in situ hybridization, light and electron microscopic immunolabeling, CART was localized to GABAergic amacrine cells in baboon retinas. The CART-positive cells had thin, varicose dendrites that gradually descended through the inner plexiform layer and ramified extensively in the innermost stratum. They resembled two types of wide-field diffuse amacrine cells that had been described previously in macaque retinas using the Golgi method and also A17, serotonin-accumulating and waterfall cells of other mammals. The CART-positive cells received synapses from rod bipolar cell axons and made synapses onto the axons in a reciprocal configuration. The CART-positive cells also received synapses from other amacrine cells. Some of these were located on their primary dendrites, and the presynaptic cells there included dopaminergic amacrine cells. Although some CART-positive somas were localized in the ganglion cell layer, they did not contain the ganglion cell marker RNA binding protein with multiple splicing (RBPMS). Based on these results and electrophysiological studies in other mammals, the CART-positive amacrine cells would be expected to play a major role in the primary rod pathway of primates, providing feedback inhibition to rod bipolar cells. PMID:27568514

Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver.

PubMed

Kakinuma, Sei; Ohta, Haruhiko; Kamiya, Akihide; Yamazaki, Yuji; Oikawa, Tsunekazu; Okada, Ken; Nakauchi, Hiromitsu

2009-07-01

Hepatic stem/progenitor cells possess active proliferative ability and the capacity for differentiation into hepatic and cholangiocytic lineages. Our group and others have shown that a prospectively defined population in mid-gestational fetal liver contains hepatic stem/progenitor cells. However, the phenotypes of such cells are incompletely elucidated. We analyzed the profile of cell-surface molecules on primary hepatic stem/progenitor cells. Expression of cell surface molecules on primary hepatic stem/progenitor cells in mouse mid-gestational fetal liver was analyzed using flow cytometric multicolor analyses and colony-formation assays. The potential of the cells for liver repopulation was examined by transplantation assay. We found that CD13 (aminopeptidase N) was detected on the cells of the previously reported (Dlk/Pref-1(+)) hepatic stem/progenitor fraction. Colony-formation assays revealed that the CD13(+) fraction, compared with the Dlk(+) fraction, of non-hematopoietic cells in fetal liver was enriched in hepatic stem/progenitor cells. Transplantation assay showed the former fraction exhibited repopulating potential in regenerating liver. Moreover, flow cytometric analysis for over 90 antigens demonstrated enrichment of hepatic stem/progenitor cells using several positive selection markers, including (hitherto unknown) CD13, CD73, CD106, and CD133. Our data indicated that CD13 is a positive selection marker for hepatic stem/progenitor cells in mid-gestational fetal liver.

Intestinal double-positive CD4+CD8+ T cells of neonatal rhesus macaques are proliferating, activated memory cells and primary targets for SIVMAC251 infection

PubMed Central

Wang, Xiaolei; Das, Arpita; Lackner, Andrew A.; Veazey, Ronald S.

2008-01-01

Peripheral blood and thymic double-positive (DP) CD4+CD8+ T cells from neonates have been described earlier, but the function and immunophenotypic characteristics of other tissue-derived DP T cells are not clearly understood. Here, we demonstrate the functional and immunophenotypic characteristics of DP cells in 6 different tissues, including thymus from normal neonatal rhesus macaques (Macaca mulatta) between 0 and 21 days of age. In general, intestinal DP T cells of neonates have higher percentages of memory markers (CD28+CD95+CD45RAlowCD62Llow) and proliferation compared with single-positive (SP) CD4+ and CD8+ T cells. In addition, percentages of DP T cells increase and CD62L expression decreases as animals mature, suggesting that DP cells mature and proliferate with maturity and/or antigen exposure. Consistent with this, intestinal DP T cells in neonates express higher levels of CCR5 and are the primary targets in simian immunodeficiency virus (SIV) infection. Finally, DP T cells produce higher levels of cytokine in response to mitogen stimulation compared with SP CD4+ or CD8+ T cells. Collectively, these findings demonstrate that intestinal DP T cells of neonates are proliferating, activated memory cells and are likely involved in regulating immune responses, in contrast to immature DP T cells in the thymus. PMID:18820133

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

Fuchigami, Takao; Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544; Kibe, Toshiro

Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactionsmore » are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave interactively via these cytokines to create a microenvironment that leads to the extension of ameloblastomas.« less

Hydrogen storage with trilithium aluminum hexahydride

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

Nathaniel, T.A.

1998-05-14

Fuel cells have good potential to replace batteries for many applications requiring moderate, portable electric power. Applications being researched can range from cellular telephones and radios to power generators for large camps. The primary advantages of fuel cells include high power density, low temperature operation, silent operation, no poisonous exhausts, high electric efficiency, and fast start-up capability. While many commercial industries are just beginning to look at the opportunities fuel cells present, the space program has driven the development of fuel cell technology. The paper discusses the status of the fuel cell and in particular, the technology for hydrogen storagemore » for fuel cell use.« less

Transferrin receptor 1 upregulation in primary tumor and downregulation in benign kidney is associated with progression and mortality in renal cell carcinoma patients

PubMed Central

Greene, Christopher J.; Attwood, Kristopher; Sharma, Nitika J.; Gross, Kenneth W.; Smith, Gary J.; Xu, Bo; Kauffman, Eric C.

2017-01-01

The central dysregulated pathway of clear cell (cc) renal cell carcinoma (RCC), the von Hippel Lindau/hypoxia inducible factor-α axis, is a key regulator of intracellular iron levels, however the role of iron uptake in human RCC tumorigenesis and progression remains unknown. We conducted a thorough, large-scale investigation of the expression and prognostic significance of the primary iron uptake protein, transferrin receptor 1 (TfR1/CD71/TFRC), in RCC patients. TfR1 immunohistochemistry was performed in over 1500 cores from 574 renal cell tumor patient tissues (primary tumors, matched benign kidneys, metastases) and non-neoplastic tissues from 36 different body sites. TfR1 levels in RCC tumors, particularly ccRCC, were significantly associated with adverse clinical prognostic features (anemia, lower body mass index, smoking), worse tumor pathology (size, stage, grade, multifocality, sarcomatoid dedifferentiation) and worse survival outcomes, including after adjustments for tumor pathology. Highest TfR1 tissue levels in the non-gravid body were detected in benign renal tubule epithelium. Opposite to TfR1 changes in the primary tumor, TfR1 levels in benign kidney dropped during tumor progression and were inversely associated with worse survival outcomes, independent of tumor pathology. Quantitative measurement of TfR1 subcellular localization in cell lines demonstrated mixed cytoplasmic and membranous expression with increased TfR1 in clusters in ccRCC versus benign renal cell lines. Results of this study support an important role for TfR1 in RCC progression and identify TfR1 as a novel RCC biomarker and therapeutic target. PMID:29291011

Cytology of Bone.

PubMed

Barger, Anne M

2017-01-01

Cytology of bone is a useful diagnostic tool. Aspiration of lytic or proliferative lesions can assist with the diagnosis of inflammatory or neoplastic processes. Bacterial, fungal, and protozoal organisms can result in significant osteomyelitis, and these organisms can be identified on cytology. Neoplasms of bone including primary bone tumors such as osteosarcoma, chondrosarcoma, fibrosarcoma, synovial cell sarcoma, and histiocytic sarcoma and tumors of bone marrow including plasma cell neoplasia and lymphoma and metastatic neoplasia can result in significant bone lysis or proliferation and can be diagnosed effectively with cytology. Copyright © 2016 Elsevier Inc. All rights reserved.

Autologous Stem Cell Transplantation Disrupts Adaptive Immune Responses during Rebound Simian/Human Immunodeficiency Virus Viremia.

PubMed

Reeves, Daniel B; Peterson, Christopher W; Kiem, Hans-Peter; Schiffer, Joshua T

2017-07-01

Primary HIV-1 infection induces a virus-specific adaptive/cytolytic immune response that impacts the plasma viral load set point and the rate of progression to AIDS. Combination antiretroviral therapy (cART) suppresses plasma viremia to undetectable levels that rebound upon cART treatment interruption. Following cART withdrawal, the memory component of the virus-specific adaptive immune response may improve viral control compared to primary infection. Here, using primary infection and treatment interruption data from macaques infected with simian/human immunodeficiency virus (SHIV), we observe a lower peak viral load but an unchanged viral set point during viral rebound. The addition of an autologous stem cell transplant before cART withdrawal alters viral dynamics: we found a higher rebound set point but similar peak viral loads compared to the primary infection. Mathematical modeling of the data that accounts for fundamental immune parameters achieves excellent fit to heterogeneous viral loads. Analysis of model output suggests that the rapid memory immune response following treatment interruption does not ultimately lead to better viral containment. Transplantation decreases the durability of the adaptive immune response following cART withdrawal and viral rebound. Our model's results highlight the impact of the endogenous adaptive immune response during primary SHIV infection. Moreover, because we capture adaptive immune memory and the impact of transplantation, this model will provide insight into further studies of cure strategies inspired by the Berlin patient. IMPORTANCE HIV patients who interrupt combination antiretroviral therapy (cART) eventually experience viral rebound, the return of viral loads to pretreatment levels. However, the "Berlin patient" remained free of HIV rebound over a decade after stopping cART. His cure is attributed to leukemia treatment that included an HIV-resistant stem cell transplant. Inspired by this case, we studied the impact of stem cell transplantation in a macaque simian/HIV (SHIV) system. Using a mechanistic mathematical model, we found that while primary infection generates an adaptive immune memory response, stem cell transplantation disrupts this learned immunity. The results have implications for HIV cure regimens based on stem cell transplantation. Copyright © 2017 American Society for Microbiology.

Autologous Stem Cell Transplantation Disrupts Adaptive Immune Responses during Rebound Simian/Human Immunodeficiency Virus Viremia

PubMed Central

Peterson, Christopher W.; Kiem, Hans-Peter

2017-01-01

ABSTRACT Primary HIV-1 infection induces a virus-specific adaptive/cytolytic immune response that impacts the plasma viral load set point and the rate of progression to AIDS. Combination antiretroviral therapy (cART) suppresses plasma viremia to undetectable levels that rebound upon cART treatment interruption. Following cART withdrawal, the memory component of the virus-specific adaptive immune response may improve viral control compared to primary infection. Here, using primary infection and treatment interruption data from macaques infected with simian/human immunodeficiency virus (SHIV), we observe a lower peak viral load but an unchanged viral set point during viral rebound. The addition of an autologous stem cell transplant before cART withdrawal alters viral dynamics: we found a higher rebound set point but similar peak viral loads compared to the primary infection. Mathematical modeling of the data that accounts for fundamental immune parameters achieves excellent fit to heterogeneous viral loads. Analysis of model output suggests that the rapid memory immune response following treatment interruption does not ultimately lead to better viral containment. Transplantation decreases the durability of the adaptive immune response following cART withdrawal and viral rebound. Our model's results highlight the impact of the endogenous adaptive immune response during primary SHIV infection. Moreover, because we capture adaptive immune memory and the impact of transplantation, this model will provide insight into further studies of cure strategies inspired by the Berlin patient. IMPORTANCE HIV patients who interrupt combination antiretroviral therapy (cART) eventually experience viral rebound, the return of viral loads to pretreatment levels. However, the “Berlin patient” remained free of HIV rebound over a decade after stopping cART. His cure is attributed to leukemia treatment that included an HIV-resistant stem cell transplant. Inspired by this case, we studied the impact of stem cell transplantation in a macaque simian/HIV (SHIV) system. Using a mechanistic mathematical model, we found that while primary infection generates an adaptive immune memory response, stem cell transplantation disrupts this learned immunity. The results have implications for HIV cure regimens based on stem cell transplantation. PMID:28404854

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.

Protein Modification: A Proposed Mechanism for the Long-Term Pathogenesis of Traumatic Brain Injury

DTIC Science & Technology

2015-06-04

two distinct phases: a first stage of “primary injury,” and subsequent “secondary injury.” Primary injury refers to the direct, physical disruption of...acid, is due to a number of post- injury effects, including the physical disruption of cell membranes and the impairment of energy-dependent...TBI. The pathophysiology of TBI is both complex and dynamic, involving physical injury, ischemia/reperfusion, hypoxia, glutamate excitotoxicity

Electromechanical Power for NASA Missions

NASA Technical Reports Server (NTRS)

Manzo, Michelle A.

2005-01-01

NASA has a wide range of missions that require electrochemical power sources. These needs are met with a variety of options that include primary and secondary cells and batteries, fuel cells, and regenerative fuel cells. This presentation wil cover an overview of NASA missions and requirements for electrochemical power sources and investigate the synergy and diversity that exist between NASA's requirements and those for military tactical power sources. Current development programs at GRC and other NASA centers, aimed at meeting NASA's future requirements will also be discussed.

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.

Apoptosis in Porcine Pluripotent Cells: From ICM to iPSCs

PubMed Central

Kim, Eunhye; Hyun, Sang-Hwan

2016-01-01

Pigs have great potential to provide preclinical models for human disease in translational research because of their similarities with humans. In this regard, porcine pluripotent cells, which are able to differentiate into cells of all three primary germ layers, might be a suitable animal model for further development of regenerative medicine. Here, we describe the current state of knowledge on apoptosis in pluripotent cells including inner cell mass (ICM), epiblast, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Information is focused on the apoptotic phenomenon in pluripotency, maintenance, and differentiation of pluripotent stem cells and reprogramming of somatic cells in pigs. Additionally, this review examines the multiple roles of apoptosis and summarizes recent progress in porcine pluripotent cells. PMID:27626414

Polyarene mediators for mediated redox flow battery

DOEpatents

Delnick, Frank M.; Ingersoll, David; Liang, Chengdu

2018-01-02

The fundamental charge storage mechanisms in a number of currently studied high energy redox couples are based on intercalation, conversion, or displacement reactions. With exception to certain metal-air chemistries, most often the active redox materials are stored physically in the electrochemical cell stack thereby lowering the practical gravimetric and volumetric energy density as a tradeoff to achieve reasonable power density. In a general embodiment, a mediated redox flow battery includes a series of secondary organic molecules that form highly reduced anionic radicals as reaction mediator pairs for the reduction and oxidation of primary high capacity redox species ex situ from the electrochemical cell stack. Arenes are reduced to stable anionic radicals that in turn reduce a primary anode to the charged state. The primary anode is then discharged using a second lower potential (more positive) arene. Compatible separators and solvents are also disclosed herein.

Regulators of Autophagosome Formation in Drosophila Muscles

PubMed Central

Zirin, Jonathan; Nieuwenhuis, Joppe; Samsonova, Anastasia; Tao, Rong; Perrimon, Norbert

2015-01-01

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function. PMID:25692684

Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research.

PubMed

Ray, Balmiki; Chopra, Nipun; Long, Justin M; Lahiri, Debomoy K

2014-09-16

Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications. This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons. We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.

Detection of the Cyanotoxins L-BMAA Uptake and Accumulation in Primary Neurons and Astrocytes.

