Sample records for primary cd34-positive cells

  1. CD34-positive interstitial cells of the human detrusor

    Rasmussen, Helle; Hansen, Alastair; Smedts, Frank;


    Interstitial cells of Cajal (ICC) are well described in the bowel wall. They are c-kit positive and play a role as pacemaker cells. Similar c-kit-positive cells have recently been described in the human bladder. The aim of this study was to characterize interstitial cells of the bladder detrusor...... using a panel of antibodies directed against CD117/c-kit, CD34, CD31, S100, tryptase, neurofilament, NSE, Factor-VIII and GFAP. A striking finding was an interstitial type of cell which is CD34 immunoreactive (CD34-ir) but CD117/c-kit negative. The cells have a tentacular morphology, enveloping...... and intermingling with individual muscle fasicles. Morphologically and immunohistochemically, they show no neurogenic, endothelial or mast cell differentiation. Transmission electron microscopy (TEM) showed the presence of interstitial cells with a round-to-oval nucleus, sparse perinuclear cytoplasm and long...

  2. CD34-positive interstitial cells of the human detrusor

    Rasmussen, Helle; Hansen, Alastair; Smedts, Frank;


    and intermingling with individual muscle fasicles. Morphologically and immunohistochemically, they show no neurogenic, endothelial or mast cell differentiation. Transmission electron microscopy (TEM) showed the presence of interstitial cells with a round-to-oval nucleus, sparse perinuclear cytoplasm and long...

  3. Intrathecal Administration of Autologous CD34 Positive Cells in Patients with Past Cerebral Infarction: A Safety Study

    Wang, Liming; Ji, Haijie; Li, Ming; Zhou, Jianjun; Bai, Wen; Zhong, Zhanqiang; Li, Na; Zhu, Delin; Zhang, Zijia; Liu, Yongjun; Wu, Mingyuan


    Regenerative strategies in treatment of stroke have great potential. The goal of the current study was to investigate safety of intrathecal administration of autologous CD34 positive cells in treatment of patients with poststroke. A total of eight male patients with a history of stroke were enrolled. The patients were treated subcutaneously with 5 μg/kg body weight rhG-CSF for 5 consecutive days, and then leukapheresis was performed to concentrate cells for CD34 positive immunoselection. All patients underwent intrathecal administration of CD34 positive cells via lumbar puncture. The primary outcome was safety evaluation for 12-month followup. In addition, behavioral function was evaluated with NIH stroke scale and Barthel index 1, 6, and 12 months after the last treatment, respectively. There were no major adverse events, and abnormal changes of blood tests during the whole treatment process included intrathecal administration and 12-month followup. The main message from the current study was that administration of G-CSF-mobilized autologous CD34 positive cells in patients with poststroke was safe. Future studies with larger population and control group are needed to confirm the safety and investigate the efficacy. PMID:24187628

  4. Circulating CD34-positive cells, glomerular filtration rate and triglycerides in relation to hypertension.

    Shimizu, Yuji; Sato, Shimpei; Koyamatsu, Jun; Yamanashi, Hirotomo; Nagayoshi, Mako; Kadota, Koichiro; Maeda, Takahiro


    Serum triglycerides have been reported to be independently associated with the development of chronic kidney disease (CKD), which is known to play a role in vascular disturbance. On the other hand, circulating CD34-positve cells, including endothelial progenitor cells, are reported to contribute to vascular repair. However, no studies have reported on the correlation between triglycerides and the number of CD34-positive cells. Since hypertension is well known factor for vascular impairment, the degree of correlation between serum triglycerides and circulating CD34-positve cells should account for hypertension status. We conducted a cross-sectional study of 274 elderly Japanese men aged ≥ 60 years (range 60-79 years) undergoing general health checkups. Multiple linear regression analysis of non-hypertensive subjects adjusting for classical cardiovascular risk factors showed that although triglyceride levels (1SD increments; 64 mg/dL) did not significantly correlate with glomerular filtration rate (GFR) (β = -2.06, p = 0.163), a significant positive correlation was seen between triglycerides and the number of circulating CD34-positive cells (β = 0.50, p = 0.004). In hypertensive subjects, a significant inverse correlation between triglycerides and GFR was observed (β = -2.66, p = 0.035), whereas no significant correlation between triglycerides and the number of circulating CD34-positive cells was noted (β = -0.004, p = 0.974). Since endothelial progenitor cells (CD34-positive cells) have been reported to contribute to vascular repair, our results indicate that in non-hypertensive subjects, triglycerides may stimulate an increase in circulating CD34-positive cells (vascular repair) by inducing vascular disturbance. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood

    Heiner Falkenberg


    Full Text Available Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF, thrombopoietin (THPO, interleukin 6 (IL-6, and fms-related tyrosine kinase 3 ligand (FLT3lg. At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

  6. Flow cytometric analysis of hemetopoietic progenitor cells in peripheral blood stem cell harvest from patients with CD34 positive acute leukemia.

    Miyazaki, T; Matsuda, I; Oguri, M; Amaya, H; Kiyosaki, M; Hamada, A; Tamaki, S; Tashiro, E; Kudo, Y; Taniguchi, O; Nakamura, T; Tomoyasu, S


    We analyzed CD34 positive cells in peripheral blood stem cell harvest (PBSCH) using flow cytometry. PBSCH from CD34 positive acute myelogeous leukemia (AML-M2) patient contained 1.87% CD34 positive cells, of which 1.21% was represented by MRD.PBSCH from CD34 positive acute lymphoblast leukemia (ALL) patient contained 3.14% CD34 positive cells, of which 0.11% was accounted for by minimal residual disease (MRD). If PBSCH from CD34 positive acute leukemia patient is analyzed for CD34 monoclonal antibody alone, the presence of CD34 positive MRD may escape attention so that CD34 positive hematopoietic progenitor cells may be overestimated. To avoid this risk, it is necessary to analyze PBSCH using both CD34 monoclonal antibody and characteristic markers of leukemia cells that were found pre-treatment.

  7. Effect of Weight Reduction on Cardiovascular Risk Factors and CD34-positive Cells in Circulation

    Nina A Mikirova, Joseph J Casciari, Ronald E Hunninghake, Margaret M Beezley


    Full Text Available Being overweight or obese is associated with an increased risk for the development of non-insulin-dependent diabetes mellitus, hypertension, and cardiovascular disease. Dyslipidemia of obesity is characterized by elevated fasting triglycerides and decreased high-density lipoprotein-cholesterol concentrations. Endothelial damage and dysfunction is considered to be a major underlying mechanism for the elevated cardiovascular risk associated with increased adiposity. Alterations in endothelial cells and stem/endothelial progenitor cell function associated with overweight and obesity predispose to atherosclerosis and thrombosis.In our study, we analyzed the effect of a low calorie diet in combination with oral supplementation by vitamins, minerals, probiotics and human chorionic gonadotropin (hCG, 125-180 IUs on the body composition, lipid profile and CD34-positive cells in circulation.During this dieting program, the following parameters were assessed weekly for all participants: fat free mass, body fat, BMI, extracellular/intracellular water, total body water and basal metabolic rate. For part of participants blood chemistry parameters and circulating CD34-positive cells were determined before and after dieting.The data indicated that the treatments not only reduced body fat mass and total mass but also improved the lipid profile. The changes in body composition correlated with the level of lipoproteins responsible for the increased cardiovascular risk factors. These changes in body composition and lipid profile parameters coincided with the improvement of circulatory progenitor cell numbers.As the result of our study, we concluded that the improvement of body composition affects the number of stem/progenitor cells in circulation.

  8. Pericardial patch angioplasty heals via an Ephrin-B2 and CD34 positive cell mediated mechanism.

    Xin Li

    Full Text Available OBJECTIVE: Pericardial patches are commonly used in vascular surgery to close arteriotomies. The mechanism of early healing after patch implantation is still not well defined. We used a rat aortic patch model to assess pericardial patch healing and examined Ephrin-B2, a marker of arterial identity, expression within the post-implantation patch. We also determined whether endothelial progenitor cells (EPC are associated with early patch healing in the arterial environment. METHODS: Wistar rats (200-250 grams underwent infrarenal aortic arteriotomy and then closure via bovine or porcine pericardial patch angioplasty. Control groups included subcutaneously implanted patches. Patches were harvested at 0-30 days and analyzed by histology, immunohistochemistry, immunofluorescence and Western blot as well as quantitative PCR. RESULTS: Prior to implantation, pericardial patches are largely composed of collagen and are acellular. Following arterial implantation, increasing numbers of CD68-positive cells as well as Ephrin-B2 and CD34 dual-positive cells are found within both bovine and porcine pericardial patches, whereas the infiltrating cells are negative for vWF and α-actin. Porcine patches have a luminal monolayer of cells at day 7, compared to bovine patches that have fewer luminal cells. Subcutaneously implanted patches do not attract Ephrin-B2/CD34-positive cells. By day 30, both bovine and porcine pericardial patches develop a neointima that contains Ephrin-B2, CD34, and VEGFR2-positive cells. CONCLUSION: Both CD68-positive and Ephrin-B2 and CD34 dual-positive cells infiltrate the pericardial patch early after implantation. Arteriotomy closure via pericardial patch angioplasty shows patch adaptation to the arterial environment that may involve a foreign body response as well as localization of EPC. Arterial remodeling of pericardial patches support endothelialization and may represent a paradigm of healing of scaffolds used for tissue engineering.

