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

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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

Cell type and transfection reagent-dependent effects on viability, cell content, cell cycle and inflammation of RNAi in human primary mesenchymal cells

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Yang, Hsiao Yin; Vonk, Lucienne A.; Licht, Ruud

2014-01-01

% amidation), for siRNA delivery into primary mesenchymal cells including nucleus pulposus cells, articular chondrocytes and mesenchymal stem cells (MSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous model gene to evaluate the extent of silencing by 20 nM or 200 nM siRNA at day...

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells.

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Ruth Merkle

2016-08-01

Full Text Available Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC, is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO. However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR. The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in

Identification of differences in gene expression in primary cell cultures of human endometrial epithelial cells and trophoblast cells following their interaction

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Høgh, Mette; Islin, Henrik; Møller, Charlotte

2006-01-01

The interaction between the cell types was simulated in vitro by growing primary cell cultures of human endometrial epithelial cells and trophoblast cells together (co-culture) and separately (control cultures). Gene expression in the cell cultures was compared using the Differential Display method and confirmed...

Primary Type3 (Non-ABC, Non-GCB Subtype of Extranodal Diffuse Large B-Cell Lymphoma of the Thyroid Bearing No MYD88 Mutation by Padlock Probe Hybridization

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Yukiko Nishi

2017-06-01

Full Text Available Primary extranodal malignant lymphoma of the thyroid is a rare entity composed of mostly neoplastic transformation of germinal center-like B cells (GCB or memory B cells. Other B-cell-type malignancies arising primarily in the thyroid have rarely been described. Immunohistochemical examination of autopsied primary malignant lymphoma of the thyroid in an 83-year-old Japanese female revealed the presence of a non-GCB subtype of diffuse large B-cell lymphoma (DLBCL without the typical codon 206 or 265 missense mutation of MYD88. The lack of the highly oncogenic MYD88 gene mutation, frequently observed in DLBCL of the activated B-cell (ABC subtype, and the detection of an extremely aggressive yet local clinical phenotype demonstrated that the present case was an exceptional entity of the type3 (non-GCB and non-ABC subtype.

Cellular lead toxicity and metabolism in primary and clonal osteoblastic bone cells

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Long, G.J.; Rosen, J.F.; Pounds, J.G.

1990-01-01

A knowledge of bone lead metabolism is critical for understanding the toxicological importance of bone lead, as a toxicant both to bone cells and to soft tissues of the body, as lead is mobilized from large reservoirs in hard tissues. To further understand the processes that mediate metabolism of lead in bone, it is necessary to determine lead metabolism at the cellular level. Experiments were conducted to determine the intracellular steady-state 210 Pb kinetics in cultures of primary and clonal osteoblastic bone cells. Osteoblastic bone cells obtained by sequential collagenase digestion of mouse calvaria or rat osteosarcoma (ROS 17/2.8) cells were labeled with 210 Pb as 5 microM lead acetate for 20 hr, and kinetic parameters were determined by measuring the efflux of 210 Pb from the cells over a 210 -min period. The intracellular metabolism of 210 Pb was characterized by three kinetic pools of 210 Pb in both cell types. Although the values of these parameters differed between the primary osteoblastic cells and ROS cells, the profile of 210 Pb was remarkably similar in both cell types. Both types exhibited one large, slowly exchanging pool (S3), indicative of mitochondrial lead. These data show that primary osteoblastic bone cells and ROS cells exhibit similar steady-state lead kinetics, and intracellular lead distribution. These data also establish a working model of lead kinetics in osteoblastic bone cells and now permit an integrated view of lead kinetics in bone

Stimulation of DNA synthesis in cultured rat alveolar type II cells

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Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

1985-01-01

Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated 3 H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of 3 H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture

Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells

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Rattner Jerome B

2009-12-01

Full Text Available Abstract Background Primary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. Defects in the formation and/or function of these structures underlie a variety of human diseases such as Alström, Bardet-Biedl, Joubert, Meckel-Gruber and oral-facial-digital type 1 syndromes. The expression and function of primary cilia in cancer cells has now become a focus of attention but has not been studied in astrocytomas/glioblastomas. To begin to address this issue, we compared the structure and expression of primary cilia in a normal human astrocyte cell line with five human astrocytoma/glioblastoma cell lines. Methods Cultured normal human astrocytes and five human astrocytoma/glioblastoma cell lines were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Monospecific antibodies were used to detect primary cilia and map the relationship between the primary cilia region and sites of endocytosis. Results We show that expression of primary cilia in normal astrocytes is cell cycle related and the primary cilium extends through the cell within a unique structure which we show to be a site of endocytosis. Importantly, we document that in each of the five astrocytoma/glioblastoma cell lines fully formed primary cilia are either expressed at a very low level, are completely absent or have aberrant forms, due to incomplete ciliogenesis. Conclusions The recent discovery of the importance of primary cilia in a variety of cell functions raises the possibility that this structure may have a role in a variety of cancers. Our finding that the formation of the primary cilium is disrupted in cells derived from astrocytoma/glioblastoma tumors provides the first

Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types.

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Gil-Ibañez, Pilar; García-García, Francisco; Dopazo, Joaquín; Bernal, Juan; Morte, Beatriz

2017-01-01

Thyroid hormones, thyroxine, and triiodothyronine (T3) are crucial for cerebral cortex development acting through regulation of gene expression. To define the transcriptional program under T3 regulation, we have performed RNA-Seq of T3-treated and untreated primary mouse cerebrocortical cells. The expression of 1145 genes or 7.7% of expressed genes was changed upon T3 addition, of which 371 responded to T3 in the presence of cycloheximide indicating direct transcriptional regulation. The results were compared with available transcriptomic datasets of defined cellular types. In this way, we could identify targets of T3 within genes enriched in astrocytes and neurons, in specific layers including the subplate, and in specific neurons such as prepronociceptin, cholecystokinin, or cortistatin neurons. The subplate and the prepronociceptin neurons appear as potentially major targets of T3 action. T3 upregulates mostly genes related to cell membrane events, such as G-protein signaling, neurotransmission, and ion transport and downregulates genes involved in nuclear events associated with the M phase of cell cycle, such as chromosome organization and segregation. Remarkably, the transcriptomic changes induced by T3 sustain the transition from fetal to adult patterns of gene expression. The results allow defining in molecular terms the elusive role of thyroid hormones on neocortical development. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Neoexpression of a functional primary cilium in colorectal cancer cells

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Blanche Sénicourt

2016-05-01

Full Text Available The Hedgehog (HH signaling pathway is involved in the maintenance of numerous cell types both during development and in the adult. Often deregulated in cancers, its involvement in colorectal cancer has come into view during the last few years, although its role remains poorly defined. In most tissues, the HH pathway is highly connected to the primary cilium (PC, an organelle that recruits functional components and regulates the HH pathway. However, normal epithelial cells of the colon display an inactive HH pathway and lack a PC. In this study, we report the presence of the PC in adenocarcinoma cells of primary colorectal tumors at all stages. Using human colorectal cancer cell lines we found a clear correlation between the presence of the PC and the expression of the final HH effector, GLI1, and provide evidence of a functional link between the two by demonstrating the recruitment of the SMO receptor to the membrane of the primary cilium. We conclude that the primary cilium directly participates in the HH pathway in colorectal cancer cells.

Metabolic responses of primary and transformed cells to intracellular Listeria monocytogenes.

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Nadine Gillmaier

Full Text Available The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using (13C-labelled glucose or glutamine as carbon tracers. The (13C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.

Clinico-Radiologic Findings in Primary Cutaneous Extranodal Natural Killer/T-Cell Lymphoma, Nasal Type Mimicking Cellulitis of the Left Arm

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Kim, Soo Hyun; Seon, Hyun Ju; Choi, Yoo Duk; Yun, Sook Jung

2015-01-01

Extranodal natural killer (NK)/T-cell lymphoma is a very rare and aggressive disease characterized histopathologically by an Epstein-Barr virus (EBV)-positive atypical lymphoid cytotoxic infiltrate, extensive vascular destruction, and prominent tissue necrosis. It commonly shows cutaneous lesions that primarily or secondarily mimic cellulitis at the primary site. We report on a very rare case of extranodal NK/T-cell lymphoma, nasal type of skin/soft tissue, in a 64-year-old man, and describe the radiological findings. The condition was misdiagnosed as cellulitis of the left arm based on initial noninvasive clinical and radiologic work-up

Tumor necrosis factor (cachetin) decreases adipose cell differentiation in primary cell culture

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Martin, R.J.; Jones, D.D.; Jewell, D.E.; Hausman, G.J.

1986-01-01

Cachetin has been shown to effect gene product expression in the established adipose cell line 3T3-L1. Expression of messenger RNA for lipoprotein lipase is suppressed in cultured adipocytes. The purpose of this study was to determine the effect of Cachetin on adipose cell differentiation in primary cell culture. Stromalvascular cells obtained from the inguinal fat pad of 4-5 week old Sprague-Dawley rats were grown in culture for two weeks. During the proliferative growth phase all cells were grown on the same medium and labelled with 3 H-thymidine. Cachetin treatment (10 -6 to 10 -10 M) was initiated on day 5, the initial phase of preadipocyte differentiation. Adipocytes and stromal cells were separated using density gradient, and 3 H-thymidine was determined for both cell types. Thymidine incorporation into adipose cells was decreased maximally (∼ 50%) at 10 -10 M. Stromalvascular cells were not influenced at any of the doses tested. Adipose cell lipid content as indicated by oil red-O staining was decreased by Cachetin. Esterase staining by adipose cells treated with Cachetin was increased indicating an increase in intracellular lipase. These studies show that Cachetin has specific effects on primary adipose cell differentiation

Primary cutaneous large B-cell lymphoma of scalp: Case report of a rare variant

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Yasmeen Khatib

2017-01-01

Full Text Available Primary cutaneous large B-cell lymphoma (Bcl is defined as a lymphoma composed of large cells constituting more than 80% of the infiltrate and absence of extracutaneous involvement after staging investigations. In the new World Health Organization/European Organization for Research and Treatment of Cancer classification, cutaneous Bcls with large cells are of three types - primary cutaneous large Bcl leg type (PCLBCLLT, primary cutaneous follicle center lymphoma diffuse type (PCFCLDT, and primary cutaneous large Bcls other (PCLBCLO. These three different types are distinct in terms of their clinicopathological features and survival. The PCLBCLO has intermediate features between those of PCLBCLLT and PCFCLDT. We present a case of PCLBCLO in a 57-year-old male who presented with a scalp swelling. Ultrasonography examination was suggestive of a sebaceous cyst. Computed tomography scan revealed the presence of an ill-defined hyperdense region in the soft tissue of the scalp region extending into the deeper layers of the scalp. Fine-needle aspiration cytology (FNAC revealed the presence of atypical lymphoid cells. Diagnosis was confirmed by biopsy and immunohistochemistry. Patient received rituximab combined with doxorubicin, vincristine, cyclophosphamide, and prednisolone regimen with complete resolution of the lesion. We present this case for its rarity, the utility of FNAC in early diagnosis, and to discuss the differential diagnosis.

Expression weighted cell type enrichments reveal genetic and cellular nature of major brain disorders

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Nathan Gerald Skene

2016-01-01

Full Text Available The cell types that trigger the primary pathology in many brain diseases remain largely unknown. One route to understanding the primary pathological cell type for a particular disease is to identify the cells expressing susceptibility genes. Although this is straightforward for monogenic conditions where the causative mutation may alter expression of a cell type specific marker, methods are required for the common polygenic disorders. We developed the Expression Weighted Cell Type Enrichment (EWCE method that uses single cell transcriptomes to generate the probability distribution associated with a gene list having an average level of expression within a cell type. Following validation, we applied EWCE to human genetic data from cases of epilepsy, Schizophrenia, Autism, Intellectual Disability, Alzheimer’s disease, Multiple Sclerosis and anxiety disorders. Genetic susceptibility primarily affected microglia in Alzheimer’s and Multiple Sclerosis; was shared between interneurons and pyramidal neurons in Autism and Schizophrenia; while intellectual disabilities and epilepsy were attributable to a range of cell-types, with the strongest enrichment in interneurons. We hypothesised that the primary cell type pathology could trigger secondary changes in other cell types and these could be detected by applying EWCE to transcriptome data from diseased tissue. In Autism, Schizophrenia and Alzheimer’s disease we find evidence of pathological changes in all of the major brain cell types. These findings give novel insight into the cellular origins and progression in common brain disorders. The methods can be applied to any tissue and disorder and have applications in validating mouse models.

Combined human papillomavirus typing and TP53 mutation analysis in distinguishing second primary tumors from lung metastases in patients with head and neck squamous cell carcinoma.

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Daher, Tamas; Tur, Mehmet Kemal; Brobeil, Alexander; Etschmann, Benjamin; Witte, Biruta; Engenhart-Cabillic, Rita; Krombach, Gabriele; Blau, Wolfgang; Grimminger, Friedrich; Seeger, Werner; Klussmann, Jens Peter; Bräuninger, Andreas; Gattenlöhner, Stefan

2018-06-01

In head and neck squamous cell carcinoma (HNSCC), the occurrence of concurrent lung malignancies poses a significant diagnostic challenge because metastatic HNSCC is difficult to discern from second primary lung squamous cell carcinoma (SCC). However, this differentiation is crucial because the recommended treatments for metastatic HNSCC and second primary lung SCC differ profoundly. We analyzed the origin of lung tumors in 32 patients with HNSCC using human papillomavirus (HPV) typing and targeted next generation sequencing of all coding exons of tumor protein 53 (TP53). Lung tumors were clearly identified as HNSCC metastases or second primary tumors in 29 patients, thus revealing that 16 patients had received incorrect diagnoses based on clinical and morphological data alone. The HPV typing and mutation analysis of all TP53 coding exons is a valuable diagnostic tool in patients with HNSCC and concurrent lung SCC, which can help to ensure that patients receive the most suitable treatment. © 2018 Wiley Periodicals, Inc.

Induction of expression of two phenotypic markers of pulmonary type II cells in a cultured cell line

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Henderson, R.F.; Waide, J.J.; Scott, G.G.

1994-01-01

The functions of pulmonary type II cells, such as synthesis of pulmonary surfactant and metabolism of inhaled xenobiotics, can be studied in primary isolates of lung cells. However, isolated type II cells, when cultured, quickly lose the phenotypic expressions characteristics of type II cells, including surfactant lipid and protein synthesis and alkaline phosphatase (AP) activity. A cultured cell line that maintained expression of type II cell markers of differentiation would be advantageous for the study of such functions as surfactant synthesis and secretion. Such a cell line would allow generation of a large number of homogeneous cells for study. The purpose of the current study was to induce markers of differentiated type II cells in a cultured cell line to facilitate studies of factors that control surfactant synthesis and secretion

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

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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.

Cell elasticity with altered cytoskeletal architectures across multiple cell types.

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Grady, Martha E; Composto, Russell J; Eckmann, David M

2016-08-01

The cytoskeleton is primarily responsible for providing structural support, localization and transport of organelles, and intracellular trafficking. The structural support is supplied by actin filaments, microtubules, and intermediate filaments, which contribute to overall cell elasticity to varying degrees. We evaluate cell elasticity in five different cell types with drug-induced cytoskeletal derangements to probe how actin filaments and microtubules contribute to cell elasticity and whether it is conserved across cell type. Specifically, we measure elastic stiffness in primary chondrocytes, fibroblasts, endothelial cells (HUVEC), hepatocellular carcinoma cells (HUH-7), and fibrosarcoma cells (HT 1080) subjected to two cytoskeletal destabilizers: cytochalasin D and nocodazole, which disrupt actin and microtubule polymerization, respectively. Elastic stiffness is measured by atomic force microscopy (AFM) and the disruption of the cytoskeleton is confirmed using fluorescence microscopy. The two cancer cell lines showed significantly reduced elastic moduli values (~0.5kPa) when compared to the three healthy cell lines (~2kPa). Non-cancer cells whose actin filaments were disrupted using cytochalasin D showed a decrease of 60-80% in moduli values compared to untreated cells of the same origin, whereas the nocodazole-treated cells showed no change in elasticity. Overall, we demonstrate actin filaments contribute more to elastic stiffness than microtubules but this result is cell type dependent. Cancer cells behaved differently, exhibiting increased stiffness as well as stiffness variability when subjected to nocodazole. We show that disruption of microtubule dynamics affects cancer cell elasticity, suggesting therapeutic drugs targeting microtubules be monitored for significant elastic changes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inefficiency in macromolecular transport of SCS-based microcapsules affects viability of primary human mesenchymal stem cells but not of immortalized cells

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Sanz-Nogués, Clara; Horan, Jason; Thompson, Kerry

2015-01-01

mesenchymal stem cells (hMSCs). Human MSCs are of interest in regenerative medicine applications due to pro-angiogenic, anti-inflammatory and immunomodulatory properties, which result from paracrine effects of this cell type. In the present work we have encapsulated primary hMSCs and hMSC-TERT immortalized...... nutrients and had a more detrimental effect on the viability of primary cell cultures compared to cell lines and immortalized cells. This article is protected by copyright. All rights reserved....

Primary cutaneous marginal zone B-cell lymphoma: clinical and histological aspects.

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Khaled, A; Sassi, S; Fazaa, B; Ben Hassouna, J; Ben Romdhane, K; Kamoun, M R

2009-02-01

According to the WHO-EORTC classification of cutaneous lymphomas, primary cutaneous marginal zone B-cell lymphoma are now well characterized. We report here a case of primary cutaneous marginal zone B-cell lymphoma in a 51 year-old man in which the diagnosis was made using both histology and immunopathology. The patient had no remarkable medical history, no history of either acute inflammation or insect bite, and presented with a 5 cm solitary asymptomatic erythematous firm, multinodular and infiltrated plaque on the back for 12 months. Histological examination and immunohistochemical study of a cutaneous biopsy provided a differential diagnosis between B cell lymphoma and lymphocytoma cutis. Full body work up revealed no signs of extracutaneous dissemination. The patient underwent surgical excision of the nodule. Histological examination showed a histological and immunophenotyping profile typical of primary cutaneous marginal zone B-cell lymphoma. The lesion was completely excised with clear margins and no recurrence occurred after a 12 month-follow-up period. Primary cutaneous marginal zone B-cell lymphoma are low-grade lymphomas that have an indolent course and a high tendency to recur. They should be differentiated from lymphocytoma cutis and from the other types of cutaneous B cell lymphomas that have a different course and prognosis.

The cytoprotective role of DJ-1 and p45 NFE2 against human primary alveolar type II cell injury and emphysema.

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Tan, Li Hui; Bahmed, Karim; Lin, Chih-Ru; Marchetti, Nathaniel; Bolla, Sudhir; Criner, Gerard J; Kelsen, Steven; Madesh, Muniswamy; Kosmider, Beata

2018-02-23

Emphysema is characterized by irreversibly enlarged airspaces and destruction of alveolar walls. One of the factors contributing to this disease pathogenesis is an elevation in extracellular matrix (ECM) degradation in the lung. Alveolar type II (ATII) cells produce and secrete pulmonary surfactants and proliferate to restore the epithelium after damage. We isolated ATII cells from control non-smokers, smokers and patients with emphysema to determine the role of NFE2 (nuclear factor, erythroid-derived 2). NFE2 is a heterodimer composed of two subunits, a 45 kDa (p45 NFE2) and 18 kDa (p18 NFE2) polypeptides. Low expression of p45 NFE2 in patients with emphysema correlated with a high ECM degradation. Moreover, we found that NFE2 knockdown increased cell death induced by cigarette smoke extract. We also studied the cross talk between p45 NFE2 and DJ-1. DJ-1 protein is a redox-sensitive chaperone that protects cells from oxidative stress. We detected that cigarette smoke significantly increased p45 NFE2 levels in DJ-1 KO mice compared to wild-type mice. Our results indicate that p45 NFE2 expression is induced by exposure to cigarette smoke, has a cytoprotective activity against cell injury, and its downregulation in human primary ATII cells may contribute to emphysema pathogenesis.

Optimizing the Primary Prevention of Type-2 Diabetes in Primary Health Care

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2017-08-18

Interprofessional Relations; Primary Health Care/Organization & Administration; Diabetes Mellitus, Type 2/Prevention & Control; Primary Prevention/Methods; Risk Reduction Behavior; Randomized Controlled Trial; Life Style

Transcriptomic profiling of primary alveolar epithelial cell differentiation in human and rat

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Crystal N. Marconett

2014-12-01

Full Text Available Cell-type specific gene regulation is a key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how changes in transcriptional activation during alveolar epithelial cell (AEC differentiation determine phenotype. We performed transcriptomic profiling using in vitro differentiation of human and rat primary AEC. This model recapitulates in vitro an in vivo process in which AEC transition from alveolar type 2 (AT2 cells to alveolar type 1 (AT1 cells during normal maintenance and regeneration following lung injury. Here we describe in detail the quality control, preprocessing, and normalization of microarray data presented within the associated study (Marconett et al., 2013. We also include R code for reproducibility of the referenced data and easily accessible processed data tables.

Genome-Wide Progesterone Receptor Binding: Cell Type-Specific and Shared Mechanisms in T47D Breast Cancer Cells and Primary Leiomyoma Cells

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Huang, Lei; Owen, Jonas K.; Xie, Anna; Navarro, Antonia; Monsivais, Diana; Coon V, John S.; Kim, J. Julie; Dai, Yang; Bulun, Serdar E.

2012-01-01

Background Progesterone, via its nuclear receptor (PR), exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma) and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. Principal Findings ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs), whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2), a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. Conclusions Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in uterine fibroids and

Lineage relationship of prostate cancer cell types based on gene expression

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Ware Carol B

2011-05-01

Full Text Available Abstract Background Prostate tumor heterogeneity is a major factor in disease management. Heterogeneity could be due to multiple cancer cell types with distinct gene expression. Of clinical importance is the so-called cancer stem cell type. Cell type-specific transcriptomes are used to examine lineage relationship among cancer cell types and their expression similarity to normal cell types including stem/progenitor cells. Methods Transcriptomes were determined by Affymetrix DNA array analysis for the following cell types. Putative prostate progenitor cell populations were characterized and isolated by expression of the membrane transporter ABCG2. Stem cells were represented by embryonic stem and embryonal carcinoma cells. The cancer cell types were Gleason pattern 3 (glandular histomorphology and pattern 4 (aglandular sorted from primary tumors, cultured prostate cancer cell lines originally established from metastatic lesions, xenografts LuCaP 35 (adenocarcinoma phenotype and LuCaP 49 (neuroendocrine/small cell carcinoma grown in mice. No detectable gene expression differences were detected among serial passages of the LuCaP xenografts. Results Based on transcriptomes, the different cancer cell types could be clustered into a luminal-like grouping and a non-luminal-like (also not basal-like grouping. The non-luminal-like types showed expression more similar to that of stem/progenitor cells than the luminal-like types. However, none showed expression of stem cell genes known to maintain stemness. Conclusions Non-luminal-like types are all representatives of aggressive disease, and this could be attributed to the similarity in overall gene expression to stem and progenitor cell types.

Helicobacter pylori Type IV Secretion System and Its Adhesin Subunit, CagL, Mediate Potent Inflammatory Responses in Primary Human Endothelial Cells

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Mona Tafreshi

2018-02-01

Full Text Available The Gram-negative bacterium, Helicobacter pylori, causes chronic gastritis, peptic ulcers, and gastric cancer in humans. Although the gastric epithelium is the primary site of H. pylori colonization, H. pylori can gain access to deeper tissues. Concurring with this notion, H. pylori has been found in the vicinity of endothelial cells in gastric submucosa. Endothelial cells play crucial roles in innate immune response, wound healing and tumorigenesis. This study examines the molecular mechanisms by which H. pylori interacts with and triggers inflammatory responses in endothelial cells. We observed that H. pylori infection of primary human endothelial cells stimulated secretion of the key inflammatory cytokines, interleukin-6 (IL-6 and interleukin-8 (IL-8. In particular, IL-8, a potent chemokine and angiogenic factor, was secreted by H. pylori-infected endothelial cells to levels ~10- to 20-fold higher than that typically observed in H. pylori-infected gastric epithelial cells. These inflammatory responses were triggered by the H. pylori type IV secretion system (T4SS and the T4SS-associated adhesin CagL, but not the translocation substrate CagA. Moreover, in contrast to integrin α5β1 playing an essential role in IL-8 induction by H. pylori upon infection of gastric epithelial cells, both integrin α5β1 and integrin αvβ3 were dispensable for IL-8 induction in H. pylori-infected endothelial cells. However, epidermal growth factor receptor (EGFR is crucial for mediating the potent H. pylori-induced IL-8 response in endothelial cells. This study reveals a novel mechanism by which the H. pylori T4SS and its adhesin subunit, CagL, may contribute to H. pylori pathogenesis by stimulating the endothelial innate immune responses, while highlighting EGFR as a potential therapeutic target for controlling H. pylori-induced inflammation.

Alginate-Poly(ethylene glycol Hybrid Microspheres for Primary Cell Microencapsulation

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Redouan Mahou

2014-01-01

Full Text Available The progress of medical therapies, which rely on the transplantation of microencapsulated living cells, depends on the quality of the encapsulating material. Such material has to be biocompatible, and the microencapsulation process must be simple and not harm the cells. Alginate-poly(ethylene glycol hybrid microspheres (alg-PEG-M were produced by combining ionotropic gelation of sodium alginate (Na-alg using calcium ions with covalent crosslinking of vinyl sulfone-terminated multi-arm poly(ethylene glycol (PEG-VS. In a one-step microsphere formation process, fast ionotropic gelation yields spherical calcium alginate gel beads, which serve as a matrix for simultaneously but slowly occurring covalent cross-linking of the PEG-VS molecules. The feasibility of cell microencapsulation was studied using primary human foreskin fibroblasts (EDX cells as a model. The use of cell culture media as polymer solvent, gelation bath, and storage medium did not negatively affect the alg-PEG-M properties. Microencapsulated EDX cells maintained their viability and proliferated. This study demonstrates the feasibility of primary cell microencapsulation within the novel microsphere type alg-PEG-M, serves as reference for future therapy development, and confirms the suitability of EDX cells as control model.

T cell-B cell interactions in primary immunodeficiencies.

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

2012-02-01

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

Cell-type specific four-component hydrogel.

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Timo Aberle

Full Text Available In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin to generate a blend (technical term: quattroGel, an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering.

Primary clear cell sarcoma of bone

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Choi, J.H.; Gu, M.J.; Kim, M.J.; Bae, Y.K.; Choi, W.H.; Shin, D.S.; Cho, K.H.

2003-01-01

Clear cell sarcoma is a rare soft tissue sarcoma of young adults with melanocytic differentiation. It occurs predominantly in the soft tissue of extremities, typically involving tendons and aponeuroses. Primary clear cell sarcoma of bone is extremely rare. We report a case of primary clear cell sarcoma of the right first metatarsal in a 48-year-old woman and provide a literature review of the entity. (orig.)

A case with primary signet ring cell adenocarcinoma of the prostate and review of the literature

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Orcun Celik

2014-06-01

Full Text Available Primary signet cell carcinoma of the prostate is a rare histological variant of prostate malignancies. It is commonly originated from the stomach, colon, pancreas, and less commonly in the bladder. Prognosis of the classical type is worse than the adenocarcinoma of the prostate. Primary signet cell adenocarcinoma is diagnosed by eliminating the adenocarcinomas of other organs such as gastrointestinal tract organs. In this case report, we present a case with primary signet cell adenocarcinoma of the prostate who received docetaxel chemotherapy because of short prostate specific antigen doubling time.

Long-Lasting Production of New T and B Cells and T-Cell Repertoire Diversity in Patients with Primary Immunodeficiency Who Had Undergone Stem Cell Transplantation: A Single-Centre Experience

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Monica Valotti

2014-01-01

Full Text Available Levels of Kappa-deleting recombination excision circles (KRECs, T-cell receptor excision circles (TRECs, and T-cell repertoire diversity were evaluated in 1038 samples of 124 children with primary immunodeficiency, of whom 102 (54 with severe combined immunodeficiency and 48 with other types of immunodeficiency underwent hematopoietic stem cell transplantation. Twenty-two not transplanted patients with primary immunodeficiency were used as controls. Only data of patients from whom at least five samples were sent to the clinical laboratory for routine monitoring of lymphocyte reconstitutions were included in the analysis. The mean time of the follow-up was 8 years. The long-lasting posttransplantation kinetics of KREC and TREC production occurred similarly in patients with severe combined immunodeficiency and with other types of immunodeficiency and, in both groups, the T-cell reconstitution was more efficient than in nontransplanted children. Although thymic output decreased in older transplanted patients, the degree of T-cell repertoire diversity, after an initial increase, remained stable during the observation period. However, the presence of graft-versus-host disease and ablative conditioning seemed to play a role in the time-related shaping of T-cell repertoire. Overall, our data suggest that long-term B- and T-cell reconstitution was equally achieved in children with severe combined immunodeficiency and with other types of primary immunodeficiency.

Transcriptomic and anatomical complexity of primary, seminal, and crown roots highlight root type-specific functional diversity in maize (Zea mays L.).

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Tai, Huanhuan; Lu, Xin; Opitz, Nina; Marcon, Caroline; Paschold, Anja; Lithio, Andrew; Nettleton, Dan; Hochholdinger, Frank

2016-02-01

Maize develops a complex root system composed of embryonic and post-embryonic roots. Spatio-temporal differences in the formation of these root types imply specific functions during maize development. A comparative transcriptomic study of embryonic primary and seminal, and post-embryonic crown roots of the maize inbred line B73 by RNA sequencing along with anatomical studies were conducted early in development. Seminal roots displayed unique anatomical features, whereas the organization of primary and crown roots was similar. For instance, seminal roots displayed fewer cortical cell files and their stele contained more meta-xylem vessels. Global expression profiling revealed diverse patterns of gene activity across all root types and highlighted the unique transcriptome of seminal roots. While functions in cell remodeling and cell wall formation were prominent in primary and crown roots, stress-related genes and transcriptional regulators were over-represented in seminal roots, suggesting functional specialization of the different root types. Dynamic expression of lignin biosynthesis genes and histochemical staining suggested diversification of cell wall lignification among the three root types. Our findings highlight a cost-efficient anatomical structure and a unique expression profile of seminal roots of the maize inbred line B73 different from primary and crown roots. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Cell surface of sea urchin micromeres and primary mesenchyme

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DeSimone, D.W.

1985-01-01

The cell surface and extracellular matrix (ECM) of the sea urchin embryo were studied during the early morphogenetic events involved in the differentiation of the micromere cell lineage. Sixteen-cell and early cleavage stage blastomeres were isolated and the protein composition of their cell surfaces examined by 125 I-labelling followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Micromere-specific cell surface proteins are reported for Arbacia punctulata, Strongylocentrotus droebachiensis, and Strongylocentrotus purpuratus. Cell surface glycoproteins were characterized on the basis of lectin binding specificity with a novel lectin affinity transfer technique. Using this procedure, cell-type specific surface proteins, which are also lectin-binding specific, can be detected. In addition, fluorescein conjugated lectins were microinjected into the blastocoels of living S. drobachiensis and Lytechinus pictus embryos and the patterns of lectin bindings observed by fluorescence microscopy. The evidence presented in this thesis suggests that the differentiation of the primary mesenchyme cells is correlated with changes in the molecular composition of the cell-surface and the ECM

Small cell type neuroendocrine carcinoma colliding with squamous cell carcinoma at esophagus

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Yang, Luoluo; Sun, Xun; Zou, Yabin; Meng, Xiangwei

2014-01-01

Collision tumor is an extremely rare tumor which defined as the concrescence of two distinct primaries neoplasms. We report here a case of collision tumor at lower third esophagus composed of small cell type neuroendocrine carcinoma (NEC), which is an very rare, highly aggressive and poorly prognostic carcinoma and squamous cell carcinoma (SqCC). In our case, pathologically, the small cell carcinoma display the characteristic of small, round, ovoid or spindle-shaped tumor cells with scant cytoplasm, which colliding with a moderately differentiated squamous cell carcinoma. Immunohistochemical staining demonstrated positive activities for CD56, synaptophysin, 34βE12, CK 5/6, ki-67 (70%-80%), but negative for CD99, chromogranin A, and TTF-1. Accurate diagnosis was made base on these findings. PMID:24817981

A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

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Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R

2018-03-01

Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

PPARgamma in immunity and inflammation: cell types and diseases.