PubMed

Tan, Vanessa X; Mazzocco, Claire; Varney, Bianca; Bodet, Dominique; Guillemin, Tristan A; Bessede, Alban; Guillemin, Gilles J

2018-01-01

We show for the first time that a newly developed polyclonal antibody (pAb) can specifically target the cyanotoxin β-methylamino-L-alanine (BMAA) and can be used to enable direct visualization of BMAA entry and accumulation in primary brain cells. We used this pAb to investigate the effect of acute and chronic accumulation, and toxicity of both BMAA and its natural isomer 2,4-diaminobutyric acid (DAB), separately or in combination, on primary cultures of rat neurons. We further present evidence that co-treatment with BMAA and DAB increased neuronal death, as measured by MAP2 fluorescence level, and appeared to reduce BMAA accumulation. DAB is likely to be acting synergistically with BMAA resulting in higher level of cellular toxicity. We also found that glial cells such as microglia and astrocytes are also able to directly uptake BMAA indicating that additional brain cell types are affected by BMAA-induced toxicity. Therefore, BMAA clearly acts at multiple cellular levels to possibly increase the risk of developing neurodegenerative diseases, including neuro- and gliotoxicity and synergetic exacerbation with other cyanotoxins.

LFA-1 expression in a series of colorectal adenocarcinomas.

PubMed

Papas, Maria G; Karatzas, Pantelis S; Papanikolaou, Ioannis S; Karamitopoulou, Evanthia; Delicha, Eumorphia M; Adler, Andreas; Triantafyllou, Konstantinos; Thomopoulou, Georgia-Heleni; Patsouris, Efstratios; Lazaris, Andreas C

2012-09-01

LFA-1 is an adhesion molecule which belongs to the β2-integrin family. Overexpression of LFA-1 in hepatic natural killer cells has been associated with increased apoptosis of neoplastic cells in colorectal cancer (CRC); moreover, studies in CRC have linked LFA-1 overexpression in neoplastic cells with vascular intrusion through adhesion to endothelial cells, thus implying a possible role in creation of metastases. We studied the expression of LFA-1 in a series of 82 patients with CRC. A standard three-step immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue sections. An IgG2a anti-CD11a monoclonal antibody was used. Cases were characterized according to clinicopathological variables including sex, age, tumor localization, size, grade, Dukes stage, wall invasion, and presence of metastatic lymph nodes (mLNs) or distal metastases. LFA-1 was expressed at the primary tumor site in 51 cases and 6/33 cases with metastatic lymphnodes. In Dukes D cases (n = 4), only one case was LFA-1(+). LFA-1 expression at the primary tumor site was associated with the absence of metastatic disease and with Dukes B stage. However, in those cases with LFA-1 expression in cancer cells in mLNs, this was associated with its expression at the primary tumor site. The positive association of LFA-1 expression in mLNs when the primary tumor site is also LFA-1(+) could imply an adaptation advantage of this specific cellular clone to its micro-environment, predisposing it to creation of mLNs, pointing to a role for LFA-1 in creation of mLNs in CRC.

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...

Inhibitory Interneurons That Express GFP in the PrP-GFP Mouse Spinal Cord Are Morphologically Heterogeneous, Innervated by Several Classes of Primary Afferent and Include Lamina I Projection Neurons among Their Postsynaptic Targets

PubMed Central

Ganley, Robert P.; Iwagaki, Noboru; del Rio, Patricia; Baseer, Najma; Dickie, Allen C.; Boyle, Kieran A.; Polgár, Erika; Watanabe, Masahiko; Abraira, Victoria E; Zimmerman, Amanda

2015-01-01

The superficial dorsal horn of the spinal cord contains numerous inhibitory interneurons, which regulate the transmission of information perceived as touch, pain, or itch. Despite the importance of these cells, our understanding of their roles in the neuronal circuitry is limited by the difficulty in identifying functional populations. One group that has been identified and characterized consists of cells in the mouse that express green fluorescent protein (GFP) under control of the prion protein (PrP) promoter. Previous reports suggested that PrP-GFP cells belonged to a single morphological class (central cells), received inputs exclusively from unmyelinated primary afferents, and had axons that remained in lamina II. However, we recently reported that the PrP-GFP cells expressed neuronal nitric oxide synthase (nNOS) and/or galanin, and it has been shown that nNOS-expressing cells are more diverse in their morphology and synaptic connections. We therefore used a combined electrophysiological, pharmacological, and anatomical approach to reexamine the PrP-GFP cells. We provide evidence that they are morphologically diverse (corresponding to “unclassified” cells) and receive synaptic input from a variety of primary afferents, with convergence onto individual cells. We also show that their axons project into adjacent laminae and that they target putative projection neurons in lamina I. This indicates that the neuronal circuitry involving PrP-GFP cells is more complex than previously recognized, and suggests that they are likely to have several distinct roles in regulating the flow of somatosensory information through the dorsal horn. PMID:25972186

Ineffective vaccination against solid tumors can be enhanced by hematopoietic cell transplantation.

PubMed

Filatenkov, Alexander; Müller, Antonia M S; Tseng, William Wei-Lin; Dejbakhsh-Jones, Sussan; Winer, Daniel; Luong, Richard; Shizuru, Judith A; Engleman, Edgar G; Strober, Samuel

2009-12-01

Vaccination with tumor Ags has not been an effective treatment for solid tumors. The goal of the current study was to determine whether a combination of vaccination and hematopoietic cell transplantation (HCT) can effectively treat primary, disseminated, or metastatic CT26 and MC38 murine colon tumors. Vaccination of tumor-bearing mice with irradiated tumor cells and CpG adjuvant failed to alter progressive tumor growth. However, mice bearing primary, disseminated lung, or metastatic liver tumors were uniformly cured after administration of total body irradiation, followed by the transplantation of hematopoietic progenitor cells and T cells from syngeneic, but not allogeneic vaccinated donors. Requirements for effective treatment of tumors included irradiation of hosts, vaccination of donors with both tumor cells and CpG, transfer of both CD4(+) and CD8(+) T cells along with progenitor cells, and ability of donor cells to produce IFN-gamma. Irradiation markedly increased the infiltration of donor T cells into the tumors, and the combined irradiation and HCT altered the balance of tumor-infiltrating cells to favor CD8(+) effector memory T cells as compared with CD4(+)CD25(+)FoxP3(+) T regulatory cells. The combination of vaccination and autologous hematopoietic cell transplantation was also effective in treating tumors. In conclusion, these findings show that otherwise ineffective vaccination to solid nonhematologic tumors can be dramatically enhanced by HCT.

Induction of anchorage-independent growth in primary human cells exposed to protons or HZE ions separately or in dual exposures.

PubMed

Sutherland, B M; Cuomo, N C; Bennett, P V

2005-10-01

Travelers on space missions will be exposed to a complex radiation environment that includes protons and heavy charged particles. Since protons are present at much higher levels than are heavy ions, the most likely scenario for cellular radiation exposure will be proton exposure followed by a hit by a heavy ion. Although the effects of individual ion species on human cells are being investigated extensively, little is known about the effects of exposure to both radiation types. One useful measure of mammalian cell damage is induction of the ability to grow in a semi-solid agar medium highly inhibitory to the growth of normal human cells, termed neoplastic transformation. Using primary human cells, we evaluated induction of soft-agar growth and survival of cells exposed to protons only or to heavy charged particles (600 MeV/nucleon silicon) only as well as of cells exposed to protons followed after a 4-day interval by silicon ions. Both ions alone efficiently transformed the human cells to anchorage-independent growth. Initial experiments indicate that the dose responses for neoplastic transformation of cells exposed to protons and then after 4 days to silicon ions appear similar to that of cells exposed to silicon ions alone.

Development of a Thin Film Solar Cell Interconnect for the Powersphere Concept

NASA Technical Reports Server (NTRS)

Simburger, Edward J.; Matsumoto, James H.; Giants, Thomas W.; Garcia, Alexander, III; Liu, Simon; Rawal, Suraj P.; Perry, Alan R.; Marshall, Craig H.; Lin, John K.; Scarborough, Stephen

2003-01-01

Progressive development of microsatellite technologies has resulted in increased demand for lightweight electrical power subsystems including solar arrays. The use of thin film photovoltaics has been recognized as a key solution to meet the power needs. The lightweight cells can generate sufficient power and still meet critical mass requirements. Commercially available solar cells produced on lightweight substrates are being studied as an option to fulfill the power needs. The commercially available solar cells are relatively inexpensive and have a high payoff potential. Commercially available thin film solar cells are primarily being produced for terrestrial applications. The need to convert the solar cell from a terrestrial to a space compatible application is the primary challenge. Solar cell contacts, grids and interconnects need to be designed to be atomic oxygen resistant and withstand rapid thermal cycling environments. A mechanically robust solar cell interconnect is also required in order to withstand handling during fabrication and survive during launch. The need to produce the solar cell interconnects has been identified as a primary goal of the Powersphere program and is the topic of this paper. Details of the trade study leading to the final design involving the solar cell wrap around contact, flex blanket, welding process, and frame will be presented at the conference.

CD44 is a direct target of miR-199a-3p and contributes to aggressive progression in osteosarcoma

PubMed Central

Gao, Yan; Feng, Yong; Shen, Jacson K.; Lin, Min; Choy, Edwin; Cote, Gregory M.; Harmon, David C.; Mankin, Henry J.; Hornicek, Francis J.; Duan, Zhenfeng

2015-01-01

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Herein, we investigated the role of cluster of differentiation 44 (CD44), a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration in osteosarcoma. We constructed a human osteosarcoma tissue microarray with 114 patient tumor specimens, including tumor tissues from primary, metastatic, and recurrent stages, and determined the expression of CD44 by immunohistochemistry. Results showed that CD44 was overexpressed in metastatic and recurrent osteosarcoma as compared with primary tumors. Higher expression of CD44 was found in both patients with shorter survival and patients who exhibited unfavorable response to chemotherapy before surgical resection. Additionally, the 3′-untranslated region of CD44 mRNA was the direct target of microRNA-199a-3p (miR-199a-3p). Overexpression of miR-199a-3p significantly inhibited CD44 expression in osteosarcoma cells. miR-199a-3p is one of the most dramatically decreased miRs in osteosarcoma cells and tumor tissues as compared with normal osteoblast cells. Transfection of miR-199a-3p significantly increased the drug sensitivity through down-regulation of CD44 in osteosarcoma cells. Taken together, these results suggest that the CD44-miR-199a-3p axis plays an important role in the development of metastasis, recurrence, and drug resistance of osteosarcoma. Developing strategies to target CD44 may improve the clinical outcome of osteosarcoma. PMID:26079799

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

groHMM: a computational tool for identifying unannotated and cell type-specific transcription units from global run-on sequencing data.

PubMed

Chae, Minho; Danko, Charles G; Kraus, W Lee

2015-07-16

Global run-on coupled with deep sequencing (GRO-seq) provides extensive information on the location and function of coding and non-coding transcripts, including primary microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and enhancer RNAs (eRNAs), as well as yet undiscovered classes of transcripts. However, few computational tools tailored toward this new type of sequencing data are available, limiting the applicability of GRO-seq data for identifying novel transcription units. Here, we present groHMM, a computational tool in R, which defines the boundaries of transcription units de novo using a two state hidden-Markov model (HMM). A systematic comparison of the performance between groHMM and two existing peak-calling methods tuned to identify broad regions (SICER and HOMER) favorably supports our approach on existing GRO-seq data from MCF-7 breast cancer cells. To demonstrate the broader utility of our approach, we have used groHMM to annotate a diverse array of transcription units (i.e., primary transcripts) from four GRO-seq data sets derived from cells representing a variety of different human tissue types, including non-transformed cells (cardiomyocytes and lung fibroblasts) and transformed cells (LNCaP and MCF-7 cancer cells), as well as non-mammalian cells (from flies and worms). As an example of the utility of groHMM and its application to questions about the transcriptome, we show how groHMM can be used to analyze cell type-specific enhancers as defined by newly annotated enhancer transcripts. Our results show that groHMM can reveal new insights into cell type-specific transcription by identifying novel transcription units, and serve as a complete and useful tool for evaluating functional genomic elements in cells.

Cell-fate specification in the epidermis: a common patterning mechanism in the root and shoot.

PubMed

Schiefelbein, John

2003-02-01

The specification of epidermal hairs in Arabidopsis provides a useful model for the study of pattern formation in plants. Although the distributions of hair cells in the root and shoot appear quite different, recent studies show that pattern formation in each relies on a common cassette of transcriptional regulators. During development in each organ, neighboring cells compete to express regulators that specify the primary cell fate (including WEREWOLF [WER]/GLABRA1 [GL1], GL3/bHLH, TRANSPARENT TESTA GLABRA [TTG], and GL2), as well as those that prevent their neighbors from adopting this fate (including CAPRICE [CPC] and TRIPTYCHON [TRY]). The basic mechanism of lateral inhibition with feedback that has been uncovered by recent studies provides a conceptual framework for understanding how patterns of cell fate in general may be specified during plant development.

Primary vascularization of allografts governs their immunogenicity and susceptibility to tolerogenesis

PubMed Central

Kant, Cavit D.; Akiyama, Yoshinobu; Tanaka, Katsunori; Shea, Susan; Connolly, Sarah E; Germana, Sharon; Winn, Henry J.; LeGuern, Christian; Tocco, Georges; Benichou, Gilles

2013-01-01

We investigated the influence of allograft primary vascularization on alloimmunity, rejection and tolerance in mice. First, we showed that fully allogeneic primarily vascularized and conventional skin transplants were rejected at the same pace. Remarkably, however, short-term treatment of mice with anti-CD40L antibodies achieved long-term survival of vascularized skin and cardiac transplants but not conventional skin grafts. Non-vascularized skin transplants triggered vigorous direct and indirect pro-inflammatory type 1 T cell responses (IL-2 and γIFN) while primarily-vascularized skin allografts failed to trigger a significant indirect alloresponse. Similar lack of indirect alloreactivity was also observed after placement of different vascularized organ transplants including hearts and kidneys while hearts placed under the skin (non-vascularized) triggers potent indirect alloresponses. Altogether, these results suggest that primary vascularization of allografts is associated with lack of indirect T cell alloreactivity. Finally, we show that long-term survival of vascularized skin allografts induced by anti-CD40L antibodies was associated with a combined lack of indirect alloresponse and a shift of the direct alloresponse towards a type 2 cytokine (IL-4, IL-10) secretion pattern but no activation/expansion of regulatory T cells. Therefore, primary vascularization of allografts governs their immunogenicity and tolerogenicity. PMID:23833234

Exfoliative cytologic findings of primary pulmonary adenoid cystic carcinom: a report of 2 cases with a review of the cytologic features.

PubMed

Daneshbod, Yahya; Modjtahedi, Ebrahim; Atefi, Sohrab; Bedayat, Gholam Reza; Daneshbod, Khosrow

2007-01-01

Adenoid cystic carcinoma is a very rare primary pulmonary neoplasm. Cytologic findings of pulmonary washing and brushing in 2 cases of primary bronchial adenoid cystic carcinoma with special histologic features are described, with an emphasis on some points that have not been reported previously, together with the diagnostic pitfalls. Two cases of primary adenoid cystic carcinoma of the lung were diagnosed on exfoliative cytology. The patients' ages were 55 and 65 years old. Cytologic findings included large and small clusters of small cells in both 2 and 3 dimensions with occasional cystlike spaces containing mucoid material. The cells were arranged in spherical, cylindrical, basaloid and rosettelike arrangements. There were also abundant small and large mucoid globules, cylinders of homogeneous, acellular, mucous material and "cannon balls." Cytoplasmic and intranuclear round inclusions were noted in case 1. Rare findings of nuclear molding were noted. In case 2, chondromyxoid material and a bimorphic population of tumor cells caused diagnostic confusion with other salivary gland-type tumors of the lung. These cases showed characteristic cytologic findings of adenoid cystic carcinoma together with rare findings of intracellular and extracellular inclusionlike bodies, myxochondroid material, bimorphic populations and nuclear molding, which can cause diagnostic confusion with other lung tumors.