  9. Combination of CD34-positive cell subsets with infarcted myocardium-like matrix stiffness: a potential solution to cell-based cardiac repair.

    Zhang, Shuning; Ma, Xin; Yao, Kang; Zhu, Hong; Huang, Zheyong; Shen, Li; Qian, Juying; Zou, Yunzeng; Sun, Aijun; Ge, Junbo


    Detection of the optimal cell transplantation strategy for myocardial infarction (MI) has attracted a great deal of attention. Commitment of engrafted cells to angiogenesis within damaged myocardium is regarded as one of the major targets in cell-based cardiac repair. Bone marrow-derived CD34-positive cells, a well-characterized population of stem cells, might represent highly functional endothelial progenitor cells and result in the formation of new blood vessels. Recently, physical microenvironment (extracellular matrix stiffness) around the engrafted cells was found to exert an essential impact on their fate. Stem cells are able to feel and respond to the tissue-like matrix stiffness to commit to a relevant lineage. Notably, the infarct area after MI experiences a time-dependent stiffness change from flexible to rigid. Our previous observations demonstrated myocardial stiffness-dependent differentiation of the unselected bone marrow-derived mononuclear cells (BMMNCs) along endothelial lineage cells. Myocardial stiffness (~42 kPa) within the optimal time domain of cell engraftment (at week 1 to 2) after MI provided a more favourable physical microenvironment for cell specification and cell-based cardiac repair. However, the difference in tissue stiffness-dependent cell differentiation between the specific cell subsets expressing and no expressing CD34 phenotype remains uncertain. We presumed that CD34-positive cell subsets facilitated angiogenesis and subsequently resulted in cardiac repair under induction of infarcted myocardium-like matrix stiffness compared with CD34-negative cells. If the hypothesis were true, it would contribute greatly to detect the optimal cell subsets for cell therapy and to establish an optimized therapy strategy for cell-based cardiac repair.

  10. Local transplantation of ex vivo expanded bone marrow-derived CD34-positive cells accelerates fracture healing.

    Kawakami, Yohei; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Matsumoto, Tomoyuki; Kuroda, Ryosuke; Shoji, Taro; Fukui, Tomoaki; Masuda, Haruchika; Akimaru, Hiroshi; Mifune, Yutaka; Kuroda, Tomoya; Horii, Miki; Yokoyama, Ayumi; Kurosaka, Masahiro; Asahara, Takayuki


    Transplantation of bone marrow (BM) CD34(+) cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34(+) cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34(+) cell expansion method. Seven-day ex vivo expansion culture of BM CD34(+) cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34(+) cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34(+) cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34(+) cells and fresh BM CD34(+) cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34(+) cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34(+) cells. In vitro, cEx-BM CD34(+) cells showed higher colony/tube-forming capacity than nonexpanded BM CD34(+) cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34(+) cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34(+) cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.

  11. CD34-positive cells and their subpopulations characterized by flow cytometry analyses on the bone marrow of healthy allogenic donors

    Jerusa Martins Carvalho

    Full Text Available CONTEXT AND OBJECTIVE: Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment. DESIGN AND SETTING: Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo. METHODS: Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment. RESULTS: In region R1, 0.1% to 2.8% of cells were CD34+/CD45+ and 1.1%, CD34+/CD45-. The main coexpressions of CD45+ cells were CD38, CD22, CD19 and CD56 in R2 and CD33, CD11c, CD14, CD15 and CD61 in R3 and R4. After sorting, 2.2x10(6 CD34+ cells were equivalent to 4.9% of total cells. Coexpression of CD34+/CD38+ and CD34+/CD117+ occurred in 94.9% and 82% of events, respectively. There was a positive relationship between CD34+ cells and engraftment. More than 80% of marrow cells expressed high Rh-123. CD34+ cell sorting showed that cells in regions of more differentiated lineages retained Rh-123 more intensively than in primitive lineage regions. CONCLUSION: We advocate that true stem cells are CD34+/CD45-/CD38-/low-Rh-123 accumulations.

  12. Human Umbilical Cord Blood CD34-Positive Cells as Predictors of the Incidence and Short-Term Outcome of Neonatal Hypoxic-Ischemic Encephalopathy: A Pilot Study

    Nasr Eldin, Mohamed Hassan; Amer, Hanaa A.; Abdelhamid, Adel E.; El Houssinie, Moustafa; Ibrahim, Abir


    Background and Purpose Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of neurological handicap in developing countries. Human umbilical cord blood (hUCB) CD34-positive (CD34+) stem cells exhibit the potential for neural repair. We tested the hypothesis that hUCB CD34+ stem cells and other cell types [leukocytes and nucleated red blood cells (NRBCs)] that are up-regulated during the acute stage of perinatal asphyxia (PA) could play a role in the early prediction of the occurrence, severity, and mortality of HIE. Methods This case-control pilot study investigated consecutive neonates exposed to PA. The hUCB CD34+ cell count in mononuclear layers was assayed using a flow cytometer. Twenty full-term neonates with PA and 25 healthy neonates were enrolled in the study. Results The absolute CD34+ cell count (p=0.02) and the relative CD34+ cell count (CD34+%) (p<0.001) in hUCB were higher in the HIE patients (n=20) than the healthy controls. The hUCB absolute CD34+ cell count (p=0.04), CD34+% (p<0.01), and Hobel risk scores (p=0.04) were higher in patients with moderate-to-severe HIE (n=9) than in those with mild HIE (n=11). The absolute CD34+ cell count was strongly correlated with CD34+% (p<0.001), Hobel risk score (p=0.04), total leukocyte count (TLC) (p<0.001), and NRBC count (p=0.01). CD34+% was correlated with TLC (p=0.02). Conclusions hUCB CD34+ cells can be used to predict the occurrence, severity, and mortality of neonatal HIE after PA. PMID:28079317

  13. Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

    Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E


    The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.

  14. Quantification of nucleated cells, CD34-positive cells and CFU-GM colonies in single bone marrow samples and bone marrow harvests derived from healthy children.

    Schündeln, Michael M; Walde, Gabriele; Basu, Oliver; Havers, Werner; Kremens, Bernhard


    Little is known regarding bone marrow (BM) cellularity, CD34+ fraction, and CFU-GM colony formation in relation to age and whether healthy children require a reference range distinct from healthy adults. We therefore analyzed a series of single BM aspirates from 45 healthy children who were evaluated as potential BM donors. Thirty-three of these children subsequently donated BM. We quantified the nucleated cell count, fraction of CD34+ cells, and number of CFU-GM colonies in single aspirates and BM harvests. Single aspirates displayed a mean nucleated cell count of 31.3 × 10(6) cells/mL, a mean fraction of 1.17% CD34+ cells, and a mean colony forming potential of 66.6 CFU-GM/10(5) cells. Harvests yielded the same number of nucleated cells but increased numbers of CD34+ cells and CFU-GM compared with single aspirates. The mean nucleated cell count in BM harvests was 31.1 × 10(6) /mL with a mean fraction of 1.95% CD34+ cells and a mean of 112.4 CFU-GM colonies/10(5) cells. The concentration of nucleated cells was elevated compared with reported adult counts, while CD34+ percentage and CFU-GM counts were similar. In this series of healthy children, the fraction of CD34+ cells, CFU-GM colonies, and nucleated cells decreased with age. We did not identify gender specific differences. To our knowledge, this represents the first comprehensive study of CD34+ cell fraction, CFU-GM counts, and nucleated cell numbers in the BM of healthy children. The findings provide valuable information for practical use for BM transplantation and contribute to the understanding of hematopoiesis from birth to adulthood.