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Széles, Lajos; Töröcsik, Dániel; Nagy, László

2007-08-01

The lipid activated transcription factor, PPARgamma appears to have multiple functions in the immune system. There are several cell types expressing the receptor, most prominently antigen presenting cells, such as macrophages and dendritic cells. The receptor's activation leads to primary transcriptional activation of many, mostly lipid metabolism-related genes. However, gene regulation also occurs on immunity and inflammation-related genes. Key questions are: in what way lipid metabolism and immune regulation are connected and how activation and/or repression of gene expression may modulate inflammatory and anti-inflammatory responses and in what way can these be utilized in therapy. Here we provide a cell type and disease centric review on the role of this lipid activated transcription factor in the various cells of the immune system it is expressed in, and in some major inflammatory diseases such as atherosclerosis, inflammatory bowel disease and rheumatoid arthritis.

Global investigation of interleukin-1β signaling in primary β-cells using quantitative phosphoproteomics

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Engholm-Keller, Kasper; Størling, Joachim; Pociot, Flemming

in β-cells by which this cytokine can modulate cell-matrix interactions during inflammation, a regulation shown in other cell types. Further data analysis is currently ongoing, and the collective results of the experiments will hopefully facilitate additional insights into the effect of IL-1β......Novel Aspect: Global phosphoproteomic analysis of cytokine signaling in primary β-cells Introduction The insulin-producing β-cells of the pancreatic islets of Langerhans are targeted by aberrant immune system responses in diabetes mellitus involving cytokines, especially interleukin-1β (IL-1 β......), which initiate apoptosis of the β-cells. As only limited amounts of primary β-cells can be isolated from model organisms like mouse and rat, global phosphoproteomic analysis of these signaling events by mass spectrometry has generally been unfeasible. We have therefore developed a strategy...

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

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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.

Delicate balance among three types of T cells in concurrent regulation of tumor immunity

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Izhak, Liat; Ambrosino, Elena; Kato, Shingo; Parish, Stanley T.; O’Konek, Jessica J.; Weber, Hannah; Xia, Zheng; Venzon, David; Berzofsky, Jay A.; Terabe, Masaki

2013-01-01

The nature of the regulatory cell types that dominate in any given tumor is not understood at present. Here we addressed this question for Tregs and type II NKT cells in syngeneic models of colorectal and renal cancer. In mice with both type I and type II NKT cells, or in mice with neither type of NKT cell, Treg depletion was sufficient to protect against tumor outgrowth. Surprisingly, in mice lacking only type I NKT cells, Treg blockade was insufficient for protection. Thus, we hypothesized that type II NKT cells may be neutralized by type I NKT cells, leaving Treg cells as the primary suppressor, whereas in mice lacking type I NKT cells, unopposed type II NKT cells could suppress tumor immunity even when Tregs were blocked. We confirmed this hypothesis in three ways by reconstituting type I NKT cells as well as selectively blocking or activating type II NKT cells with antibody or the agonist sulfatide, respectively. In this manner, we demonstrated that blockade of both type II NKT cells and Tregs is necessary to abrogate suppression of tumor immunity, but a third cell, the type I NKT cell, determines the balance between these regulatory mechanisms. As cancer patients often have deficient type I NKT cell function, managing this delicate balance among three T cell subsets may be critical for the success of immunotherapy of human cancer. PMID:23319803

Types of Stem Cells

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... Stem Cell Glossary Search Toggle Nav Types of Stem Cells Stem cells are the foundation from which all ... Learn About Stem Cells > Types of Stem Cells Stem cells Stem cells are the foundation for every organ ...

Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus.

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O'Hara, Bethany A; Gee, Gretchen V; Atwood, Walter J; Haley, Sheila A

2018-04-15

JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML. IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood

MLL-ENL cooperates with SCF to transform primary avian multipotent cells.

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Schulte, Cathleen E; von Lindern, Marieke; Steinlein, Peter; Beug, Hartmut; Wiedemann, Leanne M

2002-08-15

The MLL gene is targeted by chromosomal translocations, which give rise to heterologous MLL fusion proteins and are associated with distinct types of acute lymphoid and myeloid leukaemia. To determine how MLL fusion proteins alter the proliferation and/or differentiation of primary haematopoietic progenitors, we introduced the MLL-AF9 and MLL-ENL fusion proteins into primary chicken bone marrow cells. Both fusion proteins caused the sustained outgrowth of immature haematopoietic cells, which was strictly dependent on stem cell factor (SCF). The renewing cells have a long in vitro lifespan exceeding the Hayflick limit of avian cells. Analysis of clonal cultures identified the renewing cells as immature, multipotent progenitors, expressing erythroid, myeloid, lymphoid and stem cell surface markers. Employing a two-step commitment/differentiation protocol involving the controlled withdrawal of SCF, the MLL-ENL-transformed progenitors could be induced to terminal erythroid or myeloid differentiation. Finally, in cooperation with the weakly leukaemogenic receptor tyrosine kinase v-Sea, the MLL-ENL fusion protein gave rise to multilineage leukaemia in chicks, suggesting that other activated, receptor tyrosine kinases can substitute for ligand-activated c-Kit in vivo.

Primary orbital squamous cell carcinoma

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Ana L. Campos Arbulú

2017-02-01

Full Text Available Primary orbital squamous cell carcinoma is a rare entity. There is little published literature. We report a case of primary squamous cell carcinoma of the orbital soft tissues. Surgical resection offered the best treatment for the patient. Complete resection of the lesion was achieved. The patient received adjuvant radiotherapy due to the proximity of the lesion to the surgical margins. Surgical treatment is feasible and should be considered as part of the surgeon's arsenal. However, therapeutic decisions must be made on a case-by-case basis

Injectable scaffold materials differ in their cell instructive effects on primary human myoblasts

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Hejbøl, Eva Kildall; Sellathurai, Jeeva; Nair, Prabha Damodaran

2017-01-01

Scaffolds are materials used for delivery of cells for regeneration of tissues. They support three-dimensional organization and improve cell survival. For the repair of small skeletal muscles, injections of small volumes of cells are attractive, and injectable scaffolds for delivery of cells offer...... a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed......, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid...

Identification of XMRV infection-associated microRNAs in four cell types in culture.

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Ketha V K Mohan

Full Text Available INTRODUCTION: XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC and chronic fatigue syndrome (CFS in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA, which regulate gene expression, were so far not identified in cells infected with XMRV in culture. METHODS: Two prostate cell lines (LNCaP and DU145 and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray. RESULTS: MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs. miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types. DISCUSSION: The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1

International Nuclear Information System (INIS)

Rosa Borges, Andrew; Wieczorek, Lindsay; Johnson, Benitra; Benesi, Alan J.; Brown, Bruce K.; Kensinger, Richard D.; Krebs, Fred C.; Wigdahl, Brian; Blumenthal, Robert; Puri, Anu; McCutchan, Francine E.; Birx, Deborah L.; Polonis, Victoria R.; Schengrund, Cara-Lynne

2010-01-01

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC 50 s ranging from 0.1 to 7.4 μg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions. -- Research Highlights: →Multivalent carbohydrates (MVCs) inhibited infection of PBMCs by HIV-1. →MVCs inhibited infection by T cell line-adapted viruses. →MVCs inhibited infection by primary isolates of HIV-1. →MVCs inhibited Env-mediated membrane fusion.

Different surface sensing of the cell body and nucleus in healthy primary cells and in a cancerous cell line on nanogrooves.

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Davidson, Patricia M; Bigerelle, Maxence; Reiter, Günter; Anselme, Karine

2015-10-01

Cancer cells are known to have alterations compared to healthy cells, but can these differences extend to the way cells interact with their environment? Here, the authors focused on the alignment on an array of grooves of nanometer depth using two cell types: healthy osteoprogenitor primary cells (HOP) and a cancerous osteosarcoma (SaOs-2) cell line. Another concern was how this alignment affects the cell's interior, namely, the nucleus. Based on the results, it is proposed that these two cell types respond to different size regimes: SaOs-2 cells are more sensitive to shallow grooves while HOP cells are strongly aligned with deep grooves. As a measure of the impact of cell alignment on the nucleus the orientation and elongation of the nucleus were determined. Compared to HOP cells, the cell nucleus of SaOs-2 cells is more aligned and elongated in response to grooves, suggesting a softer nucleus and/or increased force transmission. These results support the hypothesis that cancer cells have reduced nucleus rigidity compared to healthy ones and further indicate differences in sensing, which may be important during metastasis.

Inhibition of cell growth by EGR-1 in human primary cultures from malignant glioma

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Gagliardi Franco

2004-01-01

Full Text Available Abstract Background The aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glioma cells. EGR-1 expression was examined during the early passages in vitro of 17 primary cell lines grown from 3 grade III and from 14 grade IV malignant astrocytoma explants. The explanted tumors were genetically characterized at the p53, MDM2 and INK4a/ARF loci, and fibronectin expression and growth characteristics were examined. A recombinant adenovirus overexpressing EGR-1 was tested in the primary cell lines. Results Low levels of EGR-1 protein were found in all primary cultures examined, with lower values present in grade IV tumors and in cultures carrying wild-type copies of p53 gene. The levels of EGR-1 protein were significantly correlated to the amount of intracellular fibronectin, but only in tumors carrying wild-type copies of the p53 gene (R = 0,78, p = 0.0082. Duplication time, plating efficiency, colony formation in agarose, and contact inhibition were also altered in the p53 mutated tumor cultures compared to those carrying wild-type p53. Growth arrest was achieved in both types of tumor within 1–2 weeks following infection with a recombinant adenovirus overexpressing EGR-1 but not with the control adenovirus. Conclusions Suppression of EGR-1 is a common event in gliomas and in most cases this is achieved through down-regulation of gene expression. Expression of EGR-1 by recombinant adenovirus infection almost completely abolishes the growth of tumor cells in vitro, regardless of the mutational status of the p53 gene.

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

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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.

Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

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Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

2018-02-20

Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

Delicate balance among three types of T cells in concurrent regulation of tumor immunity.

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Izhak, Liat; Ambrosino, Elena; Kato, Shingo; Parish, Stanley T; O'Konek, Jessica J; Weber, Hannah; Xia, Zheng; Venzon, David; Berzofsky, Jay A; Terabe, Masaki

2013-03-01

The nature of the regulatory cell types that dominate in any given tumor is not understood at present. Here, we addressed this question for regulatory T cells (Treg) and type II natural killer T (NKT) cells in syngeneic models of colorectal and renal cancer. In mice with both type I and II NKT cells, or in mice with neither type of NKT cell, Treg depletion was sufficient to protect against tumor outgrowth. Surprisingly, in mice lacking only type I NKT cells, Treg blockade was insufficient for protection. Thus, we hypothesized that type II NKT cells may be neutralized by type I NKT cells, leaving Tregs as the primary suppressor, whereas in mice lacking type I NKT cells, unopposed type II NKT cells could suppress tumor immunity even when Tregs were blocked. We confirmed this hypothesis in 3 ways by reconstituting type I NKT cells as well as selectively blocking or activating type II NKT cells with antibody or the agonist sulfatide, respectively. In this manner, we showed that blockade of both type II NKT cells and Tregs is necessary to abrogate suppression of tumor immunity, but a third cell, the type I NKT cell, determines the balance between these regulatory mechanisms. As patients with cancer often have deficient type I NKT cell function, managing this delicate balance among 3 T-cell subsets may be critical for the success of immunotherapy for human cancer. ©2012 AACR.

Primary central nervous system diffuse large B-cell lymphoma shows an activated B-cell-like phenotype with co-expression of C-MYC, BCL-2, and BCL-6.

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Li, Xiaomei; Huang, Ying; Bi, Chengfeng; Yuan, Ji; He, Hong; Zhang, Hong; Yu, QiuBo; Fu, Kai; Li, Dan

2017-06-01

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma, whose main prognostic factor is closely related to germinal center B-cell-like subtype (GCB- DLBCL) or activated B-cell-like type (non-GCB-DLBCL). The most common type of primary central nervous system lymphoma is diffuse large B-cell type with poor prognosis and the reason is unclear. This study aims to stratify primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) according to the cell-of-origin (COO) and to investigate the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53, further to elucidate the reason why primary central nervous system diffuse large B-cell lymphoma possesses a poor clinical outcome as well. Nineteen cases of primary central nervous system DLBCL were stratified according to immunostaining algorithms of Hans, Choi and Meyer (Tally) and we investigated the multiple proteins expression of C-MYC, BCL-6, BCL-2, TP53. The Epstein-Barr virus and Borna disease virus infection were also detected. Among nineteen cases, most (15-17 cases) were assigned to the activated B-cell-like subtype, highly expression of C-MYC (15 cases, 78.9%), BCL-2 (10 cases, 52.6%), BCL-6 (15 cases, 78.9%). Unfortunately, two cases were positive for PD-L1 while PD-L2 was not expressed in any case. Two cases infected with BDV but no one infected with EBV. In conclusion, most primary central nervous system DLBCLs show an activated B-cell-like subtype characteristic and have multiple expressions of C-MYC, BCL-2, BCL-6 protein, these features might be significant factor to predict the outcome and guide treatment of PCNS-DLBCLs. Copyright © 2017 Elsevier GmbH. All rights reserved.

Primary B cell lymphoma of the tongue base: a case report

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Bechir, Achour; Asma, Achour; Haifa, Regaieg; Nesrine, Abdessayed; Yosra, Ben Youssef; Badreddine, Sriha; Abderrahim, Khelif

2016-01-01

Primary non-Hodgkin’s lymphoma’s of the tongue is very rare and accounts for 1% of all malignant tumor of the oral cavity. Clinical features are non-specific ulcerative lesions that do not heal. In the literature, the majority of cases are diffuse large B cell type however, T cell phenotype also may occur. We describe a 77 years old man, who presented with an ulcerative mass in the left margin of the tongue the diagnosis diffuse large B cell lymphoma was confirmed. The patient is actually on treatment R-mini CEOP and has favorable evolution. PMID:28292136

Primary Cutaneous Diffuse Large B-Cell Lymphoma – a Case Report

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Milovanović Milena

2017-06-01

Full Text Available In 2005, the World Health Organization - European Organization for Research and Treatment of Cancer (WHOEORTC classified cutaneous B-cell lymphomas into 4 categories: primary cutaneous marginal zone B-cell lymphoma (PCMZL, primary cutaneous follicle center lymphoma (PCFCL, primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT, and primary cutaneous diffuse large B-cell lymphoma, other (PCDLBCL-O. The absence of evident extra-cutaneous disease is a necessary condition for the diagnosis of primary cutaneous B-cell lymphomas, because they have a completely different clinical behavior and prognosis from their nodal counterparts. PCDLBCL-O basically represents a morphological variation, lacking the typical features of PCDLBCLLT, neither confirming the definition of PCFCCL, but on the clinical ground, its behavior seems at least to partially overlap the indolent course of PCFCCL. In fact, the present WHO lymphoma classification from 2008 overcame the previous WHO-EORTC classification, including at least a part of PCDLBCL-O within the spectrum of PCFCCL. However, owing to the rarity and heterogeneity of the PCDLBCL-O, the precise clinicopathological characteristics have not been well characterized and the optimal treatment for this group of lymphomas is yet to be defined. Nevertheless, dermatologists and pathologists should be aware of this entity in order to avoid unnecessary aggressive treatment. We present a case of a 46-year-old Caucasian male with one large round-shaped tumor and a few scattered nodules localized on the back. The histopathological features of the lesion corresponded to PCDLBCL-O. The patient follow-up showed that he was disease-free three months after surgical excision of the lesions and adjuvant local radiotherapy. No additional therapy was introduced, including chemotherapy with rituximab, cyclophosphamide, doxorubicin hydrochloride, oncovin, prednisolone (R-CHOP.

Excellent outcome of immunomodulation or Bruton’s tyrosine kinase inhibition in highly refractory primary cutaneous diffuse large B-cell lymphoma, leg type

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Eva Gupta

2015-12-01

Full Text Available Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT is a rare diffuse large B-cell lymphoma confined to the skin of the legs. The typical presentation is characterized by solitary or multiple growing plaques, usually confined to one leg. We report a case of PCDLBCL-LT of activated B-cell subtype characterized by multiple local relapses in the legs, initially, and systemic relapses about seven years after the diagnosis. Local relapses were sensitive to radiation therapy. Cutaneous and systemic relapses responded well to immunomodulatory therapy with lenalidomide followed by Bruton’s tyrosine kinase inhibition with ibrutinib. Ibrutinib is the only treatment that resulted in long-lasting complete remission. Lenalidomide and especially ibrutinib appear to have a significant activity against this lymphoma and should be incorporated in the treatment of this resistant and aggressive lymphoma. To our knowledge, this is the first case of PCDLBCL-LT reported in the literature exhibiting a complete response to ibrutinib.

The influence of 60Co gamma rays to cell reproduction (An experiment using low dose levels on vero and primary monkey kidney cells)

International Nuclear Information System (INIS)

Danusupadmo, C.J. Sugiarto

1985-01-01

Vero and primary monkey kidney cells in culture were gamma irradiated with doses of 0, 0.4 and 0.8 Gy at a dose-rate of 1.30-1.45x10 3 Gy/hour. At harvest time 3 days post irradiation, 0.4 Gy proved to be able to lower the number of vero cells in such a degree that it became significantly different from the control, whereas 0.8 Gy could not suppress the number of primary cells to a level that differed significantly from its control. At harvest time of 7 days post irradiation, 0.4 Gy was found effective in lowering both vero and primary cells so that the number of the harvested cells were significantly different from the controls. At harvest time of 3 days post irradiation, 0.8 Gy caused both cell types reached levels that were not significantly different from 0.4 Gy, but at 7 days post irradiation the number of vero cells was very significantly different from that of 0.4 Gy, while the number of primary cells remained equal to that of 0.4 Gy. This phenomenon showed that irradiation could cause greater injurious effect at more advanced post irradiation times, while the more proliferative vero cells proved to be more susceptible to irradiation than primary cells, but at the same time more potential in performing repair. (author)

An optimized protocol for isolating primary epithelial cell chromatin for ChIP.

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James A Browne

Full Text Available A critical part of generating robust chromatin immunoprecipitation (ChIP data is the optimization of chromatin purification and size selection. This is particularly important when ChIP is combined with next-generation sequencing (ChIP-seq to identify targets of DNA-binding proteins, genome-wide. Current protocols refined by the ENCODE consortium generally use a two-step cell lysis procedure that is applicable to a wide variety of cell types. However, the isolation and size selection of chromatin from primary human epithelial cells may often be particularly challenging. These cells tend to form sheets of formaldehyde cross-linked material in which cells are resistant to membrane lysis, nuclei are not released and subsequent sonication produces extensive high molecular weight contamination. Here we describe an optimized protocol to prepare high quality ChIP-grade chromatin from primary human bronchial epithelial cells. The ENCODE protocol was used as a starting point to which we added the following key steps to separate the sheets of formaldehyde-fixed cells prior to lysis. (1 Incubation of the formaldehyde-fixed adherent cells in Trypsin-EDTA (0.25% room temperature for no longer than 5 min. (2 Equilibration of the fixed cells in detergent-free lysis buffers prior to each lysis step. (3 The addition of 0.5% Triton X-100 to the complete cell membrane lysis buffer. (4 Passing the cell suspension (in complete cell membrane lysis buffer through a 25-gauge needle followed by continuous agitation on ice for 35 min. Each step of the modified protocol was documented by light microscopy using the Methyl Green-Pyronin dual dye, which stains cytoplasm red (Pyronin and the nuclei grey-blue (Methyl green. This modified method is reproducibly effective at producing high quality sheared chromatin for ChIP and is equally applicable to other epithelial cell types.

Inflammatory Cell Distribution in Primary Merkel Cell Carcinoma

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Wheat, Rachel [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); Roberts, Claudia [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Waterboer, Tim [Infection and Cancer Program, DKFZ (German Cancer Research Centre), 69120 Heidelberg (Germany); Steele, Jane [Human Biomaterials Resource Centre, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); Marsden, Jerry [University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Steven, Neil M., E-mail: n.m.steven@bham.ac.uk [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Blackbourn, David J., E-mail: n.m.steven@bham.ac.uk [Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH (United Kingdom)

2014-05-06

Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus.

Inflammatory Cell Distribution in Primary Merkel Cell Carcinoma

International Nuclear Information System (INIS)

Wheat, Rachel; Roberts, Claudia; Waterboer, Tim; Steele, Jane; Marsden, Jerry; Steven, Neil M.; Blackbourn, David J.

2014-01-01

Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus

Primary Cilia, Signaling Networks and Cell Migration

DEFF Research Database (Denmark)

Veland, Iben Rønn

Primary cilia are microtubule-based, sensory organelles that emerge from the centrosomal mother centriole to project from the surface of most quiescent cells in the human body. Ciliary entry is a tightly controlled process, involving diffusion barriers and gating complexes that maintain a unique...... this controls directional cell migration as a physiological response. The ciliary pocket is a membrane invagination with elevated activity of clathrin-dependent endocytosis (CDE). In paper I, we show that the primary cilium regulates TGF-β signaling and the ciliary pocket is a compartment for CDE...... on formation of the primary cilium and CDE at the pocket region. The ciliary protein Inversin functions as a molecular switch between canonical and non-canonical Wnt signaling. In paper II, we show that Inversin and the primary cilium control Wnt signaling and are required for polarization and cell migration...

Accumulation of type VI collagen in the primary osteon of the rat femur during postnatal development.

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Kohara, Yukihiro; Soeta, Satoshi; Izu, Yayoi; Amasaki, Hajime

2015-05-01

In rodents, the long bone diaphysis is expanded by forming primary osteons at the periosteal surface of the cortical bone. This ossification process is thought to be regulated by the microenvironment in the periosteum. Type VI collagen (Col VI), a component of the extracellular matrix (ECM) in the periosteum, is involved in osteoblast differentiation at early stages. In several cell types, Col VI interacts with NG2 on the cytoplasmic membrane to promote cell proliferation, spreading and motility. However, the detailed functions of Col VI and NG2 in the ossification process in the periosteum are still under investigation. In this study, to clarify the relationship between localization of Col VI and formation of the primary osteon, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the periosteum of rat femoral diaphysis during postnatal growing periods by immunohistochemistry. Primary osteons enclosing the osteonal cavity were clearly identified in the cortical bone from 2 weeks old. The size of the osteonal cavities decreased from the outer to the inner region of the cortical bone. In addition, the osteonal cavities of newly formed primary osteons at the outermost region started to decrease in size after rats reached the age of 4 weeks. Immunohistochemistry revealed concentrated localization of Col VI in the ECM in the osteonal cavity. Col VI-immunoreactive areas were reduced and they disappeared as the osteonal cavities became smaller from the outer to the inner region. In the osteonal cavities of the outer cortical regions, Runx2-immunoreactive spindle-shaped cells and mature osteoblasts were detected in Col VI-immunoreactive areas. The numbers of Runx2-immunoreactive cells were significantly higher in the osteonal cavities than in the osteogenic layers from 2 to 4 weeks. Most of these Runx2-immunoreactive cells showed NG2-immunoreactivity. Furthermore, PCNA-immunoreactivity was detected in the Runx2-immunoreactive spindle

Analysis of histological type and CT features on patients with haemoptysis in primary lung cancer

International Nuclear Information System (INIS)

Zhang Yixing; Huang Jinhuo

2008-01-01

Objective: To explore the CT features and histological types of haemoptysis patients with primary lung cancer. Methods: 50 haemoptysis patients (group A) with primary lung cancer confirmed by bronchoendoscopic examination or operation were analyzed. 50 cases of primary lung cancer without haemoptysis were studied as the contrast group (group B). The tumor size, shape, cavity, as well as location and pathological type were compared. Results: The proportions of adenocarcinoma and squamous cell carcinoma were 48%, 26% in Group A and 23%, 31% in Group B respectively. The occurrent rate of adenocarcinoma showed significant difference between Group A and B (P>0.05). The proportion of central lung cancer and peripheral lung cancer were 54%, 50% and 46%, 50% in Group A and B respectively. No significant difference occurred in both two types of cancer between two groups (P>0.05). The average size of cancer mass were 30±2.54 mm in Group A and 32±1.93mm in group B. The occurrence rate of lung cavity was 18% and 15% respectively in Group A and B. No significant difference existed in size and the occurrence rate of lung cancer mass between Group A and B (P>0.05). Conclusion: Haemoptysis in patients of primary lung cancer was correlated with pathology type, instead of location, size and cavities. (authors)

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1

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Borges, Andrew Rosa; Wieczorek, Lindsay; Johnson, Benitra; Benesi, Alan J.; Brown, Bruce K.; Kensinger, Richard D.; Krebs, Fred C.; Wigdahl, Brian; Blumenthal, Robert; Puri, Anu; McCutchan, Francine E.; Birx, Deborah L.; Polonis, Victoria R.; Schengrund, Cara-Lynne

2010-01-01

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3’-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC50s ranging from 0.1 – 7.4 µg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions. PMID:20880566

Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications

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DH Rosenzweig

2017-03-01

Full Text Available Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.

Primary clear cell sarcoma of rib

International Nuclear Information System (INIS)

Hersekli, Murat Ali; Ozkoc, Gurkan; Akpinar, Sercan; Ozalay, Metin; Tandogan, Reha N.; Bircan, Sema; Tuncer, Ilhan

2005-01-01

Clear cell sarcoma (malignant melanoma of soft tissues) is a very rare soft tissue neoplasm. It generally arises in tendons and aponeuroses. Although metastasis of malignant melanoma to bone is not uncommon, primary clear cell sarcoma of bone is an extremely rare neoplasm. To our knowledge five cases have been reported in the English literature. We present a case of primary clear cell sarcoma of bone in a 28-year-old woman arising in the left ninth rib. We treated the patient with total excision of the mass and postoperative radiotherapy. The patient is alive and well without local recurrence or distant metastasis at 33 months after surgery. (orig.)

Multicentric primary extramammary Paget disease: a Toker cell disorder?

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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.

Identification of genuine primary pulmonary NK cell lymphoma via clinicopathologic observation and clonality assay.

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Gong, Li; Wei, Long-Xiao; Huang, Gao-Sheng; Zhang, Wen-Dong; Wang, Lu; Zhu, Shao-Jun; Han, Xiu-Juan; Yao, Li; Lan, Miao; Li, Yan-Hong; Zhang, Wei

2013-08-19

Extranodal natural killer (NK)/T-cell lymphoma, nasal type, is an uncommon lymphoma associated with the Epstein-Barr virus (EBV). It most commonly involves the nasal cavity and upper respiratory tract. Primary pulmonary NK/T cell lymphoma is extremely rare. If a patient with a NK or T-cell tumor has an unusual reaction to treatment or an unusual prognosis, it is wise to differentiate NK from T-cell tumors. The clinicopathologic characteristics, immunophenotype, EBV in situ hybridization, and T cell receptor (TCR) gene rearrangement of primary pulmonary NK cell lymphoma from a 73-year-old Chinese woman were investigated and the clonal status was determined using female X-chromosomal inactivation mosaicism and polymorphisms at the phosphoglycerate kinase (PGK) gene. The lesion showed the typical histopathologic characteristics and immunohistochemical features of NK/T cell lymphoma. However, the sample was negative for TCR gene rearrangement. A clonality assay demonstrated that the lesion was monoclonal. It is concluded that this is the first recorded case of genuine primary pulmonary NK cell lymphoma. The purpose of the present work is to recommend that pathologists carefully investigate the whole lesion to reduce the likelihood that primary pulmonary NK cell lymphoma will be misdiagnosed as an infectious lesion. In addition, TCR gene rearrangement and clonal analysis, which is based on female X-chromosomal inactivation mosaicism and polymorphisms at PGK and androgen receptor (AR) loci, were found to play important roles in differentiating NK cell lymphoma from T cell lymphoma. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5205300349457729.

Primary Hepatosplenic Large B-Cell Lymphoma

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M.R. Morales-Polanco

2008-03-01

Full Text Available Diffuse large B-cell lymphoma is the most common form of lymphoma. It usually begins in the lymph nodes; up to 40% may have an extranodal presentation. According to a definition of primary extranodal lymphoma with presentation only in extranodal sites, there are reports of large B-cell lymphomas limited to liver or spleen as separate entities, and to date there have been only three documented cases of primary hepatosplenic presentation. This paper reports a fourth case. Due to a review of the literature and the clinical course of the case reported, we conclude that primary hepatosplenic large B-cell lymphoma has been found predominantly in females older than 60 years. The patients reported had <2 months of evolution prior to diagnosis, prominent B symptoms, splenomegaly in three and hepatomegaly in two, none with lymph node involvement. All had thrombocytopenia and abnormal liver function tests; three had anemia and elevated serum lactic dehydrogenase levels, two with hemophagocytosis in bone marrow. Because of the previously mentioned data, it can be stated that primary hepatosplenic lymphoma is an uncommon and aggressive form of disease that requires immediate recognition and treatment.

A CASE REPORT OF MULTIPLE PRIMARY SQUAMOUS CELL CARCINOMAS OF THE OVARY AND SIGMOID COLON

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A. B. Villert

2016-01-01

Full Text Available Squamous cell ovarian and sigmoid colon carcinomas are extremely rare malignancies. Because of their rarity, it is difficult to investigate the clinical characteristics and prognosis of patients with theses malignancies, and therefore, the increased interest in each clinical case report is highly relevant. Multiple primary squamous cell ovarian and sigmoid colon carcinomas are the subject of discussion and differential diagnosis of sigmoid colon cancer with secondary ovarian cancer. Histopathological and clinical characteristics of the tumors were present and evidences in favor of the multiple primary malignancies were given. The association of squamous cell ovarian and sigmoid colon carcinomas with human papilloma virus type 16 was shown.

Primary Germ Cell Tumors of the Mediastinum: 10 Years of Experience in a Tertiary Teaching Hospital

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Chih-Jen Yang

2005-09-01

Full Text Available Germ cell tumors occur mostly in the gonad. Extragonadal germ cell tumors are rare, and most occur in the retroperitoneum and mediastinum. Primary mediastinal germ cell tumors are often found in the anterior portion of the mediastinum and include teratomas and non-teratomatous tumors. Non-teratomatous tumors include seminomas and malignant non-seminomatous germ cell tumors (MNSGCTs. MNSGCTs include yolk sac tumors, choriocarcinomas, embryonal carcinomas, and mixed type germ cell tumors. Teratomas are the most common germ cell tumors of the mediastinum, and seminomas are the most common non-teratomatous germ cell tumors of the mediastinum. Cases of primary mediastinal MNSGCT reported in the literature are rare. In this report, we review all primary mediastinal germ cell tumors from a 10-year period at the Chung-Ho Memorial Hospital of Kaohsiung Medical University. A total of 14 cases were reviewed, including 11 patients with mature teratomas, two with yolk sac tumors, and one with seminoma. We discuss the differences in clinical presentation, histopathologic characteristics, treatment, and prognosis.

DJ-1 Modulates Nuclear Erythroid 2-Related Factor-2-Mediated Protection in Human Primary Alveolar Type II Cells in Smokers.

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Bahmed, Karim; Messier, Elise M; Zhou, Wenbo; Tuder, Rubin M; Freed, Curt R; Chu, Hong Wei; Kelsen, Steven G; Bowler, Russell P; Mason, Robert J; Kosmider, Beata

2016-09-01

Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2-related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.

Induced Pluripotent Stem Cells: A novel frontier in the study of human primary immunodeficiencies

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Pessach, Itai M.; Ordovas-Montanes, Jose; Zhang, Shen-Ying; Casanova, Jean-Laurent; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Manos, Philip D.; Schlaeger, Thorsten M.; Park, In-Hyun; Rucci, Francesca; Agarwal, Suneet; Mostoslavsky, Gustavo; Daley, George Q.; Notarangelo, Luigi D.