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

[Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope].

PubMed

Gao, Yi-ning; Wang, Dan-ying; Pan, Zong-fu; Mei, Yu-qin; Wang, Zhi-qiang; Zhu, Dan-yan; Lou, Yi-jia

2012-07-01

To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope. Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes. The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons. Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

Induction of specific micro RNA (miRNA) species by ROS-generating metal sulfates in primary human brain cells

PubMed Central

Lukiw, Walter J.; Pogue, Aileen I.

2007-01-01

Iron- and aluminum-sulfate together, at nanomolar concentrations, trigger the production of reactive oxygen species (ROS) in cultures of human brain cells. Previous studies have shown that following ROS induction, a family of pathogenic brain genes that promote inflammatory signalling, cellular apoptosis and brain cell death is significantly over-expressed. Notably, iron- and aluminum-sulfate induce genes in cultured human brain cells that exhibit expression patterns similar to those observed to be up-regulated in moderate- to late-stage Alzheimer's disease (AD). In this study we have extended our investigations to analyze the expression of micro RNA (miRNA) populations in iron- and aluminum-sulfate treated human neural cells in primary culture. The main finding was that these ROS-generating neurotoxic metal sulfates also up-regulate a specific set of miRNAs that includes miR-9, miR-125b and miR-128. Notably, these same miRNAs are up-regulated in AD brain. These findings further support the idea that iron- and aluminum-sulfates induce genotoxicity via a ROS-mediated up-regulation of specific regulatory elements and pathogenic genes that redirect brain cell fate towards progressive dysfunction and apoptotic cell death. PMID:17629564

Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

PubMed Central

Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul

2011-01-01

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response. PMID:22072830

Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

PubMed

Lee, Yang-Jin; Park, Chang-Eun; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Jung, Suk-Yul; Shin, Ho-Joon

2011-09-01

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1β, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes.

PubMed

Yang, C; Jones, J L; Barnum, S R

1993-09-01

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

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.

Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner.

PubMed

Komatsu, Tomoko; Uchida, Kunitoshi; Fujita, Fumitaka; Zhou, Yiming; Tominaga, Makoto

2012-04-01

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is mainly expressed in primary nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent compounds such as mustard oil and cinnamaldehyde, and intracellular alkalization. Here, we show that primary alcohols, which have been reported to cause skin, eye or nasal irritation, activate human TRPA1 (hTRPA1). We measured intracellular Ca(2+) changes in HEK293 cells expressing hTRPA1 induced by 1 mM primary alcohols. Higher alcohols (1-butanol to 1-octanol) showed Ca(2+) increases proportional to the carbon chain length. In whole-cell patch-clamp recordings, higher alcohols (1-hexanol to 1-octanol) activated hTRPA1 and the potency increased with the carbon chain length. Higher alcohols evoked single-channel opening of hTRPA1 in an inside-out configuration. In addition, cysteine at 665 in the N terminus and histidine at 983 in the C terminus were important for hTRPA1 activation by primary alcohols. Furthermore, straight-chain secondary alcohols increased intracellular Ca(2+) concentrations in HEK293 cells expressing hTRPA1, and both primary and secondary alcohols showed hTRPA1 activation activities that correlated highly with their octanol/water partition coefficients. On the other hand, mouse TRPA1 did not show a strong response to 1-hexanol or 1-octanol, nor did these alcohols evoke significant pain in mice. We conclude that primary and secondary alcohols activate hTRPA1 in a carbon chain length-dependent manner. TRPA1 could be a sensor of alcohols inducing skin, eye and nasal irritation in human.

CD147 and CD98 complex-mediated homotypic aggregation attenuates the CypA-induced chemotactic effect on Jurkat T cells.

PubMed

Guo, Na; Zhang, Kui; Lv, Minghua; Miao, Jinlin; Chen, Zhinan; Zhu, Ping

2015-02-01

Homotypic cell aggregation plays important roles in physiological and pathological processes, including embryogenesis, immune responses, angiogenesis, tumor cell invasion and metastasis. CD147 has been implicated in most of these phenomena, and it was identified as a T cell activation-associated antigen due to its obvious up-regulation in activated T cells. However, the explicit function and mechanism of CD147 in T cells have not been fully elucidated. In this study, large and compact aggregates were observed in Jurkat T cells after treatment with the specific CD147 monoclonal antibody HAb18 or after the expression of CD147 was silenced by RNA interference, which indicated an inhibitory effect of CD147 in T cell homotypic aggregation. Knocking down CD147 expression resulted in a significant decrease in CD98, along with prominent cell aggregation, similar to that treated by CD98 and CD147 monoclonal antibodies. Furthermore, decreased cell chemotactic activity was observed following CD147- and CD98-mediated cell aggregation, and increased aggregation was correlated with a decrease in the chemotactic ability of the Jurkat T cells, suggesting that CD147- and CD98-mediated homotypic cell aggregation plays a negative role in T cell chemotaxis. Our data also showed that p-ERK, p-ZAP70, p-CD3ζ and p-LCK were significantly decreased in the CD147- and CD98-knocked down Jurkat T cells, which suggested that decreased CD147- and/or CD98-induced homotypic T cell aggregation and aggregation-inhibited chemotaxis might be associated with these signaling pathways. A role for CD147 in cell aggregation and chemotaxis was further indicated in primary CD4(+) T cells. Similarly, low expression of CD147 in primary T cells induced prominent cell aggregation and this aggregation attenuated primary T cell chemotactic ability in response to CypA. Our results have demonstrated the correlation between homotypic cell aggregation and the chemotactic response of T cells to CypA, and these data indicate that CD147 and CD98 might play important roles in cyclophilin-induced cell migration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Anti-allergic and anti-inflammatory effects of butanol extract from Arctium Lappa L.

PubMed

Sohn, Eun-Hwa; Jang, Seon-A; Joo, Haemi; Park, Sulkyoung; Kang, Se-Chan; Lee, Chul-Hoon; Kim, Sun-Young

2011-02-08

Atopic dermatitis is a chronic, allergic inflammatory skin disease that is accompanied by markedly increased levels of inflammatory cells, including eosinophils, mast cells, and T cells. Arctium lappa L. is a traditional medicine in Asia. This study examined whether a butanol extract of A. lappa (ALBE) had previously unreported anti-allergic or anti-inflammatory effects. This study examined the effect of ALBE on the release of β-hexosaminidase in antigen-stimulated-RBL-2H3 cells. We also evaluated the ConA-induced expression of IL-4, IL-5, mitogen-activated protein kinases (MAPKs), and nuclear factor (NF)-κB using RT-PCR, Western blotting, and ELISA in mouse splenocytes after ALBE treatment. We observed significant inhibition of β-hexosaminidase release in RBL-2H3 cells and suppressed mRNA expression and protein secretion of IL-4 and IL-5 induced by ConA-treated primary murine splenocytes after ALBE treatment. Additionally, ALBE (100 μg/mL) suppressed not only the transcriptional activation of NF-κB, but also the phosphorylation of MAPKs in ConA-treated primary splenocytes. These results suggest that ALBE inhibits the expression of IL-4 and IL-5 by downregulating MAPKs and NF-κB activation in ConA-treated splenocytes and supports the hypothesis that ALBE may have beneficial effects in the treatment of allergic diseases, including atopic dermatitis.

Anti-allergic and anti-inflammatory effects of butanol extract from Arctium Lappa L

PubMed Central

2011-01-01

Background Atopic dermatitis is a chronic, allergic inflammatory skin disease that is accompanied by markedly increased levels of inflammatory cells, including eosinophils, mast cells, and T cells. Arctium lappa L. is a traditional medicine in Asia. This study examined whether a butanol extract of A. lappa (ALBE) had previously unreported anti-allergic or anti-inflammatory effects. Methods This study examined the effect of ALBE on the release of β-hexosaminidase in antigen-stimulated-RBL-2H3 cells. We also evaluated the ConA-induced expression of IL-4, IL-5, mitogen-activated protein kinases (MAPKs), and nuclear factor (NF)-κB using RT-PCR, Western blotting, and ELISA in mouse splenocytes after ALBE treatment. Results We observed significant inhibition of β-hexosaminidase release in RBL-2H3 cells and suppressed mRNA expression and protein secretion of IL-4 and IL-5 induced by ConA-treated primary murine splenocytes after ALBE treatment. Additionally, ALBE (100 μg/mL) suppressed not only the transcriptional activation of NF-κB, but also the phosphorylation of MAPKs in ConA-treated primary splenocytes. Conclusions These results suggest that ALBE inhibits the expression of IL-4 and IL-5 by downregulating MAPKs and NF-κB activation in ConA-treated splenocytes and supports the hypothesis that ALBE may have beneficial effects in the treatment of allergic diseases, including atopic dermatitis. PMID:21303540

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

TNF-α blockade induces IL-10 expression in human CD4+ T cells

NASA Astrophysics Data System (ADS)

Evans, Hayley G.; Roostalu, Urmas; Walter, Gina J.; Gullick, Nicola J.; Frederiksen, Klaus S.; Roberts, Ceri A.; Sumner, Jonathan; Baeten, Dominique L.; Gerwien, Jens G.; Cope, Andrew P.; Geissmann, Frederic; Kirkham, Bruce W.; Taams, Leonie S.

2014-02-01

IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg-/Foxp3-independent, requires IL-10 and is overcome by IL-1β. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterized by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.

RUNX1 promotes cell growth in human T-cell acute lymphoblastic leukemia by transcriptional regulation of key target genes.

PubMed

Jenkins, Catherine E; Gusscott, Samuel; Wong, Rachel J; Shevchuk, Olena O; Rana, Gurneet; Giambra, Vincenzo; Tyshchenko, Kateryna; Islam, Rashedul; Hirst, Martin; Weng, Andrew P

2018-05-04

RUNX1 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). The spectrum of RUNX1 mutations has led to the notion that it acts as a tumor suppressor in this context; however, other studies have placed RUNX1 along with transcription factors TAL1 and NOTCH1 as core drivers of an oncogenic transcriptional program. To reconcile these divergent roles, we knocked down RUNX1 in human T-ALL cell lines and deleted Runx1 or Cbfb in primary mouse T-cell leukemias. RUNX1 depletion consistently resulted in reduced cell proliferation and increased apoptosis. RUNX1 upregulated variable sets of target genes in each cell line, but consistently included a core set of oncogenic effectors including IGF1R and NRAS. Our results support the conclusion that RUNX1 has a net positive effect on cell growth in the context of established T-ALL. Copyright © 2018. Published by Elsevier Inc.

Survival outcomes following salvage surgery for oropharyngeal squamous cell carcinoma: systematic review.

PubMed

Kao, S S; Ooi, E H

2018-04-01

Recurrent oropharyngeal squamous cell carcinoma causes great morbidity and mortality. This systematic review analyses survival outcomes following salvage surgery for recurrent oropharyngeal squamous cell carcinoma. A comprehensive search of various electronic databases was conducted. Studies included patients with recurrent or residual oropharyngeal squamous cell carcinoma treated with salvage surgery. Primary outcomes were survival rates following salvage surgery. Secondary outcomes included time to recurrence, staging at time of recurrence, post-operative complications, and factors associated with mortality and recurrence. Methodological appraisal and data extraction were conducted as per Joanna Briggs Institute methodology. Eighteen articles were included. The two- and five-year survival rates of the patients were 52 per cent and 30 per cent respectively. Improvements in treatment modalities for recurrent oropharyngeal squamous cell carcinoma were associated with improvements in two-year overall survival rates, with minimal change to five-year overall survival rates. Various factors were identified as being associated with long-term overall survival, thus assisting clinicians in patient counselling and selection for salvage surgery.

Comparison of Tumor- and Bone Marrow-Derived Mesenchymal Stromal/Stem Cells from Patients with High-Grade Osteosarcoma

PubMed Central

Le Nail, Louis-Romée; Brennan, Meadhbh; Rosset, Philippe; Piloquet, Philippe; Pichon, Olivier; Le Caignec, Cédric; Crenn, Vincent; Layrolle, Pierre; Hérault, Olivier; De Pinieux, Gonzague

2018-01-01

Osteosarcoma (OS) is suspected to originate from dysfunctional mesenchymal stromal/stem cells (MSC). We sought to identify OS-derived cells (OSDC) with potential cancer stem cell (CSC) properties by comparing OSDC to MSC derived from bone marrow of patients. This study included in vitro characterization with sphere forming assays, differentiation assays, cytogenetic analysis, and in vivo investigations of their tumorigenicity and tumor supportive capacities. Primary cell lines were isolated from nine high-grade OS samples. All primary cell lines demonstrated stromal cell characteristics. Compared to MSC, OSDC presented a higher ability to form sphere clones, indicating a potential CSC phenotype, and were more efficient at differentiation towards osteoblasts. None of the OSDC displayed the complex chromosome rearrangements typical of high grade OS and none of them induced tumors in immunodeficient mice. However, two OSDC demonstrated focused genomic abnormalities. Three out of seven, and six out of seven OSDC showed a supportive role on local tumor development, and on metastatic progression to the lungs, respectively, when co-injected with OS cells in nude mice. The observation of OS-associated stromal cells with rare genetic abnormalities and with the capacity to sustain tumor progression may have implications for future tumor treatments. PMID:29494553

Amoebal Endosymbiont Protochlamydia Induces Apoptosis to Human Immortal HEp-2 Cells

PubMed Central

Ito, Atsushi; Matsuo, Junji; Nakamura, Shinji; Yoshida, Asahi; Okude, Miho; Hayashi, Yasuhiro; Sakai, Haruna; Yoshida, Mitsutaka; Takahashi, Kaori; Yamaguchi, Hiroyuki

2012-01-01

Protochlamydia, an environmental chlamydia and obligate amoebal endosymbiotic bacterium, evolved to survive within protist hosts, such as Acanthamobae, 700 million years ago. However, these bacteria do not live in vertebrates, including humans. This raises the possibility that interactions between Protochlamydia and human cells could induce a novel cytopathic effect, leading to new insights into host-parasite relationships. Therefore, we studied the effect of Protochlamydia on the survival of human immortal cell line, HEp-2 cells and primary peripheral blood mononuclear cells (PBMC). Using mainly 4′,6-diamidino-2-phenylindole staining, fluorescent in situ hybridization, transmission electron microscopy, and also TUNEL and Transwell assays, we demonstrated that the Protochlamydia induced apoptosis in HEp-2 cells. The attachment of viable bacterial cells, but not an increase of bacterial infectious progenies within the cells, was required for the apoptosis. Other chlamydiae [Parachlamydia acanthamoebae and Chlamydia trachomatis (serovars D and L2)] did not induce the same phenomena, indicating that the observed apoptosis may be specific to the Protochlamydia. Furthermore, the bacteria had no effect on the survival of primary PBMCs collected from five volunteers, regardless of activation. We concluded that Protochlamydia induces apoptosis in human-immortal HEp-2 cells and that this endosymbiont could potentially be used as a biological tool for the elucidation of novel host-parasite relationships. PMID:22276171

Amoebal endosymbiont Protochlamydia induces apoptosis to human immortal HEp-2 cells.