  15. Loss of CD117 (c-kit)- and CD34-positive ICC and associated CD34-positive fibroblasts defines a subpopulation of chronic intestinal pseudo-obstruction.

    Streutker, C J; Huizinga, J D; Campbell, F; Ho, J; Riddell, R H


    Chronic idiopathic intestinal pseudo-obstruction is a syndrome in which symptoms of intestinal obstruction are present in the absence of mechanical obstruction. Lack of normal pacemaker activity, usually generated by the interstitial cells of Cajal (ICC), could account for the apparent obstruction. ICC are normally located around and between the myenteric plexus ganglia and within muscle and also in the deep muscular plexus of the small bowel and the submuscular plexus of the large intestine, just within the circular muscle. ICC can be demonstrated immunohistochemically with CD117 (c-kit) as well as with CD34, although this is less specific. CD34 also stains a population of fibroblasts that are intimately associated with ICC. To determine whether there is a relative deficiency of ICC and CD34-positive fibroblasts in patients with chronic idiopathic intestinal pseudo-obstruction, tissue from 30 patients of large intestine and eight patients with small intestine pseudo-obstruction was obtained. Controls (large intestinal specimens from 12 patients, small intestinal specimens from six patients) were chosen from resections for Crohn's disease and colorectal neoplasia, both with and without dilatation. Examination of pseudo-obstruction cases identified 10 patients (nine large intestinal and one small intestinal) in which both CD117 and CD34 were absent or severely reduced in all three of the examined areas. In contrast, the control cases, including those with preobstructive dilatation, showed relatively constant ICC staining. These results suggest that there is a proportion of pseudo-obstruction cases in which the ICC are markedly reduced. These results also demonstrate that, in these cases, loss of the kit immunoreactivity is correlated with the loss of CD34 staining: this indicates that both the ICC and the CD34-positive fibroblasts associated with the ICC are absent. These findings will allow surgical pathologists to identify this subpopulation of patients with CIIP

  16. Hypocellular Plaque-Like CD34-Positive Dermal Fibroma (Medallion-Like Dermal Dendrocyte Hamartoma) Presenting as a Skin-Colored Dermal Nodule.

    Mutgi, Krishna A J; Chitgopeker, Pooja; Ciliberto, Heather; Stone, Mary S


    Plaque-like CD34-positive dermal fibromas, also known as medallion-like dermal dendrocyte hamartomas (MDDHs), are a recently recognized group of congenital and acquired spindle cell neoplasms that may appear histologically similar to dermatofibrosarcoma protuberans. Recognizing the clinical heterogeneity of this neoplasm and the subtle pathologic differences are crucial to making the correct diagnosis and avoiding the aggressive surgical intervention required to treat a dermatofibrosarcoma protuberans. We present the case of a 9-year-old girl with an acquired variant of a plaque-like CD34-positive dermal fibroma without clinical epidermal change. Our case expands the clinical spectrum to include an acquired variant of a plaque-like CD34-positive dermal fibroma without clinical epidermal change. Examination of more cases is needed to determine whether all clinical variants are truly subtypes of the same neoplasm or represent distinct CD34-positive spindle cell proliferations.

  17. A novel use of growth factors, CD34 positive cells, and fibrin for fingertip injury: Description of a case

    Francesco Romano


    Full Text Available Traumatic soft tissue injuries of the finger represent a frequent hand injury challenging hand surgeons. We report a case involving a 30-year-old man with a dorsal index finger soft tissue wound failing conservative treatment. The novel use of applied fibrin membranes and concentrated growth factors yielded complete resolution of the injury in 6 months without need for skin grafting.

  18. Encapsulated feeder cells within alginate beads for ex vivo expansion of cord blood-derived CD34(+) cells

    Pan, Xiuwei; Sun, Qiong; Cai, Haibo; Gao, Yun; Tan, Wensong; Zhang, Weian


    A co-culture system based on encapsulated feeder cells within alginate beads was developed through optimizing the detailed aspects of the cell culture system to expand CD34-positive (CD34(+)) cells ex vivo...

  19. The effect of cell concentrations from different cell populations on the viability of umbilical blood stem cells.

    Hauck-Dlimi, Barbara; Dlimi, Afif; Zimmermann, Robert; Eckstein, Reinhold; Zingsem, Juergen


    In addition to bone marrow or peripheral blood derived stem cells, cord blood (CB) is an alternative source for hematopoietic stem cells. This report shows the impact of higher concentrations of leukocytes, mononu- clear cells (MNCs), and CD34-positive cells on the viability of CB derived stem cells after cryopreservation. Statistical analysis of data from 5520 CB units, prepared and cryopreserved from 2003 through 2011, was performed with appropriate software. Cell concentrations for leukocytes, platelets, red blood cells (RBCs), CD34-positive leukocytes, viable leukocytes, and MNCs were determined. The proliferation and differentiation capacity was assessed in cell culture assays. Content of leukocytes, CD34-positive leukocytes, and MNCs decreased after thawing. The recovery rate of colony forming units (CFUs) (29.05%) correlated significantly with leukocytes, platelets, RBCs, MNCs, CD34- positive leukocytes, and viable leukocytes. The recovery rate for erythroblasts (3.33%) correlated significantly with leukocytes, CD34-positive leukocytes, MNCs, and viable leukocytes. In the different cell concentration groups only RBCs showed a negative influence on viability. The concentrations of leukocytes, platelets, and CD34-positive leukocytes before cryopreservation correlated positively with the concentrations of leukocytes, CD34-positive leukocytes, MNCs as well as with the cell viability after thawing. Increased cell concentrations in CB do not limit the recovery of CD34-positive leukocytes nor the viability of leukocytes or the number of CFUs after thawing. On the contrary, CB units with high cell concentrations show a better outcome than units with low cell concentrations. Only RBCs seem to have a negative influence on CB quality.

  20. Tenascin-C and tenascin-W in whisker follicle stem cell niches: possible roles in regulating stem cell proliferation and migration.

    Tucker, Richard P; Ferralli, Jacqueline; Schittny, Johannes C; Chiquet-Ehrismann, Ruth


    The whisker follicle has CD34-positive stem cells that migrate from their niche near the bulge along the glassy membrane to the whisker bulb, where they participate in the formation of the whisker shaft. Using immunohistochemistry, we found the glycoprotein tenascin-C in the fibrous capsule of mouse whisker follicles, along the glassy membrane and in the trabecular region surrounding keratin-15-negative, CD34-positive stem cells. The related glycoprotein tenascin-W is found in the CD34-positive stem cell niche, in nearby trabeculae and along the glassy membrane. Tenascin-W is also found in the neural stem cell niche of nearby hair follicles. The formation of stress fibers and focal adhesion complexes in CD34-positive whisker-derived stem cells cultured on fibronectin was inhibited by both tenascin-C and tenascin-W, which is consistent with a role for these glycoproteins in promoting the migration of these cells from the niche to the whisker bulb. Tenascin-C, but not tenascin-W, increased the proliferation of whisker follicle stem cells in vitro. Thus, the CD34-positive whisker follicle stem cell niche contains both tenascin-C and tenascin-W, and these glycoproteins might play a role in directing the migration and proliferation of these stem cells.


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  2. Primary orbital squamous cell carcinoma

    Ana L. Campos Arbulú


    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

  3. Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells


    Background Acute myeloid leukemia (AML) is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR) than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1), DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP) was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA) against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to DNR-induced apoptosis

  4. Curcumin reduces expression of Bcl-2, leading to apoptosis in daunorubicin-insensitive CD34+ acute myeloid leukemia cell lines and primary sorted CD34+ acute myeloid leukemia cells