2010-01-01

Background The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells through the exogenous expression of transcription promises to revolutionize the study of human diseases. Objective Here we report on the generation of 25 induced pluripotent stem cell lines from 6 patients with various forms of Primary Immunodeficiencies, affecting adaptive and/or innate immunity. Methods Patients’ dermal fibroblasts were reprogrammed by expression of four transcription factors, OCT4, SOX2, KLF4, and c-MYC using a single excisable polycistronic lentiviral vector. Results Induced pluripotent stem cells derived from patients with primary immunodeficiencies show a stemness profile that is comparable to that observed in human embryonic stem cells. Following in vitro differentiation into embryoid bodies, pluripotency of the patient-derived indiced pluripotent stem cells lines was demonstrated by expression of genes characteristic of each of the three embryonic layers. We have confirmed the patient-specific origin of the induced pluripotent stem cell lines, and ascertained maintenance of karyotypic integrity. Conclusion By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of induced pluripotent stem cell lines from patients with primary immunodeficiencies represents a unique resource to investigate the pathophysiology of hematopoietic and extra-hematopoietic manifestations of these diseases, and may assist in the development of novel therapeutic approaches based on gene correction. PMID:21185069

Teaching Cell Biology in Primary Schools

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Francele de Abreu Carlan

2014-01-01

Full Text Available Basic concepts of cell biology are essential for scientific literacy. However, because many aspects of cell theory and cell functioning are quite abstract, students experience difficulties understanding them. In this study, we investigated whether diverse teaching resources such as the use of replicas of Leeuwenhoek’s microscope, visualization of cells using an optical microscope, construction of three-dimensional cell models, and reading of a comic book about cells could mitigate the difficulties encountered when teaching cell biology to 8th-grade primary school students. The results suggest that these didactic activities improve students’ ability to learn concrete concepts about cell biology, such as the composition of living beings, growth, and cicatrization. Also, the development of skills was observed, as, for example, the notion of cell size. However, no significant improvements were observed in students’ ability to learn about abstract topics, such as the structures of subcellular organelles and their functions. These results suggest that many students in this age have not yet concluded Piaget’s concrete operational stage, indicating that the concepts required for the significant learning of abstract subjects need to be explored more thoroughly in the process of designing programs that introduce primary school students to cell biology.

Primary Human Blood Dendritic Cells for Cancer Immunotherapy—Tailoring the Immune Response by Dendritic Cell Maturation

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Simone P. Sittig

2015-12-01

Full Text Available Dendritic cell (DC-based cancer vaccines hold the great promise of tipping the balance from tolerance of the tumor to rejection. In the last two decades, we have gained tremendous knowledge about DC-based cancer vaccines. The maturation of DCs has proven indispensable to induce immunogenic T cell responses. We review the insights gained from the development of maturation cocktails in monocyte derived DC-based trials. More recently, we have also gained insights into the functional specialization of primary human blood DC subsets. In peripheral human blood, we can distinguish at least three primary DC subsets, namely CD1c+ and CD141+ myeloid DCs and plasmacytoid DCs. We reflect the current knowledge on maturation and T helper polarization by these blood DC subsets in the context of DC-based cancer vaccines. The maturation stimulus in combination with the DC subset will determine the type of T cell response that is induced. First trials with these natural DCs underline their excellent in vivo functioning and mark them as promising tools for future vaccination strategies.

Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells

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Fornara, O; Bartek, J; Rahbar, A

2016-01-01

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express...... human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary...... GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co...

[Primary peripheral T-cell lymphoma of the penis: a case report and review of the literature].

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Shi, Yan-Lin; Yin, Hong-Lin; Zhou, Xiao-Jun; Zhou, Hang-Bo; Lu, Zhen-Feng

2008-11-01

To report a case of primary peripheral T-cell lymphoma of the penis. We analyzed the clinicopathological characteristics of the case of primary peripheral T-cell lymphoma using histological, cytochemical and immunohistochemical methods and by review of the literature. The patient was a 65 years old man and presented with a diffuse enlargement of the penis as the initial sign, followed by erosive ulcer in the caput penis and inguinal lymphadenectasis. The tumor was pathohistologically manifested as an epidermal ulcer, with tumorous necrosis around the capillary, infiltrative growth and atypical changes of the neoplastic cells and proliferation of capillaries. Immunohistochemically, the tumor cells were positive for CD43 and CD3, but negative for CD20, CD79a, CD34, CD30, CD56 and CD34. Clinically it responded to the chemotherapy designed for peripheral T-cell lymphoma. Primary peripheral T-cell lymphoma of the penis is an extremely rare malignant tumor, the diagnosis of which relies on histopathological examination, immunohistochemical staining and differentiation between squamous cell carcinoma and other types of lymphoma.

Primary peritoneal anaplastic giant cell carcinoma: case report of an unusual and highly malignant müllerian neoplasm.

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Lu, Xian; Zhang, Cunxian; Liu, Fang; Sung, C James; Steinhoff, Margaret M; Lawrence, W Dwayne

2008-01-01

Virtually all primary peritoneal carcinomas (PPCs) are of serous papillary type. We report an unusual histologic type of PPC composed of anaplastic giant cells, which exhibited an aggressive clinical course. A 72-year-old woman presented with lower abdominal pain. Computed tomography showed a diffuse omental thickening. The patient underwent an exploratory laparotomy with omentectomy, total hysterectomy, bilateral salpingo-oophorectomy, and appendectomy. Pathologic examination revealed extensive omental replacement by tumor but only superficial surface cortical involvement of both ovaries, a disease distribution consistent with a typical müllerian-derived PPC. However, this neoplasm was composed of diffuse anaplastic tumor giant cells, rather than serous carcinoma, which is the usual histologic type encountered in PPC. The patient died within 1 month after surgery. We report this unusual histologic variant of PPC to raise awareness that anaplastic giant cell carcinoma may arise in the pelvic peritoneum as a primary tumor.

A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

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Koban, Robert; Neumann, Markus; Daugs, Aila; Bloch, Oliver; Nitsche, Andreas; Langhammer, Stefan; Ellerbrok, Heinz

2018-02-01

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

Characteristics of primary infection of a European human immunodeficiency virus type 1 clade B isolate in chimpanzees

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Bogers, W. M.; Koornstra, W. H.; Dubbes, R. H.; ten Haaft, P. J.; Verstrepen, B. E.; Jhagjhoorsingh, S. S.; Haaksma, A. G.; Niphuis, H.; Laman, J. D.; Norley, S.; Schuitemaker, H.; Goudsmit, J.; Hunsmann, G.; Heeney, J. L.; Wigzell, H.

1998-01-01

The aim of the study was to select, from a panel of candidate European human immunodeficiency virus type 1 (HIV-1) clade B primary virus isolates, one isolate based on replication properties in chimpanzee peripheral blood mononuclear cells (PBMC). Secondly, to evaluate the in vivo kinetics of

Formation of wood secondary cell wall may involve two type cellulose synthase complexes in Populus.

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Xi, Wang; Song, Dongliang; Sun, Jiayan; Shen, Junhui; Li, Laigeng

2017-03-01

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.

Targeting of beta-arrestin2 to the centrosome and primary cilium: role in cell proliferation control.

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Anahi Molla-Herman

Full Text Available The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs. Most GPCRs are regulated by beta-arrestins, betaarr1 and betaarr2, which control both their signalling and endocytosis, suggesting that betaarrs may also function at primary cilium.In cycling cells, betaarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, betaarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, betaarr2 was found at the basal body and axoneme of primary cilia. Interestingly, betaarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, betaarrs appear to control cell cycle progression. Indeed, cells lacking betaarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.Our results show that betaarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, betaarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell "antenna".

Treatment of Primary Cutaneous CD4 Small/Medium T cell Lymphoproliferative Disorder with Intralesional Triamcinolone Acetonide.

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2018-02-15

12. REPORT TYPE 02/15/2018 Poster 4. TITLE AND SUBTITLE Treatment of Primary Cutaneous CD4+ Small/Medium T- cell Lymphoproliferative Disorder with...cutaneous CD4+ small/medium T- cell lymphoproliferative disorder (LPD) is a generally indolent cutaneous T- cell proliferation. Most cases follow a benign...lmmunohistochemistry showed diffuse CD3+ CD4+ T- cells without CD30, TIA1 or CD10. A subset of medium to large cells expressed BCL-6. Small subsets of B- cells and CDB

Pathogenesis and treatment of adult-type granulosa cell tumor of the ovary.

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Färkkilä, Anniina; Haltia, Ulla-Maija; Tapper, Johanna; McConechy, Melissa K; Huntsman, David G; Heikinheimo, Markku

2017-08-01

Adult-type granulosa cell tumor is a clinically and molecularly unique subtype of ovarian cancer. These tumors originate from the sex cord stromal cells of the ovary and represent 3-5% of all ovarian cancers. The majority of adult-type granulosa cell tumors are diagnosed at an early stage with an indolent prognosis. Surgery is the cornerstone for the treatment of both primary and relapsed tumor, while chemotherapy is applied only for advanced or non-resectable cases. Tumor stage is the only factor consistently associated with prognosis. However, every third of the patients relapse, typically in 4-7 years from diagnosis, leading to death in 50% of these patients. Anti-Müllerian Hormone and inhibin B are currently the most accurate circulating biomarkers. Adult-type granulosa cell tumors are molecularly characterized by a pathognomonic somatic missense point mutation 402C->G (C134W) in the transcription factor FOXL2. The FOXL2 402C->G mutation leads to increased proliferation and survival of granulosa cells, and promotes hormonal changes. Histological diagnosis of adult-type granulosa cell tumor is challenging, therefore testing for the FOXL2 mutation is crucial for differential diagnosis. Large international collaborations utilizing molecularly defined cohorts are essential to improve and validate new treatment strategies for patients with high-risk or relapsed adult-type granulosa cell tumor. Key Messages: Adult-type granulosa cell tumor is a unique ovarian cancer with an indolent, albeit unpredictable disease course. Adult-type granulosa cell tumors harbor a pathognomonic somatic missense mutation in transcription factor FOXL2. The key challenges in the treatment of patients with adult-type granulosa cell tumor lie in the identification and management of patients with high-risk or relapsed disease.

Differential Gene Expression of Primary Cultured Lymphatic and Blood Vascular Endothelial Cells

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Gregory M. Nelson

2007-12-01

Full Text Available Blood vascular endothelial cells (BECs and the developmentally related lymphatic endothelial cells (LECs create complementary, yet distinct vascular networks. Each endothelial cell type interacts with flowing fluid and circulating cells, yet each vascular system has evolved specialized gene expression programs and thus both cell types display different phenotypes. BECs and LECs express distinct genes that are unique to their specific vascular microenvironment. Tumors also take advantage of the molecules that are expressed in these vascular systems to enhance their metastatic potential. We completed transcriptome analyses on primary cultured LECs and BECs, where each comparative set was isolated from the same individual. Differences were resolved in the expression of several major categories, such as cell adhesion molecules (CAMs, cytokines, cytokine receptors. We have identified new molecules that are associated with BECs (e.g., claudin-9, CXCL11, neurexin-1, neurexin-2, the neuronal growth factor regulator-1 and LECs (e.g., claudin-7, CD58, hyaluronan and proteoglycan link protein 1 (HAPLN1, the poliovirus receptor-related 3 molecule that may lead to novel therapeutic treatments for diseases of lymphatic or blood vessels, including metastasis of cancer to lymph nodes or distant organs.

Homeostatic migration and distribution of innate immune cells in primary and secondary lymphoid organs with ageing.

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Nikolich-Žugich, J; Davies, J S

2017-03-01

Ageing of the innate and adaptive immune system, collectively termed immune senescence, is a complex process. One method to understand the components of ageing involves dissociating the effects of ageing on the cells of the immune system, on the microenvironment in lymphoid organs and tissues where immune cells reside and on the circulating factors that interact with both immune cells and their microenvironment. Heterochronic parabiosis, a surgical union of two organisms of disparate ages, is ideal for this type of study, as it has the power to dissociate the age of the cell and the age of the microenvironment into which the cell resides or is migrating. So far, however, it has been used sparingly to study immune ageing. Here we review the limited literature on homeostatic innate immune cell trafficking in ageing in the absence of chronic inflammation. We also review our own recent data on trafficking of innate immune subsets between primary and secondary lymphoid organs in heterochronic parabiosis. We found no systemic bias in retention or acceptance of neutrophils, macrophages, dendritic cells or natural killer cells with ageing in primary and secondary lymphoid organs. We conclude that these four innate immune cell types migrate to and populate lymphoid organs (peripheral lymph nodes, spleen and bone marrow), regardless of their own age and of the age of lymphoid organs. © 2017 British Society for Immunology.

Mantle cell lymphoma of the larynx: Primary case report

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Naciri Sarah

2012-07-01

Full Text Available Abstract Introduction Primary laryngeal lymphomas are exceedingly rare. Only about a hundred cases have been reported. They consist mainly of non-Hodgkin lymphoma, especially of diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue. We report the first case of a primary laryngeal mantle cell lymphoma. Case presentation We report a case of a primary mantle cell lymphoma of the larynx in a 70-year-old North African non-smoker male. We present a detailed report of his clinical and paraclinical data as well as treatment options. Conclusions Mantle cell lymphoma is a very aggressive lymphoma subset associated with poor prognosis. Laryngeal mantle cell lymphoma is exceedingly rare. To the best of our knowledge, this is the first case to ever be reported.

Spectrum of abdominal organ injury in a primary blast type

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Amin Abid

2009-12-01

Full Text Available Abstract Introduction Abdominal organ injury in a primary blast type is always challenging for diagnosis. Air containing abdominal viscera is most vulnerable to effects of primary blast injury. In any patient exposed to a primary blast wave who presents with an acute abdomen, an abdominal organ injury is to be kept in a clinical suspicion. Aim Study various abdominal organ injuries occurring in a primary type of blast injury. Material and methods: All those who had exploratory laparotomy for abdominal organ injuries after a primary blast injury for a period of 10 years from January 1998 - January 2008 were included in this retrospective study. Results Total 154 patients had laparotomy for abdominal organ injuries with a primary blast type of injury. Small intestine was damaged in 48 patients (31.1% followed by spleen in 22.7% cases. 54 patients (35.06% had more than one organ injured. Liver laceration was present in 30 patients (19.48%. Multiple small gut perforations were present in 37 patients (77.08%. Negative laparotomy was found in 5 patients (3.24% whereas 3 (1.94% had re-exploration. Mortality was present in 6 patients (3.89%. Conclusions Primary blast injury causes varied abdominal organ injuries. Single or multiple organ damage can be there. Small intestine is commonest viscera injured. Laparotomy gives final diagnosis.

Interfacing polymeric scaffolds with primary pancreatic ductal adenocarcinoma cells to develop 3D cancer models

NARCIS (Netherlands)

Ricci, C.; Mota, C.M.; Moscato, S.; D' Alessandro, D.; Ugel, S.; Sartoris, S.; Bronte, V.; Boggi, U.; Campani, D.; Funel, N.; Moroni, Lorenzo; Danti, S.

2014-01-01

We analyzed the interactions between human primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl

Cerebellar T-cell lymphoma: an unusual primary intracranial neoplasm

International Nuclear Information System (INIS)

Knorr, J.R.; Ragland, R.L.; Stone, B.B.; Woda, B.A.; Gelber, N.D.

1992-01-01

Primary T-cell lymphoma within the central nervous system is extremely rare. Imaging characteristics appear indistinguishable from the more common B-cell lymphoma. A case of such a primary tumor is discussed and the MRI and CT findings presented. (orig.)

When Is an Alveolar Type 2 Cell an Alveolar Type 2 Cell? A Conundrum for Lung Stem Cell Biology and Regenerative Medicine.

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Beers, Michael F; Moodley, Yuben

2017-07-01

Generating mature, differentiated, adult lung cells from pluripotent cells, such as induced pluripotent stem cells and embryonic stem cells, offers the hope of both generating disease-specific in vitro models and creating definitive and personalized therapies for a host of debilitating lung parenchymal and airway diseases. With the goal of advancing lung-regenerative medicine, several groups have developed and reported on protocols using defined media, coculture with mesenchymal components, or sequential treatments mimicking lung development, to obtain distal lung epithelial cells from stem cell precursors. However, there remains significant controversy about the degree of differentiation of these cells compared with their primary counterparts, coupled with a lack of consistency or uniformity in assessing the resultant phenotypes. Given the inevitable, exponential expansion of these approaches and the probable, but yet-to-emerge second and higher generation techniques to create such assets, we were prompted to pose the question, what makes a lung epithelial cell a lung epithelial cell? More specifically for this Perspective, we also posed the question, what are the minimum features that constitute an alveolar type (AT) 2 epithelial cell? In addressing this, we summarize a body of work spanning nearly five decades, amassed by a series of "lung epithelial cell biology pioneers," which carefully describes well characterized molecular, functional, and morphological features critical for discriminately assessing an AT2 phenotype. Armed with this, we propose a series of core criteria to assist the field in confirming that cells obtained following a differentiation protocol are indeed mature and functional AT2 epithelial cells.

Identification of cancer stem-like side population cells in purified primary cultured human laryngeal squamous cell carcinoma epithelia.

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Chun-Ping Wu

Full Text Available Cancer stem-like side population (SP cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07% was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.

Type D (distressed) personality in primary care patients with type 2 diabetes

DEFF Research Database (Denmark)

Nefs, Giesje; Pouwer, Francois; Pop, Victor J M

2012-01-01

D Scale-14 (DS14) in type 2 diabetes patients. METHODS: 1553 primary care patients with type 2 diabetes were assessed for demographic, clinical, lifestyle and psychological characteristics in 2007. A subgroup (n=1012) completed the DS14 again 1 year later. RESULTS: The two-factor model of the Type D...... or physiological risk factors, but Type D women had a more sedentary lifestyle (p=.003). Type D patients experienced less social support and more stressful life events, loneliness, and more depressed mood, anhedonia and anxiety (p0......OBJECTIVE: In cardiovascular research, Type D personality (high negative affectivity and social inhibition) has been associated with a more than 3-fold increased risk of adverse health outcomes. This study examined the validity and clinical correlates of the Type D construct as assessed by the Type...

Primary cutaneous smoldering adult T-cell leukemia/ lymphoma.

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Gittler, Julia; Martires, Kathryn; Terushkin, Vitaly; Brinster, Nooshin; Ramsay, David

2016-12-15

HTLV-1 is a virus that is endemic in southwesternJapan and the Caribbean and has been implicatedin the development of ATLL. ATLL, which is anuncommon malignant condition of peripheralT-lymphocytes, is characterized by four clinicalsubtypes, which include acute, lymphomatous,chronic, and smoldering types, that are based onLDH levels, calcium levels, and extent of organinvolvement. We present a 52-year- old woman withpruritic patches with scale on the buttocks and withtender, hyperpigmented macules and papules oftwo-years duration. Histopathologic examinationwas suggestive of mycosis fungoides, laboratoryresults showed HTLV-I and II, and the patient wasdiagnosed with primary cutaneous ATLL. We reviewthe literature on HTLV-1 and ATLL and specifically theprognosis of cutaneous ATLL. The literature suggeststhat a diagnosis of ATLL should be considered amongpatients of Caribbean origin or other endemicareas with skin lesions that suggest a cutaneousT-cell lymphoma, with clinicopathologic features ofmycosis fungoides. Differentiation between ATLLand cutaneous T-cell lymphoma is imperative as theyhave different prognoses and treatment approaches.

Increased risk of gastric adenocarcinoma after treatment of primary gastric diffuse large B-cell lymphoma

International Nuclear Information System (INIS)

Inaba, Koji; Morota, Madoka; Mayahara, Hiroshi; Ito, Yoshinori; Sumi, Minako; Uno, Takashi; Itami, Jun; Kushima, Ryoji; Murakami, Naoya; Kuroda, Yuuki; Harada, Ken; Kitaguchi, Mayuka; Yoshio, Kotaro; Sekii, Shuhei; Takahashi, Kana

2013-01-01

There have been sporadic reports about synchronous as well as metachronous gastric adenocarcinoma and primary gastric lymphoma. Many reports have dealt with metachronous gastric adenocarcinoma in mucosa-associated lymphoid tissue lymphoma of stomach. But to our knowledge, there have been no reports that document the increased incidence of metachronous gastric adenocarcinoma in patients with gastric diffuse large B-cell lymphoma. This retrospective study was conducted to estimate the incidence of metachronous gastric adenocarcinoma after primary gastric lymphoma treatment, especially in diffuse large B-cell lymphoma. The retrospective cohort study of 139 primary gastric lymphoma patients treated with radiotherapy at our hospital. Mean observation period was 61.5 months (range: 3.7-124.6 months). Patients profile, characteristics of primary gastric lymphoma and metachronous gastric adenocarcinoma were retrieved from medical records. The risk of metachronous gastric adenocarcinoma was compared with the risk of gastric adenocarcinoma in Japanese population. There were 10 (7.2%) metachronous gastric adenocarcinoma patients after treatment of primary gastric lymphomas. It was quite high risk compared with the risk of gastric carcinoma in Japanese population of 54.7/100,000. Seven patients of 10 were diffuse large B-cell lymphoma and other 3 patients were mixed type of diffuse large B-cell lymphoma and mucosa associated lymphoid tissue lymphoma. Four patients of 10 metachronous gastric adenocarcinomas were signet-ring cell carcinoma and two patients died of gastric adenocarcinoma. Metachronous gastric adenocarcinoma may have a more malignant potential than sporadic gastric adenocarcinoma. Old age, Helicobacter pylori infection and gastric mucosal change of chronic gastritis and intestinal metaplasia were possible risk factors for metachronous gastric adenocarcinoma. There was an increased risk of gastric adenocarcinoma after treatment of primary gastric lymphoma

Molecular analysis of immunoglobulin variable genes supports a germinal center experienced normal counterpart in primary cutaneous diffuse large B-cell lymphoma, leg-type.

Science.gov (United States)

Pham-Ledard, Anne; Prochazkova-Carlotti, Martina; Deveza, Mélanie; Laforet, Marie-Pierre; Beylot-Barry, Marie; Vergier, Béatrice; Parrens, Marie; Feuillard, Jean; Merlio, Jean-Philippe; Gachard, Nathalie

2017-11-01

Immunophenotype of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) suggests a germinal center-experienced B lymphocyte (BCL2+ MUM1+ BCL6+/-). As maturation history of B-cell is "imprinted" during B-cell development on the immunoglobulin gene sequence, we studied the structure and sequence of the variable part of the genes (IGHV, IGLV, IGKV), immunoglobulin surface expression and features of class switching in order to determine the PCLBCL-LT cell of origin. Clonality analysis with BIOMED2 protocol and VH leader primers was done on DNA extracted from frozen skin biopsies on retrospective samples from 14 patients. The clonal DNA IGHV sequence of the tumor was aligned and compared with the closest germline sequence and homology percentage was calculated. Superantigen binding sites were studied. Features of selection pressure were evaluated with the multinomial Lossos model. A functional monoclonal sequence was observed in 14 cases as determined for IGHV (10), IGLV (2) or IGKV (3). IGV mutation rates were high (>5%) in all cases but one (median:15.5%), with superantigen binding sites conservation. Features of selection pressure were identified in 11/12 interpretable cases, more frequently negative (75%) than positive (25%). Intraclonal variation was detected in 3 of 8 tumor specimens with a low rate of mutations. Surface immunoglobulin was an IgM in 12/12 cases. FISH analysis of IGHM locus, deleted during class switching, showed heterozygous IGHM gene deletion in half of cases. The genomic PCR analysis confirmed the deletions within the switch μ region. IGV sequences were highly mutated but functional, with negative features of selection pressure suggesting one or more germinal center passage(s) with somatic hypermutation, but superantigen (SpA) binding sites conservation. Genetic features of class switch were observed, but on the non functional allele and co-existing with primary isotype IgM expression. These data suggest that cell-of origin is

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

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Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

2015-08-01

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

Primary coolant circuits in FBR type reactors

International Nuclear Information System (INIS)

Kutani, Masushiro.

1985-01-01

Purpose: To eliminate the requirement of a pump for the forcive circulation of primary coolants and avoid the manufacturing difficulty of equipments. Constitution: In primary coolant circuits of an LMFBR type reactor having a recycling path forming a closed loop between a reactor core and a heat exchanger, coolants recycled through the recycling path are made of a magnetic fluid comprising liquid sodium incorporated with fine magnetic powder, and an electromagnet is disposed to the downstream of the heat exchanger. In the above-mentioned structure, since the magnetic fluid as the primary coolants losses its magnetic property when heated in the reactor core but recovers the property at a lower temperature after the completion of the heat exchange, the magnetic fluid can forcively be flown through the recycling path under the effect of the electromagnet disposed to the down stream of the heat exchanger to thereby forcively recycle the primary coolants. (Kawakami, Y.)

Primary radiation damage and disturbance in cell divisions

International Nuclear Information System (INIS)

Kim, Jin Kyu; Lee, Yun-Jong; Kim, Jae-Hun; Petin, Vladislav G.; Nili, Mohammad

2008-01-01

Survived cells from a homogeneous population exposed to ionizing radiation form various colonies of different sizes and morphology on a solid nutrient medium, which appear at different time intervals after irradiation. Such a phenomenon agrees well with the modern theory of microdosimetry and classical hit-and-target models of radiobiology. According to the hit-principle, individual cells exposed to the same dose of radiation are damaged in different manners. It means that the survived cells can differ in the content of sublethal damage (hits) produced by the energy absorbed into the cell and which is not enough to give rise to effective radiation damage which is responsible for cell killing or inactivation. In diploid yeast cells, the growth rate of cells from 250 colonies of various sizes appeared at different time intervals after irradiation with 600 Gy of gamma radiation from a 60 Co isotopic source was analyzed. The survival rate after irradiation was 20%. Based on the analyses results, it was possible to categorize the clones grown from irradiated cells according to the number of sub-lesions from 1 to 4. The clones with various numbers of sub-lesions were shown to be different in their viability, radiosensitivity, sensitivity to environmental conditions, and the frequency of recombination and respiratory deficient mutations. Cells from unstable clones exhibited an enhanced radiosensitivity, and an increased portion of morphologically changed cells, nonviable cells and respiration mutants, as well. The degree of expression of the foregoing effects was higher if the number of primary sublethal lesions was greater in the originally irradiated cell. Disturbance in cell division can be characterized by cell inactivation or incorrect distribution of mitochondria between daughter cells. Thus, the suggested methodology of identification of cells with a definite number of primary sublethal lesions will promote further elucidation of the nature of primary radiation

Pulmonary squamous cell carcinoma following head and neck squamous cell carcinoma: Metastasis or second primary?

NARCIS (Netherlands)

Geurts, Tom W.; Nederlof, Petra M.; van den Brekel, Michiel W. M.; van't Veer, Laura J.; de Jong, Daphne; Hart, August A. M.; van Zandwijk, Nico; Klomp, Houke; Balm, Alfons J. M.; van Velthuysen, Marie-Louise F.

2005-01-01

Purpose: To distinguish a metastasis from a second primary tumor in patients with a history of head and neck squamous cell carcinoma and subsequent pulmonary squamous cell carcinoma. Experimental Design: For 44 patients with a primary squamous cell carcinoma of the head and neck followed by a

DJ-1 Modulates Nuclear Erythroid 2–Related Factor-2–Mediated Protection in Human Primary Alveolar Type II Cells in Smokers

Science.gov (United States)

Bahmed, Karim; Messier, Elise M.; Zhou, Wenbo; Tuder, Rubin M.; Freed, Curt R.; Chu, Hong Wei; Kelsen, Steven G.; Bowler, Russell P.; Mason, Robert J.

2016-01-01

Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2–related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases. PMID:27093578

Neuroendocrine Merkel cell nodal carcinoma of unknown primary site: management and outcomes of a rare entity.

Science.gov (United States)

Kotteas, E A; Pavlidis, N

2015-04-01

Merkel cell nodal carcinoma of unknown primary (MCCUP) is a rare neuroendocrine tumour with distinct clinical and biological behaviour. We conducted a review of retrospective data extracted from 90 patients focusing on the management and outcome of this disease. We also compared life expectancy of these patients with the outcome of patients with known Merkel primaries and with neuroendocrine cancers of unidentifiable primary. There is a limited body of data for this type of malignancy, however, patients with Merkel cell nodal carcinoma of unknown primary site, seem to have better survival when treated aggressively than patients with cutaneous Merkel tumours of the same stage and equal survival with patients with low-grade neuroendocrine tumour of unknown origin. The lack of prospective trials, and the inadequate data, hamper the management of these tumours. Establishment of treatment guidelines is urgently needed. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Esophageal Large-Cell Neuroendocrine Carcinoma with Inconsistent Response to Treatment in the Primary and Metastatic Lesions

Directory of Open Access Journals (Sweden)

Takashi Tomiyama

2018-05-01

Full Text Available Esophageal large-cell neuroendocrine carcinoma (NEC is a rare malignant tumor that is characterized by high-grade malignancy and a poor prognosis. However, the rarity of esophageal NEC has prevented the development of an established treatment, and no reports have described a discrepancy in the effectiveness of cisplatin plus irinotecan between primary and metastatic lesions. A 43-year-old Japanese man was referred to our hospital with refractory epigastralgia. A previous gastrointestinal endoscopy had revealed a 50-mm type 2 tumor in the abdominal esophagus. The pathological findings indicated poorly differentiated squamous cell carcinoma. Contrast-enhanced computed tomography revealed a metastatic liver tumor. One cycle of fluorouracil and cisplatin was not effective, and endoscopy was repeatedly performed. The pathological findings indicated a large-cell malignant tumor with tumor cells that were positive for CD56, synaptophysin, and Ki-67 (> 80%. Based on a diagnosis of esophageal large-cell NEC with a metastatic liver tumor, the patient received cisplatin plus irinotecan biweekly. After 4 months, computed tomography revealed marked shrinkage of the metastatic tumor, but the patient complained of dysphagia. Endoscopy revealed enlargement of the primary tumor, which was then treated using radiotherapy plus fluorouracil and cisplatin. The primary tumor subsequently shrank, and the patient’s symptoms were relieved, but the metastatic tumor grew. Thus, chemoradiotherapy could be an option for managing a primary esophageal large-cell NEC that does not respond to chemotherapy alone. However, the possibility of an inconsistent response to therapy in primary and metastatic lesions should be considered.

Adenylate cyclase regulates elongation of mammalian primary cilia

International Nuclear Information System (INIS)

Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

2009-01-01

The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3β by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

Adenylate cyclase regulates elongation of mammalian primary cilia

Energy Technology Data Exchange (ETDEWEB)

Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J. [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada); Rattner, Jerome B. [Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada); Hoorn, Frans A. van der, E-mail: fvdhoorn@ucalgary.ca [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada)

2009-10-01

The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

Primary clear cell ductal adenocarcinoma of the pancreas: A case report and clinicopathologic literature review

Directory of Open Access Journals (Sweden)

Yashpal Modi

2014-01-01

Full Text Available We present a very rare, interesting case of a carcinoma of the pancreas with predominantly abundant clear cell morphology. According to the WHO classification, primary clear cell carcinoma of the pancreas is classified as a rare "miscellaneous" carcinoma. The tumor was observed in the distal body and tail of the pancreas of a 74-year-old woman. The histopathology of tumor cells showed well-defined cell membranes, clear cytoplasm, and prominent cell boundaries. Immunohistochemical (IHC staining showed positive reactions to antibodies against vimentin, cytokeratin 7 (CK-7, mucicarmine (MUC-1, periodic acid-Schiff (PAS, periodic acid-Schiff with diastase (PASD, carcinoembryonic antigen (CEA, and Carbohydrate Antigen 19-9 (CA 19-9. On the other hand, IHC staining was negative for alpha-fetoprotein (AFP, cytokeratin 20 (CK-20, HMB45, chromogranin, and synaptophysin. The patient was subsequently diagnosed with a primary solid-type pancreatic clear cell carcinoma with hepatic metastasis. Herein, we report this rare case and include a review of the current literature of this tumor.

Growth of primary embryo cells in a microculture system.

Science.gov (United States)

Villa, Max; Pope, Sara; Conover, Joanne; Fan, Tai-Hsi

2010-04-01

We present optimal perfusion conditions for the growth of primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) using a microfluidic perfusion culture system. In an effort to balance nutrient renewal while ensuring the presence of cell secreted factors, we found that the optimal perfusion rate for culturing primary embryonic fibroblasts (mEFs) in our experimental setting is 10 nL/min with an average flow velocity 0.55 microm/s in the microchannel. Primary mEFs may have a greater dependence on cell secreted factors when compared to their immortalized counterpart 3T3 fibroblasts cultured under similar conditions. Both the seeding density and the perfusion rate are critical for the proliferation of primary cells. A week long cultivation of mEFs and mESCs using the microculture system exhibited similar morphology and viability to those grown in a petri dish. Both mEFs and mESCs were analyzed using fluorescence immunoassays to determine their proliferative status and protein expression. Our results demonstrate that a perfusion-based microculture environment is capable of supporting the highly proliferative status of pluripotent embryonic stem cells.