PubMed

Ito, Atsushi; Matsuo, Junji; Nakamura, Shinji; Yoshida, Asahi; Okude, Miho; Hayashi, Yasuhiro; Sakai, Haruna; Yoshida, Mitsutaka; Takahashi, Kaori; Yamaguchi, Hiroyuki

2012-01-01

Protochlamydia, an environmental chlamydia and obligate amoebal endosymbiotic bacterium, evolved to survive within protist hosts, such as Acanthamobae, 700 million years ago. However, these bacteria do not live in vertebrates, including humans. This raises the possibility that interactions between Protochlamydia and human cells could induce a novel cytopathic effect, leading to new insights into host-parasite relationships. Therefore, we studied the effect of Protochlamydia on the survival of human immortal cell line, HEp-2 cells and primary peripheral blood mononuclear cells (PBMC). Using mainly 4',6-diamidino-2-phenylindole staining, fluorescent in situ hybridization, transmission electron microscopy, and also TUNEL and Transwell assays, we demonstrated that the Protochlamydia induced apoptosis in HEp-2 cells. The attachment of viable bacterial cells, but not an increase of bacterial infectious progenies within the cells, was required for the apoptosis. Other chlamydiae [Parachlamydia acanthamoebae and Chlamydia trachomatis (serovars D and L2)] did not induce the same phenomena, indicating that the observed apoptosis may be specific to the Protochlamydia. Furthermore, the bacteria had no effect on the survival of primary PBMCs collected from five volunteers, regardless of activation. We concluded that Protochlamydia induces apoptosis in human-immortal HEp-2 cells and that this endosymbiont could potentially be used as a biological tool for the elucidation of novel host-parasite relationships.

Four Projects in Electricity Suitable for Primary Schools.

ERIC Educational Resources Information Center

Ward, Alan

1983-01-01

Describes simple, electrical projects with dry cell batteries and lamps designed to teach children about conductors, insulators, and switches. Instructions for construction of circuits, the science ideas in each project, and suggestions for extending each of the investigations are included. (JM)

Viability of primary osteoblasts after treatment with tenofovir alafenamide: Lack of cytotoxicity at clinically relevant drug concentrations

PubMed Central

Callebaut, Christian; Liu, Yang; Babusis, Darius; Ray, Adrian; Miller, Michael; Kitrinos, Kathryn

2017-01-01

Tenofovir alafenamide (TAF) is a phosphonoamidate prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV). TAF is approved for the treatment of HIV-1 infection as part of the single-tablet regimen containing elvitegravir, cobicistat, emtricitabine, and TAF. When dosed once-daily, TAF results in approximately 90% lower levels of plasma TFV and a 4-fold increase in intracellular TFV-diphosphate (TFV-DP) in PBMCs compared with the TFV prodrug tenofovir disoproxil fumarate (TDF). Several antiretrovirals, including TDF, have been associated with bone mineral density decreases in patients; the effect of clinically relevant TAF concentrations on primary osteoblast viability was therefore assessed in vitro. Studies in PBMCs determined that a 2-hour TAF exposure at concentrations similar to human plasma Cmax achieved intracellular TFV-DP levels comparable to those observed after the maximum recommended human dose of 25 mg TAF. Comparable intracellular TFV-DP levels were achieved in primary osteoblasts with 2-hour TAF exposure daily for 3 days at concentrations similar to those used for PBMCs (100–400 nM). No change in cell viability was observed in either primary osteoblasts or PBMCs. The mean TAF CC50 in primary osteoblasts after 3 days of daily 2-hour pulses was >500 μM, which is >1033 times higher than the TAF maximum recommended human dose plasma Cmax. In summary, primary osteoblasts were not preferentially loaded by TAF compared with PBMCs, with comparable TFV-DP levels achieved in both cell types. Furthermore, there was no impact on osteoblast cell viability at clinically relevant TAF concentrations. PMID:28182625

Viability of primary osteoblasts after treatment with tenofovir alafenamide: Lack of cytotoxicity at clinically relevant drug concentrations.

PubMed

Callebaut, Christian; Liu, Yang; Babusis, Darius; Ray, Adrian; Miller, Michael; Kitrinos, Kathryn

2017-01-01

Tenofovir alafenamide (TAF) is a phosphonoamidate prodrug of the nucleotide HIV reverse transcriptase inhibitor tenofovir (TFV). TAF is approved for the treatment of HIV-1 infection as part of the single-tablet regimen containing elvitegravir, cobicistat, emtricitabine, and TAF. When dosed once-daily, TAF results in approximately 90% lower levels of plasma TFV and a 4-fold increase in intracellular TFV-diphosphate (TFV-DP) in PBMCs compared with the TFV prodrug tenofovir disoproxil fumarate (TDF). Several antiretrovirals, including TDF, have been associated with bone mineral density decreases in patients; the effect of clinically relevant TAF concentrations on primary osteoblast viability was therefore assessed in vitro. Studies in PBMCs determined that a 2-hour TAF exposure at concentrations similar to human plasma Cmax achieved intracellular TFV-DP levels comparable to those observed after the maximum recommended human dose of 25 mg TAF. Comparable intracellular TFV-DP levels were achieved in primary osteoblasts with 2-hour TAF exposure daily for 3 days at concentrations similar to those used for PBMCs (100-400 nM). No change in cell viability was observed in either primary osteoblasts or PBMCs. The mean TAF CC50 in primary osteoblasts after 3 days of daily 2-hour pulses was >500 μM, which is >1033 times higher than the TAF maximum recommended human dose plasma Cmax. In summary, primary osteoblasts were not preferentially loaded by TAF compared with PBMCs, with comparable TFV-DP levels achieved in both cell types. Furthermore, there was no impact on osteoblast cell viability at clinically relevant TAF concentrations.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

Mycobacterium tuberculosis Infection and Innate Responses in a New Model of Lung Alveolar Macrophages.

PubMed

Woo, Minjeong; Wood, Connor; Kwon, Doyoon; Park, Kyu-Ho Paul; Fejer, György; Delorme, Vincent

2018-01-01

Lung alveolar macrophages (AMs) are in the first line of immune defense against respiratory pathogens and play key roles in the pathogenesis of Mycobacterium tuberculosis ( Mtb ) in humans. Nevertheless, AMs are available only in limited amounts for in vitro studies, which hamper the detailed molecular understanding of host- Mtb interactions in these macrophages. The recent establishment of the self-renewing and primary Max Planck Institute (MPI) cells, functionally very close to lung AMs, opens unique opportunities for in vitro studies of host-pathogen interactions in respiratory diseases. Here, we investigated the suitability of MPI cells as a host cell system for Mtb infection. Bacterial, cellular, and innate immune features of MPI cells infected with Mtb were characterized. Live bacteria were readily internalized and efficiently replicated in MPI cells, similarly to primary murine macrophages and other cell lines. MPI cells were also suitable for the determination of anti-tuberculosis (TB) drug activity. The primary innate immune response of MPI cells to live Mtb showed significantly higher and earlier induction of the pro-inflammatory cytokines TNFα, interleukin 6 (IL-6), IL-1α, and IL-1β, as compared to stimulation with heat-killed (HK) bacteria. MPI cells previously showed a lack of induction of the anti-inflammatory cytokine IL-10 to a wide range of stimuli, including HK Mtb . By contrast, we show here that live Mtb is able to induce significant amounts of IL-10 in MPI cells. Autophagy experiments using light chain 3B immunostaining, as well as LysoTracker labeling of acidic vacuoles, demonstrated that MPI cells efficiently control killed Mtb by elimination through phagolysosomes. MPI cells were also able to accumulate lipid droplets in their cytoplasm following exposure to lipoproteins. Collectively, this study establishes the MPI cells as a relevant, versatile host cell model for TB research, allowing a deeper understanding of AMs functions in this pathology.

Analysis of Fusobacterium persistence and antibiotic response in colorectal cancer.

PubMed

Bullman, Susan; Pedamallu, Chandra S; Sicinska, Ewa; Clancy, Thomas E; Zhang, Xiaoyang; Cai, Diana; Neuberg, Donna; Huang, Katherine; Guevara, Fatima; Nelson, Timothy; Chipashvili, Otari; Hagan, Timothy; Walker, Mark; Ramachandran, Aruna; Diosdado, Begoña; Serna, Garazi; Mulet, Nuria; Landolfi, Stefania; Ramon Y Cajal, Santiago; Fasani, Roberta; Aguirre, Andrew J; Ng, Kimmie; Élez, Elena; Ogino, Shuji; Tabernero, Josep; Fuchs, Charles S; Hahn, William C; Nuciforo, Paolo; Meyerson, Matthew

2017-12-15

Colorectal cancers comprise a complex mixture of malignant cells, nontransformed cells, and microorganisms. Fusobacterium nucleatum is among the most prevalent bacterial species in colorectal cancer tissues. Here we show that colonization of human colorectal cancers with Fusobacterium and its associated microbiome-including Bacteroides , Selenomonas , and Prevotella species-is maintained in distal metastases, demonstrating microbiome stability between paired primary and metastatic tumors. In situ hybridization analysis revealed that Fusobacterium is predominantly associated with cancer cells in the metastatic lesions. Mouse xenografts of human primary colorectal adenocarcinomas were found to retain viable Fusobacterium and its associated microbiome through successive passages. Treatment of mice bearing a colon cancer xenograft with the antibiotic metronidazole reduced Fusobacterium load, cancer cell proliferation, and overall tumor growth. These observations argue for further investigation of antimicrobial interventions as a potential treatment for patients with Fusobacterium -associated colorectal cancer. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Predictive Outcomes for HER2-enriched Cancer Using Growth and Metastasis Signatures Driven By SPARC.

PubMed

Güttlein, Leandro N; Benedetti, Lorena G; Fresno, Cristóbal; Spallanzani, Raúl G; Mansilla, Sabrina F; Rotondaro, Cecilia; Raffo Iraolagoitia, Ximena L; Salvatierra, Edgardo; Bravo, Alicia I; Fernández, Elmer A; Gottifredi, Vanesa; Zwirner, Norberto W; Llera, Andrea S; Podhajcer, Osvaldo L

2017-03-01

Understanding the mechanism of metastatic dissemination is crucial for the rational design of novel therapeutics. The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein which has been extensively associated with human breast cancer aggressiveness although the underlying mechanisms are still unclear. Here, shRNA-mediated SPARC knockdown greatly reduced primary tumor growth and completely abolished lung colonization of murine 4T1 and LM3 breast malignant cells implanted in syngeneic BALB/c mice. A comprehensive study including global transcriptomic analysis followed by biological validations confirmed that SPARC induces primary tumor growth by enhancing cell cycle and by promoting a COX-2-mediated expansion of myeloid-derived suppressor cells (MDSC). The role of SPARC in metastasis involved a COX-2-independent enhancement of cell disengagement from the primary tumor and adherence to the lungs that fostered metastasis implantation. Interestingly, SPARC-driven gene expression signatures obtained from these murine models predicted the clinical outcome of patients with HER2-enriched breast cancer subtypes. In total, the results reveal that SPARC and its downstream effectors are attractive targets for antimetastatic therapies in breast cancer. Implications: These findings shed light on the prometastatic role of SPARC, a key protein expressed by breast cancer cells and surrounding stroma, with important consequences for disease outcome. Mol Cancer Res; 15(3); 304-16. ©2016 AACR . ©2016 American Association for Cancer Research.

Bioreducible Zinc(II)-Coordinative Polyethylenimine with Low Molecular Weight for Robust Gene Delivery of Primary and Stem Cells.

PubMed

Liu, Shuai; Zhou, Dezhong; Yang, Jixiang; Zhou, Hao; Chen, Jiatong; Guo, Tianying

2017-03-30

To transform common low-molecular-weight (LMW) cationic polymers, such as polyethylenimine (PEI), to highly efficient gene vectors would be of great significance but remains challenging. Because LMW cationic polymers perform far less efficiently than their high-molecular-weight counterparts, mainly due to weaker nucleic acid encapsulation, herein we report the design and synthesis of a dipicolylamine-based disulfide-containing zinc(II) coordinative module (Zn-DDAC), which is used to functionalize LMW PEI (M w ≈ 1800 Da) to give a non-viral vector (Zn-PD) with high efficiency and safety in primary and stem cells. Given its high phosphate binding affinity, Zn-DDAC can significantly promote the DNA packaging functionality of PEI 1.8k and improve the cellular uptake of formulated polyplexes, which is particularly critical for hard-to-transfect cell types. Furthermore, Zn-PD polymer can be cleaved by glutathione in cytoplasm to facilitate DNA release post internalization and diminish the cytotoxicity. Consequently, the optimal Zn-PD mediates 1-2 orders of magnitude higher gluciferase activity than commercial transfection reagents, Xfect and PEI 25k , across diverse cell types, including primary and stem cells. Our findings provide a valuable insight into the exploitation of LMW cationic polymers for gene delivery and demonstrate great promise for the development of next-generation non-viral vectors for clinically viable gene therapy.

Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

PubMed

Rimann, Markus; Bono, Epifania; Annaheim, Helene; Bleisch, Matthias; Graf-Hausner, Ursula

2016-08-01

Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application. © 2015 Society for Laboratory Automation and Screening.

Advances in primary recovery: centrifugation and membrane technology.

PubMed

Roush, David J; Lu, Yuefeng

2008-01-01

Significant and continual improvements in upstream processing for biologics have resulted in challenges for downstream processing, both primary recovery and purification. Given the high cell densities achievable in both microbial and mammalian cell culture processes, primary recovery can be a significant bottleneck in both clinical and commercial manufacturing. The combination of increased product titer and low viability leads to significant relative increases in the levels of process impurities such as lipids, intracellular proteins and nucleic acid versus the product. In addition, cell culture media components such as soy and yeast hydrolysates have been widely applied to achieve the cell culture densities needed for higher titers. Many of the process impurities can be negatively charged at harvest pH and can form colloids during the cell culture and harvest processes. The wide size distribution of these particles and the potential for additional particles to be generated by shear forces within a centrifuge may result in insufficient clarification to prevent fouling of subsequent filters. The other residual process impurities can lead to precipitation and increased turbidity during processing and even interference with the performance of the capturing chromatographic step. Primary recovery also poses significant challenges owing to the necessity to execute in an expedient manner to minimize both product degradation and bioburden concerns. Both microfiltration and centrifugation coupled with depth filtration have been employed successfully as primary recovery processing steps. Advances in the design and application of membrane technology for microfiltration and dead-end filtration have contributed to significant improvements in process performance and integration, in some cases allowing for a combination of multiple unit operations in a given step. Although these advances have increased productivity and reliability, the net result is that optimization of primary recovery processes has become substantially more complicated. Ironically, the application of classical chemical engineering approaches to overcome issues in primary recovery and purification (e.g., turbidity and trace impurity removal) are just recently gaining attention. Some of these techniques (e.g., membrane cascades, pretreatment, precipitation, and the use of affinity tags) are now seen almost as disruptive technologies. This paper will review the current and potential future state of research on primary recovery, including relevant papers presented at the 234th American Chemical Society (ACS) National Meeting in Boston.

A reporter mouse model for in vivo tracing and in vitro molecular studies of melanocytic lineage cells and their diseases.

PubMed

Crawford, Melissa; Leclerc, Valerie; Dagnino, Lina

2017-08-15

Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations. This is due, in part, to differences in the conditions used to isolate, purify and culture these cells in individual studies. By breeding ROSA mT/mG and Tyr::CreER T2 mice, we generated animals in which melanocytic lineage cells are identified through expression of green fluorescent protein. We also used defined conditions to systematically investigate the proliferation and migration responses of primary melanocytes on various extracellular matrix (ECM) substrates. Under our culture conditions, mouse melanocytes exhibit doubling times in the range of 10 days, and retain exponential proliferative capacity for 50-60 days. In culture, these melanocytes showed distinct responses to different ECM substrates. Specifically, laminin-332 promoted cell spreading, formation of dendrites, random motility and directional migration. In contrast, low or intermediate concentrations of collagen I promoted adhesion and acquisition of a bipolar morphology, and interfered with melanocyte forward movements. Our systematic evaluation of primary melanocyte responses emphasizes the importance of clearly defining culture conditions for these cells. This, in turn, is essential for the interpretation of melanocyte responses to extracellular cues and to understand the molecular basis of disorders involving the melanocytic cell lineage. © 2017. Published by The Company of Biologists Ltd.