    Huang Sheng-Shan


    Full Text Available Abstract Background Acute myeloid leukemia (AML is an immunophenotypically heterogenous malignant disease, in which CD34 positivity is associated with poor prognosis. CD34+ AML cells are 10-15-fold more resistant to daunorubicin (DNR than CD34- AML cells. Curcumin is a major component of turmeric that has shown cytotoxic activity in multiple cancers; however, its anti-cancer activity has not been well studied in DNR-insensitive CD34+ AML cells. The aim of this study was to therefore to explore curcumin-induced cytotoxicity in DNR-insensitive CD34+ AML cell lines (KG1a, Kasumi-1, DNR-sensitive U937 AML cells, and primary CD34+ AML bone-marrow-derived cells. Methods Primary human CD34+ cells were isolated from peripheral blood mononuclear cells or bone marrow mononuclear cells using a CD34 MicroBead kit. The growth inhibitory effects of curcumin were evaluated by MTT and colony-formation assays. Cell cycle distribution was examined by propidium iodide (PI assay. Apoptosis was analyzed by Wright-Giemsa, Hoechst 33342 and Annexin-V/PI staining assays. The change in mitochondrial membrane potential (MMP was examined by JC-1 staining and flow cytometry. Expression of apoptosis-related proteins was determined by reverse transcription-polymerase chain reaction and Western blotting. Short interfering RNA (siRNA against Bcl-2 was used in CD34+ KG1a and Kasumi-1 cells incubated with/without DNR. Results Curcumin inhibited proliferation and induced apoptosis and G1/S arrest in both DNR-insensitive KG1a, Kasumi-1 and DNR-sensitive U937 cells. Curcumin-induced apoptosis was associated with reduced expression of both Bcl-2 mRNA and protein, subsequent loss of MMP, and activation of caspase-3 followed by PARP degradation. Curcumin synergistically enhanced the cytotoxic effect of DNR in DNR-insensitive KG1a and Kasumi-1 cells, consistent with decreased Bcl-2 expression. Accordingly, siRNA against Bcl-2 increased the susceptibility of KG1a and Kasumi-1 cells to

  5. Correlation between expression of cyclooxygenase-2 and the presence of inflammatory cells in human primary hepatocellular carcinoma: Possible role in tumor promotion and angiogenesis

    Melchiorre Cervello; Daniela Foderà; Ada Maria Florena; Maurizio Soresi; Claudio Tripodo; Natale D'Alessandro; Giuseppe Montalto


    AIM: To investigate the association of cyclooxygenase-2(COX-2) expression with angiogenesis and the number and type of inflammatory cells (macrophages/Kupffer cells;mast cells) within primary hepatocellular carcinoma (HCC)tissues and adjacent non-tumorous (MT) tissues.METHODS: Immunohistochemistry for COX-2, CD34,CD68 and mast cell tryptase (MCT) was performed on 14 well-characterized series of liver-cirrhosis-associated HCC patients. COX-2 expression and the number of inflammatory cells in tumor lesions and surrounding liver tissues of each specimen were compared. Moreover,COX-2, CD34 staining and the number of inflammatory cells in areas with different histological degrees within each tumor sample were comparatively analyzed.RESULTS: The percentage of COX-2 positive cells was significantly higher in NT tissues than in tumors. COX-2 expression was higher in well-differentiated HCC than in poorly-differentiated tissues. Few mast cells were observed within the tumor mass, whereas a higher number was observed in the surrounding tissue, especially in peri-portal spaces of NT tissues. Abundant macrophages/Kupffer cells were observed in NT tissues, whereas the number of cells was significantly lower in the tumor mass.However, a higher cell number was observed in the well-differentiated tumor and progressively decreased in relation to the differentiation grade. Within the tumor, a positive correlation was found between COX-2 expression and the number of macrophages/Kupffer cells and mast cells. Moreover, there was a positive correlation between CD34 and COX-2 expression in tumor tissues. Comparison between well- and poorly-differentiated HCC showed that the number of CD34-positive cells decreased with dedifferentiation. However, COX-2 was the only independent variable showing a positive correlation with CD34 in a multivariate analysis.CONCLUSION: The presence of inflammatory cells and COX-2 expression in liver tumor suggests a possible relationship with tumor

  6. Induction of embryonic stem cells to hematopoietic cells in vitro


    In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright's staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34+ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34-CD38+. Wright's staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.

  7. Primary lithium cell life studies

    Capulli, John; Donley, Sam; Deligiannis, Frank; Shen, David


    One solution for providing a truly independent power source is to package, within the critical subsystem element, a primary battery that can remain dormant for time periods as long as the mission life, which can be 10-15 years, maximum. When primary power from the spacecraft solar array/battery system is interrupted, the backup battery system, which is connected through a diode to the power input line, would automatically support the load to avoid a power interruption to the critical load for a time period long enough to ensure that ground control could access the satellite and correct the anomaly by sending appropriate commands to the spacecraft. Critical subsystems identified for the application are telemetry and command circuits, volatile computer memory, attitude control circuits, and some critical payloads. Due to volume packaging and weight restrictions that exist on most spacecraft, coupled with the long storage periods required, lithium cell technology was selected for the backup power source. Because of the high energy density (200-400 Wh/kg), long shelf life, and load capability, soluble cathode primary lithium technology was chosen. The most important lithium cell properties that require detail characterization for this application are capacity loss, shelf life, and the voltage delay mechanism. These are functions of storage time and temperature. During storage, a passive film builds up on the lithium electrode. The film protects the lithium electrode from progressive capacity decay but requires time to break down when a load is applied. This phenomenon results in a depressed voltage during the period of film breakdown which can last from fractions of a second to minutes.

  8. Molluscan cells in culture: primary cell cultures and cell lines


    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  9. Molluscan cells in culture: primary cell cultures and cell lines.

    Yoshino, T P; Bickham, U; Bayne, C J


    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  10. Primary Culture of Porcine Pancreatic Acinar Cells


    OBJECTIVE: To develop a method for the primary culture of porcine pancreatic acinar cells. INTERVENTIONS: Dispersed pancreatic acinar cells available utilizing RPMI-1640 medium containing collagenase III. After purification, the isolated acinar cells were cultured in RPMI-1640 medium with the addition of 2.5% fetal bovine serum. MAIN OUTCOME MEASURES: The morphological characteristics of acinar cells were described. (3)H-thymidine incorporation of acinar cells and the activity of amylase or l...

  11. Cell therapy of primary myopathies.

    Sampaolesi, M; Biressi, S; Tonlorenzi, R; Innocenzi, A; Draghici, E; Cusella de Angelis, M G; Cossu, G


    Mesoangioblasts are multipotent progenitors of mesodermal tissues. In vitro mesoangioblasts differentiate into many mesoderm cell types, such as smooth, cardiac and striated muscle, bone and endothelium. After transplantation mesoangioblasts colonize mostly mesoderm tissues and differentiate into many cell types of the mesoderm. When delivered through the arterial circulation, mesoangioblasts significantly restore skeletal muscle structure and function in a mouse model of muscular dystrophy. Their ability to extensively self-renew in vitro, while retaining multipotency, qualifies mesoangioblasts as a novel class of stem cells. Phenotype, properties and possible origin of mesoangioblasts are addressed in the first part of this paper. In the second part we will focus on the cell therapy approach for the treatment of Muscular Dystrophy and we will describe why mesangioblasts appear to be promising candidates for this strategy.

  12. Primary cortical brain cells influence osteoblast activity.

    Anissian, Lucas; Kirby, Michael; Stark, André


    The presence of neuropeptides and neuroreceptors in the bone have been reported in several studies. Bone turn-over seems to be controlled by the nervous system. The actual pathway or the control mechanism is still under investigation. In this study we investigate the changes in osteoblast cells if they are in co-culture with primary cortical brain cells. After seven days in co-culture with the primary fetal brain cells the osteoblast cells exhibited hypertrophic morphological changes and showed stronger ALP activity.

  13. Primary Cilia, Signaling Networks and Cell Migration

    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...

  14. Primary Cilia, Signaling Networks and Cell Migration

    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...

  15. Primary Culture of Porcine Pancreatic Acinar Cells

    Zhao X


    Full Text Available OBJECTIVE: To develop a method for the primary culture of porcine pancreatic acinar cells. INTERVENTIONS: Dispersed pancreatic acinar cells available utilizing RPMI-1640 medium containing collagenase III. After purification, the isolated acinar cells were cultured in RPMI-1640 medium with the addition of 2.5% fetal bovine serum. MAIN OUTCOME MEASURES: The morphological characteristics of acinar cells were described. (3H-thymidine incorporation of acinar cells and the activity of amylase or lipase were determined during the culture process. RESULTS: There were no remarkable morphological changes in the pancreatic acinar cells during the 20 days culture. The acini showed a tendency to gather but did not attach to the walls of the culture disks. A good (3H-thymidine incorporation of acinar cells in the primary culture was maintained. The secretion of amylase or lipase from the acini decreased with the length of time of the culture. DISCUSSION: The primary culture of acinar cells from a porcine pancreas which was carried out in this study maintained the normal morphology of the acinar cells and their ability to grow but not their secretion of amylase or lipase. The method would benefit by the further experiments on acini of porcine pancreas.