Radiosensitivity of primary cultured fish cells with different ploidy

International Nuclear Information System (INIS)

Mitani, Hiroshi; Egami, Nobuo; Kobayashi, Hiromu.

1986-01-01

The radiosensitivity of primary cultured goldfish cells (Carassius auratus) was investigated by colony formation assay. The radiosensitivity of cells from two varieties of goldfish, which show different sensitivity to lethal effect of ionizing radiation in vivo, was almost identical. Primary cultured cells from diploid, triploid and tetraploid fish retained their DNA content as measured by microfluorometry, and the nuclear size increases as ploidy increases. However, radiosensitivity was not related to ploidy. (author)

Primary B-cell Lymphoma of the Thyroid Featuring the Different Ultrasonographic Findings

Energy Technology Data Exchange (ETDEWEB)

Kim, Ji Na; Choi, Yoon Jung; Kim, Dong Hoon [Kangbuk Samsung Medical Center, Seoul (Korea, Republic of)

2009-06-15

We review here 3 cases of primary thyroid lymphoma that we experienced during the past 5 years (age range: 39-55, all of the patients were female). The clinical and various ultrasonographic characteristics together with the other imaging modalities of primary thyroid lymphomas are described. The clinical features at presentation for one patient were a goiter with rapid growth and this was accompanied by compressive symptoms. The tumors of the other 2 patients were incidentally found during screening thyroid ultrasound exams. The pathologic studies of 2 cases showed a diffuse B-cell lymphoma with associated Hashimoto's thyroiditis and one case was a B-cell lymphoma of the MALT type. An extra-thyroid extension was shown in one case. The treatments included surgery alone for two cases, and chemotherapy and radiation therapy for one case. A US exam of thyroid lymphoma can show various morphological features, and US-CNB is helpful for diagnosing thyroid lymphoma.

Biophysical effects in off-resonant gold nanoparticle mediated (GNOME) laser transfection of cell lines, primary- and stem cells using fs laser pulses.

Science.gov (United States)

Schomaker, Markus; Killian, Doreen; Willenbrock, Saskia; Heinemann, Dag; Kalies, Stefan; Ngezahayo, Anaclet; Nolte, Ingo; Ripken, Tammo; Junghanß, Christian; Meyer, Heiko; Murua Escobar, Hugo; Heisterkamp, Alexander

2015-08-01

Gold nanoparticle mediated (GNOME) laser transfection is a powerful technique to deliver small biologically relevant molecules into cells. However, the transfection of larger and especially negatively charged DNA remains challenging. The efficiency for pDNA was 0.57% using parameter that does not influence the endo- and exogenous DNA. In order to gain a deeper understanding of the actual molecule uptake process, the uptake efficiency was determined using molecules of different sizes. It was evaluated that uncharged dextran molecules (2000 kDa) were delivered with an efficiency of 68%. The intracellular distribution of injected molecules was visualized and larger molecules were primary found in the cytoplasm. Patch clamp measurements suggested a permeabilization time up to 15 minutes. The uptake efficiency depended on the size and charge of the molecule to deliver as well as the cell size. A minor role for transfection plays the cell type since primary stem cells were successfully transfected. The perforation efficiency of semi-adherent and suspension cells is influenced by the cell and molecule size. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

Energy Technology Data Exchange (ETDEWEB)

Salazar-García, Samuel; Silva-Ramírez, Ana Sonia; Ramirez-Lee, Manuel A.; Rosas-Hernandez, Hector [Universidad Autonoma de San Luis Potosi, Facultad de Ciencias Quimicas (Mexico); Rangel-López, Edgar [Instituto Nacional de Neurologia y Neurocirugia Manuel Velasco Suárez, Laboratorio de Aminoacidos Excitadores (Mexico); Castillo, Claudia G. [Facultad de Medicina, Universidad Autonoma de San Luis Potosi (Mexico); Santamaría, Abel [Instituto Nacional de Neurologia y Neurocirugia Manuel Velasco Suárez, Laboratorio de Aminoacidos Excitadores (Mexico); Martinez-Castañon, Gabriel A. [Universidad Autonoma de San Luis Potosi, Facultad de Estomatologia (Mexico); Gonzalez, Carmen, E-mail: cgonzalez.uaslp@gmail.com, E-mail: gonzalez.castillocarmen@fcq.uaslp.mx [Universidad Autonoma de San Luis Potosi, Facultad de Ciencias Quimicas (Mexico)

2015-11-15

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 (AgNO{sub 3}) 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.

Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

Science.gov (United States)

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.

Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

International Nuclear Information System (INIS)

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

2015-01-01

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 (AgNO 3 ) 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

Response of Primary Human Bone Marrow Mesenchymal Stromal Cells and Dermal Keratinocytes to Thermal Printer Materials In Vitro.

Science.gov (United States)

Schmelzer, Eva; Over, Patrick; Gridelli, Bruno; Gerlach, Jörg C

Advancement in thermal three-dimensional printing techniques has greatly increased the possible applications of various materials in medical applications and tissue engineering. Yet, potential toxic effects on primary human cells have been rarely investigated. Therefore, we compared four materials commonly used in thermal printing for bioengineering, namely thermally printed acrylonitrile butadiene styrene, MED610, polycarbonate, and polylactic acid, and investigated their effects on primary human adult skin epidermal keratinocytes and bone marrow mesenchymal stromal cells (BM-MSCs) in vitro. We investigated indirect effects on both cell types caused by potential liberation of soluble substances from the materials, and also analyzed BM-MSCs in direct contact with the materials. We found that even in culture without direct contact with the materials, the culture with MED610 (and to a lesser extent acrylonitrile butadiene styrene) significantly affected keratinocytes, reducing cell numbers and proliferation marker Ki67 expression, and increasing glucose consumption, lactate secretion, and expression of differentiation-associated genes. BM-MSCs had decreased metabolic activity, and exhibited increased cell death in direct culture on the materials. MED610 and acrylonitrile butadiene styrene induced the strongest expression of genes associated to differentiation and estrogen receptor activation. In conclusion, we found strong cell-type-specific effects of the materials, suggesting that materials for applications in regenerative medicine should be carefully selected not only based on their mechanical properties but also based on their cell-type-specific biological effects.

Gene expression relationship between prostate cancer cells of Gleason 3, 4 and normal epithelial cells as revealed by cell type-specific transcriptomes

International Nuclear Information System (INIS)

Pascal, Laura E; Liu, Alvin Y; Vêncio, Ricardo ZN; Page, Laura S; Liebeskind, Emily S; Shadle, Christina P; Troisch, Pamela; Marzolf, Bruz; True, Lawrence D; Hood, Leroy E

2009-01-01

Prostate cancer cells in primary tumors have been typed CD10 - /CD13 - /CD24 hi /CD26 + /CD38 lo /CD44 - /CD104 - . This CD phenotype suggests a lineage relationship between cancer cells and luminal cells. The Gleason grade of tumors is a descriptive of tumor glandular differentiation. Higher Gleason scores are associated with treatment failure. CD26 + cancer cells were isolated from Gleason 3+3 (G3) and Gleason 4+4 (G4) tumors by cell sorting, and their gene expression or transcriptome was determined by Affymetrix DNA array analysis. Dataset analysis was used to determine gene expression similarities and differences between G3 and G4 as well as to prostate cancer cell lines and histologically normal prostate luminal cells. The G3 and G4 transcriptomes were compared to those of prostatic cell types of non-cancer, which included luminal, basal, stromal fibromuscular, and endothelial. A principal components analysis of the various transcriptome datasets indicated a closer relationship between luminal and G3 than luminal and G4. Dataset comparison also showed that the cancer transcriptomes differed substantially from those of prostate cancer cell lines. Genes differentially expressed in cancer are potential biomarkers for cancer detection, and those differentially expressed between G3 and G4 are potential biomarkers for disease stratification given that G4 cancer is associated with poor outcomes. Differentially expressed genes likely contribute to the prostate cancer phenotype and constitute the signatures of these particular cancer cell types

Differential heat shock response of primary human cell cultures and established cell lines

DEFF Research Database (Denmark)

Richter, W W; Issinger, O G

1986-01-01

degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

Primary Squamous Cell Carcinoma of Stomach: A Rare Entity ...

African Journals Online (AJOL)

Schmidt C, Schmid A, Lüttges JE, Kremer B, Henne-Bruns D. Primary squamous cell carcinoma of the stomach. Report of a case and review of literature. Hepatogastroenterology 2001;48:1033-6. 5. Muto M, Hasebe T, Muro K, Boku N, Ohtsu A, Fujii T, et al. Primary squamous cell carcinoma of the stomach: A case report with ...

Changing practice: red blood cell typing by molecular methods for patients with sickle cell disease.

Science.gov (United States)

Casas, Jessica; Friedman, David F; Jackson, Tannoa; Vege, Sunitha; Westhoff, Connie M; Chou, Stella T

2015-06-01

Extended red blood cell (RBC) antigen matching is recommended to limit alloimmunization in patients with sickle cell disease (SCD). DNA-based testing to predict blood group phenotypes has enhanced availability of antigen-negative donor units and improved typing of transfused patients, but replacement of routine serologic typing for non-ABO antigens with molecular typing for patients has not been reported. This study compared the historical RBC antigen phenotypes obtained by hemagglutination methods with genotype predictions in 494 patients with SCD. For discrepant results, repeat serologic testing was performed and/or investigated by gene sequencing for silent or variant alleles. Seventy-one typing discrepancies were identified among 6360 antigen comparisons (1.1%). New specimens for repeat serologic testing were obtained for 66 discrepancies and retyping agreed with the genotype in 64 cases. One repeat Jk(b-) serologic phenotype, predicted Jk(b+) by genotype, was found by direct sequencing of JK to be a silenced allele, and one N typing discrepancy remains under investigation. Fifteen false-negative serologic results were associated with alleles encoding weak antigens or single-dose Fy(b) expression. DNA-based RBC typing provided improved accuracy and expanded information on RBC antigens compared to hemagglutination methods, leading to its implementation as the primary method for extended RBC typing for patients with SCD at our institution. © 2015 AABB.

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

Science.gov (United States)

Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

2015-01-01

Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

Survival after bone metastasis by primary cancer type

DEFF Research Database (Denmark)

Svensson, Elisabeth; Christiansen, Christian F; Ulrichsen, Sinna P

2017-01-01

%, 11% to 14%). The risk of mortality was increased for the majority of cancer types among patients with bone and synchronous metastases compared with bone only (adjusted relative risk 1.29-1.57), except for cervix, ovarian and bladder cancer. CONCLUSIONS: While patients with bone metastases after most......OBJECTIVE: In the 10 most common primary types with bone metastases, we aimed to examine survival, further stratifying on bone metastases only or with additional synchronous metastases. METHODS: We included all patients aged 18 years and older with incident hospital diagnosis of solid cancer...... between 1994 and 2010, subsequently diagnosed with BM until 2012. We followed patients from date of bone metastasis diagnosis until death, emigration or 31 December 2012, whichever came first. We computed 1-year, 3-year and 5-year survival (%) and the corresponding 95% CIs stratified on primary cancer...

CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells.

Directory of Open Access Journals (Sweden)

Laurie A Steiner

Full Text Available CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC and primary human erythroid cells from single donors.Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner.These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis.

Effect of cell phone-like electromagnetic radiation on primary human thyroid cells.

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Silva, Veronica; Hilly, Ohad; Strenov, Yulia; Tzabari, Cochava; Hauptman, Yirmi; Feinmesser, Raphael

2016-01-01

To evaluate the potential carcinogenic effects of radiofrequency energy (RFE) emitted by cell phones on human thyroid primary cells. Primary thyroid cell culture was prepared from normal thyroid tissue obtained from patients who underwent surgery at our department. Subconfluent thyroid cells were irradiated under different conditions inside a cell incubator using a device that simulates cell phone-RFE. Proliferation of control and irradiated cells was assessed by the immunohistochemical staining of antigen Kiel clone-67 (Ki-67) and tumor suppressor p53 (p53) expression. DNA ploidy and the stress biomarkers heat shock protein 70 (HSP70) and reactive oxygen species (ROS) was evaluated by fluorescence-activated cell sorting (FACS). Our cells highly expressed thyroglobulin (Tg) and sodium-iodide symporter (NIS) confirming the origin of the tissue. None of the irradiation conditions evaluated here had an effect neither on the proliferation marker Ki-67 nor on p53 expression. DNA ploidy was also not affected by RFE, as well as the expression of the biomarkers HSP70 and ROS. Our conditions of RFE exposure seem to have no potential carcinogenic effect on human thyroid cells. Moreover, common biomarkers usually associated to environmental stress also remained unchanged. We failed to find an association between cell phone-RFE and thyroid cancer. Additional studies are recommended.

Cell type discovery using single-cell transcriptomics: implications for ontological representation.

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Aevermann, Brian D; Novotny, Mark; Bakken, Trygve; Miller, Jeremy A; Diehl, Alexander D; Osumi-Sutherland, David; Lasken, Roger S; Lein, Ed S; Scheuermann, Richard H

2018-05-01

Cells are fundamental function units of multicellular organisms, with different cell types playing distinct physiological roles in the body. The recent advent of single-cell transcriptional profiling using RNA sequencing is producing 'big data', enabling the identification of novel human cell types at an unprecedented rate. In this review, we summarize recent work characterizing cell types in the human central nervous and immune systems using single-cell and single-nuclei RNA sequencing, and discuss the implications that these discoveries are having on the representation of cell types in the reference Cell Ontology (CL). We propose a method, based on random forest machine learning, for identifying sets of necessary and sufficient marker genes, which can be used to assemble consistent and reproducible cell type definitions for incorporation into the CL. The representation of defined cell type classes and their relationships in the CL using this strategy will make the cell type classes being identified by high-throughput/high-content technologies findable, accessible, interoperable and reusable (FAIR), allowing the CL to serve as a reference knowledgebase of information about the role that distinct cellular phenotypes play in human health and disease.

Clinical Significance of "Double-hit" and "Double-protein" expression in Primary Gastric B-cell Lymphomas.

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He, Miaoxia; Chen, Keting; Li, Suhong; Zhang, Shimin; Zheng, Jianming; Hu, Xiaoxia; Gao, Lei; Chen, Jie; Song, Xianmin; Zhang, Weiping; Wang, Jianmin; Yang, Jianmin

2016-01-01

Primary gastric B-cell lymphoma is the second most common malignancy of the stomach. There are many controversial issues about its diagnosis, treatment and clinical management. "Double-hit" and "double-protein" involving gene rearrangement and protein expression of c-Myc and bcl2/bcl6 are the most used terms to describe DLBCL poor prognostic factors in recent years. However, very little is known about the role of these prognostic factors in primary gastric B-cell lymphomas. This study aims to obtain a molecular pathology prognostic model of gastric B-cell lymphoma for clinical stratified management by evaluating how the "double-hit" and "double-protein" in tumor cells as well as microenvironmental reaction of tumor stromal tissue affect clinical outcome in primary gastric B-cell lymphomas. Data and tissues of 188 cases diagnosed with gastric B-cell lymphomas were used in this study. Tumor tissue microarray (TMA) of formalin fixed and paraffin embedded (FFPE) tissues was constructed for fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis with a serial of biomarkers containing MYC, BCL2, BCL6, CD31, SPARC, CD10, MUM1 and Ki-67. Modeled period analysis was used to estimate 3-year and 5-year overall survival (OS) and disease-free survival (DFS) distributions. There was no definite "double-hit" case though the gene rearrangement of c-Myc (5.9%), bcl2 (0.1%) and bcl6 (7.4%) was found in gastric B-cell lymphomas. The gene amplification or copy gains of c-Myc (10.1%), bcl-2 (17.0%) and bcl-6 (0.9%) were present in these lymphomas. There were 12 cases of the lymphomas with the "double-protein" expression of MYC and BCL2/BCL6. All patients with "double-protein" gastric B-cell lymphomas had poor outcome compared with those without. More importantly, "MYC-BCL2-BCL6" negative group of gastric B-cell lymphoma patients had favorable clinical outcome regardless clinical stage, pathological types and therapeutic modalities. And the similar better

Arachidonate metabolism increases as rat alveolar type II cells differentiate in vitro

International Nuclear Information System (INIS)

Lipchik, R.J.; Chauncey, J.B.; Paine, R.; Simon, R.H.; Peters-Golden, M.

1990-01-01

Rat type II alveolar epithelial cells are known to undergo morphological and functional changes when maintained in culture for several days. Having previously demonstrated that these cells can deacylate free arachidonic acid (AA) and metabolize it to products of the cyclooxygenase pathway, the present study was undertaken to determine whether in vitro differentiation was accompanied by alterations in the availability and metabolism of AA. We assessed the constitutive and ionophore A23187-induced deacylation and metabolism of endogenous AA, as well as the metabolism of exogenously supplied AA, in primary cultures of rat type II cells at days 2, 4, and 7 after isolation. Levels of free endogenous AA were increased at day 4, whereas eicosanoid synthesis, predominantly prostaglandin E2 and prostacyclin, increased markedly only at day 7. A similar time course of augmentation of prostanoid release was seen in response to exogenous AA. Type II cells cultured on fibronectin, intended to hasten cell flattening and spreading, demonstrated accelerated increases in available free AA in response to A23187; cells cultured on basement membrane derived from Engelbreth-Holm-Swarm mouse sarcoma, known to maintain the type II phenotype, exhibited diminished levels of available free AA. From these findings, we conclude that alterations in arachidonate metabolism are linked to alterations in cellular phenotype. The potentiation of eicosanoid synthesis accompanying in vitro differentiation suggests a possible role for the alveolar epithelium in the modulation of inflammation and fibrosis in the distal lung

Induction of chromosomal aberrations in human primary fibroblasts and immortalized cancer cells exposed to extremely-low-frequency electromagnetic fields

International Nuclear Information System (INIS)

Seyyedi, S. S.; Mozdarani, H.; Rezaei Tavirani, M.; Heydari, S.

2010-01-01

Rapidly increasing possibilities of exposure to environmental extremely low-frequency electromagnetic fields have become a topic of worldwide investigation. Epidemiological and laboratory studies suggest that exposure to extremely low-frequency electromagnetic fields may increase cancer risk therefore assessment of chromosomal damage in various cell lines might be of predictive value for future risk estimation. Materials and Methods: Primary cultures of fibroblasts from human skin biopsy were exposed to continuous extremely low-frequency electromagnetic fields (3, 50 and 60 Hz, sinusoidal, 3h, and 4 m T). Also immortalized cell lines, SW480, MCF-7 and 1321N1 were exposed to continuous extremely low-frequency electromagnetic fields (50 Hz, sinusoidal, 3 h, 4 m T). Metaphase plates Were prepared according to standard methods and stained in 5% Giemsa solution. Chromosomal aberrations of both chromosome and chromatid types were scored to evaluate the effects of extremely low-frequency electromagnetic fields on primary or established cell lines. Results: Results indicate that by increasing the frequency of extremely low-frequency electromagnetic fields, chromosomal aberrations were increased up to 7-fold above background levels in primary human fibroblast cells. In addition, continuous exposure to a 50 Hz electromagnetic field led to a significant increase in chromosomal aberrations in SW480, MCF-7 and 1321N1 cell lines compared to sham control. Conclusion: Results obtained indicate that extremely low-frequency electromagnetic fields has the potential for induction of chromosomal aberrations in all cell types.

Long-term in vitro, cell-type-specific genome-wide reprogramming of gene expression

International Nuclear Information System (INIS)

Hakelien, Anne-Mari; Gaustad, Kristine G.; Taranger, Christel K.; Skalhegg, Bjorn S.; Kuentziger, Thomas; Collas, Philippe

2005-01-01

We demonstrate a cell extract-based, genome-wide and heritable reprogramming of gene expression in vitro. Kidney epithelial 293T cells have previously been shown to take on T cell properties following a brief treatment with an extract of Jurkat T cells. We show here that 293T cells exposed for 1 h to a Jurkat cell extract undergo genome-wide, target cell-type-specific and long-lasting transcriptional changes. Microarray analyses indicate that on any given week after extract treatment, ∼2500 genes are upregulated >3-fold, of which ∼900 are also expressed in Jurkat cells. Concomitantly, ∼1500 genes are downregulated or repressed, of which ∼500 are also downregulated in Jurkat cells. Gene expression changes persist for over 30 passages (∼80 population doublings) in culture. Target cell-type specificity of these changes is shown by the lack of activation or repression of Jurkat-specific genes by extracts of 293T cells or carcinoma cells. Quantitative RT-PCR analysis confirms the long-term transcriptional activation of genes involved in key T cell functions. Additionally, growth of cells in suspended aggregates, expression of CD3 and CD28 T cell surface markers, and interleukin-2 secretion by 293T cells treated with extract of adult peripheral blood T cells illustrate a functional nuclear reprogramming. Therefore, target cell-type-specific and heritable changes in gene expression, and alterations in cell function, can be promoted by extracts derived from transformed cells as well as from adult primary cells

Bronchoalveolar lavage fluid from normal rats stimulates DNA synthesis in rat alveolar type II cells

International Nuclear Information System (INIS)

Leslie, C.C.; McCormick-Shannon, K.; Mason, R.J.

1989-01-01

Proliferation of alveolar type II cells after lung injury is important for the restoration of the alveolar epithelium. Bronchoalveolar lavage fluid (BALF) may represent an important source of growth factors for alveolar type II cells. To test this possibility, BALF fluid was collected from normal rats, concentrated 10-fold by Amicon filtration, and tested for its ability to stimulate DNA synthesis in rat alveolar type II cells in primary culture. BALF induced a dose-dependent increase in type II cell DNA synthesis resulting in a 6-fold increase in [3H]thymidine incorporation. Similar doses also stimulated [3H]thymidine incorporation into rat lung fibroblasts by 6- to 8-fold. Removal of pulmonary surface active material by centrifugation did not significantly reduce the stimulatory activity of BALF for type II cells. The stimulation of type II cell DNA synthesis by BALF was reduced by 100% after heating at 100 degrees C for 10 min, and by approximately 80% after reduction with dithiothreitol, and after trypsin treatment. Dialysis of BALF against 1 N acetic acid resulted in a 27% reduction in stimulatory activity. The effect of BALF in promoting type II cell DNA synthesis was more pronounced when tested in the presence of serum, although serum itself has very little effect on type II cell DNA synthesis. When BALF was tested in combination with other substances that stimulate type II cell DNA synthesis (cholera toxin, insulin, epidermal growth factor, and acidic fibroblast growth factor), additive effects or greater were observed. When BALF was chromatographed over Sephadex G150, the activity eluted with an apparent molecular weight of 100 kDa

Primary mantle cell lymphoma of tonsil: Case report

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Knežević Snežana B.

2017-01-01

Full Text Available Introduction: Mantle cell lymphoma is rare type of the mature B cell lymphoma. It includes 4% - 6% of all Non Hodgkin's Lymphomas. Compared to the other subtypes of lymphoma it develops more often in older men, and the median age of patients is 65 years. Primary tonsillar lymphoma accounts for less than 1% of head and neck malignancies. Method: Data obtained from medical records of the patient. Objective: Emphasize the importance of early and accurate diagnosis and early treatment of malignant diseases. Case report: Patient RP, 63 years old, presents with difficult swallowing, hoarseness, enlarged tonsils, snoring. Left tonsil almost sets into the right tonsillar vine, displaces the uvula and covers the isthmus. Respiratory sound is normal, with rhythmic action of the heart and soft abdomen. Good general condition. Echo: enlarged and actively altered lymph glands of the middle right jugular chain, the largest 148x77 mm, on the left side lymph nodes are enlarged, the largest is 143x72 mm. Echo of the abdomen inconspicuous. Lab: WBC 5.9, RBC 5.2, Hb 152, Hct 0.44, SE 10, CK 129, LDH 331, CRP 4.6, ALP 61, fibrinogen 2.4, Ca2+ 2.3, phosphate 0.8; BK, HCV, HBsAg, EB, HIV negative. X-ray of the chest inconspicuous. Admitted to the hematology department of the General Hospital. PH: Immunoproliferative disease. Immunohistochemistry, at the institute of Pathology: IHH CK AE1-AE3, PAX5 +, CD20 +, CD3, bcl2 +, bcl6-, CyklinD1 +, CD23-, CD43 +, MUM1 - / +, Ki67 + in about 20% of the tumor cells. Morphological and immunohistochemical findings: Mantle cell lymphoma. MSCD of the neck, chest and upper abdomen: Left tonsil diameter is 28x32 mm and length is 36mm, with lobular contour and heterogeneous structure, asymmetrically narrowing lumen of the airways to 7 mm. pathologically enlarged submandibular and par jugular lymph nodes (10-15 mm diameter on the left. There were no pathological findings in the lung parenchyma. Abdominal and retroperitoneal lymph nodes

A Case Report of Primary B–Cell Lymphoma of the Urinary Bladder

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M. Abbasi

2004-07-01

Full Text Available Primary malignant lymphoma of the urinary bladder is very rare. Less than 100 cases have been reported. The best treatment approach for this disease remained unknown.In this article we reported a 41-year-old-female who was admitted to Sina hospital with the chief complaint of macrohematuria that was followed by dysuria , frequency , noturia and urgency. Other examinations were normal and there was no organomegaly and lymphadenopathy.In ultrasonography the thickening of trigone zone of the urinary bladder was reported. The patient underwent a transurethral biopsy of the bladder that revealed malignant lymphoma , intermediate grade , diffuse mixed small and large cell type ( B-cell lymphoma. The reports of computed tomography scan of the thorax , abdomen and pelvis and bonemarrow biopsy were normal and results of metastatic work up were negative.Primary lymphoma of the urinary bladder was diagnosed and a combination of systemic chemotherapy and relatively low dose irradiation were done for the patient. The patient is in complete remission with this kind of treatment now.

Primary Small Cell Neuroendocrine Carcinoma of Vagina: A Rare Case Report

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Jignasa N. Bhalodia

2011-01-01

Full Text Available Primary small cell neuroendocrine carcinoma of vagina is an extremely rare disease. There have been only 26 previously reported cases in literature. Here, we report a case of primary small cell neuroendocrine carcinoma of vagina. Immunohistochemistry (IHC showed tumor cells positive for synaptophysin, chromogranin, and neuron-specific enolase (NSE.

Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis.

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Barnay-Verdier, Stéphanie; Dall'osso, Diane; Joli, Nathalie; Olivré, Juliette; Priouzeau, Fabrice; Zamoum, Thamilla; Merle, Pierre-Laurent; Furla, Paola

2013-10-01

The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5 μm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition.

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Gervin, Kristina; Page, Christian Magnus; Aass, Hans Christian D; Jansen, Michelle A; Fjeldstad, Heidi Elisabeth; Andreassen, Bettina Kulle; Duijts, Liesbeth; van Meurs, Joyce B; van Zelm, Menno C; Jaddoe, Vincent W; Nordeng, Hedvig; Knudsen, Gunn Peggy; Magnus, Per; Nystad, Wenche; Staff, Anne Cathrine; Felix, Janine F; Lyle, Robert

2016-09-01

Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation

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Marconett, Crystal N.; Zhou, Beiyun; Rieger, Megan E.; Selamat, Suhaida A.; Dubourd, Mickael; Fang, Xiaohui; Lynch, Sean K.; Stueve, Theresa Ryan; Siegmund, Kimberly D.; Berman, Benjamin P.

2013-01-01

Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation. PMID:23818859

Establishment and evaluation of a stable cattle type II alveolar epithelial cell line.

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Feng Su

Full Text Available Macrophages and dendritic cells are recognized as key players in the defense against mycobacterial infection. Recent research has confirmed that alveolar epithelial cells (AECs also play important roles against mycobacterium infections. Thus, establishing a stable cattle AEC line for future endogenous immune research on bacterial invasion is necessary. In the present study, we first purified and immortalized type II AECs (AEC II cells by transfecting them with a plasmid containing the human telomerase reverse trancriptase gene. We then tested whether or not the immortalized cells retained the basic physiological properties of primary AECs by reverse-transcription polymerase chain reaction and Western blot. Finally, we tested the secretion capacity of immortalized AEC II cells upon stimulation by bacterial invasion. The cattle type II alveolar epithelial cell line (HTERT-AEC II that we established retained lung epithelial cell characteristics: the cells were positive for surfactants A and B, and they secreted tumor necrosis factor-α and interleukin-6 in response to bacterial invasion. Thus, the cell line we established is a potential tool for research on the relationship between AECs and Mycobacterium tuberculosis.

Human antibodies that neutralize primary human immunodeficiency virus type 1 in vitro do not provide protection in an in vivo model.

NARCIS (Netherlands)

M. Schutten (Martin); K. Tenner-Racz; P. Racz; D.W. van Bekkum (Dirk); A.D.M.E. Osterhaus (Albert)

1996-01-01

textabstractRecently, conflicting data have been published about the ability of antibodies which efficiently neutralize T cell-adapted human immunodeficiency virus type 1 (HIV-1) strains to neutralize primary HIV-1 strains in vitro and in vivo. Here we present data indicating that such antibodies

Molecular Mechanisms of Disease Progression in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type during Ibrutinib Therapy

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Lucy C. Fox

2018-06-01

Full Text Available Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT is one of the well-recognized extranodal lymphomas commonly addicted to the B-cell receptor-MYD88 superpathway. We aimed to describe the genomic changes in a patient who progressed through treatment with ibrutinib, a Bruton’s tyrosine kinase (BTK inhibitor. An 80-year-old woman presented with multiply relapsed PCDLBCL-LT after multiple lines of chemoimmunotherapy and radiotherapy. Pre-treatment testing of the localized cutaneous tumor lesion on a lymphoid amplicon panel demonstrated an MYD88 p.L265P mutation. Ibrutinib therapy was subsequently commenced, resulting in complete resolution of the skin disease. Despite an ongoing skin response, the patient developed progressive nodal disease at two months. Genomic analysis of the cutaneous tumor sample at baseline was compared to that of the inguinal lymph node upon progression, and revealed the acquisition of multiple genomic changes. These included several aberrations expected to bypass BTK inhibition, including two CARD11-activating mutations, and a deleterious mutation in the nuclear factor kappa B (NF-κB negative regulator, NFKBIE. In addition, an IgH-IRF8 translocation was detected (which brings the IRF8 transcription factor under control of the immunoglobulin heavy chain locus, representing a third plausible mechanism contributing to ibrutinib resistance. Several copy-number changes occurred in both samples, including an amplification of 18q, which encodes the anti-apoptotic protein BCL2. We describe the first case of novel genomic changes of PCDLBCL-LT that occurred while on ibrutinib, providing important mechanistic insights into both pathogenesis and drug resistance.

Longevity in vivo of primary cell wall cellulose synthases.

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Hill, Joseph Lee; Josephs, Cooper; Barnes, William J; Anderson, Charles T; Tien, Ming

2018-02-01

Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

Cell longevity and sustained primary growth in palm stems.

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Tomlinson, P Barry; Huggett, Brett A

2012-12-01

Longevity, or organismal life span, is determined largely by the period over which constituent cells can function metabolically. Plants, with modular organization (the ability continually to develop new organs and tissues) differ from animals, with unitary organization (a fixed body plan), and this difference is reflected in their respective life spans, potentially much longer in plants than animals. We draw attention to the observation that palm trees, as a group of monocotyledons without secondary growth comparable to that of lignophytes (plants with secondary growth from a bifacial cambium), retain by means of sustained primary growth living cells in their trunks throughout their organismal life span. Does this make palms the longest-lived trees because they can grow as individuals for several centuries? No conventional lignophyte retains living metabolically active differentiated cell types in its trunk for this length of time, even though the tree as a whole can exist for millennia. Does this contrast also imply that the long-lived cells in a palm trunk have exceptional properties, which allows this seeming immortality? We document the long-life of many tall palm species and their inherent long-lived stem cell properties, comparing such plants to conventional trees. We provide a summary of aspects of cell age and life span in animals and plants. Cell replacement is a feature of animal function, whereas conventional trees rely on active growth centers (meristems) to sustain organismal development. However, the long persistence of living cells in palm trunks is seen not as evidence for unique metabolic processes that sustain longevity, but is a consequence of unique constructional features. This conclusion suggests that the life span of plant cells is not necessarily genetically determined.

Receptor for advanced glycation end-products is a marker of type I lung alveolar cells.