A reporter mouse model for in vivo tracing and in vitro molecular studies of melanocytic lineage cells and their diseases

PubMed Central

Crawford, Melissa; Leclerc, Valerie

2017-01-01

ABSTRACT Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations. This is due, in part, to differences in the conditions used to isolate, purify and culture these cells in individual studies. By breeding ROSAmT/mG and Tyr::CreERT2 mice, we generated animals in which melanocytic lineage cells are identified through expression of green fluorescent protein. We also used defined conditions to systematically investigate the proliferation and migration responses of primary melanocytes on various extracellular matrix (ECM) substrates. Under our culture conditions, mouse melanocytes exhibit doubling times in the range of 10 days, and retain exponential proliferative capacity for 50-60 days. In culture, these melanocytes showed distinct responses to different ECM substrates. Specifically, laminin-332 promoted cell spreading, formation of dendrites, random motility and directional migration. In contrast, low or intermediate concentrations of collagen I promoted adhesion and acquisition of a bipolar morphology, and interfered with melanocyte forward movements. Our systematic evaluation of primary melanocyte responses emphasizes the importance of clearly defining culture conditions for these cells. This, in turn, is essential for the interpretation of melanocyte responses to extracellular cues and to understand the molecular basis of disorders involving the melanocytic cell lineage. PMID:28642245

Directing adult human periodontal ligament-derived stem cells to retinal fate.

PubMed

Huang, Li; Liang, Jiajian; Geng, Yiqun; Tsang, Wai-Ming; Yao, Xiaowu; Jhanji, Vishal; Zhang, Mingzhi; Cheung, Herman S; Pang, Chi Pui; Yam, Gary Hin-Fai

2013-06-06

To investigate the retinal fate competence of human postnatal periodontal ligament (PDL)-derived stem cells (PDLSC) through a directed differentiation mimicking mammalian retinogenesis. Human teeth were collected from healthy subjects younger than 35 years old. Primary PDLSC were isolated by collagenase digestion and cultivated. PDLSC at passage 3 were cultured in the induction media containing Noggin (antagonist of bone morphogenic protein) and Dkk-1 (antagonist of Wnt/β-catenin signaling). Gene expression of neural crest cells, retinal progenitors, and retinal neurons, including photoreceptors, was revealed by RNA analyses, immunofluorescence, and flow cytometry. The neuronal-like property of differentiated cells in response to excitatory glutamate was examined by fluo-4-acetoxymethyl calcium imaging assay. Primary human PDLSC stably expressed marker genes for neural crest (Notch1, BMP2, Slug, Snail, nestin, and Tuj1), mesenchymal stem cell (CD44, CD90, and vimentin), and embryonic stem cell (c-Myc, Klf4, Nanog, and SSEA4). Under low attachment culture, PDLSC generated neurospheres expressing nestin, p75/NGFR, Pax6, and Tuj1 (markers of neural progenitors). When neurospheres were plated on Matrigel-coated surface, they exhibited rosette-like outgrowth. They expressed eye field transcription factors (Pax6, Rx, Lhx, Otx2). By flow cytometry, 94% of cells were Pax6(nuclear)Rx(+), indicative of retinal progenitors. At prolonged induction, they expressed photoreceptor markers (Nrl, rhodopsin and its kinase) and showed significant responsiveness to excitatory glutamate. Primary human PDLSC could be directed to retinal progenitors with competence for photoreceptor differentiation. Human neural crest-derived PDL is readily accessible and can be an ample autologous source of undifferentiated cells for retinal cell regeneration.

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

PubMed Central

Cao, Yuhong; Chen, Haodong; Birey, Fikri; Leal-Ortiz, Sergio A.; Han, Crystal M.; Santiago, Juan G.; Paşca, Sergiu P.; Wu, Joseph C.; Melosh, Nicholas A.

2017-01-01

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation. PMID:28223521

Scanning and transmission electron microscopy of the damage to small intestinal mucosa following X irradiation or hyperthermia

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

Carr, K.E.; Hume, S.P.; Marigold, J.C.

Scanning and transmission electron microscopy (S.E.M. and T.E.M.) and resin histology have been used to investigate the effects on mouse small intestinal villi of heating at 43 degrees C for 20 minutes and of irradiation with 10 Gy X-rays. Damage after irradiation included conical villi and giant cells. Damage after heating included the production of conical and rudimentary villi and the stacking of enterocytes. Individual cells showed signs of abnormalities in their cell membranes, nuclei and cytoplasmic components. The differences in the response after irradiation and hyperthermia are linked to the fact that heating has a primary effect on villousmore » structure, whereas irradiation mainly affects the proliferative pool of crypt cells.« less

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.

Conditional deletion of p53 and Rb in the renin-expressing compartment of the pancreas leads to a highly penetrant metastatic pancreatic neuroendocrine carcinoma

PubMed Central

Glenn, Sean T.; Jones, Craig A.; Sexton, Sandra; LeVea, Charles M.; Caraker, Susan M.; Hajduczok, George; Gross, Kenneth W.

2014-01-01

Efforts to model human pancreatic neuroendocrine tumors (PanNET) in animals have been moderately successful, with minimal evidence for glucagonomas or metastatic spread. The renin gene while classically associated with expression in the kidney is also expressed in many other extra-renal tissues including the pancreas. To induce tumorigenesis within renin specific tissues, floxed alleles of p53 and Rb were selectively abrogated using Cre-recombinase driven by the renin promoter. The primary neoplasm generated is a highly metastatic islet cell carcinoma of the pancreas. Lineage tracing identifies descendants of renin-expressing cells as pancreatic alpha cells despite a lack of active renin expression in the mature pancreas. Both primary and metastatic tumors express high levels of glucagon, furthermore an increased level of glucagon is found in the serum identifying the pancreatic cancer as a functional glucagonoma. This new model is highly penetrant and exhibits robust frequency of metastases to lymph nodes and liver, mimicking human disease and provides a useful platform for better understanding pancreatic endocrine differentiation and development, as well as islet cell carcinogenesis. The use of fluorescent reporters for lineage tracing of the cells contributing to disease initiation and progression provides a unique opportunity to dissect the timeline of disease, examining mechanisms of the metastatic process, as well as recovering primary and metastatic cells for identifying co-operating mutations that are necessary for progression of disease. PMID:24292676

The Androgen-Regulated Calcium-Activated Nucleotidase 1 (CANT1) Is Commonly Overexpressed in Prostate Cancer and Is Tumor-Biologically Relevant in Vitro

PubMed Central

Gerhardt, Josefine; Steinbrech, Corinna; Büchi, Oralea; Behnke, Silvia; Bohnert, Annette; Fritzsche, Florian; Liewen, Heike; Stenner, Frank; Wild, Peter; Hermanns, Thomas; Müntener, Michael; Dietel, Manfred; Jung, Klaus; Stephan, Carsten; Kristiansen, Glen

2011-01-01

Previously, we identified the calcium-activated nucleotidase 1 (CANT1) transcript as up-regulated in prostate cancer. Now, we studied CANT1 protein expression in a large cohort of nearly 1000 prostatic tissue samples including normal tissue, prostatic intraepithelial neoplasia (PIN), primary carcinomas, metastases, and castrate-resistant carcinomas, and further investigated its functional relevance. CANT1 displayed predominantly a Golgi-type immunoreactivity with additional and variable cytoplasmic staining. In comparison to normal tissues, the staining intensity was significantly increased in PIN lesions and cancer. In cancer, high CANT1 levels were associated with a better prognosis, and castrate-resistant carcinomas commonly showed lower CANT1 levels than primary carcinomas. The functional role of CANT1 was investigated using RNA interference in two prostate cancer cell lines with abundant endogenous CANT1 protein. On CANT1 knockdown, a significantly diminished cell number and DNA synthesis rate, a cell cycle arrest in G1 phase, and a strong decrease of cell transmigration rate and wound healing capacity of CANT1 knockdown cells was found. However, on forced CANT1 overexpression, cell proliferation and migration remained unchanged. In summary, CANT1 is commonly overexpressed in the vast majority of primary prostate carcinomas and in the precursor lesion PIN and may represent a novel prognostic biomarker. Moreover, this is the first study to demonstrate a functional involvement of CANT1 in tumor biology. PMID:21435463

Conditional deletion of p53 and Rb in the renin-expressing compartment of the pancreas leads to a highly penetrant metastatic pancreatic neuroendocrine carcinoma.

PubMed

Glenn, S T; Jones, C A; Sexton, S; LeVea, C M; Caraker, S M; Hajduczok, G; Gross, K W

2014-12-11

Efforts to model human pancreatic neuroendocrine tumors (PanNETs) in animals have been moderately successful, with minimal evidence for glucagonomas or metastatic spread. The renin gene, although classically associated with expression in the kidney, is also expressed in many other extrarenal tissues including the pancreas. To induce tumorigenesis within rennin-specific tissues, floxed alleles of p53 and Rb were selectively abrogated using Cre-recombinase driven by the renin promoter. The primary neoplasm generated is a highly metastatic islet cell carcinoma of the pancreas. Lineage tracing identifies descendants of renin-expressing cells as pancreatic alpha cells despite a lack of active renin expression in the mature pancreas. Both primary and metastatic tumors express high levels of glucagon; furthermore, an increased level of glucagon is found in the serum, identifying the pancreatic cancer as a functional glucagonoma. This new model is highly penetrant and exhibits robust frequency of metastases to the lymph nodes and the liver, mimicking human disease, and provides a useful platform for better understanding pancreatic endocrine differentiation and development, as well as islet cell carcinogenesis. The use of fluorescent reporters for lineage tracing of the cells contributing to disease initiation and progression provides an unique opportunity to dissect the timeline of disease, examining mechanisms of the metastatic process, as well as recovering primary and metastatic cells for identifying cooperating mutations that are necessary for progression of disease.

Epigenetic determinants of ovarian clear cell carcinoma biology

PubMed Central

Yamaguchi, Ken; Huang, Zhiqing; Matsumura, Noriomi; Mandai, Masaki; Okamoto, Takako; Baba, Tsukasa; Konishi, Ikuo; Berchuck, Andrew; Murphy, Susan K.

2015-01-01

Targeted approaches have revealed frequent epigenetic alterations in ovarian cancer, but the scope and relation of these changes to histologic subtype of disease is unclear. Genome-wide methylation and expression data for 14 clear cell carcinoma (CCC), 32 non-CCC, and 4 corresponding normal cell lines were generated to determine how methylation profiles differ between cells of different histological derivations of ovarian cancer. Consensus clustering showed that CCC is epigenetically distinct. Inverse relationships between expression and methylation in CCC were identified, suggesting functional regulation by methylation, and included 22 hypomethylated (UM) genes and 276 hypermethylated (HM) genes. Categorical and pathway analyses indicated that the CCC-specific UM genes were involved in response to stress and many contain hepatocyte nuclear factor (HNF) 1 binding sites, while the CCC-specific HM genes included members of the estrogen receptor alpha (ERalpha) network and genes involved in tumor development. We independently validated the methylation status of 17 of these pathway-specific genes, and confirmed increased expression of HNF1 network genes and repression of ERalpha pathway genes in CCC cell lines and primary cancer tissues relative to non-CCC specimens. Treatment of three CCC cell lines with the demethylating agent Decitabine significantly induced expression for all five genes analyzed. Coordinate changes in pathway expression were confirmed using two primary ovarian cancer datasets (p<0.0001 for both). Our results suggest that methylation regulates specific pathways and biological functions in CCC, with hypomethylation influencing the characteristic biology of the disease while hypermethylation contributes to the carcinogenic process. PMID:24382740

TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

PubMed

Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

2010-01-01

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

Inhibition of SIRT1 Catalytic Activity Increases p53 Acetylation but Does Not Alter Cell Survival following DNA Damage

PubMed Central

Solomon, Jonathan M.; Pasupuleti, Rao; Xu, Lei; McDonagh, Thomas; Curtis, Rory; DiStefano, Peter S.; Huber, L. Julie

2006-01-01

Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells. PMID:16354677

A Metabolic Biofuel Cell: Conversion of Human Leukocyte Metabolic Activity to Electrical Currents

PubMed Central

2011-01-01

An investigation of the electrochemical activity of human white blood cells (WBC) for biofuel cell (BFC) applications is described. WBCs isolated from whole human blood were suspended in PBS and introduced into the anode compartment of a proton exchange membrane (PEM) fuel cell. The cathode compartment contained a 50 mM potassium ferricyanide solution. Average current densities between 0.9 and 1.6 μA cm-2 and open circuit potentials (Voc) between 83 and 102 mV were obtained, which were both higher than control values. Cyclic voltammetry was used to investigate the electrochemical activity of the activated WBCs in an attempt to elucidate the mechanism of electron transfer between the cells and electrode. Voltammograms were obtained for the WBCs, including peripheral blood mononuclear cells (PBMCs - a lymphocyte-monocyte mixture isolated on a Ficoll gradient), a B lymphoblastoid cell line (BLCL), and two leukemia cell lines, namely K562 and Jurkat. An oxidation peak at about 363 mV vs. SCE for the PMA (phorbol ester) activated primary cells, with a notable absence of a reduction peak was observed. Oxidation peaks were not observed for the BLCL, K562 or Jurkat cell lines. HPLC confirmed the release of serotonin (5-HT) from the PMA activated primary cells. It is believed that serotonin, among other biochemical species released by the activated cells, contributes to the observed BFC currents. PMID:21569243

Bio optofluidics cell sorter: cell-BOCS concept and applications

NASA Astrophysics Data System (ADS)

Roth, Tue; Glückstad, Jesper

2012-03-01

The cell-BOCS is a novel microfluidics based cell-sorting instrument utilizing next generation optical trapping technology developed at the Technical University of Denmark. It is targeted emerging bio-medical research and diagnostics markets where it for certain applications offers a number of advantages over conventional fluorescence activated cell-sorting (FACSTM) technology. Advantages include gentle handling of cells, sterile sorting, easy operation, small footprint and lower cost allowing out-of-core-facility use. Application examples are found within sorting of fragile transfected cells, high value samples and primary cell lines, where traditional FACS technology has limited application due to it's droplet-based approach to cell-sorting. In the diagnostics field, in particular applying the cell-BOCS for isolating pure populations of circulating tumor cells is an area that has generated a lot of interest.

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.

CAVEOLINS AND LUNG FUNCTION

PubMed Central

Maniatis, Nikolaos A.; Chernaya, Olga; Shinin, Vasily; Minshall, Richard D.

2012-01-01

The primary function of the mammalian lung is to facilitate diffusion of oxygen to venous blood and to ventilate carbon dioxide produced by catabolic reactions within cells. However, it is also responsible for a variety of other important functions, including host defense and production of vasoactive agents to regulate not only systemic blood pressure, but also water, electrolyte and acid-base balance. Caveolin-1 is highly expressed in the majority of cell types in the lung, including epithelial, endothelial, smooth muscle, connective tissue cells, and alveolar macrophages. Deletion of caveolin-1 in these cells results in major functional aberrations, suggesting that caveolin-1 may be crucial to lung homeostasis and development. Furthermore, generation of mutant mice that under-express caveolin-1 results in severe functional distortion with phenotypes covering practically the entire spectrum of known lung diseases, including pulmonary hypertension, fibrosis, increased endothelial permeability, and immune defects. In this Chapter, we outline the current state of knowledge regarding caveolin-1-dependent regulation of pulmonary cell functions and discuss recent research findings on the role of caveolin-1 in various pulmonary disease states, including obstructive and fibrotic pulmonary vascular and inflammatory diseases. PMID:22411320

Identification of BCAP, a new protein associated with basal bodies and centrioles.