  16. Primary mantle cell lymphoma of the trachea.

    Guddati, Achuta K; Marak, Creticus P


    Primary mantle cell lymphoma (MCL) is a controversial entity. It is difficult to diagnose MCL in a single organ without lymph node involvement. However, with the advent of PET-CT scans and large panels of immunohistochemistry markers, there have been increasing reports of primary MCL detected in various organs of which the GI tract is the most common. In this case report, we describe the diagnosis and clinical course of a patient who presented with "B symptoms" and respiratory distress. On further investigation, he was found to have a mass in his trachea, which was diagnosed as primary MCL.

  17. Teaching Cell Biology in Primary Schools

    Francele de Abreu Carlan


    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.

  18. T cell-B cell interactions in primary immunodeficiencies.

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


    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.

  19. Failure of cell cleavage induces senescence in tetraploid primary cells.

    Panopoulos, Andreas; Pacios-Bras, Cristina; Choi, Justin; Yenjerla, Mythili; Sussman, Mark A; Fotedar, Rati; Margolis, Robert L


    Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

  20. Primary Hepatosplenic Large B-Cell Lymphoma

    M.R. Morales-Polanco


    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.

  1. Inflammatory Cell Distribution in Primary Merkel Cell Carcinoma

    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: [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: [Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH (United Kingdom)


    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.

  2. Merkel cell carcinoma versus metastatic small cell primary bronchogenic carcinoma

    Katya Lisette Velasquez Cantillo


    Full Text Available Merkel cell carcinoma (MCC of the skin is a rare, aggressive, malignant neuroendocrine neoplasm. The tumor classically demonstrates positive immunohistochemistry (IHC staining for chromogranin A(ChrA, cytokeratin 20 (CK20, neuron specific enolase (NSE and/or achaete-acute complex-like 1 (MASH1. The newly identified Merkel cell polyomavirus (MCPyV has been found to be associated with most MCC cases. The primary histologic differential diagnoses of cutaneous MCC is small cell primary bronchogenic carcinoma (SCLC; moreover, both are of neuroendocrine origin. SCLC accounts for approximately 10-15% of all primary lung cancer cases; this histologic subtype is a distinct entity with biological and oncological features distinct from non-small cell lung cancer (NSCLC. In contradistinction to MCC, SCLC is classically IHC positive for cytokeratin 7 (CK7 and transcription factor (TTF-1. Similar to SCLC, MCC cell lines may be classified into two different biochemical subgroups designated as Classic and Variant. In our review and case report, we aim to emphasize the importance of a multidisciplinary approach to the approach to this difficult differential diagnosis. We also aim to comment about features of the cells of origin of MCC and SCLC; to summarize the microscopic features of both tumors; and to review their respective epidemiologic, clinical, prognostic and treatment features. We want to emphasize the initial workup study of the differential diagnosis patient, including evaluating clinical lymph nodes, a clinical history of any respiratory abnormality, and chest radiogram. If a diagnosis of primary cutaneous MCC is confirmed, classic treatment includes excision of the primary tumor with wide margins, excision of a sentinel lymph node, and computed tomography, positron emission tomography and/or Fluorine-18-fluorodeoxyglucose positron emission tomography scan studies

  3. Gene therapy of primary T cell immunodeficiencies.

    Fischer, Alain; Hacein-Bey-Abina, Salima; Cavazzana-Calvo, Marina


    Gene therapy of severe combined immunodeficiencies has been proven to be effective to provide sustained correction of the T cell immunodeficiencies. This has been achieved for 2 forms of SCID, i.e SCID-X1 (γc deficiency) and adenosine deaminase deficiency. Occurrence of gene toxicity generated by integration of first generation retroviral vectors, as observed in the SCID-X1 trials has led to replace these vectors by self inactivated (SIN) retro(or lenti) viruses that may provide equivalent efficacy with a better safety profile. Results of ongoing clinical studies in SCID as well as in other primary immunodeficiencies, such as the Wiskott Aldrich syndrome, will be thus very informative.

  4. Primary Plasma Cell Leukemia: Identity Card 2016.

    Musto, Pellegrino; Simeon, Vittorio; Todoerti, Katia; Neri, Antonino


    Primary plasma cell leukemia (PPCL) is an aggressive and rare variant of multiple myeloma (MM), characterized by peculiar adverse clinical and biological features. Though the poor outcome of PPCL has been slightly improved by novel treatments during the last 10 years, due to the limited number of available studies in this uncommon disease, optimal therapy remains a classic unmet clinical need. Anyway, in the real-life practice, induction with a bortezomib-based three-drug combination, including dexamethasone and, possibly, lenalidomide, or, alternatively, thalidomide, cyclophosphamide, or doxorubicin, is a reasonable first-line option. This approach may be particularly advisable for patients with adverse cytogenetics, hyperleucocytosis, and rapidly progressive disease, in whom a fast response is required, or for those with suboptimal renal function, where, however, lenalidomide should be used with caution until renal activity is restored. In younger subjects, leukemia/lymphoma-like more intensive regimens, including hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone or continue-infusion cisplatin, doxorubicin, cyclophosphamide, and etoposide, may be also combined with bortezomib +/- thalidomide. Treatment must be started immediately after a diagnosis of PPCL is made to avoid the risk of irreversible disease complications and, in such a context, the prevention of tumor lysis syndrome is mandatory. In patients eligible for autologous stem cell transplantation (AuSCT), other alkylating agents, in particular melphalan, should be initially avoided in order to allow adequate collections of CD34+ peripheral blood stem cells (PBSC). A combination of lenalidomide and dexamethasone may be a valuable alternative option to manage older or unfit patients or those with slower disease evolution or with signs of neuropathy, contraindicating the use of bortezomib. Patients not suitable for transplant procedures should continue the treatment, if a

  5. Left Supraclavicular Spindle Cell Lipoma

    Oladejo Olaleye; Bertram Fu; Ram Moorthy; Charles Lawson; Myles Black; David Mitchell


    Background. Spindle cell lipoma (SCL) is a benign lipomatous tumour, typically occurring in the posterior neck, shoulder or upper back of elderly males. They compose of fat, CD34 positive spindle cells, and ropey collagen on a myxoid matrix. This case highlights a rare presentation of SCL and the need for pre-operative diagnosis. Case Report. A 63-year-old gentleman presented with a pre-existing left supraclavicular mass that had recently increased in size. FNA and CT Scans were performed and...

  6. Primary marginal zone B-cell lymphoma of appendix

    Radha S


    Full Text Available Primary lymphomas of appendix are extremely rare tumors. The first case of primary lymphoma of appendix was reported by Warren in the year 1898. Incidence of primary lymphoma of appendix is 0.015% of all gastrointestinal lymphomas. This is a report of primary marginal zone B-cell lymphoma of appendix which presented as appendicular mass. As some cases are incidentally discovered, this case emphasizes that histological examination of all appendicectomy specimens is mandatory.

  7. In vitro methods to culture primary human breast epithelial cells.

    Raouf, Afshin; Sun, Yu Jia


    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  8. Management of primary small cell carcinoma of the esophagus

    SUN Ke-lin; HE Jie; CHENG Gui-yu; CHAI Li-xun


    Background Primary small cell carcinoma of the esophagus is rare. Although surgery is successful in eradicating local tumor, the five-year survival rate of patients with primary small cell carcinoma of the esophagus after resection is lower than that of patients with primary squamous cell carcinoma of the esophagus. The purpose of this study was to analyze the clinical manifestations, pathological features and treatment of primary small cell carcinoma of the esophagus.Methods A total of 73 patients with primary small cell carcinoma of the esophagus who had been treated by surgery from 1984 to 2003 were analyzed retrospectively.Results In this series, the overall resection rate was 94.5% (69/73), the radical resection rate 89.0% (65/73) and the operative mortality 1.4% (1/73). The 1-, 3- and 5-year survival rates of patients were 50.7%, 13.7% and 8.2%,respectively.Conclusions Primary small cell carcinoma of the esophagus is rare with a poor prognosis. Surgical resection is the leading method for patients with stage Ⅰ or Ⅱ primary small cell carcinoma of the esophagus. Postoperative chemotherapy is beneficial to these patients. The patients of stage Ⅲ or Ⅳ should be given chemotherapy and radiation therapy.

  9. Primary Testicular B-cell Lymphoma

    Aykut Buğra Şentürk


    Full Text Available Primary testicular lymphoma constitutes only 1-7% of all testicular neoplasms and less than 1% of all non-Hodgkin lymphoma. We report a 69-year-old man who presented with a painful right testicular mass. Treatment modalities consist of surgical excision, chemotherapy and radiation therapy, however there are no standardized treatment options.