Science.gov (United States)

Shirasawa, Madoka; Fujiwara, Naoyuki; Hirabayashi, Susumu; Ohno, Hideki; Iida, Junko; Makita, Koshi; Hata, Yutaka

2004-02-01

Lung alveolar epithelial cells are comprised of type I (ATI) and type II (ATII) cells. ATI cells are polarized, although they have very flat morphology. The identification of marker proteins for apical and basolateral membranes of ATI cells is important to investigate into the differentiation of ATI cells. In this paper, we characterized receptor for advanced glycation end-products (RAGE) as a marker for ATI cells. RAGE was localized on basolateral membranes of ATI cells in the immunoelectron microscopy and its expression was enhanced in a parallel manner to the differentiation of ATI cells in vivo and in primary cultures of ATII cells. RAGE and T1 alpha, a well-known ATI marker protein, were targeted to basolateral and apical membranes, respectively, when expressed in polarized Madine Darby canine kidney cells. Moreover, RAGE was expressed in ATI cells after T1 alpha in vivo and in ex in vivo organ cultures. In conclusion, RAGE is a marker for basolateral membranes of well-differentiated ATI cells. ATI cells require some signal provided by the in vivo environment to express RAGE.

Epithelial cells derived from human embryonic stem cells display p16INK4A senescence, hypermotility, and differentiation properties shared by many P63+ somatic cell types

DEFF Research Database (Denmark)

Dabelsteen, Sally; Hercule, Paula; Barron, Patricia

2009-01-01

hESderK cells and keratinocytes a substantially extended lifespan. When exposed to transforming growth factor beta or to an incompletely processed form of Laminin-332, three lifespan-extended or immortalized hESderK lines that we studied became directionally hypermotile, a wound healing and invasion......(+)/K14(+) urothelial and tracheobronchial epithelial cells. Primary and immortalized lines of these cell types had growth requirements and hypermotility responses similar to keratinocytes and bmi1 expression facilitated their immortalization by engineering to express the catalytic subunit of telomerase...

Transfection in Primary Cultured Neuronal Cells.

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Marwick, Katie F M; Hardingham, Giles E

2017-01-01

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here, we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

A Web-Server of Cell Type Discrimination System

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Anyou Wang

2014-01-01

Full Text Available Discriminating cell types is a daily request for stem cell biologists. However, there is not a user-friendly system available to date for public users to discriminate the common cell types, embryonic stem cells (ESCs, induced pluripotent stem cells (iPSCs, and somatic cells (SCs. Here, we develop WCTDS, a web-server of cell type discrimination system, to discriminate the three cell types and their subtypes like fetal versus adult SCs. WCTDS is developed as a top layer application of our recent publication regarding cell type discriminations, which employs DNA-methylation as biomarkers and machine learning models to discriminate cell types. Implemented by Django, Python, R, and Linux shell programming, run under Linux-Apache web server, and communicated through MySQL, WCTDS provides a friendly framework to efficiently receive the user input and to run mathematical models for analyzing data and then to present results to users. This framework is flexible and easy to be expended for other applications. Therefore, WCTDS works as a user-friendly framework to discriminate cell types and subtypes and it can also be expended to detect other cell types like cancer cells.

Reliability Evaluation of Primary Cells | Anyaka | Nigerian Journal of ...

African Journals Online (AJOL)

Evaluation of the reliability of a primary cell took place in three stages: 192 cells went through a slow-discharged test. A designed experiment was conducted on 144 cells; there were three factors in the experiment: Storage temperature (three levels), thermal shock (two levels) and date code (two levels). 16 cells ...

Primary Small Cell Carcinoma of the Upper Urinary Tract

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Victor Ka-Siong Kho

2010-03-01

Full Text Available We report a case of primary extrapulmonary small cell carcinoma of the distal ureter, with a synchronous small cell carcinoma of the ipsilateral renal pelvis. These tumors, rarely reported in the urinary tract, are locally aggressive and have a poor prognosis. A 77-year-old male bedridden patient presented with fever and chills with left side-flank pain for 3 days. Following a diagnosis of ureteral urothelial carcinoma, hand-assisted laparoscopic nephroureterectomy with bladder cuff excision was carried out. Adjuvant chemotherapy was given after pathologic report of primary small cell carcinoma of the distal ureter and a synchronous small cell carcinoma of the ipsilateral renal pelvis. After 3 cycles of combination chemotherapy, the patient died 4 months postoperatively due to sepsis.

Hypersensitivity to DNA-damaging agents in primary degenerations of excitable tissue

International Nuclear Information System (INIS)

Robbins, J.H.

1983-01-01

Defects in DNA-repair mechanisms render xeroderma pigmentosum cells hypersensitive to killing by the uv-type of DNA-damaging agent. Some xeroderma pigmentosum patients develop a primary neuronal degeneration, and cell lines from patients with the earliest onset of neurodegeneration are the most sensitive to killing by uv radiation. These findings led to the neuronal DNA integrity theory which holds that when the integrity of neuronal DNA is destroyed by the accumulation of unrepaired DNA damaged spontaneously or by endogenous metabolites, the neurons will undergo a primary degeneration. Cells from patients with Cockayne syndrome, a demyelinating disorder with a primary retinal degeneration, are also hypersensitive to the uv-type of DNA-damaging agent. Cells from patients with the primary neuronal degeneration of ataxia telangiectasia are hypersensitive to the x-ray-type of DNA-damaging agent. Cells from other patients with primary degeneration of excitable tissue also have hypersensitivity to the x-ray-type of DNA-damaging agent. These disorders include (1) primary neuronal degenerations which are either genetic (e.g., Huntington disease, familial dysautonomia, Friedreich ataxia) or sporadic (e.g., Alzheimer disease, Parkinson disease), (2) primary muscle degenerations (e.g., Duchenne muscular dystrophy), and (3) a primary retinal degeneration (Usher syndrome). Death of excitable tissue in vivo in these radiosensitive diseases may result from unrepaired DNA. This hypersensitivity provides the basis for developing suitable presymptomatic and prenatal tests for these diseases, for elucidating their pathogenesis, and for developing future therapies. 119 references, 3 figures, 3 tables

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

Science.gov (United States)

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

2010-01-25

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

Energy Technology Data Exchange (ETDEWEB)

Henderson, R.F.; Waide, J.J.; Lechner, J.F.

1995-12-01

A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture.

Science.gov (United States)

Nakanishi, Takeo; Fukushi, Akimasa; Sato, Masanobu; Yoshifuji, Mayuko; Gose, Tomoka; Shirasaka, Yoshiyuki; Ohe, Kazuyo; Kobayashi, Masato; Kawai, Keiichi; Tamai, Ikumi

2011-12-05

Since in vitro cell culture models often show altered apical transporter expression, they are not necessarily suitable for the analysis of renal transport processes. Therefore, we aimed here to investigate the usefulness of primary-cultured rat proximal tubular cells (PTCs) for this purpose. After isolation of renal cortical cells from rat kidneys, PTCs were enriched and the gene expression and function of apical transporters were analyzed by means of microarray, RT-PCR and uptake experiments. RT-PCR confirmed that the major apical transporters were expressed in rat PTCs. Na(+)-dependent uptake of α-methyl-d-glucopyranoside (αMG), ergothioneine and carnitine by the PTCs suggests functional expression of Sglts, Octn1 and Octn2, respectively. Inhibition of pH-dependent glycylsarcosine uptake by low concentration of cephalexin, which is a β-lactam antibiotics recognized by Pepts, indicates a predominant role of high affinity type Pept2, but not low affinity type Pept1, in the PTCs. Moreover, the permeability ratio of [(14)C]αMG (apical to basolateral/basolateral to apical) across PTCs was 4.3, suggesting that Sglt-mediated reabsorptive transport is characterized. In conclusion, our results indicate that rat PTCs in primary culture are found to be a promising in vitro model to evaluate reabsorption processes mediated at least by Sglts, Pept2, Octn1 and Octn2.

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours.

Science.gov (United States)

Bate-Eya, Laurel T; Ebus, Marli E; Koster, Jan; den Hartog, Ilona J M; Zwijnenburg, Danny A; Schild, Linda; van der Ploeg, Ida; Dolman, M Emmy M; Caron, Huib N; Versteeg, Rogier; Molenaar, Jan J

2014-02-01

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models. Copyright © 2013 Elsevier Ltd. All rights reserved.

Graphene Films Show Stable Cell Attachment and Biocompatibility with Electrogenic Primary Cardiac Cells

OpenAIRE

Kim, Taeyong; Kahng, Yung Ho; Lee, Takhee; Lee, Kwanghee; Kim, Do Han

2013-01-01

Graphene has attracted substantial attention due to its advantageous materialistic applicability. In the present study, we tested the biocompatibility of graphene films synthesized by chemical vapor deposition with electrogenic primary adult cardiac cells (cardiomyocytes) by measuring the cell properties such as cell attachment, survival, contractility and calcium transients. The results show that the graphene films showed stable cell attachment and excellent biocompatibility with the electro...

Efficacy of pegaspargase in extra nodal natural killer/T-cell lymphoma nasal type: A case report from China

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Xingan Xiong

2015-01-01

Full Text Available Extranodal natural killer (NK/T-cell lymphoma, nasal type, is a rare and highly aggressive disease with a grim prognosis. There is no known satisfactory treatment. The author herein to report one case of L-asparaginase extranodal NK/T-cell lymphoma primary treated with L-asparaginase methotrexate and dexamethasone.

Location of tumor affects local and distant immune cell type and number.

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Hensel, Jonathan A; Khattar, Vinayak; Ashton, Reading; Lee, Carnellia; Siegal, Gene P; Ponnazhagan, Selvarangan

2017-03-01

Tumors comprise heterogeneous populations of cells, including immune infiltrates that polarize during growth and metastasis. Our preclinical studies on breast cancer (BCa) identified functional differences in myeloid-derived suppressor cells based on tumor microenvironment (TME), prompting variations in host immune response to tumor growth, and dissemination based on tissue type. In order to understand if such variations existed among other immune cells, and if such alteration occurs in response to tumor growth at the primary site or due to bone dissemination, we characterized immune cells, examining localized growth and in the tibia. In addition, immune cells from the spleen were examined from animals of both tumor locations by flow cytometry. The study demonstrates that location of tumor, and not simply the tumor itself, has a definitive role in regulating immune effectors. Among all immune cells characterized, macrophages were decreased and myeloid dendritic cell were increased in both tumor locations. This difference was more evident in subcutaneous tumors. Additionally, spleens from mice with subcutaneous tumors contained greater increases in both macrophages and myeloid dendritic cells than in mice with bone tumors. Furthermore, in subcutaneous tumors there was an increase in CD4 + and CD8 + T-cell numbers, which was also observed in their spleens. These data indicate that alterations in tumor-reactive immune cells are more pronounced at the primary site, and exert a similar change at the major secondary lymphoid organ than in the bone TME. These findings could provide translational insight into designing therapeutic strategies that account for location of metastatic foci.

Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells

International Nuclear Information System (INIS)

Ray, Ratna B.; Basu, Arnab; Steele, Robert; Beyene, Aster; McHowat, Jane; Meyer, Keith; Ghosh, Asish K.; Ray, Ranjit

2004-01-01

Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences

Radiation Gene-expression Signatures in Primary Breast Cancer Cells.

Science.gov (United States)

Minafra, Luigi; Bravatà, Valentina; Cammarata, Francesco P; Russo, Giorgio; Gilardi, Maria C; Forte, Giusi I

2018-05-01

In breast cancer (BC) care, radiation therapy (RT) is an efficient treatment to control localized tumor. Radiobiological research is needed to understand molecular differences that affect radiosensitivity of different tumor subtypes and the response variability. The aim of this study was to analyze gene expression profiling (GEP) in primary BC cells following irradiation with doses of 9 Gy and 23 Gy delivered by intraoperative electron radiation therapy (IOERT) in order to define gene signatures of response to high doses of ionizing radiation. We performed GEP by cDNA microarrays and evaluated cell survival after IOERT treatment in primary BC cell cultures. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate candidate genes. We showed, for the first time, a 4-gene and a 6-gene signature, as new molecular biomarkers, in two primary BC cell cultures after exposure at 9 Gy and 23 Gy respectively, for which we observed a significantly high survival rate. Gene signatures activated by different doses of ionizing radiation may predict response to RT and contribute to defining a personalized biological-driven treatment plan. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Alveolar epithelial type II cells induce T cell tolerance to specific antigen

DEFF Research Database (Denmark)

Lo, Bernice; Hansen, Søren; Evans, Kathy

2008-01-01

The lungs face the immunologic challenge of rapidly eliminating inhaled pathogens while maintaining tolerance to innocuous Ags. A break in this immune homeostasis may result in pulmonary inflammatory diseases, such as allergies or asthma. The observation that alveolar epithelial type II cells (Type...... II) constitutively express the class II MHC led us to hypothesize that Type II cells play a role in the adaptive immune response. Because Type II cells do not express detectable levels of the costimulatory molecules CD80 and CD86, we propose that Type II cells suppress activation of naive T cells...

Imprint lithography provides topographical nanocues to guide cell growth in primary cortical cell culture

NARCIS (Netherlands)

Xie, S.; Luttge, R.

2014-01-01

In this paper, we describe a technology platform to study the effect of nanocues on the cell growth direction in primary cortical cell culture. Topographical cues to cells are provided using nanoscale features created by Jet and Flash Imprint Lithography, coated with polyethylenimine. We

Glucose-mediated control of ghrelin release from primary cultures of gastric mucosal cells

Science.gov (United States)

Sakata, Ichiro; Park, Won-Mee; Walker, Angela K.; Piper, Paul K.; Chuang, Jen-Chieh; Osborne-Lawrence, Sherri

2012-01-01

The peptide hormone ghrelin is released from a distinct group of gastrointestinal cells in response to caloric restriction, whereas its levels fall after eating. The mechanisms by which ghrelin secretion is regulated remain largely unknown. Here, we have used primary cultures of mouse gastric mucosal cells to investigate ghrelin secretion, with an emphasis on the role of glucose. Ghrelin secretion from these cells upon exposure to different d-glucose concentrations, the glucose antimetabolite 2-deoxy-d-glucose, and other potential secretagogues was assessed. The expression profile of proteins involved in glucose transport, metabolism, and utilization within highly enriched pools of mouse ghrelin cells and within cultured ghrelinoma cells was also determined. Ghrelin release negatively correlated with d-glucose concentration. Insulin blocked ghrelin release, but only in a low d-glucose environment. 2-Deoxy-d-glucose prevented the inhibitory effect of high d-glucose exposure on ghrelin release. mRNAs encoding several facilitative glucose transporters, hexokinases, the ATP-sensitive potassium channel subunit Kir6.2, and sulfonylurea type 1 receptor were expressed highly within ghrelin cells, although neither tolbutamide nor diazoxide exerted direct effects on ghrelin secretion. These findings suggest that direct exposure of ghrelin cells to low ambient d-glucose stimulates ghrelin release, whereas high d-glucose and glucose metabolism within ghrelin cells block ghrelin release. Also, low d-glucose sensitizes ghrelin cells to insulin. Various glucose transporters, channels, and enzymes that mediate glucose responsiveness in other cell types may contribute to the ghrelin cell machinery involved in regulating ghrelin secretion under these different glucose environments, although their exact roles in ghrelin release remain uncertain. PMID:22414807

Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line.

Science.gov (United States)

Meng, Ran; Zhou, Jin; Sui, Meng; Li, ZhiYong; Feng, GuoSheng; Yang, BaoFeng

2010-01-01

This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment.

Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9.

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Enas R Yassin

2009-08-01

Full Text Available NUP98-HOXA9 is the prototype of a group of oncoproteins associated with acute myeloid leukemia. It consists of an N-terminal portion of NUP98 fused to the homeodomain of HOXA9 and is believed to act as an aberrant transcription factor that binds DNA through the homeodomain. Here we show that NUP98-HOXA9 can regulate transcription without binding to DNA. In order to determine the relative contributions of the NUP98 and HOXA9 portions to the transforming ability of NUP98-HOXA9, the effects of NUP98-HOXA9 on primary human CD34+ cells were dissected and compared to those of wild-type HOXA9. In contrast to previous findings in mouse cells, HOXA9 had only mild effects on the differentiation and proliferation of primary human hematopoietic cells. The ability of NUP98-HOXA9 to disrupt the differentiation of primary human CD34+ cells was found to depend primarily on the NUP98 portion, whereas induction of long-term proliferation required both the NUP98 moiety and an intact homeodomain. Using oligonucleotide microarrays in primary human CD34+ cells, a group of genes was identified whose dysregulation by NUP98-HOXA9 is attributable primarily to the NUP98 portion. These include RAP1A, HEY1, and PTGS2 (COX-2. Their functions may reflect the contribution of the NUP98 moiety of NUP98-HOXA9 to leukemic transformation. Taken together, these results suggest that the effects of NUP98-HOXA9 on gene transcription and cell transformation are mediated by at least two distinct mechanisms: one that involves promoter binding through the homeodomain with direct transcriptional activation, and another that depends predominantly on the NUP98 moiety and does not involve direct DNA binding.

Immunocytochemical characterization of primary cell culture in canine transmissible venereal tumor

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Luis M.M. Flórez

Full Text Available Abstract: Immunochemistry with anti-vimentin, anti-lysozyme, anti-alpha 1 antitrypsin, anti-CD3 and anti-CD79α antibodies has been used for characterization of primary cell culture in the transmissible venereal tumor (TVT. Samples for primary cell culture and immunohistochemistry assays were taken from eight dogs with cytological and clinical diagnosis of TVT. To validate the immunochemical results in the primary cell culture of TVT, a chromosome count was performed. For the statistical analysis, the Mann-Whitney test with p<0.05 was used. TVT tissues and culture cells showed intense anti-vimentin immunoreactivity, lightly to moderate immunoreactivity for anti-lysozyme, and mild for anti-alpha-antitrypsin. No marking was achieved for CD3 and CD79α. All culture cells showed chromosomes variable number of 56 to 68. This is the first report on the use of immunocytochemical characterization in cell culture of TVT. Significant statistic difference between immunochemistry in tissue and culture cell was not established, what suggests that the use of this technique may provide greater certainty for the confirmation of tumors in the primary culture. This fact is particularly important because in vitro culture of tumor tissues has been increasingly used to provide quick access to drug efficacy and presents relevant information to identify potential response to anticancer medicine; so it is possible to understand the behavior of the tumor.

Cytokine-induced killer cells are type II natural killer T cells

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Schmidt-Wolf, Ingo G.H.

2007-09-01

Full Text Available Background: Until now, cytokine-induced killer (CIK cells were assumed to be part of the type I natural killer T (NKT cell population, but it was not yet investigated if this is correct. Methods: For analysis, CIK cells were generated by various culture conditions. Human type I NKT cells express a T cell receptor (TCR composed of an invariant Vα24-JαQ chain combined with one of several Vβ chains. The Vα24 is a reliable marker for the presence of these TCRs. Results: While comparing cultures stimulated with different substances, we observed the lack of any Vα24 on the surface of CIK culture cells. Conclusion: We conclude that CIK cells do not belong to the type I NKT cells.

Inhibition of type I NKT cells by retinoids or following sulfatide-mediated activation of type II NKT cells attenuates alcoholic liver disease

Science.gov (United States)

Maricic, Igor; Sheng, Huiming; Marrero, Idania; Seki, Ehikiro; Kisseleva, Tatiana; Chaturvedi, Som; Molle, Natasha; Mathews, K. Stephanie; Gao, Bin; Kumar, Vipin

2015-01-01

Innate immune mechanisms leading to liver injury following chronic alcohol ingestion are poorly understood. Natural killer T (NKT) cells, enriched in the liver and comprised of at least two distinct subsets, type I and type II, recognize different lipid antigens presented by CD1d molecules. We have investigated whether differential activation of NKT cell subsets orchestrates inflammatory events leading to alcoholic liver disease (ALD). We found that following chronic plus binge feeding of Lieber-DeCarli liquid diet in male C57BL/6 mice, type I but not type II NKT cells are activated leading to recruitment of inflammatory Gr-1highCD11b+ cells into liver. A central finding is that liver injury following alcohol feeding is dependent upon type I NKT cells. Thus liver injury is significantly inhibited in Jα18−/− mice deficient in type I NKT cells as well as following their inactivation by sulfatide-mediated activation of type II NKT cells. Furthermore we have identified a novel pathway involving all-trans retinoic acid (ATRA) and its receptor RARγ signaling that inhibits type I NKT cells and consequently ALD. A semi-quantitative PCR analysis of hepatic gene expression of some of the key proinflammatory molecules shared in human disease indicated that their upregulation in ALD is dependent upon type I NKT cells. Conclusion Type I but not type II NKT cells become activated following alcohol feeding. Type I NKT cells-induced inflammation and neutrophil recruitment results in liver tissue damage while type II NKT cells protect from injury in ALD. Inhibition of type I NKT cells by retinoids or by sulfatide prevents ALD. Since the CD1d pathway is highly conserved between mice and humans, NKT cell subsets might be targeted for potential therapeutic intervention in ALD. PMID:25477000

Primary clear cell renal carcinoma cells display minimal mitochondrial respiratory capacity resulting in pronounced sensitivity to glycolytic inhibition by 3-Bromopyruvate.

Science.gov (United States)

Nilsson, H; Lindgren, D; Mandahl Forsberg, A; Mulder, H; Axelson, H; Johansson, M E

2015-01-08

Changes of cellular metabolism are an integral property of the malignant potential of most cancer cells. Already in the 1930s, Otto Warburg observed that tumor cells preferably utilize glycolysis and lactate fermentation for energy production, rather than the mitochondrial oxidative phosphorylation dominating in normal cells, a phenomenon today known as the Warburg effect. Even though many tumor types display a high degree of aerobic glycolysis, they still retain the activity of other energy-producing metabolic pathways. One exception seems to be the clear cell variant of renal cell carcinoma, ccRCC, where the activity of most other pathways than that of glycolysis has been shown to be reduced. This makes ccRCC a promising candidate for the use of glycolytic inhibitors in treatment of the disease. However, few studies have so far addressed this issue. In this report, we show a strikingly reduced mitochondrial respiratory capacity of primary human ccRCC cells, resulting in enhanced sensitivity to glycolytic inhibition by 3-Bromopyruvate (3BrPA). This effect was largely absent in established ccRCC cell lines, a finding that highlights the importance of using biologically relevant models in the search for new candidate cancer therapies. 3BrPA markedly reduced ATP production in primary ccRCC cells, followed by cell death. Our data suggest that glycolytic inhibitors such as 3BrPA, that has been shown to be well tolerated in vivo, should be further analyzed for the possible development of selective treatment strategies for patients with ccRCC.

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

Science.gov (United States)

Shamim, Thorakkal

2009-07-01

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

Sulfatide-activated type II NKT cells prevent allergic airway inflammation by inhibiting type I NKT cell function in a mouse model of asthma.

Science.gov (United States)

Zhang, Guqin; Nie, Hanxiang; Yang, Jiong; Ding, Xuhong; Huang, Yi; Yu, Hongying; Li, Ruyou; Yuan, Zhuqing; Hu, Suping

2011-12-01

Asthma is a common chronic inflammatory disease involving many different cell types. Recently, type I natural killer T (NKT) cells have been demonstrated to play a crucial role in the development of asthma. However, the roles of type II NKT cells in asthma have not been investigated before. Interestingly, type I and type II NKT cells have been shown to have opposing roles in antitumor immunity, antiparasite immunity, and autoimmunity. We hypothesized that sulfatide-activated type II NKT cells could prevent allergic airway inflammation by inhibiting type I NKT cell function in asthma. Strikingly, in our mouse model, activation of type II NKT cells by sulfatide administration and adoptive transfer of sulfatide-activated type II NKT cells result in reduced-inflammation cell infiltration in the lung and bronchoalveolar lavage fluid, decreased levels of IL-4 and IL-5 in the BALF; and decreased serum levels of ovalbumin-specific IgE and IgG1. Furthermore, it is found that the activation of sulfatide-reactive type II NKT cells leads to the functional inactivation of type I NKT cells, including the proliferation and cytokine secretion. Our data reveal that type II NKT cells activated by glycolipids, such as sulfatide, may serve as a novel approach to treat allergic diseases and other disorders characterized by inappropriate type I NKT cell activation.

[3H]GABA uptake as a marker for cell type in primary cultures of cerebellum and olfactory bulb

International Nuclear Information System (INIS)

Currie, D.N.; Dutton, G.R.

1980-01-01

Uptake of [ 3 H]GABA into cell cultures of rat cerebellum and olfactory bulb was studied by autoradiography, using β-alanine and aminocyclohexane carboxylic acid to distinguish neuronal-specific and glial-specific uptake. Neurons and astrocytes were also labelled by tetanus toxin and anti-GFAP respectively. This combination of markers allowed identification and quantification of several cell types. Cerebellar cultures were found to contain 77% granule neurons, 7.5% inhibitory neurons (probably stellate and basket cells) and 15% astrocytes. Olfactory bulb cultures were over 50% in small neurons which accumulated GABA, the olfactory bulb granule neuron being GABAergic in vivo. (Auth.)

A 3-D Model of a Perennial Ryegrass Primary Cell Wall and Its Enzymatic Degradation

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Indrakumar Vetharaniam

2014-05-01

Full Text Available We have developed a novel 3-D, agent-based model of cell-wall digestion to improve our understanding of ruminal cell-wall digestion. It offers a capability to study cell walls and their enzymatic modification, by providing a representation of cellulose microfibrils and non-cellulosic polysaccharides and by simulating their spatial and catalytic interactions with enzymes. One can vary cell-wall composition and the types and numbers of enzyme molecules, allowing the model to be applied to a range of systems where cell walls are degraded and to the modification of cell walls by endogenous enzymes. As a proof of principle, we have modelled the wall of a mesophyll cell from the leaf of perennial ryegrass and then simulated its enzymatic degradation. This is a primary, non-lignified cell wall and the model includes cellulose, hemicelluloses (glucuronoarabinoxylans, 1,3;1,4-β-glucans, and xyloglucans and pectin. These polymers are represented at the level of constituent monosaccharides, and assembled to form a 3-D, meso-scale representation of the molecular structure of the cell wall. The composition of the cell wall can be parameterised to represent different walls in different cell types and taxa. The model can contain arbitrary combinations of different enzymes. It simulates their random diffusion through the polymer networks taking collisions into account, allowing steric hindrance from cell-wall polymers to be modelled. Steric considerations are included when target bonds are encountered, and breakdown products resulting from enzymatic activity are predicted.

Industrial n-type solar cells with >20% cell efficiency

Energy Technology Data Exchange (ETDEWEB)

Romijn, I.G.; Anker, J.; Burgers, A.R.; Gutjahr, A.; Koppes, M.; Kossen, E.J.; Lamers, M.W.P.E.; Heurtault, Benoit; Saynova-Oosterling, D.S.; Tool, C.J.J. [ECN Solar Energy, Petten (Netherlands)

2013-03-15

To realize high efficiencies at low costs, ECN has developed the n-Pasha solar cell concept. The n-Pasha cell concept is a bifacial solar cell concept on n-Cz base material, with which average efficiencies of above 20% have been demonstrated. In this paper recent developments at ECN to improve the cost of ownership (lower Euro/Wp) of the n-Pasha cell concept are discussed. Two main drivers for the manufacturing costs of n-type solar cells are addressed: the n-type Cz silicon material and the silver consumption. We show that a large resistivity range between 2 and 8 cm can be tolerated for high cell efficiency, and that the costs due to the silver metallization can be significantly reduced while increasing the solar cell efficiency. Combining the improved efficiency and cost reduction makes the n-Pasha cell concept a very cost effective solution to manufacture high efficient solar cells and modules.

Immune cell-poor melanomas benefit from PD-1 blockade after targeted type I IFN activation.

Science.gov (United States)

Bald, Tobias; Landsberg, Jennifer; Lopez-Ramos, Dorys; Renn, Marcel; Glodde, Nicole; Jansen, Philipp; Gaffal, Evelyn; Steitz, Julia; Tolba, Rene; Kalinke, Ulrich; Limmer, Andreas; Jönsson, Göran; Hölzel, Michael; Tüting, Thomas

2014-06-01

Infiltration of human melanomas with cytotoxic immune cells correlates with spontaneous type I IFN activation and a favorable prognosis. Therapeutic blockade of immune-inhibitory receptors in patients with preexisting lymphocytic infiltrates prolongs survival, but new complementary strategies are needed to activate cellular antitumor immunity in immune cell-poor melanomas. Here, we show that primary melanomas in Hgf-Cdk4(R24C) mice, which imitate human immune cell-poor melanomas with a poor outcome, escape IFN-induced immune surveillance and editing. Peritumoral injections of immunostimulatory RNA initiated a cytotoxic inflammatory response in the tumor microenvironment and significantly impaired tumor growth. This critically required the coordinated induction of type I IFN responses by dendritic, myeloid, natural killer, and T cells. Importantly, antibody-mediated blockade of the IFN-induced immune-inhibitory interaction between PD-L1 and PD-1 receptors further prolonged the survival. These results highlight important interconnections between type I IFNs and immune-inhibitory receptors in melanoma pathogenesis, which serve as targets for combination immunotherapies. Using a genetically engineered mouse melanoma model, we demonstrate that targeted activation of the type I IFN system with immunostimulatory RNA in combination with blockade of immune-inhibitory receptors is a rational strategy to expose immune cell-poor tumors to cellular immune surveillance. ©2014 American Association for Cancer Research.

The preventive role of type 2 NKT cells in the development of type 1 diabetes.

Science.gov (United States)

Sørensen, Jakob Ørskov; Buschard, Karsten; Brogren, Carl-Henrik

2014-03-01

In the last two decades, natural killer T (NKT) cells have emerged as an important factor in preventing type 1 diabetes (T1D) when investigated in the experimental non-obese diabetic (NOD) mouse model. So far, investigations have largely focused on type 1 NKT cells with invariant T-cell receptors, whereas the role of type 2 NKT cells with diverse T-cell receptors is less well understood. However, there have been several findings which indicate that in fact type 2 NKT cells may regulate the progression of type 1 diabetes in NOD mice, including a fraction of these cells which recognize β-cell-enriched sulfatide. Therefore, the focus for this review is to present the current evidence of the effect of type 2 NKT cells on the development of T1D. In general, there is still uncertainty surrounding the mechanism of activation and function of NKT cells. Here, we present two models of the effector mechanisms, respectively, Th1/Th2 polarization and the induction of tolerogenic dendritic cells (DC). In conclusion, this review points to the importance of immunoregulation by type 2 NKT cells in preventing the development of T1D and highlights the induction of tolerogenic DC as a likely mechanism. The possible therapeutic role of type 1 and type 2 NKT cells are evaluated and future experiments concerning type 2 NKT cells and T1D are proposed. © 2013 APMIS. Published by John Wiley & Sons Ltd.

3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

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Yeonhee Kim

Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

Systems-level comparison of host responses induced by pandemic and seasonal influenza A H1N1 viruses in primary human type I-like alveolar epithelial cells in vitro

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Guan Yi

2010-10-01

Full Text Available Abstract Background Pandemic influenza H1N1 (pdmH1N1 virus causes mild disease in humans but occasionally leads to severe complications and even death, especially in those who are pregnant or have underlying disease. Cytokine responses induced by pdmH1N1 viruses in vitro are comparable to other seasonal influenza viruses suggesting the cytokine dysregulation as seen in H5N1 infection is not a feature of the pdmH1N1 virus. However a comprehensive gene expression profile of pdmH1N1 in relevant primary human cells in vitro has not been reported. Type I alveolar epithelial cells are a key target cell in pdmH1N1 pneumonia. Methods We carried out a comprehensive gene expression profiling using the Affymetrix microarray platform to compare the transcriptomes of primary human alveolar type I-like alveolar epithelial cells infected with pdmH1N1 or seasonal H1N1 virus. Results Overall, we found that most of the genes that induced by the pdmH1N1 were similarly regulated in response to seasonal H1N1 infection with respect to both trend and extent of gene expression. These commonly responsive genes were largely related to the interferon (IFN response. Expression of the type III IFN IL29 was more prominent than the type I IFN IFNβ and a similar pattern of expression of both IFN genes was seen in pdmH1N1 and seasonal H1N1 infection. Genes that were significantly down-regulated in response to seasonal H1N1 but not in response to pdmH1N1 included the zinc finger proteins and small nucleolar RNAs. Gene Ontology (GO and pathway over-representation analysis suggested that these genes were associated with DNA binding and transcription/translation related functions. Conclusions Both seasonal H1N1 and pdmH1N1 trigger similar host responses including IFN-based antiviral responses and cytokine responses. Unlike the avian H5N1 virus, pdmH1N1 virus does not have an intrinsic capacity for cytokine dysregulation. The differences between pdmH1N1 and seasonal H1N1 viruses

Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines.