PubMed

Ponsard, Cecile; Seltzer, Virginie; Perret, Eric; Tournier, Frederic; Middendorp, Sandrine

2007-05-01

Cilia exert critical functions in numerous organisms, including that of cell motility, fluid transport and protozoan locomotion. Defects in this organelle can lead to lethal pathologies in humans, including primary ciliary dyskinesia. An understanding of the cilia formation process would lead to better characterization of defects involved in such pathologies. In the present study, we identified a gene encoding a novel human protein, BCAP for Basal body Centriole-Associated Protein, which shares homologies with a previously described protein, Outer Dense Fiber 2 (ODF2). ODF2, a major component of the sperm tail cytoskeleton, is required for the formation of mother centriole distal/subdistal appendages and the generation of primary cilia. Here, we show that the bcap gene contains 18 alternatively spliced exons and encodes five different isoforms, three long and two short ones. BCAP is preferentially expressed in cilia/flagella containing tissues. Moreover, its expression is correlated with cilia formation during mucociliary differentiation of human nasal epithelial cells. Using immunofluorescence analyses, BCAP was localized within basal bodies of ciliated cells and within centrioles of proliferating cells. In light of the several spliced isoforms of BCAP and the particular localization of the protein, BCAP isoforms could play distinct roles in cilia and in centrosomes.

ToxCast Phase I

EPA Pesticide Factsheets

Background: Chemical toxicity testing is being transformed by advances in biology and computer modeling, concerns over animal use and the thousands of environmental chemicals lacking toxicity data. EPA's ToxCast program aims to address these concerns by screening and prioritizing chemicals for potential human toxicity using in vitro assays and in silico approaches. Objectives: This project aims to evaluate the use of in vitro assays for understanding the types of molecular and pathway perturbations caused by environmental chemicals and to build initial prioritization models of in vivo toxicity. Methods: We tested 309 mostly pesticide active chemicals in 467 assays across 9 technologies, including high-throughput cell-free assays and cell-based assays in multiple human primary cells and cell lines, plus rat primary hepatocytes. Both individual and composite scores for effects on genes and pathways were analyzed. Results: Chemicals display a broad spectrum of activity at the molecular and pathway levels. Many expected interactions are seen, including endocrine and xenobiotic metabolism enzyme activity. Chemicals range in promiscuity across pathways, from no activity to affecting dozens of pathways. We find a statistically significant inverse association between the number of pathways perturbed by a chemical at low in vitro concentrations and the lowest in vivo dose at which a chemical causes toxicity. We also find associations between a small set in vitro ass

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.

Differential gene expression profiling of matched primary renal cell carcinoma and metastases reveals upregulation of extracellular matrix genes.

PubMed

Ho, T H; Serie, D J; Parasramka, M; Cheville, J C; Bot, B M; Tan, W; Wang, L; Joseph, R W; Hilton, T; Leibovich, B C; Parker, A S; Eckel-Passow, J E

2017-03-01

The majority of renal cell carcinoma (RCC) studies analyze primary tumors, and the corresponding results are extrapolated to metastatic RCC tumors. However, it is unknown if gene expression profiles from primary RCC tumors differs from patient-matched metastatic tumors. Thus, we sought to identify differentially expressed genes between patient-matched primary and metastatic RCC tumors in order to understand the molecular mechanisms underlying the development of RCC metastases. We compared gene expression profiles between patient-matched primary and metastatic RCC tumors using a two-stage design. First, we used Affymetrix microarrays on 15 pairs of primary RCC [14 clear cell RCC (ccRCC), 1 papillary] tumors and patient-matched pulmonary metastases. Second, we used a custom NanoString panel to validate seven candidate genes in an independent cohort of 114 ccRCC patients. Differential gene expression was evaluated using a mixed effect linear model; a random effect denoting patient was included to account for the paired data. Third, The Cancer Genome Atlas (TCGA) data were used to evaluate associations with metastasis-free and overall survival in primary ccRCC tumors. We identified and validated up regulation of seven genes functionally involved in the formation of the extracellular matrix (ECM): DCN, SLIT2, LUM, LAMA2, ADAMTS12, CEACAM6 and LMO3. In primary ccRCC, CEACAM6 and LUM were significantly associated with metastasis-free and overall survival (P < 0.01). We evaluated gene expression profiles using the largest set to date, to our knowledge, of patient-matched primary and metastatic ccRCC tumors and identified up regulation of ECM genes in metastases. Our study implicates up regulation of ECM genes as a critical molecular event leading to visceral, bone and soft tissue metastases in ccRCC. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Genomic Instability and Telomere Fusion of Canine Osteosarcoma Cells

PubMed Central

Maeda, Junko; Yurkon, Charles R.; Fujisawa, Hiroshi; Kaneko, Masami; Genet, Stefan C.; Roybal, Erica J.; Rota, Garrett W.; Saffer, Ethan R.; Rose, Barbara J.; Hanneman, William H.; Thamm, Douglas H.; Kato, Takamitsu A.

2012-01-01

Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA. PMID:22916246

Randomized Phase III Trial of Adjuvant Pazopanib Versus Placebo After Nephrectomy in Patients With Localized or Locally Advanced Renal Cell Carcinoma.

PubMed

Motzer, Robert J; Haas, Naomi B; Donskov, Frede; Gross-Goupil, Marine; Varlamov, Sergei; Kopyltsov, Evgeny; Lee, Jae Lyun; Melichar, Bohuslav; Rini, Brian I; Choueiri, Toni K; Zemanova, Milada; Wood, Lori A; Reaume, M Neil; Stenzl, Arnulf; Chowdhury, Simon; Lim, Ho Yeong; McDermott, Ray; Michael, Agnieszka; Bao, Weichao; Carrasco-Alfonso, Marlene J; Aimone, Paola; Voi, Maurizio; Doehn, Christian; Russo, Paul; Sternberg, Cora N

2017-12-10

Purpose This phase III trial evaluated the efficacy and safety of pazopanib versus placebo in patients with locally advanced renal cell carcinoma (RCC) at high risk for relapse after nephrectomy. Patients and Methods A total of 1,538 patients with resected pT2 (high grade) or ≥ pT3, including N1, clear cell RCC were randomly assigned to pazopanib or placebo for 1 year; 403 patients received a starting dose of 800 mg or placebo. To address toxicity attrition, the 800-mg starting dose was lowered to 600 mg, and the primary end point analysis was changed to disease-free survival (DFS) for pazopanib 600 mg versus placebo (n = 1,135). Primary analysis was performed after 350 DFS events in the intent-to-treat (ITT) pazopanib 600 mg group (ITT 600mg ), and DFS follow-up analysis was performed 12 months later. Secondary end point analyses included DFS with ITT pazopanib 800 mg (ITT 800mg ) and safety. Results The primary analysis results of DFS ITT 600mg favored pazopanib but did not show a significant improvement over placebo (hazard ratio [HR], 0.86; 95% CI, 0.70 to 1.06; P = .165). The secondary analysis of DFS in ITT 800mg (n = 403) yielded an HR of 0.69 (95% CI, 0.51 to 0.94). Follow-up analysis in ITT 600mg yielded an HR of 0.94 (95% CI, 0.77 to 1.14). Increased ALT and AST were common adverse events leading to treatment discontinuation in the pazopanib 600 mg (ALT, 16%; AST, 5%) and 800 mg (ALT, 18%; AST, 7%) groups. Conclusion The results of the primary DFS analysis of pazopanib 600 mg showed no benefit over placebo in the adjuvant setting.

Non-aqueous primary cell

NASA Astrophysics Data System (ADS)

James, S. D.; Smith, P. H.; Oneill, K. M.; Wilson, M. H.

1986-05-01

This patent application relates to electrochemical cells and especially to high-energy, liquid cathode, non-aqueous lithium electrochemical cells free from highly toxic materials. A non-aqueous lithium electrochemical cell is described which includes a halocarbon cathode depolarizer which is 1,2-dichloroethane, 1.1,2-trichloroethane, 1,1,2,2-tetrachloroethane, 1,2-dichloro-1,1-difluoroethane or mixtures thereof and a cathode catalyst which is copper, rhodium, palladium, cobalt phthalocyanine, nickel phthalocyanine, iron phthalocyanine, a cobalt tetraaza-(14)-annulene, a nickel tetraaza-(14)-annulene, a iron tetraaza-(14)-annulene, a cobalt porphyrin, a nickel porphyrin, a iron porphyrin, or a mixture thereof.

Silencing the Snail-dependent RNA splice regulator ESRP1 drives malignant transformation of human pulmonary epithelial cells. | Office of Cancer Genomics

Cancer.gov

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated including in non-small cell lung cancer (NSCLC). Here we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo.

Acute Leukemia and Concurrent Mediastinal Germ Cell Tumor: Case Report and Literature Review.

PubMed

Maese, Luke; Li, K David; Xu, Xinjie; Afify, Zeinab; Paxton, Christian N; Putnam, Angelica

2017-04-01

There is a known association of primary nonseminomatous mediastinal germ cell tumors (NSMGCT) and hematologic malignancy in younger males not linked to treatment. When combined these two rare entities convey a very poor prognosis. Here we report a 16-year-old male with an anterior mediastinal mass diagnosed as a malignant germ cell tumor based on elevation of serologic markers. He was found to have acute leukemia with megakaryocytic differentiation several days later. We focus our report on the pathologic findings, including a review of the literature, and a novel molecular analysis of the germ cell tumor.

Pluripotency of Stem Cells from Human Exfoliated Deciduous Teeth for Tissue Engineering

PubMed Central

Rosa, Vinicius; Dubey, Nileshkumar; Islam, Intekhab; Min, Kyung-San; Nör, Jacques E.

2016-01-01

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative pluripotent cells that can be retrieved from primary teeth. Although SHED are isolated from the dental pulp, their differentiation potential is not limited to odontoblasts only. In fact, SHED can differentiate into several cell types including neurons, osteoblasts, adipocytes, and endothelial cells. The high plasticity makes SHED an interesting stem cell model for research in several biomedical areas. This review will discuss key findings about the characterization and differentiation of SHED into odontoblasts, neurons, and hormone secreting cells (e.g., hepatocytes and islet-like cell aggregates). The outcomes of the studies presented here support the multipotency of SHED and their potential to be used for tissue engineering-based therapies. PMID:27313627

Primary Central Nervous System T-Cell Lymphoma With Aberrant Expression of CD20 and CD79a: A Diagnostic Pitfall.

PubMed

Gupta, Neha; Nasim, Mansoor; Spitzer, Silvia G; Zhang, Xinmin

2017-10-01

Primary central nervous system T-cell lymphoma (PCNSTCL) is rare, accounting for 2% of CNS lymphomas. We report the first case of PCNSTCL with aberrant expression of CD20 and CD79a in an 81-year-old man with a left periventricular brain mass. A biopsy revealed dense lymphoid infiltrate consisting of medium-sized cells in a background of gliosis and many histiocytes. The lymphoid cells were positive for CD2, CD3, CD7, CD8, T-cell intracellular antigen-1, granzyme B, CD20, and CD79a and negative for CD4, CD5, PAX-5, OCT-2, BOB-1, human herpes virus-8, and Epstein-Barr virus-encoded small RNAs. Molecular studies revealed clonal TCR-β and TCR-γ gene rearrangements and negative immunoglobulin gene rearrangements. The patient was treated with chemotherapy (vincristine and methotrexate) and rituximab, but he died 1 month after the diagnosis. This is a unique case that emphasizes the use of a multimodal approach, including a broad immunohistochemical panel and molecular studies in lineage determination for lymphomas with ambiguous phenotype.

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis.

PubMed

Paschall, Amy V; Liu, Kebin

2016-08-14

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 10(4) cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

Infection rate and tissue localization of murine IL-12p40-producing monocyte-derived CD103(+) lung dendritic cells during pulmonary tuberculosis.

PubMed

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103(+) dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40(+) cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype.

Infection Rate and Tissue Localization of Murine IL-12p40-Producing Monocyte-Derived CD103+ Lung Dendritic Cells during Pulmonary Tuberculosis

PubMed Central

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D.; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype. PMID:23861965

Primary plasma cell leukemia 2.0: advances in biology and clinical management.

PubMed

Neri, Antonino; Todoerti, Katia; Lionetti, Marta; Simeon, Vittorio; Barbieri, Marzia; Nozza, Filomena; Vona, Gabriella; Pompa, Alessandra; Baldini, Luca; Musto, Pellegrino

2016-11-01

Primary plasma cell leukemia (PPCL) is a rare and aggressive variant of multiple myeloma. The introduction of novel agents and modern technologies has recently partially changed the clinical and biological scenario of this malignancy, allowing limited, but not negligible, progresses. Areas covered: We will discuss: the complex landscape of genetic alterations in PPCL, derived from conventional and high-throughput technologies; the best available treatments for PPCL; the possible future therapeutic perspectives. Expert commentary: PPCL requires an immediate and intensive multi-phase treatment with short therapy-free intervals, which should include novel agents and autologous stem cell transplantation in eligible patients. Allogeneic transplantation should be considered in selected cases. In older and/or frailer individuals, personalized approaches should be applied. Integrated treatments with next generation proteasome inhibitors/IMIDs and monoclonal antibodies are currently planned or under investigation. The identification of novel genomic biomarkers may be potentially helpful for risk stratification and future personalized therapies.

Prognostic Factors for Survival in Patients Treated With Stereotactic Radiosurgery for Recurrent Brain Metastases After Prior Whole Brain Radiotherapy

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

Caballero, Jorge A.; Sneed, Penny K., E-mail: psneed@radonc.ucsf.edu; Lamborn, Kathleen R.

2012-05-01

Purpose: To evaluate prognostic factors for survival after stereotactic radiosurgery (SRS) for new, progressive, or recurrent brain metastases (BM) after prior whole brain radiotherapy (WBRT). Methods and Materials: Patients treated between 1991 and 2007 with Gamma Knife SRS for BM after prior WBRT were retrospectively reviewed. Potential prognostic factors were analyzed overall and by primary site using univariate and stepwise multivariate analyses and recursive partitioning analysis, including age, Karnofsky performance status (KPS), primary tumor control, extracranial metastases, number of BM treated, total SRS target volume, and interval from WBRT to SRS. Results: A total of 310 patients were analyzed, includingmore » 90 breast, 113 non-small-cell lung, 31 small-cell lung, 42 melanoma, and 34 miscellaneous patients. The median age was 56, KPS 80, number of BM treated 3, and interval from WBRT to SRS 8.1 months; 76% had controlled primary tumor and 60% had extracranial metastases. The median survival was 8.4 months overall and 12.0 vs. 7.9 months for single vs. multiple BM treated (p = 0.001). There was no relationship between number of BM and survival after excluding single-BM patients. On multivariate analysis, favorable prognostic factors included age <50, smaller total target volume, and longer interval from WBRT to SRS in breast cancer patients; smaller number of BM, KPS >60, and controlled primary in non-small-cell lung cancer patients; and smaller total target volume in melanoma patients. Conclusions: Among patients treated with salvage SRS for BM after prior WBRT, prognostic factors appeared to vary by primary site. Although survival time was significantly longer for patients with a single BM, the median survival time of 7.9 months for patients with multiple BM seems sufficiently long for salvage SRS to appear to be worthwhile, and no evidence was found to support the use of a cutoff for number of BM appropriate for salvage SRS.« less

Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

PubMed

Esch, Mandy B; Prot, Jean-Matthieu; Wang, Ying I; Miller, Paula; Llamas-Vidales, Jose Ricardo; Naughton, Brian A; Applegate, Dawn R; Shuler, Michael L

2015-05-21

We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs.