    The HOPE (Hydrogen-Oxygen Primary Extraterrestrial) Fuel Cell Program is a multi-phase effort to advance the state-of-the-art of fuel cells by...configuration fuel cell module. The HOPE spacecraft, fuel supply tanks, pneumatics, and thermal systems were designed and fabricated to provide...verify water removal, thermal design, and 30-day shelf-life of the fuel cell . The 35-cell module was subjected to a series of performance tests

  11. Apoptosis of human primary gastric carcinoma cells induced by genistein

    Hai-Bo Zhou; Juan-Juan Chen; Wen-Xia Wang; Jian-Ting Cai; Qin Du


    AIM: To investigate the apoptosis in primary gastric cancer cells induced by genistein, and the relationship between this apoptosis and expression of bcl-2 and bax.METHODS: MTT assay was used to determine the cell growth inhibitory rate in vitro. Transmission electron microscope and TUNEL staining were used to quantitatively and qualitatively detect the apoptosis of primary gastric cancer cells before and after genistein treatment. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosisassociated genes bcl-2 and bax.RESULTS: Genistein inhibited the growth of primary gastric cancer cells in dose-and time-dependent manner. Genistein induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the apoptotic rates of primary gastric cancer cells increased time-dependently. Immunohistochemical staining showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the positivity rates of Bcl-2 proteins were apparently reduced with time and the positivity rates of Bax proteins were apparently increased with time. After exposed to genistein at 20 μmol/L for 24,48, 72 and 96 respectively, the density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time.CONCLUSION: Genistein is able to induce the apoptosis in primary gastric cancer cells. This apoptosis may be mediated by down-regulating the apoptosis- associated bcl-2 gene and up-regulating the expression of apoptosis-associated bax gene.

  12. Analysis of primary cilia in directional cell migration in fibroblasts

    Christensen, Søren Tvorup; Veland, Iben; Schwab, Albrecht;


    Early studies of migrating fibroblasts showed that primary cilia orient in front of the nucleus and point toward the leading edge. Recent work has shown that primary cilia coordinate a series of signaling pathways critical to fibroblast cell migration during development and in wound healing. In p...

  13. Sealed Primary Lithium-Inorganic Electrolyte Cell


    Battery , Thionyl Chloride , Lithium , Lithium Aluminum Chloride , Hermetic Lithium Battery , D Cell, Voltage-Delay, Shelf Life, High Energy Density Battery ... lithium - thionyl chloride , inorganic electrclyte system is one of the highest energy density systems known to date (1-4). The cells contain an Li anoae, a...However, this is not tne case with te thionyl chloride system. A completely discharged battery , while sitting on

  14. Primary cell culture of human adenocarcinomas--practical considerations.

    Lerescu, Lucian; Tucureanu, Cătălin; Caraş, Iuliana; Neagu, Stefan; Melinceanu, Laura; Sălăgeanu, Aurora


    Cell culture is one of the major tools for oncology research, being an excellent system in which to study the biochemistry and molecular biology associated with individual cancer types and to understand cancer cell physiology. Progress in understanding the biology of any type of carcinoma has been impeded by the inability to culture adequately malignant cells from most epithelial tissues. The ultimate in vitro tumor model would completely reflect the in vivo tumor microenvironment in function and mechanism. Unfortunately, such a model does not currently exist. Homogeneous cell lines that can be continuously propagated on plastic surfaces have been extensively used as a surrogate for tumor environment; however they are very different from the in vivo tumor cells. Model systems involving primary culture represent the situation most closely related to the original tissue although they have a number of disadvantages over cell lines, such as the limited ability to repeat studies with a well characterized culture system that can be used in multiple laboratories. The primary culture may contain many types of stromal and infiltrating cell types potentially complicating the interpretation of data. Yet, their properties better reflect the cellular interactions present in intact tissue. The present article reviews the critical steps in obtaining, routine maintenance and cryopreservation of primary tumor cell cultures, based on information from literature and personal experience on the subject. The article also includes an updated protocol for primary tumor cell isolation and culture.

  15. Primary Immunodeficiency Diseases and Hematopoietic Stem Cell Transplantation

    Ayse Ozkan


    Full Text Available Hematopoietic stem cell transplantation (HSCT is the only curative therapy for primary immunodeficiency diseases. Early diagnosis, including prenatally, and early transplantation improve HSCT outcomes. Survival rates improve with advances in the methods of preparing hosts and donor cells, and in supportive and conditioning regimes.

  16. The exocyst localizes to the primary cilium in MDCK cells.

    Rogers, Katherine K; Wilson, Patricia D; Snyder, Richard W; Zhang, Xiaoyu; Guo, Wei; Burrow, Christopher R; Lipschutz, Joshua H


    Primary cilia play a role in the maintenance of tubular epithelial differentiation and ciliary dysfunction can result in abnormal cyst formation, such as occurs in autosomal dominant polycystic kidney disease (ADPKD). We previously showed that the exocyst, an eight-protein complex involved in the biogenesis of polarity from yeast to mammals, is centrally involved in cyst formation [Mol. Biol. Cell. 11 (2000) 4259]. Here we show that the exocyst complex localizes to the primary cilium in Madin-Darby canine kidney (MDCK) tubular epithelial cells. We further show that the exocyst is overexpressed in both cell lines and primary cell cultures of ADPKD origin, suggesting that the exocyst may be involved in the pathogenesis of ADPKD.

  17. Left supraclavicular spindle cell lipoma.

    Olaleye, Oladejo; Fu, Bertram; Moorthy, Ram; Lawson, Charles; Black, Myles; Mitchell, David


    Background. Spindle cell lipoma (SCL) is a benign lipomatous tumour, typically occurring in the posterior neck, shoulder or upper back of elderly males. They compose of fat, CD34 positive spindle cells, and ropey collagen on a myxoid matrix. This case highlights a rare presentation of SCL and the need for pre-operative diagnosis. Case Report. A 63-year-old gentleman presented with a pre-existing left supraclavicular mass that had recently increased in size. FNA and CT Scans were performed and results discussed in the mutidisciplinary team meeting. Excisional biopsy was recommended. Radiology. CT neck showed a left supraclavicular mass of fatty density with fine internal septations. A low-grade liposarcoma could not be excluded. Histopathology. FNA was indeterminate. Histology of specimen showed bland spindle cells with no evidence of malignancy. Immuno-histochemistry showed SCL with CD34 positivity and negative staining on CDK4 and p16. Management. Excision biopsy of the mass was performed which was technically difficult as the mass invaginated around the brachial plexus. The patient recovered well post-operatively with no neurological deficits. Conclusion. Spindle cell lipoma is a rare benign tumour and a pre-operative diagnosis based on the clinical context, imaging and immuno-histochemistry is crucial to management.

  18. Control of hair cell excitability by vestibular primary sensory neurons.

    Brugeaud, Aurore; Travo, Cécile; Demêmes, Danielle; Lenoir, Marc; Llorens, Jordi; Puel, Jean-Luc; Chabbert, Christian


    International audience; In the rat utricle, synaptic contacts between hair cells and the nerve fibers arising from the vestibular primary neurons form during the first week after birth. During that period, the sodium-based excitability that characterizes neonate utricle sensory cells is switched off. To investigate whether the establishment of synaptic contacts was responsible for the modulation of the hair cell excitability, we used an organotypic culture of rat utricle in which the setting ...

  19. Primary culture of Rhodnius prolixus (Hemiptera: Reduviidae salivary gland cells

    Fernanda F Rocha


    Full Text Available In the present paper, we developed a primary culture of Rhodnius prolixus salivary gland and main salivary canal cells. Cells remained viable in culture for 30 days. Three types of cells were indentified in the salivary gland cultures, with binuclear cells being the most abundant. The supernatants of salivary cultures contained mainly 16-24 kDa proteins and presented anticoagulant and apyrase activities. Secretion vesicles were observed budding from the cellular monolayer of the main salivary canal cells. These results indicate that R. prolixus salivary proteins may be produced in vitro and suggest that the main salivary canal may have a possible secretory role.