Science.gov (United States)

Adalsteinsson, Viktor A; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B; Huang, Cindy; Bowman, Brittany; Williamson, Christina A; Kwon, Douglas S; Wittrup, K Dane; Love, J Christopher

2013-10-01

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

Enteroendocrine cell types revisited

DEFF Research Database (Denmark)

Engelstoft, Maja S; Egerod, Kristoffer Lihme; Lund, Mari L

2013-01-01

The GI-tract is profoundly involved in the control of metabolism through peptide hormones secreted from enteroendocrine cells scattered throughout the gut mucosa. A large number of recently generated transgenic reporter mice have allowed for direct characterization of biochemical and cell...... biological properties of these previously highly elusive enteroendocrine cells. In particular the surprisingly broad co-expression of six functionally related hormones in the intestinal enteroendocrine cells indicates that it should be possible to control not only the hormone secretion but also the type...... and number of enteroendocrine cells. However, this will require a more deep understanding of the factors controlling differentiation, gene expression and specification of the enteroendocrine cells during their weekly renewal from progenitor cells in the crypts of the mucosa....

Cell type-specific neuroprotective activity of untranslocated prion protein.

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Elena Restelli

2010-10-01

Full Text Available A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP. However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy

NARCIS (Netherlands)

Dahnrich, C.; Komorowski, L.; Probst, C.; Seitz-Polski, B.; Esnault, V.; Wetzels, J.F.M.; Hofstra, J.M.; Hoxha, E.; Stahl, R.A.K.; Lambeau, G.; Stocker, W.; Schlumberger, W.

2013-01-01

BACKGROUND: Autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) are specific markers for primary membranous nephropathy (pMN) and anti-PLA2R1 serum levels may be useful to monitor disease activity. So far, a recombinant cell-based indirect immunofluorescence assay (RC-IFA) using

Vaginal immunization to elicit primary T-cell activation and dissemination.

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Elena Pettini

Full Text Available Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4(+ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4(+ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.

Polyglandular Autoimmune Syndrome Type III with Primary Hypoparathyroidism

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Sang Jin Kim

2013-09-01

Full Text Available Polyglandular autoimmune syndrome is defined as multiple endocrine gland insufficiencies accompanied by autoimmune diseases of the endocrine and nonendocrine system. After Schmidt introduced a case of nontuberculosis adrenal gland dysfunction with thyroiditis in 1926, Neufeld defined polyglandular autoimmune syndrome by I, II, and III subtypes in 1980 by their presentation of occurrence age, heredity methods, relationship with human leukocyte antigen, and accompanying diseases. We report a case of a 32-year-old female with polyglandular autoimmune syndrome III accompanied by type 1 diabetes mellitus that was treated with insulin (36 units per day for 11 years. She had insulin deficiency and Hashimoto thyroiditis as an autoimmune disorder. In addition, she had several features similar to Albright's hereditary osteodystrophy including short stature, truncal obesity, round face, short neck, low intelligence (full IQ 84, and decreased memory. Although Albright's hereditary osteodystrophy is morphological evidence of pseudohypoparathyroidism or pseudopseudohypoparathyroidism, she had primary hypoparathyroidism on laboratory results. Here, we report a case of polyglandular autoimmune syndrome III with type 1 diabetes mellitus, autoimmune thyroiditis, and primary hypoparathyroidism, accompanied by clinical features similar to Albright's hereditary osteodystrophy.

EXPLICIT AND IMPLICIT TYPES OF CORRECTIVE FEEDBACK IN TURKISH PRIMARY EDUCATION

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M. Pınar BABANOĞLU

2015-08-01

Full Text Available Corrective feedback (CF has become a significant notion in EFL learning as it is seen as a facilitator to enhance L2 learning. CF is basically refer to any feedback provided to learner containing evidence of learner error of language form (Russel & Spada, 2006, or an immediate response by teacher to learner utterances containing error explained which is also theoretically a complex phenomenon with several functions (Lyster, et.al., 2012. A distinction can be drawn whether CF types are being implicit or explicit that there is not a clear indication of error in implicit CF whereas there is in explicit CF types. This study investigates the explicit and implicit CF types used by teachers in EFL classrooms in primary levels aiming at to see whether there are quantitative differences in the preference of CF types being explicit/implicit. Data of the study gathered from EFL classroom teachers’ course session observations at various levels of primary school. Methodology depends on frequency analysis of CF types employed by teachers during EFL course sessions. Results favored explicit CF types by teachers during EFL sessions.

Equine Herpesvirus Type 1 Enhances Viral Replication in CD172a+ Monocytic Cells upon Adhesion to Endothelial Cells.

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Laval, Kathlyn; Favoreel, Herman W; Poelaert, Katrien C K; Van Cleemput, Jolien; Nauwynck, Hans J

2015-11-01

Equine herpesvirus type 1 (EHV-1) is a main cause of respiratory disease, abortion, and encephalomyelopathy in horses. Monocytic cells (CD172a(+)) are the main carrier cells of EHV-1 during primary infection and are proposed to serve as a "Trojan horse" to facilitate the dissemination of EHV-1 to target organs. However, the mechanism by which EHV-1 is transferred from CD172a(+) cells to endothelial cells (EC) remains unclear. The aim of this study was to investigate EHV-1 transmission between these two cell types. We hypothesized that EHV-1 employs specific strategies to promote the adhesion of infected CD172a(+) cells to EC to facilitate EHV-1 spread. Here, we demonstrated that EHV-1 infection of CD172a(+) cells resulted in a 3- to 5-fold increase in adhesion to EC. Antibody blocking experiments indicated that α4β1, αLβ2, and αVβ3 integrins mediated adhesion of infected CD172a(+) cells to EC. We showed that integrin-mediated phosphatidylinositol 3-kinase (PI3K) and ERK/MAPK signaling pathways were involved in EHV-1-induced CD172a(+) cell adhesion at early times of infection. EHV-1 replication was enhanced in adherent CD172a(+) cells, which correlates with the production of tumor necrosis factor alpha (TNF-α). In the presence of neutralizing antibodies, approximately 20% of infected CD172a(+) cells transferred cytoplasmic material to uninfected EC and 0.01% of infected CD172a(+) cells transmitted infectious virus to neighboring cells. Our results demonstrated that EHV-1 infection induces adhesion of CD172a(+) cells to EC, which enhances viral replication, but that transfer of viral material from CD172a(+) cells to EC is a very specific and rare event. These findings give new insights into the complex pathogenesis of EHV-1. Equine herpesvirus type 1 (EHV-1) is a highly prevalent pathogen worldwide, causing frequent outbreaks of abortion and myeloencephalopathy, even in vaccinated horses. After primary replication in the respiratory tract, EHV-1 disseminates

Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine

DEFF Research Database (Denmark)

Jeppesen, Maria; Hagel, Grith; Glenthoj, Anders

2017-01-01

Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approac...... and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine....... for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient's tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal...... cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days...

Self-renewing Monolayer of Primary Colonic or Rectal Epithelial CellsSummary

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Yuli Wang

2017-07-01

Full Text Available Background & Aims: Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D tissue cultured from primary colon cells has not been accomplished. Methods: The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified. Results: The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. Conclusions: This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies. Keywords: Colonic Epithelial Cells, Monolayer, Organoids, Compound Screening

Concurrent primary hyperparathyroidism and pheochromocytoma in a Chinese lady with neurofibromatosis type 1

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Cheuk-Lik Wong

2018-04-01

Full Text Available We report a case of elderly Chinese lady with neurofibromatosis type-1 presenting with longstanding palpitation, paroxysmal hypertension and osteoporosis. Biochemical testing showed mild hypercalcaemia with non-suppressed parathyroid hormone level suggestive of primary hyperparathyroidism, and mildly elevated urinary fractionated normetanephrine and plasma-free normetanephrine pointing to a catecholamine-secreting pheochromocytoma/paraganglioma. Further scintigraphic investigation revealed evidence of a solitary parathyroid adenoma causing primary hyperparathyroidism and a left pheochromocytoma. Resection of the parathyroid adenoma and pheochromocytoma resulted in normalization of biochemical abnormalities and hypertension. The rare concurrence of primary hyperparathyroidism and pheochromocytoma in neurofibromatosis type-1 is discussed.

Choline Deficiency Causes Colonic Type II Natural Killer T (NKT) Cell Loss and Alleviates Murine Colitis under Type I NKT Cell Deficiency.

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Sagami, Shintaro; Ueno, Yoshitaka; Tanaka, Shinji; Fujita, Akira; Niitsu, Hiroaki; Hayashi, Ryohei; Hyogo, Hideyuki; Hinoi, Takao; Kitadai, Yasuhiko; Chayama, Kazuaki

2017-01-01

Serum levels of choline and its derivatives are lower in patients with inflammatory bowel disease (IBD) than in healthy individuals. However, the effect of choline deficiency on the severity of colitis has not been investigated. In the present study, we investigated the role of choline deficiency in dextran sulfate sodium (DSS)-induced colitis in mice. Methionine-choline-deficient (MCD) diet lowered the levels of type II natural killer T (NKT) cells in the colonic lamina propria, peritoneal cavity, and mesenteric lymph nodes, and increased the levels of type II NKT cells in the livers of wild-type B6 mice compared with that in mice fed a control (CTR) diet. The gene expression pattern of the chemokine receptor CXCR6, which promotes NKT cell accumulation, varied between colon and liver in a manner dependent on the changes in the type II NKT cell levels. To examine the role of type II NKT cells in colitis under choline-deficient conditions, we assessed the severity of DSS-induced colitis in type I NKT cell-deficient (Jα18-/-) or type I and type II NKT cell-deficient (CD1d-/-) mice fed the MCD or CTR diets. The MCD diet led to amelioration of inflammation, decreases in interferon (IFN)-γ and interleukin (IL)-4 secretion, and a decrease in the number of IFN-γ and IL-4-producing NKT cells in Jα18-/- mice but not in CD1d-/- mice. Finally, adaptive transfer of lymphocytes with type II NKT cells exacerbated DSS-induced colitis in Jα18-/- mice with MCD diet. These results suggest that choline deficiency causes proinflammatory type II NKT cell loss and alleviates DSS-induced colitis. Thus, inflammation in DSS-induced colitis under choline deficiency is caused by type II NKT cell-dependent mechanisms, including decreased type II NKT cell and proinflammatory cytokine levels.

Results of radiotherapy for primary subglottic squamous cell carcinoma

International Nuclear Information System (INIS)

Paisley, Sonya; Warde, Padraig R.; O'Sullivan, Brian; Waldron, John; Gullane, Patrick J.; Payne, David; Liu, F.-F.; Bayley, Andrew; Ringash, Jolie; Cummings, Bernard J.

2002-01-01

Purpose: To retrospectively evaluate the outcome after radical radiotherapy (RT) and surgical salvage and assess the risk of late toxicity for patients with primary subglottic squamous cell carcinoma treated at our center. Methods and Materials: Between 1971 and 1996, 43 patients with primary squamous cell carcinoma of the subglottis (35 men, 8 women) were treated with radical RT. All received megavoltage irradiation, most commonly to a dose of 50-52 Gy in 20 fractions during 4 weeks (39 patients). The median follow-up was 4.2 years. Results: Local control was achieved with RT alone in 24 (56%) of the 43 patients: 7 of 11 with T1, 8 of 12 with T2, 4 of 8 with T3, and 5 of 12 with T4. The 5-year actuarial local relapse-free rate was 52%. Subsequent local control was achieved in 11 of the 13 patients with failed RT and attempted surgical salvage, for an ultimate local control rate of 81.4% (35 of 43). The 5-year overall and cause-specific actuarial survival rate was 50.3% and 66.9%, respectively. No patients developed Grade 3 or 4 late radiation morbidity. Conclusion: These data support the use of primary RT in the treatment of patients with primary squamous cell carcinoma of the subglottis as an appropriate treatment approach providing an option for laryngeal conservation

Electromigration of cadmium in contaminated soils driven by single and multiple primary cells

International Nuclear Information System (INIS)

Yuan Songhu; Wu Chan; Wan Jinzhong; Lu Xiaohua

2008-01-01

This study tentatively used an iron (Fe) and carbon (C) primary cell, instead of dc electric power, to drive the electromigration of cadmium in contaminated soils. The addition of acid to C compartment increased the electric potential, while the addition of acid to Fe compartment had a slight influence on the potential. It was feasible using the primary cell to drive the electromigration of cadmium in kaolin. The electromigration efficiencies were highly related to the soil pH. Lower pH led to greater migration efficiency. The mechanisms involved the desorption of cadmium from soils to pore solution and the electromigration of cadmium in the pore solution. The desorption was critical to the electromigration process. The series of primary cells could expand the treatment area, but the electromigration efficiencies of cadmium in each cell were less than that achieved by single primary cell. Since the potential gradient produced by the primary cell was rather low, the electromigration rate of pollutants was very low and remediation duration was long. The application would be acceptable in some specific sites, such as acidic soils or artificially controlled acid conditions so that heavy metals have been desorbed from soils

Primary signet cell adenocarcinoma of bladder

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Prateek Kinra

2017-01-01

Full Text Available Primary signet cell cancer of the urinary bladder is a relatively rare entity. Since there is no mucinous epithelium in the bladder, It is proposed that the tumor arises from metaplastic urothelium. Two thirds of the tumours are mucin secreting, in most of which the site of the deposition is either extracellular or intracellular displacing the nucleus to a peripheral crescent, giving the cells a signet ring appearance. The tumours are most often infiltrative and diffusely involving the majority of the bladder akin to its name sake in stomach. It is essential to distinguish this carcinoma from gastrointestinal metastases as different therapeutic strategies are often necessary.

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

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Lojk J

2015-02-01

Full Text Available Jasna Lojk,1 Vladimir B Bregar,1 Maruša Rajh,1 Katarina Miš,2 Mateja Erdani Kreft,3 Sergej Pirkmajer,2 Peter Veranič,3 Mojca Pavlin1 1Group for Nano and Biotechnological Applications, Faculty of Electrical Engineering, 2Institute of Pathophysiology, Faculty of Medicine, 3Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Abstract: Magnetic nanoparticles (NPs are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA in three cell types: Chinese Hamster Ovary (CHO, mouse melanoma (B16 cell line, and primary human myoblasts (MYO. We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

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Gebhard Daniel

2010-06-01

Full Text Available Abstract Background The Host Cell Reactivation Assay (HCRA is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances. A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing. Results In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis. The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts. Conclusions The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity

Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

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Jan Pavel Kucera

2015-09-01

Full Text Available Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs. However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands.CV was significantly lower in strands composed purely of SCMs (5.5±1.5 cm/s, n=11 as compared to PCMs (34.9±2.9 cm/s, n=21 at similar refractoriness (100% SCMs: 122±25 ms, n=9; 100% PCMs: 139±67 ms, n=14. In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV.These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

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

Science.gov (United States)

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

Resveratrol differentially regulates NAMPT and SIRT1 in Hepatocarcinoma cells and primary human hepatocytes.

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Susanne Schuster

Full Text Available 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.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

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Gina D Kusuma

Full Text Available Mesenchymal stem/stromal cells (MSCs exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25 human telomerase reverse transcriptase (hTERT transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs and decidua basalis (DMSCs, respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells

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Lee, Jonghyeob; Snyder, Emily R.; Liu, Yinghua; Gu, Xueying; Wang, Jing; Flowers, Brittany M.; Kim, Yoo Jung; Park, Sangbin; Szot, Gregory L.; Hruban, Ralph H.; Longacre, Teri A.; Kim, Seung K.

2017-01-01

Development of systems that reconstitute hallmark features of human pancreatic intraepithelial neoplasia (PanINs), the precursor to pancreatic ductal adenocarcinoma, could generate new strategies for early diagnosis and intervention. However, human cell-based PanIN models with defined mutations are unavailable. Here, we report that genetic modification of primary human pancreatic cells leads to development of lesions resembling native human PanINs. Primary human pancreas duct cells harbouring...

Membrane-type-3 matrix metalloproteinase (MT3-MMP functions as a matrix composition-dependent effector of melanoma cell invasion.

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Olga Tatti

Full Text Available In primary human melanoma, the membrane-type matrix metalloproteinase, MT3-MMP, is overexpressed in the most aggressive nodular-type tumors. Unlike MT1-MMP and MT2-MMP, which promote cell invasion through basement membranes and collagen type I-rich tissues, the function of MT3-MMP in tumor progression remains unclear. Here, we demonstrate that MT3-MMP inhibits MT1-MMP-driven melanoma cell invasion in three-dimensional collagen, while yielding an altered, yet MT1-MMP-dependent, form of expansive growth behavior that phenocopies the formation of nodular cell colonies. In melanoma cell lines originating from advanced primary or metastatic lesions, endogenous MT3-MMP expression was associated with limited collagen-invasive potential. In the cell lines with highest MT3-MMP expression relative to MT1-MMP, collagen-invasive activity was increased following stable MT3-MMP gene silencing. Consistently, MT3-MMP overexpression in cells derived from less advanced superficially spreading melanoma lesions, or in the MT3-MMP knockdown cells, reduced MT1-MMP-dependent collagen invasion. Rather than altering MT1-MMP transcription, MT3-MMP interacted with MT1-MMP in membrane complexes and reduced its cell surface expression. By contrast, as a potent fibrinolytic enzyme, MT3-MMP induced efficient invasion of the cells in fibrin, a provisional matrix component frequently found at tumor-host tissue interfaces and perivascular spaces of melanoma. Since MT3-MMP was significantly upregulated in biopsies of human melanoma metastases, these results identify MT3-MMP as a matrix-dependent modifier of the invasive tumor cell functions during melanoma progression.

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

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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

Primary orbital precursor T-cell lymphoblastic lymphoma

DEFF Research Database (Denmark)

Stenman, Lisa; Persson, Marta; Enlund, Fredrik

2016-01-01

Primary T-cell lymphoblastic lymphoma (T-LBL) in the eye region is very rare. The present study described a unique case of T-LBL involving the extraocular muscles. A 22-year-old male patient presented with a 3-week history of headache, reduced visual acuity and edema of the left eye. Clinical...... knowledge, this is the first report of a case of T-LBL involving the extraocular muscles. Although primary T-LBL in the eye region is very rare, our findings demonstrate that lymphoma should be considered in the differential diagnosis of patients with similar symptoms....

Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

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Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

2016-05-10

Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

Development of Eimeria nieschulzi (Coccidia, Apicomplexa Gamonts and Oocysts in Primary Fetal Rat Cells

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Hong Chen

2013-01-01

Full Text Available The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

Activity deprivation induces neuronal cell death: mediation by tissue-type plasminogen activator.

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Eldi Schonfeld-Dado

Full Text Available Spontaneous activity is an essential attribute of neuronal networks and plays a critical role in their development and maintenance. Upon blockade of activity with tetrodotoxin (TTX, neurons degenerate slowly and die in a manner resembling neurodegenerative diseases-induced neuronal cell death. The molecular cascade leading to this type of slow cell death is not entirely clear. Primary post-natal cortical neurons were exposed to TTX for up to two weeks, followed by molecular, biochemical and immunefluorescence analysis. The expression of the neuronal marker, neuron specific enolase (NSE, was down-regulated, as expected, but surprisingly, there was a concomitant and striking elevation in expression of tissue-type plasminogen activator (tPA. Immunofluorescence analysis indicated that tPA was highly elevated inside affected neurons. Transfection of an endogenous tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1, protected the TTX-exposed neurons from dying. These results indicate that tPA is a pivotal player in slowly progressing activity deprivation-induced neurodegeneration.

Type II NKT cells: a distinct CD1d-restricted immune regulatory NKT cell subset.

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Dasgupta, Suryasarathi; Kumar, Vipin

2016-08-01

Type II natural killer T cells (NKT) are a subset of the innate-like CD1d-restricted lymphocytes that are reactive to lipid antigens. Unlike the type I NKT cells, which express a semi-invariant TCR, type II NKT cells express a broader TCR repertoire. Additionally, other features, such as their predominance over type I cells in humans versus mice, the nature of their ligands, CD1d/lipid/TCR binding, and modulation of immune responses, distinguish type II NKT cells from type I NKT cells. Interestingly, it is the self-lipid-reactivity of type II NKT cells that has helped define their physiological role in health and in disease. The discovery of sulfatide as one of the major antigens for CD1d-restricted type II NKT cells in mice has been instrumental in the characterization of these cells, including the TCR repertoire, the crystal structure of the CD1d/lipid/TCR complex, and their function. Subsequently, several other glycolipids and phospholipids from both endogenous and microbial sources have been shown to activate type II NKT cells. The activation of a specific subset of type II NKT cells following administration with sulfatide or lysophosphatidylcholine (LPC) leads to engagement of a dominant immunoregulatory pathway associated with the inactivation of type I NKT cells, conventional dendritic cells, and inhibition of the proinflammatory Th1/Th17 cells. Thus, type II NKT cells have been shown to be immunosuppressive in autoimmune diseases, inflammatory liver diseases, and in cancer. Knowing their relatively higher prevalence in human than type I NKT cells, understanding their biology is imperative for health and disease.

A-type and B-type lamins initiate layer assembly at distinct areas of the nuclear envelope in living cells

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Furukawa, Kazuhiro, E-mail: furukawa@chem.sc.niigata-u.ac.jp [Department of Chemistry, Faculty of Science, Niigata University, Niigata 950-2181 (Japan); Ishida, Kazuya; Tsunoyama, Taka-aki; Toda, Suguru; Osoda, Shinichi; Horigome, Tsuneyoshi [Department of Chemistry, Faculty of Science, Niigata University, Niigata 950-2181 (Japan); Fisher, Paul A. [Department of Pharmacological Sciences, School of Medicine, University Medical Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651 (United States); Sugiyama, Shin [Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602 (Japan)

2009-04-15

To investigate nuclear lamina re-assembly in vivo, Drosophila A-type and B-type lamins were artificially expressed in Drosophila lamin Dm{sub 0}null mutant brain cells. Both exogenous lamin C (A-type) and Dm{sub 0} (B-type) formed sub-layers at the nuclear periphery, and efficiently reverted the abnormal clustering of the NPC. Lamin C initially appeared where NPCs were clustered, and subsequently extended along the nuclear periphery accompanied by the recovery of the regular distribution of NPCs. In contrast, lamin Dm{sub 0} did not show association with the clustered NPCs during lamina formation and NPC spacing recovered only after completion of a closed lamin Dm{sub 0} layer. Further, when lamin Dm{sub 0} and C were both expressed, they did not co-polymerize, initiating layer formation in separate regions. Thus, A and B-type lamins reveal differing properties during lamina assembly, with A-type having the primary role in organizing NPC distribution. This previously unknown complexity in the assembly of the nuclear lamina could be the basis for intricate nuclear envelope functions.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

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Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

2009-07-01

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

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Malgorzata Bienkowska-Haba

2009-07-01

Full Text Available Following attachment to primary receptor heparan sulfate proteoglycans (HSPG, human papillomavirus type 16 (HPV16 particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Primary Hyperparathyroidism in Patients with Multiple Endocrine Neoplasia Type 1

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Grzegorz Piecha

2010-01-01

Full Text Available Primary hyperparathyroidism may occur as a part of an inherited syndrome in a combination with pancreatic endocrine tumours and/or pituitary adenoma, which is classified as Multiple Endocrine Neoplasia type 1 (MEN-1. This syndrome is caused by a germline mutation in MEN-1 gene encoding a tumour-suppressor protein, menin. Primary hyperparathyroidism is the most frequent clinical presentation of MEN-1, which usually appears in the second decade of life as an asymptomatic hypercalcemia and progresses through the next decades. The most frequent clinical presentation of MEN-1-associated primary hyperparathyroidism is bone demineralisation and recurrent kidney stones rarely followed by chronic kidney disease. The aim of this paper is to present the pathomechanism, screening procedures, diagnosis, and management of primary hyperparathyroidism in the MEN-1 syndrome. It also summarises the recent advances in the pharmacological therapy with a new group of drugs—calcimimetics.

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

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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.

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

International Nuclear Information System (INIS)

Cha, Zhanshan; Qian, Guangfang; Zang, Yan; Gu, Haihui; Huang, Yanyan; Zhu, Lishuang; Li, Jinqi; Liu, Yang; Tu, Xiaohua; Song, Haihan; Qian, Baohua

2017-01-01

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

Mechanisms of growth inhibition of primary prostate epithelial cells following gamma irradiation or photodynamic therapy include senescence, necrosis, and autophagy, but not apoptosis

International Nuclear Information System (INIS)

Frame, Fiona M.; Savoie, Huguette; Bryden, Francesca; Giuntini, Francesca; Mann, Vincent M.; Simms, Matthew S.; Boyle, Ross W.; Maitland, Norman J.

2015-01-01

In comparison to more differentiated cells, prostate cancer stem-like cells are radioresistant, which could explain radio-recurrent prostate cancer. Improvement of radiotherapeutic efficacy may therefore require combination therapy. We have investigated the consequences of treating primary prostate epithelial cells with gamma irradiation and photodynamic therapy (PDT), both of which act through production of reactive oxygen species (ROS). Primary prostate epithelial cells were cultured from patient samples of benign prostatic hyperplasia and prostate cancer prior to treatment with PDT or gamma irradiation. Cell viability was measured using MTT and alamar blue assay, and cell recovery by colony-forming assays. Immunofluorescence of gamma-H2AX foci was used to quantify DNA damage, and autophagy and apoptosis were assessed using Western blots. Necrosis and senescence were measured by propidium iodide staining and beta-galactosidase staining, respectively. Both PDT and gamma irradiation reduced the colony-forming ability of primary prostate epithelial cells. PDT reduced the viability of all types of cells in the cultures, including stem-like cells and more differentiated cells. PDT induced necrosis and autophagy, whereas gamma irradiation induced senescence, but neither treatment induced apoptosis. PDT and gamma irradiation therefore inhibit cell growth by different mechanisms. We suggest these treatments would be suitable for use in combination as sequential treatments against prostate cancer

Targeting natural killer cell reactivity by employing antibody to NKp46: implications for type 1 diabetes.

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Rami Yossef

Full Text Available Natural killer (NK cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D. Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down-regulate the surface expression of NKp46 on primary murine NK cells following antibody injection in vivo. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose streptozotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15-treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.

Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

DEFF Research Database (Denmark)

Hellsten, Ylva; Frandsen, Ulrik

1997-01-01

1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P Skeletal muscle cells were...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P contracted, but not in the moderately contracted muscle cells...

Unleashing the potential of the root hair cell as a single plant cell type model in root systems biology

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Zhenzhen eQiao

2013-11-01

Full Text Available Plant root is an organ composed of multiple cell types with different functions. This multicellular complexity limits our understanding of root biology because –omics studies performed at the level of the entire root reflect the average responses of all cells composing the organ. To overcome this difficulty and allow a more comprehensive understanding of root cell biology, an approach is needed that would focus on one single cell type in the plant root. Because of its biological functions (i.e. uptake of water and various nutrients; primary site of infection by nitrogen-fixing bacteria in legumes, the root hair cell is an attractive single cell model to study root cell response to various stresses and treatments. To fully study their biology, we have recently optimized procedures in obtaining root hair cell samples. We culture the plants using an ultrasound aeroponic system maximizing root hair cell density on the entire root systems and allowing the homogeneous treatment of the root system. We then isolate the root hair cells in liquid nitrogen. Isolated root hair yields could be up to 800 to 1000 mg of plant cells from 60 root systems. Using soybean as a model, the purity of the root hair was assessed by comparing the expression level of genes previously identified as soybean root hair specific between preparations of isolated root hair cells and stripped roots, roots devoid in root hairs. Enlarging our tests to include other plant species, our results support the isolation of large quantities of highly purified root hair cells which is compatible with a systems biology approach.

Cytotoxicity and accumulation of ergot alkaloids in human primary cells.

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Mulac, Dennis; Humpf, Hans-Ulrich

2011-04-11

Ergot alkaloids are secondary metabolites produced by fungi of the species Claviceps. Toxic effects after consumption of contaminated grains are described since mediaeval times. Of the more than 40 known ergot alkaloids six are found predominantly. These are ergotamine, ergocornine, ergocryptine, ergocristine, ergosine and ergometrine, along with their corresponding isomeric forms (-inine-forms). Toxic effects are known to be induced by an interaction of the ergot alkaloids as neurotransmitters, like dopamine or serotonin. Nevertheless data concerning cytotoxic effects are missing and therefore a screening of the six main ergot alkaloids was performed in human primary cells in order to evaluate the toxic potential. As it is well known that ergot alkaloids isomerize easily the stability was tested in the cell medium. Based on these results factors were calculated to correct the used concentration values to the biologically active lysergic (-ine) form. These factors range from 1.4 for the most stable compound ergometrine to 5.0 for the most unstable ergot alkaloid ergocristine. With these factors, reflecting the instability, several controverse literature data concerning the toxicity could be explained. To evaluate the cytotoxic effects of ergot alkaloids, human cells in primary culture were used. These cells remain unchanged in contrast to cell lines and the data allow a better comparison to the in vivo situation than using immortalized cell lines. To characterize the effects on primary cells, renal proximal tubule epithelial cells (RPTEC) and normal human astrocytes (NHA) were used. The parameters necrosis (LDH-release) and apoptosis (caspase-3-activation, DNA condensation and fragmentation) were distinguished. The results show that depending on the individual structure of the peptide ergot alkaloids the toxic properties change. While ergometrine as a lysergic acid amide did not show any effect, the peptide ergot alkaloids revealed a different toxic potential. Of

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio)

International Nuclear Information System (INIS)

Eide, Marta; Rusten, Marte; Male, Rune; Jensen, Knut Helge Midtbø; Goksøyr, Anders

2014-01-01

Highlights: •The ZFL cell line and primary hepatocytes were characterized. •Basic and induced expression of nuclear receptors and target genes were found. •The ZFL cell line expresses very low basic levels of most genes. •The ZFL cells have low induction of gene expression following exposures. •Primary hepatocytes show large sex-dependent differences in gene expression. -- Abstract: 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

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio)

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Eide, Marta, E-mail: marta.eide@bio.uib.no [Department of Biology, University of Bergen, Bergen (Norway); Rusten, Marte; Male, Rune [Department of Molecular Biology, University of Bergen, Bergen (Norway); Jensen, Knut Helge Midtbø; Goksøyr, Anders [Department of Biology, University of Bergen, Bergen (Norway)

2014-02-15

Highlights: •The ZFL cell line and primary hepatocytes were characterized. •Basic and induced expression of nuclear receptors and target genes were found. •The ZFL cell line expresses very low basic levels of most genes. •The ZFL cells have low induction of gene expression following exposures. •Primary hepatocytes show large sex-dependent differences in gene expression. -- Abstract: 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

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer

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Chiara Francavilla

2017-03-01

Full Text Available Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer.

Functional cell types in taste buds have distinct longevities.

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Isabel Perea-Martinez

Full Text Available Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.

Functional cell types in taste buds have distinct longevities.

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Perea-Martinez, Isabel; Nagai, Takatoshi; Chaudhari, Nirupa

2013-01-01

Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU) to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor) taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic) taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.

Primary Cutaneous Carcinosarcoma of the Basal Cell Subtype Should Be Treated as a High-Risk Basal Cell Carcinoma.

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Bourgeault, Emilie; Alain, Jimmy; Gagné, Eric

2015-01-01

Cutaneous carcinosarcoma is a rare primary tumor of the skin, characterized by biphasic epithelial and mesenchymal differentiation. Due to the limited number of cases reported, there is no consensus regarding treatment and prognosis. Some authors suggest that cutaneous carcinosarcomas should be viewed as aggressive tumors, with ancillary imaging used to evaluate potential metastatic disease. Other reports demonstrate an indolent disease course, especially with epidermal-type cutaneous carcinosarcomas. We report a case of cutaneous carcinosarcoma, which we treated with electrodessication and curettage following a shave biopsy. The tumor had an epithelial component resembling a basal cell carcinoma and a fibrosarcomatous stroma. At 1-year follow-up, our patient did not show evidence of recurrence or metastasis. Our case suggests that a cutaneous carcinosarcoma with an epithelial component composed of basal cell carcinoma can be regarded as a high-risk nonmelanoma skin cancer. © The Author(s) 2015.