Effects of 5-fluorouracil in nuclear and cellular morphology, proliferation, cell cycle, apoptosis, cytoskeletal and caveolar distribution in primary cultures of smooth muscle cells.

PubMed

Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

2013-01-01

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer.

Effects of 5-Fluorouracil in Nuclear and Cellular Morphology, Proliferation, Cell Cycle, Apoptosis, Cytoskeletal and Caveolar Distribution in Primary Cultures of Smooth Muscle Cells

PubMed Central

Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

2013-01-01

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer. PMID:23646193

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.

Surgical Management of Metastatic Disease.

PubMed

Keung, Emily Z; Fairweather, Mark; Raut, Chandrajit P

2016-10-01

Sarcomas are rare cancers of mesenchymal cell origin that include many histologic subtypes and molecularly distinct entities. For primary resectable sarcoma, surgery is the mainstay of treatment. Despite treatment, approximately 50% of patients with soft tissue sarcoma are diagnosed with or develop distant metastases, significantly affecting their survival. Although systemic therapy with conventional chemotherapy remains the primary treatment modality for those with metastatic sarcoma, increased survival has been achieved in select patients who receive multimodality therapy, including surgery, for their metastatic disease. This article provides an overview of the literature on surgical management of pulmonary and hepatic sarcoma metastases. Copyright © 2016 Elsevier Inc. All rights reserved.

A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles.

PubMed

Singh, Bir; Avci, Utku; Eichler Inwood, Sarah E; Grimson, Mark J; Landgraf, Jeff; Mohnen, Debra; Sørensen, Iben; Wilkerson, Curtis G; Willats, William G T; Haigler, Candace H

2009-06-01

Cotton (Gossypium hirsutum) provides the world's dominant renewable textile fiber, and cotton fiber is valued as a research model because of its extensive elongation and secondary wall thickening. Previously, it was assumed that fibers elongated as individual cells. In contrast, observation by cryo-field emission-scanning electron microscopy of cotton fibers developing in situ within the boll demonstrated that fibers elongate within tissue-like bundles. These bundles were entrained by twisting fiber tips and consolidated by adhesion of a cotton fiber middle lamella (CFML). The fiber bundles consolidated via the CFML ultimately formed a packet of fiber around each seed, which helps explain how thousands of cotton fibers achieve their great length within a confined space. The cell wall nature of the CFML was characterized using transmission electron microscopy, including polymer epitope labeling. Toward the end of elongation, up-regulation occurred in gene expression and enzyme activities related to cell wall hydrolysis, and targeted breakdown of the CFML restored fiber individuality. At the same time, losses occurred in certain cell wall polymer epitopes (as revealed by comprehensive microarray polymer profiling) and sugars within noncellulosic matrix components (as revealed by gas chromatography-mass spectrometry analysis of derivatized neutral and acidic glycosyl residues). Broadly, these data show that adhesion modulated by an outer layer of the primary wall can coordinate the extensive growth of a large group of cells and illustrate dynamic changes in primary wall structure and composition occurring during the differentiation of one cell type that spends only part of its life as a tissue.

Emergent Behaviors from a Cellular Automaton Model for Invasive Tumor Growth in Heterogeneous Microenvironments

PubMed Central

Jiao, Yang; Torquato, Salvatore

2011-01-01

Understanding tumor invasion and metastasis is of crucial importance for both fundamental cancer research and clinical practice. In vitro experiments have established that the invasive growth of malignant tumors is characterized by the dendritic invasive branches composed of chains of tumor cells emanating from the primary tumor mass. The preponderance of previous tumor simulations focused on non-invasive (or proliferative) growth. The formation of the invasive cell chains and their interactions with the primary tumor mass and host microenvironment are not well understood. Here, we present a novel cellular automaton (CA) model that enables one to efficiently simulate invasive tumor growth in a heterogeneous host microenvironment. By taking into account a variety of microscopic-scale tumor-host interactions, including the short-range mechanical interactions between tumor cells and tumor stroma, degradation of the extracellular matrix by the invasive cells and oxygen/nutrient gradient driven cell motions, our CA model predicts a rich spectrum of growth dynamics and emergent behaviors of invasive tumors. Besides robustly reproducing the salient features of dendritic invasive growth, such as least-resistance paths of cells and intrabranch homotype attraction, we also predict nontrivial coupling between the growth dynamics of the primary tumor mass and the invasive cells. In addition, we show that the properties of the host microenvironment can significantly affect tumor morphology and growth dynamics, emphasizing the importance of understanding the tumor-host interaction. The capability of our CA model suggests that sophisticated in silico tools could eventually be utilized in clinical situations to predict neoplastic progression and propose individualized optimal treatment strategies. PMID:22215996

[The significance of pedigree genetic screening and rapid immunological parameters in the diagnosis of primary hemophagocytic lymphohistiocytosis].

PubMed

Zhang, J; Wang, Y N; Wang, J S; Wu, L; Wei, N; Fu, L; Gao, Z; Chen, J H; Pei, R J; Wang, Z

2016-07-01

To investigate the significance of pedigree genetic screening and rapid immunological parameters in the diagnosis of primary hemophagocytic lymphohistiocytosis (HLH). Four cases of primary HLH patients with PRF1, UNC13D and SH2D1A gene mutations were conducted pedigree investigation, including family genetic screening and detections of immunological parameters (NK cell activity, CD107a degranulation and expression of HLH related defective protein), to evaluate the significance of these different indicators in the diagnosis of primary HLH and explore their correlations. The DNA mutations of the four families included missense mutation c.T172C (p.S58P) and non- frameshift deletions c.1083_1094del (p.361_365del), missense mutation c.C1349T (p.T450M) and frameshift mutation c.1090_1091delCT (p.T364fsX93) in PRF1 gene, missense mutation c.G2588A (p.G863D) in UNC13D gene and hemizygous mutation c.32T>G (p.I11S) in SH2D1A gene. The patients and their family members presented decreased NK cell activities. Individuals who carried mutations of PRF1 gene and SH2D1A gene showed low expression of perforin (PRF1) and signaling lymphocytic activation molecule associated protein (SAP). And the patient with UNC13D gene mutation and his family member with identical mutation showed significant reducing cytotoxic degranulation function (expression of CD107a). Pedigree genetic screening and rapid detection of immunological parameters might play an important role in the diagnosis of primary HLH, and both of them had good consistency. As an efficient detection means, the rapid immunological detection indicators would provide reliable basis for the early diagnosis of the primary HLH.

The graphic cell method: a new look at digitizing geologic maps

USGS Publications Warehouse

Hanley, J.T.

1982-01-01

The graphic cell method is an alternative method of digitizing areal geologic information. It involves a discrete-point sampling scheme in which the computer establishes a matrix of cells over the map. Each cell and the whole cell is assigned the identity or value of the geologic information that is recognized at its center. Cell size may be changed to suit the needs of the user. The computer program resolves the matrix and identifies potential errors such as multiple assignments. Input includes the digitized boundaries of each geologic formation. This method should eliminate a primary bottleneck in the creation and testing of geomathematical models in such disciplines as resource appraisal. ?? 1982.

The Business Case for Fuel Cells: Delivering Sustainable Value

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

Curtin, Sandra; Gangi, Jennifer

This report, written and compiled by Argonne National Laboratory and the Fuel Cell and Hydrogen Energy Association with support from the Fuel Cell Technologies Office, provides an overview of private sector fuel cell installations at U.S. businesses as of December 31, 2016. Over the past few decades, hundreds of thousands of fuel cells have been installed around the world, for primary or backup power, as well as in various other applications including portable and emergency backup power. Fuel cells have also been deployed in other applications such as heat and electricity for homes and apartments, material handling, passenger vehicles, buses,more » and remote, off-grid sites.« less

New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells.

PubMed

Xiao, Wenjin; Perry, Guillaume; Komori, Kikuo; Sakai, Yasuyuki

2015-11-01

To develop an in vitro liver tissue equivalent, hepatocytes should be cocultured with liver non-parenchymal cells to mimic the in vivo physiological microenvironments. In this work, we describe a physiologically-relevant liver tissue model by hierarchically organizing layers of primary rat hepatocytes and human liver sinusoidal endothelial cells (TMNK-1) on an oxygen-permeable polydimethylsiloxane (PDMS) membrane, which facilitates direct oxygenation by diffusion through the membrane. This in vivo-mimicking hierarchical coculture was obtained by simply proceeding the overlay of TMNK-1 cells on the hepatocyte layer re-formed on the collagen immobilized PDMS membranes. The comparison of hepatic functionalities was achieved between coculture and sandwich culture with Matrigel, in the presence and absence of direct oxygenation. A complete double-layered structure of functional liver cells with vertical contact between hepatocytes and TMNK-1 was successfully constructed in the coculture with direct oxygen supply and was well-maintained for 14 days. The hepatocytes in this hierarchical culture exhibited improved survival, functional bile canaliculi formation, cellular level polarization and maintenance of metabolic activities including Cyp1A1/2 activity and albumin production. By contrast, the two cell populations formed discontinuous monolayers on the same surfaces in the non-oxygen-permeable cultures. These results demonstrate that (i) the direct oxygenation through the PDMS membranes enables very simple formation of a hierarchical structure consisting of a hepatocyte layer and a layer of TMNK-1 and (ii) we may include other non-parenchymal cells in this format easily, which can be widely applicable to other epithelial organs.

Preferential and Increased Uptake of Hydroxyl-Terminated PAMAM Dendrimers by Activated Microglia in Rabbit Brain Mixed Glial Culture.

PubMed

Alnasser, Yossef; Kambhampati, Siva P; Nance, Elizabeth; Rajbhandari, Labchan; Shrestha, Shiva; Venkatesan, Arun; Kannan, Rangaramanujam M; Kannan, Sujatha

2018-04-27

Polyamidoamine (PAMAM) dendrimers are multifunctional nanoparticles with tunable physicochemical features, making them promising candidates for targeted drug delivery in the central nervous system (CNS). Systemically administered dendrimers have been shown to localize in activated glial cells, which mediate neuroinflammation in the CNS. These dendrimers delivered drugs specifically to activated microglia, producing significant neurological improvements in multiple brain injury models, including in a neonatal rabbit model of cerebral palsy. To gain further insight into the mechanism of dendrimer cell uptake, we utilized an in vitro model of primary glial cells isolated from newborn rabbits to assess the differences in hydroxyl-terminated generation 4 PAMAM dendrimer (D4-OH) uptake by activated and non-activated glial cells. We used fluorescently-labelled D4-OH (D-Cy5) as a tool for investigating the mechanism of dendrimer uptake. D4-OH PAMAM dendrimer uptake was determined by fluorescence quantification using confocal microscopy and flow cytometry. Our results indicate that although microglial cells in the mixed cell population demonstrate early uptake of dendrimers in this in vitro system, activated microglia take up more dendrimer compared to resting microglia. Astrocytes showed delayed and limited uptake. We also illustrated the differences in mechanism of uptake between resting and activated microglia using different pathway inhibitors. Both resting and activated microglia primarily employed endocytotic pathways, which are enhanced in activated microglial cells. Additionally, we demonstrated that hydroxyl terminated dendrimers are taken up by primary microglia using other mechanisms including pinocytosis, caveolae, and aquaporin channels for dendrimer uptake.

Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system

PubMed Central

Stoff-Khalili, Mariam A; Stoff, Alexander; Rivera, Angel A; Banerjee, Nilam S; Everts, Maaike; Young, Scott; Siegal, Gene P; Richter, Dirk F; Wang, Minghui; Dall, Peter; Mathis, J Michael; Zhu, Zeng B; Curiel, David T

2005-01-01

Introduction In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Adenoviral vector mediated strategies for breast cancer gene therapy and virotherapy are a promising novel therapeutic platform for the treatment of breast cancer. However, the promiscuous tropism of adenoviruses (Ads) is a major concern. Employing tissue specific promoters (TSPs) to restrict transgene expression or viral replication is an effective way to increase specificity towards tumor tissues and to reduce adverse effects in non-target tissues such as the liver. In this regard, candidate breast cancer TSPs include promoters of the genes for the epithelial glycoprotein 2 (EGP-2), cyclooxygenase-2 (Cox-2), α-chemokine SDF-1 receptor (stromal-cell-derived factor, CXCR4), secretory leukoprotease inhibitor (SLPI) and survivin. Methods We employed E1-deleted Ads that express the reporter gene luciferase under the control of the promoters of interest. We evaluated this class of vectors in various established breast cancer cell lines, primary breast cancer cells and finally in the most stringent preclinical available substrate system, constituted by precision cut tissue slices of human breast cancer and liver. Results Overall, the CXCR4 promoter exhibited the highest luciferase activity in breast cancer cell lines, primary breast cancer cells and breast cancer tissue slices. Importantly, the CXCR4 promoter displayed a very low activity in human primary fibroblasts and human liver tissue slices. Interestingly, gene expression profiles correlated with the promoter activities both in breast cancer cell lines and primary breast cancer cells. Conclusion These data suggest that the CXCR4 promoter has an ideal 'breast cancer-on/liver-off' profile, and could, therefore, be a powerful tool in Ad vector based gene therapy or virotherapy of the carcinoma of the breast. PMID:16457694

Spinal intradural primary germ cell tumour--review of literature and case report.

PubMed

Biswas, Ahitagni; Puri, Tarun; Goyal, Shikha; Gupta, Ruchika; Eesa, Muneer; Julka, Pramod Kumar; Rath, Goura Kishor

2009-03-01

Primary spinal cord germ cell tumour is a rare tumour. We herein review the tumour characteristics, associated risk factors, treatment policy, and patterns of failure of primary intradural germ cell tumour. We conducted a PUBMED search using a combination of keywords such as "spinal germ cell tumor," "germinoma," "extradural," "intradural," "intramedullary," "extramedullary," and identified 19 cases of primary spinal germ cell tumour. Clinical features, pathologic characteristics, and treatment details of these patients including status at follow-up were noted from respective case reports. We also describe a case of a young Indian patient of intradural extramedullary germ cell tumour treated with a combination of surgery, chemotherapy, and radiotherapy. The median age at presentation was 24 years. The most common location of the tumour was thoracic (40%). Beta-HCG overproduction was noted in 40% of the patients. Most patients were treated with a combination of surgery, radiation therapy, and systemic chemotherapy. Median follow-up was 16.5 months. Recurrence was observed in 10% of the patients, all in beta-HCG over-producing tumours. The illustrative case was a 28-year male, presenting with pain in lower back and both lower limbs for 2 months. Magnetic resonance imaging spine showed an inhomogeneous hyperintense soft tissue mass at L(2)-L(4) spinal level. He was treated with complete surgical excision and four cycles of chemotherapy with BEP regimen following a histological diagnosis of non-seminomatous germ cell tumour. Palliative irradiation to the lumbar spine was given on progression at 3 months. The patient eventually succumbed to his condition, due to compressive transverse myelitis possibly due to cervical cord metastasis. Limited surgery followed by upfront radiation therapy and adjuvant chemotherapy is the optimal management of this rare group of tumour. Omission of radiation therapy from the treatment armamentarium might engender local recurrence and spinal dissemination at first failure.