  20. Male Pelvic Squamous Cell Carcinoma of Unknown Primary Origin

    Lauren Chiec


    Full Text Available Pelvic squamous cell carcinoma of unknown primary origin has been described in several case reports of female patients. However, there have been no published reports describing male patients with pelvic squamous cell cancer of unknown primary origin. Our case describes a 52-year-old man who presented with right buttock pain, rectal urgency, and constipation. His physical examination demonstrated tenderness to palpation around his gluteal folds. Computed tomography scan of his abdomen and pelvis demonstrated a large mass in his retroperitoneum. The mass was determined to be squamous cell carcinoma of unknown primary origin. Additionally, the patient had small nodules in his right lower lung lobe and right hepatic lobe. The patient was treated with concomitant chemoradiation, including cisplatin and intensity-modulated radiation therapy, followed by carboplatin and paclitaxel. The patient achieved partial remission, in which he remained one year after his presentation. Our case is consistent with the literature which suggests that squamous cell carcinoma of unknown primary origin occurring outside of the head and neck region may have a more favorable prognosis than other carcinomas of unknown primary origin. Further studies are necessary to determine the most appropriate work-up, diagnosis, and optimal treatment strategies.

  1. Primary cutaneous blastoid mantle cell lymphoma-case report.

    Estrozi, Bruna; Sanches, José A; Varela, Paulo C S; Bacchi, Carlos E


    Mantle cell lymphoma (MCL) commonly involves extranodal sites, usually as a manifestation of disseminated disease. In rare cases, MCLs may arise as a primary tumor in the skin. Blastoid mantle cell lymphoma (BV-MCL) is a rare variant and has a more aggressive clinical course. The phenotype of BV-MCL is characterized as CD20+, CD5+, cyclin D1+, CD23-, and CD10-. Interphase fluorescence in situ hybridization shows a characteristic t(11;14) fusion pattern. We report a case of a BV-MCL arising in skin as primary cutaneous MCL with the characteristic immunophenotype and translocation.

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

    Richter, W W; Issinger, O G


    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...

  3. Primary small cell carcinoma of skin. Histogenetical study.

    Cachaza, J A; Garcia del Moral, R; López Caballero, J; Caracuel Ruiz, M; Caballero Morales, T


    Four cases of primary small cell carcinoma of the skin (PSCCS) are presented (ages ranging from 60 to 68 years). Ultrastructurally, two cell types were identified, with both presenting electron-dense secretory granules and paranuclear intermediate filaments. Argyrophylia was positive in one case. Intense solar elastosis in two cases and actinic keratosis in one case suggest a possible role from solar damage in the pathogenesis of this tumor. According to comparative ultrastructural features, different histogenetic possibilities in Merkel cells (MC), peripheral neuroblastic tissue, and totipotential cells are discussed. Some neurosecretory-like granules were observed in basal cell carcinoma (BCC). We consider that PSCCS reproduces cells similar to MC and probably originates in stem cells with totipotential capacity.

  4. Gene transfection in primary stem-like cells of giant cell tumor of bone.

    Singh, Shalini; Mak, Isabella; Power, Patricia; Cunningham, Melissa; Cunnigham, Melissa; Turcotte, Robert; Ghert, Michelle


    The neoplastic stem-like stromal cell of giant cell tumor of bone (GCT) survives for multiple passages in primary culture with a stable phenotype, and exhibits multipotent characteristics. The pathophysiology of this tumor has been studied through the primary culture of these cells. However, successful gene transfer of these cells has not been reported to date. In this short report, we describe the development of the first reported technique that results in efficient gene transfection in primary stem-like cells of GCT.

  5. Uptake of gold nanoparticles in primary human endothelial cells

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


    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....... Uptake of AuNPs in HUVECs occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures in the form of single particles and agglomerates of 2–3 particles....

  6. The Primary Cilium in Cell Signaling and Cancer

    Michaud III, Edward J [ORNL; Yoder, Bradley [University of Alabama, Birmingham


    The primary cilium is a microtubule-based antenna-like structure that emanates from the surface of virtually all cells in the mammalian body. It is anchored to the cell by the basal body, which develops from the mother centriole of the centrosome in a manner that is coordinately regulated with the cell cycle. The primary cilium is a sensory organelle that receives both mechanical and chemical signals from other cells and the environment, and transmits these signals to the nucleus to elicit a cellular response. Recent studies revealed that multiple components of the Sonic hedgehog and plateletderived growth factor receptor-A signal transduction pathways localize to the primary cilium, and that loss of the cilium blocks ligand-induced signaling by both pathways. In light of the major role that these pathways play in numerous types of cancer, we anticipate that the emerging discoveries being made about the function of the primary cilium in signaling pathways that are critical for embryonic development and tissue homeostasis in adults will also provide novel insights into the molecular mechanisms of carcinogenesis. (Cancer Res 2006; 66 13): 6463-7)

  7. Pulp tissue from primary teeth: new source of stem cells

    Paloma Dias Telles


    Full Text Available SHED (stem cells from human exfoliated deciduous teeth represent a population of postnatal stem cells capable of extensive proliferation and multipotential differentiation. Primary teeth may be an ideal source of postnatal stem cells to regenerate tooth structures and bone, and possibly to treat neural tissue injury or degenerative diseases. SHED are highly proliferative cells derived from an accessible tissue source, and therefore hold potential for providing enough cells for clinical applications. In this review, we describe the current knowledge about dental pulp stem cells and discuss tissue engineering approaches that use SHED to replace irreversibly inflamed or necrotic pulps with a healthy and functionally competent tissue that is capable of forming new dentin.

  8. Clear cell sarcoma: A case mimicking primary cutaneous malignant melanoma

    Rodriguez-Martin M


    Full Text Available Clear cell sarcoma (CCS is a recently described variant of sarcoma characterized by prominent clear cells showing features similar to clear cell melanoma. This neoplasm was first described by Dr. Franz M. Erzinger. Primary CCS usually arises in deeper soft tissues, in association with fascia, tendons, or aponeuroses. Characteristic translocation t(12;22 (q13;q12 has been considered pathognomonic for CCS. Prognosis is related to tumor size. An early recognition and initial radical surgery is the key to a favourable outcome. We present a patient with an unusual neoplasm that resembled malignant melanoma.


    Samanta DR, Bose Chaitali, Panda Sasmita, Upadhaya Ashis, Das Abhijit, Senapati SN


    Full Text Available Primary squamous cell carcinoma of renal pelvis is rare clinical entity with only few cases have been reported in the literature. It is usually associated with long standing renal calculi. Insidious onset of symptom and inconclusive clinical and radiological features leads to locally advanced or metastatic disease at presentation; resulting in poor prognosis. Here we are reporting two cases of squamous cell carcinoma of kidney having renal calculi to highlight its clinical presentation and to document the association of squamous cell carcinoma in longstanding nephrolithiasis due to its rarity.

  10. Single-cell qPCR on dispersed primary pituitary cells -an optimized protocol

    Haug Trude M


    Full Text Available Abstract Background The incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries. Results Harvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency. Conclusion Depending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.

  11. Primary cells and stem cells in drug discovery: emerging tools for high-throughput screening.

    Eglen, Richard; Reisine, Terry


    Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest. Furthermore, stem cells, of either embryonic or adult origin, as well as those derived from differentiated cells, are now finding a place in drug discovery. Collectively, these cells are expanding the utility of authentic human cells, either as screening tools or as therapeutics, as well as providing cells derived directly from patients. Nonetheless, the growing use of phenotypically relevant cells (including primary cells or stem cells) is not without technical difficulties, particularly when their envisioned use lies in high-throughput screening (HTS) protocols. In particular, the limited availability of homogeneous primary or stem cell populations for HTS mandates that novel technologies be developed to accelerate their adoption. These technologies include detection of responses with very few cells as well as protocols to generate cell lines in abundant, homogeneous populations. In parallel, the growing use of changes in cell phenotype as the assay readout is driving greater use of high-throughput imaging techniques in screening. Taken together, the greater availability of novel primary and stem cell phenotypes as well as new detection technologies is heralding a new era of cellular screening. This convergence offers unique opportunities to identify drug candidates for disorders at which few therapeutics are presently available.

  12. Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells.

    Kassmer, Susannah H; Bruscia, Emanuela M; Zhang, Ping-Xia; Krause, Diane S


    Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.

  13. Primary Non-Hodgkin B Cell Lymphoma in a Man

    Sh.M.I. Alhabshi


    Full Text Available Malignant breast lymphoma is a rare condition and primary breast lymphoma is extremely rare in"nthe male population. We present a case of a 26-year-old man (transgender who presented with a large palpable mass in the right breast. This mass was rapidly growing in size associated with right axillary lymphadenopathy. Ultrasound and MRI findings were consistent with BIRADS IV lesion which was suspicious of malignancy. Core biopsy was performed and histopathology confirmed the diagnosis of primary non Hodgkin B cell lymphoma of the breast.