Primary Diffuse Large Cell Lymphoma of the Bladder: Case Report and Literature Review

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Mansour Ansari

2017-01-01

Full Text Available Most bladder tumors are epithelial in origin. Nonepithelial cancers are rarely located in the bladder. Sarcomas are the most common malignancies among nonepithelial cancers. Primary bladder lymphoma is rare and mostly low grade. Here, we have reported a case of diffuse large cell lymphoma of the bladder. The patient, a 64-year-old man, had urinary frequency for 18 months. Abdominal sonography indicated a thick bladder wall and transurethral biopsy showed diffuse large cell lymphoma. Immunohistochemistry (IHC results showed that the tumor was positive for CD20, CD45, and Pax-5 and negative for BCL-2, cytokeratin, and S100. He had a normal bone marrow biopsy, abdominal, pelvic and chest CT scans. He had no B symptoms. The patient received 6 cycles of R-CHOP followed by radiotherapy (36 Gy to the pelvis. Six months after treatment, the patient is well and has returned to work. We have searched PubMed for primary diffuse large cell lymphoma. Primary diffuse large cell lymphoma of the bladder is best treated according to treatment for diffuse large cell lymphoma of other sites, which includes chemotherapy and radiotherapy. As seen in our review, primary diffuse large cell lymphoma of the bladder has a similar clinical course to diffuse large cell lymphoma of other sites.

A Case of Successful Remission of Extensive Primary Gastric Diffuse Large B Cell Lymphoma: Radiologic, Endoscopic and Pathologic Evidence

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Mike M. Bismar

2014-04-01

Full Text Available Though rare amongst stomach neoplasms, primary gastric diffuse large B cell lymphoma is one of the commonest extranodal non-Hodgkin lymphomas. If left untreated, it can have a devastating progression and life-threatening consequences. We present the case of a successfully treated large antral ulcer confirmed to be large B cell lymphoma as evidenced by radiologic, endoscopic and histopathologic findings. A brief discussion about the types of gastric lymphoma, their Helicobacter pylori relation and therapeutic modalities follows.

High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

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Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

2014-08-01

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Primary central nervous system B-cell lymphoma in a young dog

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Kim, Na-Hyun; Ciesielski, Thomas; Kim, Jung H.; Yhee, Ji-Young; Im, Keum-Soon; Nam, Hae-Mi; Kim, Il-Hwan; Kim, Jong-Hyuk; Sur, Jung-Hyang

2012-01-01

This report describes a primary central nervous system B-cell lymphoma in a 3-year-old intact female Maltese dog. Canine primary central nervous system lymphomas constitute about 4% of all intracranial primary neoplasms, but comprehensive histopathologic classifications have rarely been carried out. This is the first report of this disease in a young adult dog. PMID:23115372

Primary endometrial squamous cell carcinoma with extensive squamous metaplasia and dysplasia

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Bagga Permeet

2008-04-01

Full Text Available Primary squamous cell carcinoma of endometrium is a rare entity. Only 64 cases have been documented in the literature. We report a case of 60-year-old postmenopausal woman who presented with abdominal distention and blood-stained vaginal discharge for 6-7 months. Clinically, chronic pyometra was considered. Total abdominal hysterectomy was performed and histopathologically, it was diagnosed as a case of primary squamous cell carcinoma of endometrium with extensive squamous metaplasia and dysplasia.

Histone deacetylase inhibitors epigenetically promote reparative events in primary dental pulp cells

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Duncan, Henry F., E-mail: Hal.Duncan@dental.tcd.ie [Division of Restorative Dentistry and Periodontology, Dublin Dental University Hospital, Trinity College Dublin, Lincoln Place, Dublin 2 (Ireland); Smith, Anthony J. [Oral Biology, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham (United Kingdom); Fleming, Garry J.P. [Material Science Unit, Division of Oral Biosciences, Dublin Dental University Hospital, Trinity College Dublin, Dublin (Ireland); Cooper, Paul R. [Oral Biology, School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham (United Kingdom)

2013-06-10

Application of histone deacetylase inhibitors (HDACi) to cells epigenetically alters their chromatin structure and induces transcriptional and cellular reparative events. This study investigated the application of two HDACi, valproic acid (VPA) and trichostatin A (TSA) on the induction of repair-associated responses in primary dental pulp cell (DPC) cultures. Flow cytometry demonstrated that TSA (100 nM, 400 nM) significantly increased cell viability. Neither HDACi was cytotoxic, although cell growth analysis revealed significant anti-proliferative effects at higher concentrations for VPA (>0.5 mM) and TSA (>50 nM). While high-content-analysis demonstrated that HDACi did not significantly induce caspase-3 or p21 activity, p53-expression was increased by VPA (3 mM, 5 mM) at 48 h. HDACi-exposure induced mineralization per cell dose-dependently to a plateau level (VPA-0.125 mM and TSA-25 nM) with accompanying increases in mineralization/dentinogenic-associated gene expression at 5 days (DMP-1, BMP-2/-4, Nestin) and 10 days (DSPP, BMP-2/-4). Both HDACis, at a range of concentrations, significantly stimulated osteopontin and BMP-2 protein expression at 10 and 14 days further supporting the ability of HDACi to promote differentiation. HDACi exert different effects on primary compared with transformed DPCs and promote mineralization and differentiation events without cytotoxic effects. These novel data now highlight the potential in restorative dentistry for applying low concentrations of HDACi in vital pulp treatment. -- Highlights: • Valproic acid and trichostatin A promoted mineralization in primary pulp cells. • Cell viability, apoptosis, caspase-3, p21 unaltered; p53 increased by valproic acid. • Trichostatin A increased cell viability at 24 h at selected concentrations. • Altered cell toxicity and differentiation between primary and transformed cells. • HDACi-induced the differentiation marker proteins osteopontin and BMP-2.

Histone deacetylase inhibitors epigenetically promote reparative events in primary dental pulp cells

International Nuclear Information System (INIS)

Duncan, Henry F.; Smith, Anthony J.; Fleming, Garry J.P.; Cooper, Paul R.

2013-01-01

Application of histone deacetylase inhibitors (HDACi) to cells epigenetically alters their chromatin structure and induces transcriptional and cellular reparative events. This study investigated the application of two HDACi, valproic acid (VPA) and trichostatin A (TSA) on the induction of repair-associated responses in primary dental pulp cell (DPC) cultures. Flow cytometry demonstrated that TSA (100 nM, 400 nM) significantly increased cell viability. Neither HDACi was cytotoxic, although cell growth analysis revealed significant anti-proliferative effects at higher concentrations for VPA (>0.5 mM) and TSA (>50 nM). While high-content-analysis demonstrated that HDACi did not significantly induce caspase-3 or p21 activity, p53-expression was increased by VPA (3 mM, 5 mM) at 48 h. HDACi-exposure induced mineralization per cell dose-dependently to a plateau level (VPA-0.125 mM and TSA-25 nM) with accompanying increases in mineralization/dentinogenic-associated gene expression at 5 days (DMP-1, BMP-2/-4, Nestin) and 10 days (DSPP, BMP-2/-4). Both HDACis, at a range of concentrations, significantly stimulated osteopontin and BMP-2 protein expression at 10 and 14 days further supporting the ability of HDACi to promote differentiation. HDACi exert different effects on primary compared with transformed DPCs and promote mineralization and differentiation events without cytotoxic effects. These novel data now highlight the potential in restorative dentistry for applying low concentrations of HDACi in vital pulp treatment. -- Highlights: • Valproic acid and trichostatin A promoted mineralization in primary pulp cells. • Cell viability, apoptosis, caspase-3, p21 unaltered; p53 increased by valproic acid. • Trichostatin A increased cell viability at 24 h at selected concentrations. • Altered cell toxicity and differentiation between primary and transformed cells. • HDACi-induced the differentiation marker proteins osteopontin and BMP-2

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture.

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Valencia, L; Bidet, M; Martial, S; Sanchez, E; Melendez, E; Tauc, M; Poujeol, C; Martin, D; Namorado, M D; Reyes, J L; Poujeol, P

2001-05-01

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).

Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis.

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Dequina Nicholas

Full Text Available Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96 analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.

Primary mucinous adenocarcinoma of the bladder with signet-ring cells: case report

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Marcelo Lorenzi Marques

Full Text Available CONTEXT: Primary adenocarcinomas of the bladder are uncommon and usually occur by contiguity with or hematogenic dissemination of other adenocarcinomas such as colorectal, prostate and gynecological tract carcinomas. Mucinous and signet-ring cell histological patterns are even rarer and it is often difficult to morphologically distinguish them from metastatic colorectal adenocarcinoma. CASE REPORT: We present and discuss a rare case of primary mucinous adenocarcinoma of the bladder with signet-ring cells in a 57-year-old male patient. Other primary sites for the tumor had been excluded and, in the absence of digestive tract tumor and for confirmation that it was a primary bladder tumor, an immunohistochemistry study was performed.

Survival after bone metastasis by primary cancer type

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Svensson, Elisabeth; Christiansen, Christian F; Ulrichsen, Sinna P

2017-01-01

OBJECTIVE: In the 10 most common primary types with bone metastases, we aimed to examine survival, further stratifying on bone metastases only or with additional synchronous metastases. METHODS: We included all patients aged 18 years and older with incident hospital diagnosis of solid cancer...... between 1994 and 2010, subsequently diagnosed with BM until 2012. We followed patients from date of bone metastasis diagnosis until death, emigration or 31 December 2012, whichever came first. We computed 1-year, 3-year and 5-year survival (%) and the corresponding 95% CIs stratified on primary cancer...... prostate (34%), breast (22%) and lung (20%). One-year survival after bone metastasis diagnosis was lowest in patients with lung cancer (10%, 95% CI 9% to 11%) and highest in patients with breast cancer (51%, 50% to 53%). At 5 years of follow-up, only patients with breast cancer had over 10% survival (13...

Primary focal T-cell lymphoma of the liver: a case report and review of the literature.

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Cerban, Razvan; Gheorghe, Liana; Becheanu, Gabriel; Serban, Valentin; Gheorghe, Cristian

2012-06-01

We present the case of a previously healthy 62 year old man who developed primary non-Hodgkin lymphoma of the liver. Biopsy confirmed that it was a diffuse large anaplastic T-cell lymphoma of an extremely rare type. The diagnosis of this type of lesions is suggested by the presence of a hepatic mass without lymphadenopathy, splenomegaly or bone marrow involvement associated with normal tumor markers (carcinoembryonic antigen, alpha-fetoprotein and CA 19-9 levels). Histological examination of tissue is essential to confirm the diagnosis. Treatment options are surgical resection and/or chemotherapy but the rate of response to treatment varies widely. Some patients can achieve prolonged remission.

Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry

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Etienne Caron

2017-03-01

Full Text Available Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. Given differential protein expression across tissues and developmental stages, the architecture and dynamics of signaling interaction proteomes is, likely, highly context dependent. However, current interaction information has been almost exclusively obtained from transformed cells. In this study, we applied an advanced and robust workflow combining mouse genetics and affinity purification (AP-SWATH mass spectrometry to profile the dynamics of 53 high-confidence protein interactions in primary T cells, using the scaffold protein GRB2 as a model. The workflow also provided a sufficient level of robustness to pinpoint differential interaction dynamics between two similar, but functionally distinct, primary T cell populations. Altogether, we demonstrated that precise and reproducible quantitative measurements of protein interaction dynamics can be achieved in primary cells isolated from mammalian tissues, allowing resolution of the tissue-specific context of cell-signaling events.

Primary clear cell sarcoma of bone: a unique site of origin

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Gelczer, R.K.; Wenger, D.E.; Wold, L.E.

1999-01-01

Clear cell sarcoma is a rare soft tissue neoplasm, accounting for less than 1% of soft tissue sarcomas. We are presenting a case of a clear cell sarcoma of bone which, to our knowledge, is the only report of a primary clear cell sarcoma of bone. (orig.)

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

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Cha, Zhanshan [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Qian, Guangfang [Department of Endocrinology, Zhangqiu Municipal Hospital of Traditional Chinese Medicine, Zhangqiu, Shandong 250200 (China); Zang, Yan; Gu, Haihui; Huang, Yanyan; Zhu, Lishuang; Li, Jinqi; Liu, Yang; Tu, Xiaohua [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Song, Haihan [Emergency Center, East Hospital, Shanghai 200120 (China); Qian, Baohua, E-mail: qianbhl963@163.com [Department of Transfusion, Changhai Hospital, Second Military Medical University, Shanghai 200433 (China)

2017-01-01

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

Primary mesenchymal stem cells in human transplanted lungs are CD90/CD105 perivascularly located tissue-resident cells

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Rolandsson, Sara; Andersson Sjöland, Annika; Brune, Jan C

2014-01-01

BACKGROUND: Mesenchymal stem cells (MSC) have not only been implicated in the development of lung diseases, but they have also been proposed as a future cell-based therapy for lung diseases. However, the cellular identity of the primary MSC in human lung tissues has not yet been reported. This st......BACKGROUND: Mesenchymal stem cells (MSC) have not only been implicated in the development of lung diseases, but they have also been proposed as a future cell-based therapy for lung diseases. However, the cellular identity of the primary MSC in human lung tissues has not yet been reported...

Primary hyperoxaluria type 1: clinical manifestations in infancy and prenatal diagnosis

DEFF Research Database (Denmark)

Illum, N; Lavard, L; Danpure, C J

1992-01-01

biopsy demonstrated complete absence of alanine: glyoxylate aminotransferase catalytic activity and immunoreactive protein compatible with a diagnosis of primary hyperoxaluria type 1. He died at the age of 11 months, just before liver transplantation was made possible. Fetal liver biopsy in the mother......A 9-month-old Pakistani boy of consanguineous parents presented with uraemia preceded by pyuria from 5 weeks of age. He had no history of renal calculi or macroscopic haematuria. Renal biopsy revealed severe calcium oxalate deposition in the tubuli and fibrosis of the interstitial tissue. Liver......'s subsequent pregnancy showed normal enzymatic activity. Early detection and early replacement of the missing enzyme by liver transplantation are considered to be crucial for the survival of severely affected infants with the acute neonatal form of primary hyperoxaluria type 1. Persistent pyuria could...

Quantitative analysis of rat adipose tissue cell recovery, and non-fat cell volume, in primary cell cultures

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Floriana Rotondo

2016-11-01

Full Text Available Background White adipose tissue (WAT is a complex, diffuse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defense, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT by breaking up the matrix of collagen fibres; however, it is unclear to what extent adipocyte number in primary cultures correlates with their number in intact WAT, since recovery and viability are often unknown. Experimental Design Epididymal WAT of four young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of glucose uptake and lactate, glycerol and NEFA excretion rates up to 48 h. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes were measured. The presence of non-nucleated cells (erythrocytes was also estimated. Results Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70–75%. Cells showed even higher metabolic activity in the second than in the first day of incubation. Adipocytes were 7%, erythrocytes 66% and other stromal (nucleated cells 27% of total WAT cells. However, their overall volumes were 90%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 1.3% of WAT. Conclusions The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have also found that the “live cell mass” of adipose tissue is very small: about 13 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood (the rats were killed by exsanguination. These data translate (with

Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector

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Lauro Augusto de Oliveira

2010-10-01

Full Text Available PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80% confluency prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS. We evaluated the transduction efficiency (TE over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells. RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1% and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7%. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

A Case of Primary Gastric Small-Cell Carcinoma in an Elderly Patient

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Fa-Chang Yu

2012-03-01

Full Text Available We report a case of primary small-cell carcinoma of the stomach in a 75-year-old man. The patient was admitted to our hospital with a 1-week history of intermittent tarry stool. An upper gastrointestinal examination revealed a large stage A2 ulcer in the greater curvature of the body of the stomach, and pathological findings from biopsy specimens revealed small-cell carcinoma. The tumor cells were small-sized, composed of hyperchromatic nuclei with scant cytoplasm, and stained positive for cytokeratin, synaptophysin, and chromogranin A. The patient was diagnosed with primary small-cell carcinoma of the stomach. He declined further evaluation and received palliative management. This is a rare carcinoma of the stomach, with aggressive manifestations and a poor prognosis. The mean survival of patients with primary gastric small-cell carcinoma is reported to be 7 months. The choice of treatment for this disease is still controversial. This rare gastric tumor should be listed in the differential diagnosis of gastric carcinoma in the elderly.

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

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Szilvia Veszelka

2018-05-01

Full Text Available 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

Nanoscaffold's stiffness affects primary cortical cell network formation

NARCIS (Netherlands)

Xie, Sijia; Schurink, Bart; Wolbers, F.; Lüttge, Regina; Hassink, Gerrit Cornelis

2014-01-01

Networks of neurons cultured on-chip can provide insights into both normal and disease-state brain function. The ability to guide neuronal growth in specific, artificially designed patterns allows us to study how brain function follows form. Primary cortical cells cultured on nanograting scaffolds,

Synchronous sigmoid and caecal cancers together with a primary renal cell carcinoma.

LENUS (Irish Health Repository)

Bhargava, A

2012-06-01

Multiple primary neoplasms, a common clinical entity, can be classified as synchronous or metachronous. Renal cell carcinoma, in particular, is associated with a high rate of multiple primary neoplasms.

The MEK1/2-ERK Pathway Inhibits Type I IFN Production in Plasmacytoid Dendritic Cells

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Vaclav Janovec

2018-02-01

Full Text Available Recent studies have reported that the crosslinking of regulatory receptors (RRs, such as blood dendritic cell antigen 2 (BDCA-2 (CD303 or ILT7 (CD85g, of plasmacytoid dendritic cells (pDCs efficiently suppresses the production of type I interferons (IFN-I, α/β/ω and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9 ligands. The exact mechanism of how this B cell receptor (BCR-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer.

Science.gov (United States)

Francavilla, Chiara; Lupia, Michela; Tsafou, Kalliopi; Villa, Alessandra; Kowalczyk, Katarzyna; Rakownikow Jersie-Christensen, Rosa; Bertalot, Giovanni; Confalonieri, Stefano; Brunak, Søren; Jensen, Lars J; Cavallaro, Ugo; Olsen, Jesper V

2017-03-28

Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Establishment of primary bovine intestinal epithelial cell culture and clone method.

Science.gov (United States)

Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

2017-01-01

The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

International Nuclear Information System (INIS)

Cuthill, S.; Poellinger, L.

1988-01-01

The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [ 3 H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin

Primary signet ring cell carcinoma of the appendix mimicking acute appendicitis

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Mario Fusari

2012-10-01

Full Text Available Primary signet ring cell carcinoma of the appendix is a very rare neoplasm that usually presents with signs and symptoms of acute appendicitis and in particular with a right lower abdominal pain. Preoperative imaging detection of appendiceal adenocarcinoma has an important value because it may result in an appropriate surgical procedure. We report a rare case of primary signet ring cell carcinoma of the vermiform appendix in an 80-year-old man who was misdiagnosed on computed tomography (CT scan as acute appendicitis.

Development of Functional Microfold (M Cells from Intestinal Stem Cells in Primary Human Enteroids.

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Joshua D Rouch

Full Text Available Intestinal microfold (M cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs, and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting.Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium.Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2 in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells.Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an

Proliferation and mineralization ability of dental pulp cells derived from primary and permanent teeth

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Suttatip Kamolmatyakul

2011-04-01

Full Text Available The aims of this study were to compare the proliferation and mineralization ability of CFU-F selected dental pulp cellsderived from primary and permanent teeth. Those cells were isolated by enzyme digestion and analyzed for their colonyformingcapacity. The cell proliferation was measured by the MTT assay on day 1, day 7, and day14. Alizarin Red S stainingwas used to detect mineralized nodule formation of the cells on day 7, 14, 21, and 28. Proliferation of CFU-F selected pulpcells from primary teeth was significantly higher than that of CFU-F selected pulp cells from permanent teeth in all periods ofthe experiment. Upon cultured cells in mineralization inducing media, the mineralized nodules appeared as early as day 14 inCFU-F selected pulp cells from primary teeth and MG-63, whereas those of CFU-F selected pulp cells from permanent teethcan be found at day 21. On day 21 and day 28, the mineralized nodules of the CFU-F selected pulp cells from the primaryteeth group were more than those in the CFU-F selected pulp cells from the permanent teeth group. Mineralized noduleformation in the CFU-F selected pulp cells from the permanent teeth group appeared later and were less than those ofCFU-F selected pulp cells from primary teeth. However, mineralized nodules in CFU-F selected pulp cells from the permanentteeth group increased very fast after their appearance. Those results suggest that CFU-F selected pulp cells from primaryteeth had a higher proliferation rate and mineralization rate when compared to CFU-F selected pulp cells from permanentteeth.

Primary tonsillar mast cell tumour in a dog.

Science.gov (United States)

Shekell, C C; Thomson, M J; Miller, R I; Mackie, J T

2018-05-01

A 6-year-old speyed female Bull Arab-cross dog was found to have a small tonsillar nodule. Histological examination revealed a well-differentiated mast cell tumour (MCT). At initial staging, no evidence of concurrent cutaneous or visceral MCTs was found on a complete blood count, a single lateral thoracic radiograph, abdominal ultrasound or cytology of the spleen and regional lymph nodes. A diagnosis of primary tonsillar MCT was made. At 40 months postoperatively, the dog is alive with no evidence of gross tumour progression, in contrast to some previous reports of rapid disease progression and metastasis in dogs with primary oral MCTs. To the authors' knowledge, no previous reports of a primary MCT of the tonsil in dogs exist in the veterinary literature. © 2018 Australian Veterinary Association.

Early host cell targets of Yersinia pestis during primary pneumonic plague.

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Roger D Pechous

Full Text Available Inhalation of Yersinia pestis causes primary pneumonic plague, a highly lethal syndrome with mortality rates approaching 100%. Pneumonic plague progression is biphasic, with an initial pre-inflammatory phase facilitating bacterial growth in the absence of host inflammation, followed by a pro-inflammatory phase marked by extensive neutrophil influx, an inflammatory cytokine storm, and severe tissue destruction. Using a FRET-based probe to quantitate injection of effector proteins by the Y. pestis type III secretion system, we show that these bacteria target alveolar macrophages early during infection of mice, followed by a switch in host cell preference to neutrophils. We also demonstrate that neutrophil influx is unable to limit bacterial growth in the lung and is ultimately responsible for the severe inflammation during the lethal pro-inflammatory phase.

Primary intraosseous squamous cell carcinoma mimicking periapical disease: a case report

Energy Technology Data Exchange (ETDEWEB)

Choi, Yoon Joo; Oh, Song Hee; Kang, Ju Han; Choi, Hwa Young; Kim, Gyu Tae; Choi, Yong Suk; Hwang, Eui Hwan [Dept. of Oral and Maxillofacial Radiology, School of Dentistry, Kyung Hee University, Seoul (Korea, Republic of); Yu, Jae June [Dept. of Oral and Maxillofacial Radiology, Kangdong Sacred Heart Hospital, Hallym Medical Center, Seoul (Korea, Republic of)

2012-09-15

Primary intraosseous squamous cell carcinoma (PIOSCC) is a rare carcinoma, which arises within the jaws without connection to the oral mucosa and presumably develops from a remnant of odontogenic epithelium. We present a case of solid type PIOSCC in a 52-year-old male patient complaining of dull pain on his left lower molar. In this case, early stage PIOSCC mimicking a periapical lesion might lead to a one-year delay in treatment due to the misdiagnosis of osteomyelitis after extraction of the third molar. The clinical, radiological, and histologic features are described. In this case, there was initial radiographic evidence for PIOSCC mimicking a periapical lesion. Incautious radiographic interpretation and treatment procedures had delayed the correct diagnosis and resulted in extensive bony destruction during the patient's disease progression.

Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

International Nuclear Information System (INIS)

L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon

2007-01-01

NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application

Amiloride-sensitive channels in type I fungiform taste cells in mouse

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Clapp Tod R

2008-01-01

Full Text Available Abstract Background Taste buds are the sensory organs of taste perception. Three types of taste cells have been described. Type I cells have voltage-gated outward currents, but lack voltage-gated inward currents. These cells have been presumed to play only a support role in the taste bud. Type II cells have voltage-gated Na+ and K+ current, and the receptors and transduction machinery for bitter, sweet, and umami taste stimuli. Type III cells have voltage-gated Na+, K+, and Ca2+ currents, and make prominent synapses with afferent nerve fibers. Na+ salt transduction in part involves amiloride-sensitive epithelial sodium channels (ENaCs. In rodents, these channels are located in taste cells of fungiform papillae on the anterior part of the tongue innervated by the chorda tympani nerve. However, the taste cell type that expresses ENaCs is not known. This study used whole cell recordings of single fungiform taste cells of transgenic mice expressing GFP in Type II taste cells to identify the taste cells responding to amiloride. We also used immunocytochemistry to further define and compare cell types in fungiform and circumvallate taste buds of these mice. Results Taste cell types were identified by their response to depolarizing voltage steps and their presence or absence of GFP fluorescence. TRPM5-GFP taste cells expressed large voltage-gated Na+ and K+ currents, but lacked voltage-gated Ca2+ currents, as expected from previous studies. Approximately half of the unlabeled cells had similar membrane properties, suggesting they comprise a separate population of Type II cells. The other half expressed voltage-gated outward currents only, typical of Type I cells. A single taste cell had voltage-gated Ca2+ current characteristic of Type III cells. Responses to amiloride occurred only in cells that lacked voltage-gated inward currents. Immunocytochemistry showed that fungiform taste buds have significantly fewer Type II cells expressing PLC signalling

Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

Science.gov (United States)

Yamamoto, H; Karasawa, J; Sagi, N; Takahashi, S; Horikomi, K; Okuyama, S; Nukada, T; Sora, I; Yamamoto, T

2001-08-03

Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

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Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: Jamboor.vishwanatha@unthsc.edu [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

2011-11-04

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high ({approx}97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

Science.gov (United States)

Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

2011-11-01

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

International Nuclear Information System (INIS)

Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K

2011-01-01

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (∼97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

In Vitro Efficient Expansion of Tumor Cells Deriving from Different Types of Human Tumor Samples

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Ilaria Turin

2014-03-01

Full Text Available Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.

Retroviral-mediated gene therapy for the differentiation of primary cells into a mineralizing osteoblastic phenotype.

Science.gov (United States)

Phillips, Jennifer E; García, Andrés J

2008-01-01

Bone tissue engineering has emerged as a promising strategy for the repair of critical-sized skeletal fractures. However, the clinical application of this approach has been limited by the availability of a robust mineralizing cell source. Non-osteogenic cells, such as skin fibroblasts, are an attractive cell-source alternative because they are easy to harvest from autologous donor skin biopsies and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer. Notably, sustained expression of Runx2 was not sufficient to promote functional osteogenesis in these cells, and co-treatment with the steroid hormone dexamethasone was required to induce deposition of biologically-equivalent matrix mineralization. On the basis of these results, we then investigated the osteogenic capacity of these genetically engineered fibroblasts when seeded on polymeric scaffolds in vitro and in vivo. These experiments demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days in vivo implantation in a subcutaneous, heterotopic site. Overall, these results offer evidence that transcription factor-based gene therapy may be a powerful strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. This concept may also be applied to control functional differentiation in a broad range of cell types and tissue engineering applications. The chapter below outlines detailed methods for the isolation and ex vivo genetic modification of primary dermal fibroblasts using retroviral-mediated delivery of the Runx2 transgene in both monolayer culture and three-dimensional scaffolds.

Primary intestinal T cell lymphomas in Indian patients - In search of enteropathic T cell lymphoma

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Shet Tanuja

2010-07-01

Full Text Available Objective: This series of six intestinal T cell lymphomas (ITCL attempts to document enteropathy-associated T cell lymphoma (EATCL in India. Materials and Methods: A total of six ITCL were selected from 170 gastrointestinal lymphomas in last 10 years. Results: The cases studied included EATCL (4, ITCL with a CD4 positive phenotype (1 and ITCL NK/T cell type (1. Of the four EATCL, two occurred in the ileum, one in right colon and one in duodenum. In three EATCL cases, there was history of celiac disease or lactose intolerance and enteropathic changes were noted in the adjacent mucosa. These tumors had CD3+/CD8+/CD56 (+/-/CD4-/ Granzyme B+ immunophenotype. One EATCL was monomorphic small cell type (type II EATCL with a CD3+/CD8-CD56+/CD4-/ Granzyme B+ phenotype. EBER- ISH (Epstein Barr virus coded RNA′s- in situ hybridization revealed positive tumor cells in ITCL NK/T cell type and in bystander cells in three EATCL. Conclusion: ITCL are rare in Indian patients but do occur and comprise a mixture of the enteropathic and non-enteropathic subtypes.

Crosstalk between type II NKT cells and T cells leads to spontaneous chronic inflammatory liver disease.

Science.gov (United States)

Weng, Xiufang; He, Ying; Visvabharathy, Lavanya; Liao, Chia-Min; Tan, Xiaosheng; Balakumar, Arjun; Wang, Chyung-Ru

2017-10-01

Natural killer T (NKT) cells are CD1d-restricted innate-like T cells that modulate innate and adaptive immune responses. Unlike the well-characterized invariant/type I NKT cells, type II NKT cells with a diverse T cell receptor repertoire are poorly understood. This study defines the pathogenic role of type II NKT cells in the etiology of chronic liver inflammation. Transgenic mice with the Lck promoter directing CD1d overexpression on T cells in Jα18 wild-type (Lck-CD1dTgJα18 + ; type I NKT cell sufficient) and Jα18-deficient (Lck-CD1dTgJα18 o , type I NKT cell deficient) mice were analyzed for liver pathology and crosstalk between type II NKT cells and conventional T cells. CD1d expression on T cells in peripheral blood samples and liver sections from autoimmune hepatitis patients and healthy individuals were also examined. Lck-CD1dTgJα18 o and Lck-CD1dTgJα18 + mice developed similar degrees of liver pathology resembling chronic autoimmune hepatitis in humans. Increased CD1d expression on T cells promoted the activation of type II NKT cells and other T cells. This resulted in T h 1-skewing and impaired T h 2 cytokine production in type II NKT cells. Dysfunction of type II NKT cells was accompanied by conventional T cell activation and pro-inflammatory cytokine production, leading to a hepatic T/B lymphocyte infiltration, elevated autoantibodies and hepatic injury in Lck-CD1dTg mice. A similar mechanism could be extended to humans as CD1d expression is upregulated on activated human T cells and increased presence of CD1d-expressing T cells was observed in autoimmune hepatitis patients. Our data reveals enhanced crosstalk between type II NKT cells and conventional T cells, leading to a T h 1-skewed inflammatory milieu, and consequently, to the development of chronic autoimmune liver disease. Lay summary: CD1d overexpression on T cells enhances crosstalk between type II NKT cells and T cells, resulting in their aberrant activation and leading to the

Uptake of gold nanoparticles in primary human endothelial cells

DEFF Research Database (Denmark)

Klingberg, Henrik; Oddershede, Lene B.; Löschner, Katrin

2015-01-01

Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy......–3 or more particles. Pre-treatment with chlorpromazine inhibited the AuNP-uptake in HUVECs, indicating that internalisation occurred mainly by clathrin-mediated endocytosis. Cell activation by exposure to tumour necrosis factor or lipopolysaccharide had a slight or no effect on the uptake of Au...

Targeting dysfunctional beta-cell signaling for the potential treatment of type 1 diabetes mellitus.

Science.gov (United States)

Fenske, Rachel J; Kimple, Michelle E

2018-03-01

Since its discovery and purification by Frederick Banting in 1921, exogenous insulin has remained almost the sole therapy for type 1 diabetes mellitus. While insulin alleviates the primary dysfunction of the disease, many other aspects of the pathophysiology of type 1 diabetes mellitus are unaffected. Research aimed towards the discovery of novel type 1 diabetes mellitus therapeutics targeting different cell signaling pathways is gaining momentum. The focus of these efforts has been almost entirely on the impact of immunomodulatory drugs, particularly those that have already received FDA-approval for other autoimmune diseases. However, these drugs can often have severe side effects, while also putting already immunocompromised individuals at an increased risk for other infections. Potential therapeutic targets in the insulin-producing beta-cell have been largely ignored by the type 1 diabetes mellitus field, save the glucagon-like peptide 1 receptor. While there is preliminary evidence to support the clinical exploration of glucagon-like peptide 1 receptor-based drugs as type 1 diabetes mellitus adjuvant therapeutics, there is a vast space for other putative therapeutic targets to be explored. The alpha subunit of the heterotrimeric G z protein (Gα z ) has been shown to promote beta-cell inflammation, dysfunction, death, and failure to replicate in the context of diabetes in a number of mouse models. Genetic loss of Gα z or inhibition of the Gα z signaling pathway through dietary interventions is protective against the development of insulitis and hyperglycemia. The multifaceted effects of Gα z in regards to beta-cell health in the context of diabetes make it an ideal therapeutic target for further study. It is our belief that a low-risk, effective therapy for type 1 diabetes mellitus will involve a multidimensional approach targeting a number of regulatory systems, not the least of which is the insulin-producing beta-cell. Impact statement The expanding

Systematic analysis of protein turnover in primary cells.