Conditional immortalization of Gunn rat hepatocytes: an ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer.

PubMed

Fox, I J; Chowdhury, N R; Gupta, S; Kondapalli, R; Schilsky, M L; Stockert, R J; Chowdhury, J R

1995-03-01

Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGR)-mediated endocytosis are being developed to transfer genes for the correction of bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT) deficiency. Ex vivo evaluation of these gene transfer vectors would be facilitated by a cell system that lacks bilirubin-UGT, but expresses differentiated liver functions, including ASGR. We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58). At 33 degrees C, the immortalized hepatocyte clones expressed SV40 large T antigen, synthesized DNA, and doubled in number every 2 to 3 days. At this temperature, differentiated hepatocyte markers, e.g., albumin, ASGR, and androsterone-UGT, were expressed at 5% to 10% of the levels found in primary hepatocytes maintained in culture for 24 hours. Glutathione-S-transferase Yp (GST-Yp), an oncofetal protein, was expressed in these cells at 33 degrees C, but was undetectable in primary hepatocytes. In contrast, when the cells were cultured at 39 degrees C or 37 degrees C, the large T antigen was degraded, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were observed. The expression of albumin, ASGR, and androsterone-UGT, and their corresponding mRNAs, increased to 25% to 40% of the level in primary hepatocytes, whereas GST-Yp expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red-labeled asialoorosomucoid, and binding and degradation of 125I-asialoorosomucoid. After liposome-mediated transfer of a plasmid containing the coding region of human bilirubin-UGT1, driven by the SV40 large T promoter, active human bilirubin-UGT1 was expressed in these cells. The immortalized cells were not tumorigenic after transplantation into severe combined immunodeficiency mice. These conditionally immortalized cells will be useful for ex vivo evaluation of bilirubin-UGT gene transfer vectors.

Engineering the Future: Cell 6

NASA Technical Reports Server (NTRS)

Stahl, P. H.

2010-01-01

This slide presentation reviews the development of the James Webb Space Telescope (JWST), explaining the development using a systems engineering methodology. Included are slides showing the organizational chart, the JWST Science Goals, the size of the primary mirror, and full scale mockups of the JSWT. Also included is a review of the JWST Optical Telescope Requirements, a review of the preliminary design and analysis, the technology development required to create the JWST, with particular interest in the specific mirror technology that was required, and views of the mirror manufacturing process. Several slides review the process of verification and validation by testing and analysis, including a diagram of the Cryogenic Test Facility at Marshall, and views of the primary mirror while being tested in the cryogenic facility.

Cellular tropism of human enterovirus D species serotypes EV-94, EV-70, and EV-68 in vitro: implications for pathogenesis.

PubMed

Smura, Teemu; Ylipaasto, Petri; Klemola, Päivi; Kaijalainen, Svetlana; Kyllönen, Lauri; Sordi, Valeria; Piemonti, Lorenzo; Roivainen, Merja

2010-11-01

Enterovirus 94 (EV-94) is an enterovirus serotype described recently which, together with EV-68 and EV-70, forms human enterovirus D species. This study investigates the seroprevalences of these three serotypes and their abilities to infect, replicate, and damage cell types considered to be essential for enterovirus-induced diseases. The cell types studied included human leukocyte cell lines, primary endothelial cells, and pancreatic islets. High prevalence of neutralizing antibodies against EV-68 and EV-94 was found in the Finnish population. The virus strains studied had wide leukocyte tropism. EV-94 and EV-68 were able to produce infectious progeny in leukocyte cell lines with monocytic, granulocytic, T-cell, or B-cell characteristics. EV-94 and EV-70 were capable of infecting primary human umbilical vein endothelial cells, whereas EV-68 had only marginal progeny production and did not induce cytopathic effects in these cells. Intriguingly, EV-94 was able to damage pancreatic islet β-cells, to infect, replicate, and cause necrosis in human pancreatic islets, and to induce proinflammatory and chemoattractive cytokine expression in endothelial cells. These results suggest that HEV-D viruses may be more prevalent than has been thought previously, and they provide in vitro evidence that EV-94 may be a potent pathogen and should be considered a potentially diabetogenic enterovirus type. © 2010 Wiley-Liss, Inc.

The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells

PubMed Central

Zhang, Jenny; Jima, Dereje; Moffitt, Andrea B.; Liu, Qingquan; Czader, Magdalena; Hsi, Eric D.; Fedoriw, Yuri; Dunphy, Cherie H.; Richards, Kristy L.; Gill, Javed I.; Sun, Zhen; Love, Cassandra; Scotland, Paula; Lock, Eric; Levy, Shawn; Hsu, David S.; Dunson, David; Dave, Sandeep S.

2014-01-01

In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer. PMID:24682267

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

PubMed Central

Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

Influenza A Virus Infection of Human Respiratory Cells Induces Primary MicroRNA Expression*

PubMed Central

Buggele, William A.; Johnson, Karen E.; Horvath, Curt M.

2012-01-01

The cellular response to virus infection is initiated by recognition of the invading pathogen and subsequent changes in gene expression mediated by both transcriptional and translational mechanisms. In addition to well established means of regulating antiviral gene expression, it has been demonstrated that RNA interference (RNAi) can play an important role in antiviral responses. Virus-derived small interfering RNA (siRNA) is a primary antiviral response exploited by plants and invertebrate animals, and host-encoded microRNA (miRNA) species have been clearly implicated in the regulation of innate and adaptive immune responses in mammals and other vertebrates. Examination of miRNA abundance in human lung cell lines revealed endogenous miRNAs, including miR-7, miR-132, miR-146a, miR-187, miR-200c, and miR-1275, to specifically accumulate in response to infection with two influenza A virus strains, A/Udorn/72 and A/WSN/33. Known antiviral response pathways, including Toll-like receptor, RIG-I-like receptor, and direct interferon or cytokine stimulation did not alter the abundance of the tested miRNAs to the extent of influenza A virus infection, which initiates primary miRNA transcription via a secondary response pathway. Gene expression profiling identified 26 cellular mRNAs targeted by these miRNAs, including IRAK1, MAPK3, and other components of innate immune signaling systems. PMID:22822053

Gene Expression Networks Underlying Ovarian Development in Wild Largemouth Bass (Micropterus salmoides)

PubMed Central

Martyniuk, Christopher J.; Prucha, Melinda S.; Doperalski, Nicholas J.; Antczak, Philipp; Kroll, Kevin J.; Falciani, Francesco; Barber, David S.; Denslow, Nancy D.

2013-01-01

Background Oocyte maturation in fish involves numerous cell signaling cascades that are activated or inhibited during specific stages of oocyte development. The objectives of this study were to characterize molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in wild female largemouth bass to improve understanding of the molecular sequence of events underlying oocyte maturation. Methods Transcriptomic analysis was performed on eight morphologically diverse stages of the ovary, including primary and secondary stages of oocyte growth, ovulation, and atresia. Ovary histology, plasma vitellogenin, 17β-estradiol, and testosterone were also measured to correlate with gene networks. Results Global expression patterns revealed dramatic differences across ovarian development, with 552 and 2070 genes being differentially expressed during both ovulation and atresia respectively. Gene set enrichment analysis (GSEA) revealed that early primary stages of oocyte growth involved increases in expression of genes involved in pathways of B-cell and T-cell receptor-mediated signaling cascades and fibronectin regulation. These pathways as well as pathways that included adrenergic receptor signaling, sphingolipid metabolism and natural killer cell activation were down-regulated at ovulation. At atresia, down-regulated pathways included gap junction and actin cytoskeleton regulation, gonadotrope and mast cell activation, and vasopressin receptor signaling and up-regulated pathways included oxidative phosphorylation and reactive oxygen species metabolism. Expression targets for luteinizing hormone signaling were low during vitellogenesis but increased 150% at ovulation. Other networks found to play a significant role in oocyte maturation included those with genes regulated by members of the TGF-beta superfamily (activins, inhibins, bone morphogenic protein 7 and growth differentiation factor 9), neuregulin 1, retinoid X receptor, and nerve growth factor family. Conclusions This study offers novel insight into the gene networks underlying vitellogenesis, ovulation and atresia and generates new hypotheses about the cellular pathways regulating oocyte maturation. PMID:23527095

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex.

PubMed

Wise, Jillian F; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K; Tao, Rong-Hua; Sabichi, Anita L; Ao, Xue; Maeng, Hoyoung; Samaniego, Felipe

2013-06-06

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

Assembly of Hepatitis Delta Virus: Particle Characterization, Including the Ability To Infect Primary Human Hepatocytes▿

PubMed Central

Gudima, Severin; He, Yiping; Meier, Anja; Chang, Jinhong; Chen, Rongji; Jarnik, Michal; Nicolas, Emmanuelle; Bruss, Volker; Taylor, John

2007-01-01

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells. PMID:17229685

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex

PubMed Central

Wise, Jillian F.; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K.; Tao, Rong-Hua; Sabichi, Anita L.; Ao, Xue; Maeng, Hoyoung

2013-01-01

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL–Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target. PMID:23599269

Effects of glucose on lactose synthesis in mammary epithelial cells from dairy cow.

PubMed

Lin, Ye; Sun, Xiaoxu; Hou, Xiaoming; Qu, Bo; Gao, Xuejun; Li, Qingzhang

2016-05-26

Lactose, as the primary osmotic component in milk, is the major determinant of milk volume. Glucose is the primary precursor of lactose. However, the effect of glucose on lactose synthesis in dairy cow mammary glands and the mechanism governing this process are poorly understood. Here we showed that glucose has the ability to induce lactose synthesis in dairy cow mammary epithelial cells, as well as increase cell viability and proliferation. A concentration of 12 mM glucose was the optimum concentration to induce cell growth and lactose synthesis in cultured dairy cow mammary epithelial cells. In vitro, 12 mM glucose enhanced lactose content, along with the expression of genes involved in glucose transportation and the lactose biosynthesis pathway, including GLUT1, SLC35A2, SLC35B1, HK2, β4GalT-I, and AKT1. In addition, we found that AKT1 knockdown inhibited cell growth and lactose synthesis as well as expression of GLUT1, SLC35A2, SLC35B1, HK2, and β4GalT-I. Glucose induces cell growth and lactose synthesis in dairy cow mammary epithelial cells. Protein kinase B alpha acts as a regulator of metabolism in dairy cow mammary gland to mediate the effects of glucose on lactose synthesis.

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

Heterogeneity of Estrogen Receptor Expression in Circulating Tumor Cells from Metastatic Breast Cancer Patients

PubMed Central

Babayan, Anna; Hannemann, Juliane; Spötter, Julia; Müller, Volkmar

2013-01-01

Background Endocrine treatment is the most preferable systemic treatment in metastatic breast cancer patients that have had an estrogen receptor (ER) positive primary tumor or metastatic lesions, however, approximately 20% of these patients do not benefit from the therapy and demonstrate further metastatic progress. One reason for failure of endocrine therapy might be the heterogeneity of ER expression in tumor cells spreading from the primary tumor to distant sites which is reflected in detectable circulating tumor cells (CTCs). Methods A sensitive and specific staining protocol for ER, keratin 8/18/19, CD45 was established. Peripheral blood from 35 metastatic breast cancer patients with ER-positive primary tumors was tested for the presence of CTCs. Keratin 8/18/19 and DAPI positive but CD45 negative cells were classified as CTCs and evaluated for ER staining. Subsequently, eight individual CTCs from four index patients (2 CTCs per patient) were isolated and underwent whole genome amplification and ESR1 gene mutation analysis. Results CTCs were detected in blood of 16 from 35 analyzed patients (46%), with a median of 3 CTCs/7.5 ml. In total, ER-negative CTCs were detected in 11/16 (69%) of the CTC positive cases, including blood samples with only ER-negative CTCs (19%) and samples with both ER-positive and ER-negative CTCs (50%). No correlation was found between the intensity and/or percentage of ER staining in the primary tumor with the number and ER status of CTCs of the same patient. ESR1 gene mutations were not found. Conclusion CTCs frequently lack ER expression in metastatic breast cancer patients with ER-positive primary tumors and show a considerable intra-patient heterogeneity, which may reflect a mechanism to escape endocrine therapy. Provided single cell analysis did not support a role of ESR1 mutations in this process. PMID:24058649

Discovery of a stem-like multipotent cell fate.

PubMed

Paffhausen, Emily S; Alowais, Yasir; Chao, Cara W; Callihan, Evan C; Creswell, Karen; Bracht, John R

2018-01-01

Adipose derived stem cells (ASCs) can be obtained from lipoaspirates and induced in vitro to differentiate into bone, cartilage, and fat. Using this powerful model system we show that after in vitro adipose differentiation a population of cells retain stem-like qualities including multipotency. They are lipid (-), retain the ability to propagate, express two known stem cell markers, and maintain the capacity for trilineage differentiation into chondrocytes, adipocytes, and osteoblasts. However, these cells are not traditional stem cells because gene expression analysis showed an overall expression profile similar to that of adipocytes. In addition to broadening our understanding of cellular multipotency, our work may be particularly relevant to obesity-associated metabolic disorders. The adipose expandability hypothesis proposes that inability to differentiate new adipocytes is a primary cause of metabolic syndrome in obesity, including diabetes and cardiovascular disease. Here we have defined a differentiation-resistant stem-like multipotent cell population that may be involved in regulation of adipose expandability in vivo and may therefore play key roles in the comorbidities of obesity.

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

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

Li Hongzhen; Zhou Jianjun; Miki, Jun

2008-01-01

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetalmore » bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.« less

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

Application of single-cell sequencing in human cancer.

PubMed

Rantalainen, Mattias

2017-11-02

Precision medicine is emerging as a cornerstone of future cancer care with the objective of providing targeted therapies based on the molecular phenotype of each individual patient. Traditional bulk-level molecular phenotyping of tumours leads to significant information loss, as the molecular profile represents an average phenotype over large numbers of cells, while cancer is a disease with inherent intra-tumour heterogeneity at the cellular level caused by several factors, including clonal evolution, tissue hierarchies, rare cells and dynamic cell states. Single-cell sequencing provides means to characterize heterogeneity in a large population of cells and opens up opportunity to determine key molecular properties that influence clinical outcomes, including prognosis and probability of treatment response. Single-cell sequencing methods are now reliable enough to be used in many research laboratories, and we are starting to see applications of these technologies for characterization of human primary cancer cells. In this review, we provide an overview of studies that have applied single-cell sequencing to characterize human cancers at the single-cell level, and we discuss some of the current challenges in the field. © The Author 2017. Published by Oxford University Press.

Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment

PubMed Central

Li, Dongqi; Li, Huiling; Ren, Mingyan; Liao, Yedan; Yu, Shunling; Chen, Yanjin; Yang, Yihao; Zhang, Ya

2016-01-01

Osteosarcoma is the most common primary bone tumor in children and adolescents. Although combined therapy including surgery and multi-agent chemotherapy have resulted in great improvements in the overall survival of patients, chemoresistance remains an obstacle for the treatment of osteosarcoma. Molecular targets or effective agents that are actively involved in cell death including apoptosis, autophagy and necroptosis have been studied. We summarized how these agents (novel compounds, miRNAs, or proteins) regulate apoptotic, autophagic and necroptotic pathways; and discussed the current knowledge on the role of these new agents in chemotherapy resistance in osteosarcoma. PMID:27007056

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