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Mathieson, Toby; Franken, Holger; Kosinski, Jan; Kurzawa, Nils; Zinn, Nico; Sweetman, Gavain; Poeckel, Daniel; Ratnu, Vikram S; Schramm, Maike; Becher, Isabelle; Steidel, Michael; Noh, Kyung-Min; Bergamini, Giovanna; Beck, Martin; Bantscheff, Marcus; Savitski, Mikhail M

2018-02-15

A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000-6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types.

Cutaneous features seen in primary liver cell (Hepatocellular ...

African Journals Online (AJOL)

Primary liver cell carcinoma (PLCC), predominantly hepatocellular carcinoma is a killer. In the southwestern region of Nigeria it occupies the second position, behind prostate cancer in males. Females account for about a third of diagnosed cases. Children are not spared. Over 80 % of PLCC cases present to the hospital at ...

Cell type-specific genetic and optogenetic tools reveal hippocampal CA2 circuits.

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Kohara, Keigo; Pignatelli, Michele; Rivest, Alexander J; Jung, Hae-Yoon; Kitamura, Takashi; Suh, Junghyup; Frank, Dominic; Kajikawa, Koichiro; Mise, Nathan; Obata, Yuichi; Wickersham, Ian R; Tonegawa, Susumu

2014-02-01

The formation and recall of episodic memory requires precise information processing by the entorhinal-hippocampal network. For several decades, the trisynaptic circuit entorhinal cortex layer II (ECII)→dentate gyrus→CA3→CA1 and the monosynaptic circuit ECIII→CA1 have been considered the primary substrates of the network responsible for learning and memory. Circuits linked to another hippocampal region, CA2, have only recently come to light. Using highly cell type-specific transgenic mouse lines, optogenetics and patch-clamp recordings, we found that dentate gyrus cells, long believed to not project to CA2, send functional monosynaptic inputs to CA2 pyramidal cells through abundant longitudinal projections. CA2 innervated CA1 to complete an alternate trisynaptic circuit, but, unlike CA3, projected preferentially to the deep, rather than to the superficial, sublayer of CA1. Furthermore, contrary to existing knowledge, ECIII did not project to CA2. Our results allow a deeper understanding of the biology of learning and memory.

Types and distribution of mucous cells of the abalone Haliotis ...

African Journals Online (AJOL)

user

2012-05-08

May 8, 2012 ... Key words: Haliotis diversicolor; mucous cells, types, distribution. .... Figure 2. The shape of the mucous cells. O (oval or circle-like); c (cup- like); s ... (J) mucous cells at base of gill filaments, small cells, type II and type III; (K) a ...

The regulated secretory pathway in CD4(+ T cells contributes to human immunodeficiency virus type-1 cell-to-cell spread at the virological synapse.

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Clare Jolly

2011-09-01

Full Text Available Direct cell-cell spread of Human Immunodeficiency Virus type-1 (HIV-1 at the virological synapse (VS is an efficient mode of dissemination between CD4(+ T cells but the mechanisms by which HIV-1 proteins are directed towards intercellular contacts is unclear. We have used confocal microscopy and electron tomography coupled with functional virology and cell biology of primary CD4(+ T cells from normal individuals and patients with Chediak-Higashi Syndrome and report that the HIV-1 VS displays a regulated secretion phenotype that shares features with polarized secretion at the T cell immunological synapse (IS. Cell-cell contact at the VS re-orientates the microtubule organizing center (MTOC and organelles within the HIV-1-infected T cell towards the engaged target T cell, concomitant with polarization of viral proteins. Directed secretion of proteins at the T cell IS requires specialized organelles termed secretory lysosomes (SL and we show that the HIV-1 envelope glycoprotein (Env localizes with CTLA-4 and FasL in SL-related compartments and at the VS. Finally, CD4(+ T cells that are disabled for regulated secretion are less able to support productive cell-to-cell HIV-1 spread. We propose that HIV-1 hijacks the regulated secretory pathway of CD4(+ T cells to enhance its dissemination.

Isolation and characterization of a primary proximal tubular epithelial cell model from human kidney by CD10/CD13 double labeling.

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Cynthia Van der Hauwaert

Full Text Available Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.

Type two innate lymphoid cells; the Janus cells in health and disease

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Maazi, Hadi; Akbari, Omid

2017-01-01

Summary Innate lymphoid cells are functionally diverse subsets of immune cells including the conventional natural killer cells, lymphoid tissue inducers, type 1, 2 and 3 with significant roles in immunity and pathogenesis of inflammatory diseases. Type 2 innate lymphoid cells (ILC2s) resemble type 2 helper (Th2) cells in cytokine production and contribute to anti-helminth immunity, maintaining mucosal tissue integrity and adipose tissue browning. ILC2s play important roles in the pathogenesis of allergic diseases and asthma. Studying the pathways of activation and regulation of ILC2s are currently a priority for giving a better understanding of pathogenesis of diseases with immunological roots. Recently, our laboratory and others have shown several pathways of regulation of ILC2s by costimulatory molecules such as ICOS, regulatory T cells and by compounds such as nicotine. In this review, we summarize the current understanding of the mechanisms of activation and regulation of ILC2s and the role of these cells in health and disease. PMID:28658553

Type two innate lymphoid cells: the Janus cells in health and disease.

Science.gov (United States)

Maazi, Hadi; Akbari, Omid

2017-07-01

Innate lymphoid cells are functionally diverse subsets of immune cells including the conventional natural killer cells, lymphoid tissue inducers, type 1, 2, and 3 with significant roles in immunity and pathogenesis of inflammatory diseases. Type 2 innate lymphoid cells (ILC2s) resemble type 2 helper (Th2) cells in cytokine production and contribute to anti-helminth immunity, maintaining mucosal tissue integrity, and adipose tissue browning. ILC2s play important roles in the pathogenesis of allergic diseases and asthma. Studying the pathways of activation and regulation of ILC2s are currently a priority for giving a better understanding of pathogenesis of diseases with immunological roots. Recently, our laboratory and others have shown several pathways of regulation of ILC2s by co-stimulatory molecules such as ICOS, regulatory T cells and by compounds such as nicotine. In this review, we summarize the current understanding of the mechanisms of activation and regulation of ILC2s and the role of these cells in health and disease. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Oscillatory fluid flow influences primary cilia and microtubule mechanics.

Science.gov (United States)

Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

2014-07-01

Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity. Copyright © 2014 Wiley Periodicals, Inc.

Radiation effects on polyethylene foam of open cell type

International Nuclear Information System (INIS)

Tang Beilin; Kanako Kaji; Iwao Yoshizawa; Choji Kohara; Motoyoshi Hatada

1991-01-01

The effects of electron beam irradiation on polyethylene foam of open cell type have been studied. Experiments for determining of gel fraction and physical-mechanical properties of irradiated polyethylene foam of open cell type as a function of dose, respectively, were carried out. The dimensional stability of irradiated specimens at elevated temperatures was measured. It was found that tensile strength did not change and gel fraction increased when the specimen was irradiated in nitrogen atmosphere with increasing dose up to 300 kGy. The result shows that dimensional stability of polyethylene foam of open cell type after being kept in an oven at 70 deg C and 110 deg C for 22 h is improved by irradiation in nitrogen atmosphere. The similar results of irradiated EVA foam of open cell type irradiated foam of open cell type were obtained

Analysis of T Cell Subsets in Adult Primary/Idiopathic Minimal Change Disease: A Pilot Study

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Francisco Salcido-Ochoa

2017-01-01

Full Text Available Aim. To characterise infiltrating T cells in kidneys and circulating lymphocyte subsets of adult patients with primary/idiopathic minimal change disease. Methods. In a cohort of 9 adult patients with primary/idiopathic minimal change recruited consecutively at disease onset, we characterized (1 infiltrating immune cells in the kidneys using immunohistochemistry and (2 circulating lymphocyte subsets using flow cytometry. As an exploratory analysis, association of the numbers and percentages of both kidney-infiltrating immune cells and the circulating lymphocyte subsets with kidney outcomes including deterioration of kidney function and proteinuria, as well as time to complete clinical remission up to 48 months of follow-up, was investigated. Results. In the recruited patients with primary/idiopathic minimal change disease, we observed (a a dominance of infiltrating T helper 17 cells and cytotoxic cells, comprising cytotoxic T cells and natural killer cells, over Foxp3+ Treg cells in the renal interstitium; (b an increase in the circulating total CD8+ T cells in peripheral blood; and (c an association of some of these parameters with kidney function and proteinuria. Conclusions. In primary/idiopathic minimal change disease, a relative numerical dominance of effector over regulatory T cells can be observed in kidney tissue and peripheral blood. However, larger confirmatory studies are necessary.

Posttransplantation primary cutaneous CD30 (Ki-1)-positive large-cell lymphoma.

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Seçkin, D; Demirhan, B; Oğuz Güleç, T; Arikan, U; Haberal, M

2001-12-01

We describe the case of a 51-year-old female renal transplant recipient with primary cutaneous CD30-positive large-cell lymphoma of T-cell origin. Cutaneous T-cell lymphomas are rarely reported in organ transplant recipients, and we believe they should be considered in the differential diagnosis of cutaneous neoplastic and infectious diseases affecting this patient group.

Identification and characterization of cells with cancer stem cell properties in human primary lung cancer cell lines.

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Ping Wang

Full Text Available Lung cancer (LC with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC, large cell carcinoma (LCC, squamous cell carcinoma (SCC and adenocarcinoma (AC. We identified a small population of cells strongly positive for CD44 (CD44(high and a main population which was either weakly positive or negative for CD44 (CD44(low/-. Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44(highCD90(+ sub-population. Moreover, these CD44(highCD90(+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44(highCD90(+ population a good candidate for the lung CSCs. Both CD44(highCD90(+ and CD44(highCD90(- cells in the PLCCL derived from SCC formed spheroids, whereas the CD44(low/- cells were lacking this potential. These results indicate that CD44(highCD90(+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44(high sub-population.

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

Science.gov (United States)

Suo, Zhenhe; Munthe, Else; Solberg, Steinar; Ma, Liwei; Wang, Mengyu; Westerdaal, Nomdo Anton Christiaan; Kvalheim, Gunnar; Gaudernack, Gustav

2013-01-01

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population. PMID:23469181

Simultaneous Expression of Cancer Stem Cell-Like Properties and Cancer-Associated Fibroblast-Like Properties in a Primary Culture of Breast Cancer Cells

Energy Technology Data Exchange (ETDEWEB)

Ishikawa, Mami; Inoue, Takahiro; Shirai, Takuma; Takamatsu, Kazuhiko; Kunihiro, Shiori; Ishii, Hirokazu [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Nishikata, Takahito, E-mail: nisikata@konan-u.ac.jp [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe 650-0047 (Japan)

2014-07-31

The importance of cancer-associated fibroblasts (CAFs) in cancer biology has been recently highlighted owing to their critical roles in cancer growth, progression, metastasis, and therapeutic resistance. We have previously established a primary culture of breast cancer cells, which showed epithelial-mesenchymal transition and cancer stem cell-like properties. In this study, we found that the primary culture also showed CAF-like properties. For example, hypoxia inducible factor 1α (HIF1A) and its downstream genes, nuclear factor-kappa B2 (NF-κB2) and BCL2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), and many enzymes involved in glycolysis, such as GAPDH, LDH, PGAM1, and PKM2, were highly overexpressed in the primary culture. Moreover, media conditioned with the primary culture cells enhanced the growth of breast cancer cells. Similar to previous CAF studies, this enhancement suggested to be occurred through fibroblast growth factor signaling. This MCKH primary culture cell, which showed simultaneous expression of tumorigenic and CAF properties, offers a unique experimental system for studying the biology of CAFs.

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

Science.gov (United States)

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 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.

A co-culture system with three different primary human cell populations reveals that biomaterials and MSC modulate macrophage-driven fibroblast recruitment.

Science.gov (United States)

Caires, Hugo R; Barros da Silva, Patrícia; Barbosa, Mário A; Almeida, Catarina R

2018-03-01

The biological response to implanted biomaterials is a complex and highly coordinated phenomenon involving many different cell types that interact within 3D microenvironments. Here, we increased the complexity of a 3D platform to include at least 3 cell types that play a role in the host response upon scaffold implantation. With this system, it was possible to address how immune responses triggered by 3D biomaterials mediate recruitment of stromal cells that promote tissue regeneration, mesenchymal stromal/stem cells (MSC), or a foreign body response, fibroblasts. Primary human macrophages yielded the highest fibroblast recruitment when interacting with chitosan scaffolds but not polylactic acid. Interestingly, when there were MSC and fibroblasts in the same environment, macrophages in chitosan scaffolds again promoted a significant increase on fibroblast recruitment, but not of MSC. However, macrophages that were firstly allowed to interact with MSC within the scaffolds were no longer able to recruit fibroblasts. This study illustrates the potential to use different scaffolds to regulate the dynamics of recruitment of proregenerative or fibrotic cell types through immunomodulation. Overall, this work strengths the idea that ex vivo predictive systems need to consider the different players involved in the biological response to biomaterials and that timing of arrival of specific cell types will affect the outcome. Copyright © 2017 John Wiley & Sons, Ltd.

The Functions of Type I and Type II Natural Killer T (NKT) Cells in Inflammatory Bowel Diseases

Science.gov (United States)

Liao, Chia-Min; Zimmer, Michael I.; Wang, Chyung-Ru

2013-01-01

CD1d-restricted natural killer T (NKT) cells are a distinct subset of T cells that rapidly produce an array of cytokines upon activation and play a critical role in regulating various immune responses. NKT cells are classified into two groups based on differences in T cell receptor (TCR) usage. Type I NKT cells have an invariant TCRα-chain and are readily detectable by α-galactosylceramide (α-GalCer)-loaded CD1d tetramers. Type II NKT cells have a more diverse TCR repertoire and cannot be directly identified. Both types of NKT cells as well as multiple CD1d-expressing cell types are present in the intestine and their interactions are likely to be modulated by pathogenic and commensal microbes, which in turn contribute to the intestinal immune responses in health and disease. Indeed, in several animal models of inflammatory bowel disease (IBD), Type I NKT cells have been shown to make both protective and pathogenic contributions to disease. In contrast, in human patients suffering from ulcerative colitis (UC), and a mouse model in which both CD1d expression and the frequency of Type II NKT cells are increased, Type II NKT cells appear to promote intestinal inflammation. In this review, we summarize present knowledge on the antigen recognition, activation and function of NKT cells with a particular focus on their role in IBD, and discuss factors that may influence the functional outcome of NKT cell responses in intestinal inflammation. PMID:23518808

Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation.

Science.gov (United States)

Nair, Shiny; Boddupalli, Chandra Sekhar; Verma, Rakesh; Liu, Jun; Yang, Ruhua; Pastores, Gregory M; Mistry, Pramod K; Dhodapkar, Madhav V

2015-02-19

Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human βGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, βGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human βGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders. © 2015 by The American Society of Hematology.

Evaluating the uptake and intracellular fate of polystyrene nanoparticles by primary and hepatocyte cell lines in vitro

International Nuclear Information System (INIS)

Johnston, Helinor J.; Semmler-Behnke, Manuela; Brown, David M.; Kreyling, Wolfgang; Tran, Lang; Stone, Vicki

2010-01-01

Nanoparticles (NPs) are being used within diverse applications such as medicines, clothing, cosmetics and food. In order to promote the safe development of such nanotechnologies it is essential to assess the potential adverse health consequences associated with human exposure. The liver is recognised as a target site for NP toxicity, due to NP accumulation within this organ subsequent to injection, inhalation or instillation. The uptake of fluorescent polystyrene carboxylated particles (20 nm or 200 nm diameter) by hepatocytes was determined using confocal microscopy; with cells imaged 'live' during particle exposure or after exposure within fixed cells. Comparisons between the uptake of polystyrene particles by primary rat hepatocytes, and human hepatocyte cell lines (C3A and HepG2) were made. Uptake of particles by hepatocytes was size, time, and serum dependent. Specifically, the uptake of 200 nm particles was limited, but 20 nm NPs were internalised by all cell types from 10 min onwards. At 10 min, 20 nm NP fluorescence co-localised with the tubulin cytoskeleton staining; after 30 min NP fluorescence compartmentalised into structures located within and/or between cells. The fate of internalised NPs was considered and they were not contained within early endosomes or lysosomes, but within mitochondria of cell lines. NPs accumulated within bile canaliculi to a limited extent, which suggests that NPs can be eliminated within bile. This is in keeping with the finding that gold NPs were eliminated in bile following intravenous injection into rats. The findings were, in the main, comparable between primary rat hepatocytes and the different human hepatocyte cell lines.

Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties

International Nuclear Information System (INIS)

Busch, Wibke; Bastian, Susanne; Trahorsch, Ulrike; Iwe, Maria; Kühnel, Dana; Meißner, Tobias; Springer, Armin; Gelinsky, Michael; Richter, Volkmar; Ikonomidou, Chrysanthy; Potthoff, Annegret; Lehmann, Irina; Schirmer, Kristin

2011-01-01

Cellular internalisation of industrial engineered nanoparticles is undesired and a reason for concern. Here we investigated and compared the ability of seven different mammalian cell cultures in vitro to incorporate six kinds of engineered nanoparticles, focussing on the role of cell type and particle properties in particle uptake. Uptake was examined using light and electron microscopy coupled with energy dispersive X-ray spectroscopy (EDX) for particle element identification. Flow cytometry was applied for semi-quantitative analyses of particle uptake and for exploring the influence on uptake by the phagocytosis inhibitor Cytochalasin D (CytoD). All particles studied were found to enter each kind of cultured cells. Yet, particles were never found within cell nuclei. The presence of the respective particles within the cells was confirmed by EDX. Live-cell imaging revealed the time-dependent process of internalisation of technical nanoparticles, which was exemplified by tungsten carbide particle uptake into the human skin cells, HaCaT. Particles were found to co-localise with lysosomal structures within the cells. The incorporated nanoparticles changed the cellular granularity, as measured by flow cytometry, already after 3 h of exposure in a particle specific manner. By correlating particle properties with flow cytometry data, only the primary particle size was found to be a weakly influential property for particle uptake. CytoD, an inhibitor of actin filaments and therewith of phagocytosis, significantly inhibited the internalisation of particle uptake in only two of the seven investigated cell cultures. Our study, therefore, supports the notion that nanoparticles can enter mammalian cells quickly and easily, irrespective of the phagocytic ability of the cells.

Feedback control of primary circulation pump of PIUS-Type reactor

International Nuclear Information System (INIS)

Fujii, Mikiya; Anoda, Yoshinari; Murata, Hideo; Yonomoto, Taisuke; Kukita, Yutaka; Tasaka, Kanji.

1991-05-01

In operating the PIUS-Type reactor, it is required to keep stationary density interfaces between the primary loop hot water and the poison tank cold, borated water by maintaining pressure balance between the primary-loop and the poison-tank. The authors have developed a primary circulation pump speed control system and tested it in small-scale experiments. This control system regulates the pump speed based on measurements of the density lock differential pressure which is proportional to the elevation of the interface in the density lock. This pump speed control facilitated the normal plant operation which included core power changes. However, the elevation of the density interface indicated oscillatory behavior when the pump speed was regulated as a linear function of the density lock differential pressure. The mechanism responsible for such oscillatory behavior was found to be manometric oscillations that could be eliminated by adding a damping term to compensate for the mechanical delay of the primary pump speed. The passive shutdown function of the reactor was retained by setting an upper limit to the pump speed. This was confirmed in a loss-of-feedwater abnormal transient test. (author)

A Case of Mature Natural Killer-Cell Neoplasm Manifesting Multiple Choroidal Lesions: Primary Intraocular Natural Killer-Cell Lymphoma

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Yoshiaki Tagawa

2015-11-01

Full Text Available Purpose: Natural killer (NK cell neoplasm is a rare disease that follows an acute course and has a poor prognosis. It usually emerges from the nose and appears in the ocular tissue as a metastasis. Herein, we describe a case of NK-cell neoplasm in which the eye was considered to be the primary organ. Case: A 50-year-old female displayed bilateral anterior chamber cells, vitreous opacity, bullous retinal detachment, and multiple white choroidal mass lesions. Although malignant lymphoma or metastatic tumor was suspected, various systemic examinations failed to detect any positive results. A vitrectomy was performed OS; however, histocytological analyses from the vitreous sample showed no definite evidence of malignancy, and IL-10 concentration was low. Enlarged choroidal masses were fused together. Three weeks after the first visit, the patient suddenly developed an attack of fever, night sweat, and hepatic dysfunction, and 5 days later, she passed away due to multiple organ failure. Immunohistochemisty and in situ hybridization revealed the presence of atypical cells positive for CD3, CD56, and Epstein-Barr virus-encoded RNAs, resulting in the diagnosis of NK-cell neoplasm. With the characteristic clinical course, we concluded that this neoplasm was a primary intraocular NK-cell lymphoma. Conclusions: This is the first report to describe primary intraocular NK-cell neoplasm. When we encounter atypical choroidal lesions, we should consider the possibility of NK-cell lymphoma, even though it is a rare disease.

Sequential immunization with V3 peptides from primary human immunodeficiency virus type 1 produces cross-neutralizing antibodies against primary isolates with a matching narrow-neutralization sequence motif.

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Eda, Yasuyuki; Takizawa, Mari; Murakami, Toshio; Maeda, Hiroaki; Kimachi, Kazuhiko; Yonemura, Hiroshi; Koyanagi, Satoshi; Shiosaki, Kouichi; Higuchi, Hirofumi; Makizumi, Keiichi; Nakashima, Toshihiro; Osatomi, Kiyoshi; Tokiyoshi, Sachio; Matsushita, Shuzo; Yamamoto, Naoki; Honda, Mitsuo

2006-06-01

An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the "PGR" motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.

Community governance in primary health care: towards an international Ideal Type.

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Meads, Geoffrey; Russell, Grant; Lees, Amanda

2017-10-01

Against a global background of increased resource management responsibilities for primary health care agencies, general medical practices, in particular, are increasingly being required to demonstrate the legitimacy of their decision making in market oriented environments. In this context a scoping review explores the potential utility for health managers in primary health care of community governance as a policy concept. The review of recent research suggests that applied learning from international health systems with enhanced approaches to public and patient involvement may contribute to meeting this requirement. Such approaches often characterise local health systems in Latin America and North West Europe where innovative models are beginning to respond effectively to the growing demands on general practice. The study design draws on documentary and secondary data analyses to identify common components of community governance from the countries in these regions, supplemented by other relevant international studies and sources where appropriate. Within a comprehensive framework of collaborative governance the components are aggregated in an Ideal Type format to provide a point of reference for possible adaptation and transferable learning across market oriented health systems. Each component is illustrated with international exemplars from recent organisational practices in primary health care. The application of community governance is considered for the particular contexts of GP led Clinical Commissioning Groups in England and Primary Health Networks in Australia. Some components of the Ideal Type possess potentially powerful negative as well as positive motivational effects, with PPI at practice levels sometimes hindering the development of effective local governance. This highlights the importance of careful and competent management of the growing resources attributed to primary health care agencies, which possess an increasingly diverse range of non

Human B cells fail to secrete type I interferons upon cytoplasmic DNA exposure.

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Gram, Anna M; Sun, Chenglong; Landman, Sanne L; Oosenbrug, Timo; Koppejan, Hester J; Kwakkenbos, Mark J; Hoeben, Rob C; Paludan, Søren R; Ressing, Maaike E

2017-11-01

Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes

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Higashi Richard M

2008-10-01

Full Text Available Abstract Background The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30 and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis. Results The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells. Conclusion The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells

Early events associated with infection of Epstein-Barr virus infection of primary B-cells.

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Sabyasachi Halder

2009-09-01

Full Text Available Epstein Barr virus (EBV is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology was used to introduce an expression cassette of green fluorescent protein (GFP by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.

Evaluation of silk biomaterials in combination with extracellular matrix coatings for bladder tissue engineering with primary and pluripotent cells.

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Franck, Debra; Gil, Eun Seok; Adam, Rosalyn M; Kaplan, David L; Chung, Yeun Goo; Estrada, Carlos R; Mauney, Joshua R

2013-01-01

Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of α-actin and SM22α mRNA and protein expression levels following TGFβ1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results

Metastatic Signet-Ring Cell Gastric Carcinoma Masquerading as Breast Primary

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Dinesh Chandra Doval

2009-03-01

Full Text Available Metastasis to the breast from an extra-mammary primary is a rare phenomenon; metastasis from gastric carcinoma to the breast is extremely so. We report a case who initially presented as mucin-secreting and signet-ring cell tumor of the ovary, and after an interval of 8 months with breast and chest wall metastatic nodules. The covert gastric primary eluded the oncologists at both presentations.

β-Arrestin-2-Dependent Signaling Promotes CCR4-mediated Chemotaxis of Murine T-Helper Type 2 Cells.

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Lin, Rui; Choi, Yeon Ho; Zidar, David A; Walker, Julia K L

2018-06-01

Allergic asthma is a complex inflammatory disease that leads to significant healthcare costs and reduction in quality of life. Although many cell types are implicated in the pathogenesis of asthma, CD4 + T-helper cell type 2 (Th2) cells are centrally involved. We previously reported that the asthma phenotype is virtually absent in ovalbumin-sensitized and -challenged mice that lack global expression of β-arrestin (β-arr)-2 and that CD4 + T cells from these mice displayed significantly reduced CCL22-mediated chemotaxis. Because CCL22-mediated activation of CCR4 plays a role in Th2 cell regulation in asthmatic inflammation, we hypothesized that CCR4-mediated migration of CD4 + Th2 cells to the lung in asthma may use β-arr-dependent signaling. To test this hypothesis, we assessed the effect of various signaling inhibitors on CCL22-induced chemotaxis using in vitro-polarized primary CD4 + Th2 cells from β-arr2-knockout and wild-type mice. Our results show, for the first time, that CCL22-induced, CCR4-mediated Th2 cell chemotaxis is dependent, in part, on a β-arr2-dependent signaling pathway. In addition, we show that this chemotactic signaling mechanism involves activation of P-p38 and Rho-associated protein kinase. These findings point to a proinflammatory role for β-arr2-dependent signaling and support β-arr2 as a novel therapeutic target in asthma.

A Computational Analysis of the Function of Three Inhibitory Cell Types in Contextual Visual Processing

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Jung H. Lee

2017-04-01

Full Text Available Most cortical inhibitory cell types exclusively express one of three genes, parvalbumin, somatostatin and 5HT3a. We conjecture that these three inhibitory neuron types possess distinct roles in visual contextual processing based on two observations. First, they have distinctive synaptic sources and targets over different spatial extents and from different areas. Second, the visual responses of cortical neurons are affected not only by local cues, but also by visual context. We use modeling to relate structural information to function in primary visual cortex (V1 of the mouse, and investigate their role in contextual visual processing. Our findings are three-fold. First, the inhibition mediated by parvalbumin positive (PV cells mediates local processing and could underlie their role in boundary detection. Second, the inhibition mediated by somatostatin-positive (SST cells facilitates longer range spatial competition among receptive fields. Third, non-specific top-down modulation to interneurons expressing vasoactive intestinal polypeptide (VIP, a subclass of 5HT3a neurons, can selectively enhance V1 responses.

Cell-type-specific gene delivery into neuronal cells in vitro and in vivo

International Nuclear Information System (INIS)

Parveen, Zahida; Mukhtar, Muhammad; Rafi, Mohammed; Wenger, David A.; Siddiqui, Khwaja M.; Siler, Catherine A.; Dietzschold, Bernhard; Pomerantz, Roger J.; Schnell, Matthias J.; Dornburg, Ralph

2003-01-01

The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain

Bystander-mediated genomic instability after high LET radiation in murine primary haemopoietic stem cells

International Nuclear Information System (INIS)

Bowler, Deborah A.; Moore, Stephen R.; Macdonald, Denise A.; Smyth, Sharon H.; Clapham, Peter; Kadhim, Munira A.

2006-01-01

Communication between irradiated and unirradiated (bystander) cells can result in responses in unirradiated cells that are similar to responses in their irradiated counterparts. The purpose of the current experiment was to test the hypothesis that bystander responses will be similarly induced in primary murine stem cells under different cell culture conditions. The experimental systems used here, co-culture and media transfer, are similar in that they both restrict communication between irradiated and bystander cells to media borne factors, but are distinct in that with the media transfer technique, cells can only communicate after irradiation, and with co-culture, cells can communication before, during and after irradiation. In this set of parallel experiments, cell type, biological endpoint, and radiation quality and dose, were kept constant. In both experimental systems, clonogenic survival was significantly decreased in all groups, whether irradiated or bystander, suggesting a substantial contribution of bystander effects (BE) to cell killing. Genomic instability (GI) was induced under all radiation and bystander conditions in both experiments, including a situation where unirradiated cells were incubated with media that had been conditioned for 24 h with irradiated cells. The appearance of delayed aberrations (genomic instability) 10-13 population doublings after irradiation was similar to the level of initial chromosomal damage, suggesting that the bystander factor is able to induce chromosomal alterations soon after irradiation. Whether these early alterations are related to those observed at later timepoints remains unknown. These results suggest that genomic instability may be significantly induced in a bystander cell population whether or not cells communicate during irradiation

X-ray induction of immortalization in primary rat embryo cells associated with and without tumorigenicity

International Nuclear Information System (INIS)

Sierra, E.; Oberley, L.W.; Guernsey, D.L.

1985-01-01

Cultures of primary rat embryo fibroblasts were irradiated with X-rays (3 Gy). After 14 days the majority of colonies in both irradiated and control plates had senesced. Surviving clones were ring isolated from irradiated and control plates and grown in culture. A phase of rapid proliferation after isolation was observed, followed by a decline (crisis) leading to senescence. Several clones from the irradiated plates were able to recover from this crisis and gave rise to continuous cell lines, while all colonies from control plates senesced. Three types of cells have been identified among the irradiated survivors: (1) immortal fully transformed, capable of growth in soft agar (Aga/sup +/) and tumor formation, (2) immortal normal, not able to grow in soft agar (Aga/sup -/) and nontumorigenic, and (3) immortal Aga/sup -/ cells which progressed to malignancy (Aga/sup +/, tumorigenicity) after further sub-culture. These data support the suggestion that X-rays can induce immortalization of mammalian cells in the absence of tumorigenicity, in addition to (and separate from) the fully tumorigenetic state

Management of obesity in patients with type 2 diabetes mellitus in primary care.

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Mohammad, Shoaib; Ahmad, Jamal

2016-01-01

Obesity and being overweight is the most powerful risk factor accounting for 80-90% of patients with type 2 diabetes mellitus (T2DM). The epidemic of obesity is driving the diabetes epidemic to alarming levels and primary care is becoming an important setting for obesity management in T2DM in India. Yet many primary care providers feel ill-equipped or inadequately supported to address obesity in patients with diabetes. This article reviews the most recent and strongest evidence-based strategies that may aid physicians in management of obesity in patients with T2DM in primary care. A systematic literature search of MEDLINE using the search terms Obesity, Obesity in T2DM, weight loss and Primary Care was conducted. The American Diabetes Association, National Institute for Health, National Institute of Health and Excellence (NICE), Scottish Intercollegiate Guidelines Network (SIGN) and World Health Organization websites were also searched. Most studies in this area are observational in design with few randomized controlled trials (RCTs). Articles and studies involving meta-analysis or RCTs were preferred over other types. Effective weight management treatment in T2DM patient can be implemented in the primary care setting. Evidence based individualized lifestyle and pharmacologic measures supported by behavioral intervention and counseling with appropriate and informed surgical referrals has the potential to improve the success of weight management within primary care. Copyright © 2016 Diabetes India. Published by Elsevier Ltd. All rights reserved.

Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

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Kubis, Hans-Peter; Scheibe, Renate J; Decker, Brigitte; Hufendiek, Karsten; Hanke, Nina; Gros, Gerolf; Meissner, Joachim D