  14. Primary antitumor immune response mediated by CD4+ T cells.

    Corthay, Alexandre; Skovseth, Dag K; Lundin, Katrin U; Røsjø, Egil; Omholt, Hilde; Hofgaard, Peter O; Haraldsen, Guttorm; Bogen, Bjarne


    Gene-targeted mice have recently revealed a role for lymphocytes and interferon-gamma (IFNgamma) in conferring protection against cancer, but the mechanisms remain unclear. Here, we have characterized a successful primary antitumor immune response initiated by naive CD4+ T cells. Major histocompatibility complex class II (MHC-II)-negative myeloma cells injected subcutaneously into syngeneic mice were surrounded within 3 days by macrophages that captured tumor antigens. Within 6 days, naive myeloma-specific CD4+ T cells became activated in draining lymph nodes and subsequently migrated to the incipient tumor site. Upon recognition of tumor-derived antigenic peptides presented on MHC-II by macrophages, the myeloma-specific CD4+ T cells were reactivated and started to secrete cytokines. T cell-derived IFNgamma activated macrophages in close proximity to the tumor cells. Tumor cell growth was completely inhibited by such locally activated macrophages. These data indicate a mechanism for immunosurveillance of MHC-II-negative cancer cells by tumor-specific CD4+ T cells through collaboration with macrophages.




    Full Text Available A 38-year-old female presented with a history of progressively enlarging abdominal mass. Abdominal computed tomography showed a pelvic mass involving both the ovaries and omentum. CA-125 was normal. Staging surgery was performed and the histopathological diagnosis of Transitional Cell Carcinoma was made and later confirmed by immuno-histochemistry. Transitional cell carcinoma of the ovary is a rare subtype of epithelial ovarian cancer. Surgical resection is the primary therapeutic approach, and patient’s outcomes after chemotherapy are better than for other types of ovarian cancers.

  16. Primary T-cell lymphoblastic lymphoma in the middle ear.

    Li, Bo; Liu, Shixi; Yang, Hui; Wang, Weiya


    T-cell lymphoblastic lymphoma (T-LBL) is a highly aggressive lymphoma characterized by precursor T-cell malignancy and lymphadenopathy or mediastinal involvement. We present the case of an 11-year-old boy with a diagnosis of middle ear T-LBL, which manifested as a headache, hearing loss and peripheral facial paralysis. The child was given intensive chemotherapy and had a complete response. To our knowledge, this is the first case reported in the literature of T-LBL originating in the middle ear. This case aims to help clinicians to be vigilant about the possibility of primary lesions at atypical sites in some special diseases.

  17. Primary Cell Wall Structure in the Evolution of Land Plants


    Investigation of the primary cell walls of lower plants improves our understanding of the cell biology of these organisms but also has the potential to improve our understanding of cell wall structure and function in angiosperms that evolved from lower plants. Cell walls were prepared from eight species, ranging from a moss to advanced gymnosperms, and subjected to sequential chemical extraction to separate the main polysaccharide fractions. The glycosyl compositions of these fractions were then determined by gas chromatography. The results were compared among the eight plants and among data from related studies reported in the existing published reports to identify structural features that have been either highly conserved or clearly modified during evolution. Among the highly conserved features are the presence of a cellulose framework, the presence of certain hemicelluloses such as xyloglucan, and the presence of rhamnogalacturonan Ⅱ, a domain in pectic polysaccharides. Among the modified features are the abundance of mannosyl-containing hemicelluloses and the presence of methylated sugars.

  18. A case of Primary Bone Anaplastic Large Cell Lymphoma

    Kim, Kyung Hyun; Jung, Yun Hwa; Han, Chi Wha; Woo, In Sook; Son, Jong ho


    Patient: Female, 52 Final Diagnosis: Primary bone anaplastic large cell lymphoma Symptoms: Bone pain Medication: — Clinical Procedure: — Specialty: Oncology Objective: Unusual clinical course Background: Anaplastic large cell lymphoma (ALCL) is a relatively rare subtype of non-Hodgkin’s lymphoma (NHL). Like other types of NHL, ALCL primarily involves the nodal area, and sometimes it can involve several extra-nodal sites such as skin, soft tissue, and lungs. However, extensive bone involvement in cases of ALCL is very rare whether it is primary or secondary. Without nodular involvement, ALCL can be misdiagnosed as bone tumor or metastatic carcinoma such as lung, breast, or prostate cancer, which frequently spread to bone. Case Report: A 52-year-old woman with generalized pain and 2 months of fever of unknown origin presented to our institution. After extensive evaluation, only multiple osteolytic bone lesions with periosteal soft tissue reaction were identified. Repeated core needle biopsy revealed only inflammatory cells with histiocytic reactions. After pathologic and chromosomal analysis of sufficient tissue, which was acquired from incisional biopsy, primary bone ALCL was confirmed. Conclusions: Clinicians should keep in mind that ALCL can present with extensive bone involvement without nodal involvement. PMID:27729639

  19. Erythropoietin (EPO-receptor signaling induces cell death of primary myeloma cells in vitro

    Thea Kristin Våtsveen


    Full Text Available Abstract Background Multiple myeloma is an incurable complex disease characterized by clonal proliferation of malignant plasma cells in a hypoxic bone marrow environment. Hypoxia-dependent erythropoietin (EPO-receptor (EPOR signaling is central in various cancers, but the relevance of EPOR signaling in multiple myeloma cells has not yet been thoroughly investigated. Methods Myeloma cell lines and malignant plasma cells isolated from bone marrow of myeloma patients were used in this study. Transcript levels were analysed by quantitative PCR and cell surface levels of EPOR in primary cells by flow cytometry. Knockdown of EPOR by short interfering RNA was used to show specific EPOR signaling in the myeloma cell line INA-6. Flow cytometry was used to assess viability in primary cells treated with EPO in the presence and absence of neutralizing anti-EPOR antibodies. Gene expression data for total therapy 2 (TT2, total therapy 3A (TT3A trials and APEX 039 and 040 were retrieved from NIH GEO omnibus and EBI ArrayExpress. Results We show that the EPOR is expressed in myeloma cell lines and in primary myeloma cells both at the mRNA and protein level. Exposure to recombinant human EPO (rhEPO reduced viability of INA-6 myeloma cell line and of primary myeloma cells. This effect could be partially reversed by neutralizing antibodies against EPOR. In INA-6 cells and primary myeloma cells, janus kinase 2 (JAK-2 and extracellular signal regulated kinase 1 and 2 (ERK-1/2 were phosphorylated by rhEPO treatment. Knockdown of EPOR expression in INA-6 cells reduced rhEPO-induced phospo-JAK-2 and phospho-ERK-1/2. Co-cultures of primary myeloma cells with bone marrow-derived stroma cells did not protect the myeloma cells from rhEPO-induced cell death. In four different clinical trials, survival data linked to gene expression analysis indicated that high levels of EPOR mRNA were associated with better survival. Conclusions Our results demonstrate for the first time

  20. Erythropoietin (EPO)-receptor signaling induces cell death of primary myeloma cells in vitro.

    Våtsveen, Thea Kristin; Sponaas, Anne-Marit; Tian, Erming; Zhang, Qing; Misund, Kristine; Sundan, Anders; Børset, Magne; Waage, Anders; Brede, Gaute


    Multiple myeloma is an incurable complex disease characterized by clonal proliferation of malignant plasma cells in a hypoxic bone marrow environment. Hypoxia-dependent erythropoietin (EPO)-receptor (EPOR) signaling is central in various cancers, but the relevance of EPOR signaling in multiple myeloma cells has not yet been thoroughly investigated. Myeloma cell lines and malignant plasma cells isolated from bone marrow of myeloma patients were used in this study. Transcript levels were analysed by quantitative PCR and cell surface levels of EPOR in primary cells by flow cytometry. Knockdown of EPOR by short interfering RNA was used to show specific EPOR signaling in the myeloma cell line INA-6. Flow cytometry was used to assess viability in primary cells treated with EPO in the presence and absence of neutralizing anti-EPOR antibodies. Gene expression data for total therapy 2 (TT2), total therapy 3A (TT3A) trials and APEX 039 and 040 were retrieved from NIH GEO omnibus and EBI ArrayExpress. We show that the EPOR is expressed in myeloma cell lines and in primary myeloma cells both at the mRNA and protein level. Exposure to recombinant human EPO (rhEPO) reduced viability of INA-6 myeloma cell line and of primary myeloma cells. This effect could be partially reversed by neutralizing antibodies against EPOR. In INA-6 cells and primary myeloma cells, janus kinase 2 (JAK-2) and extracellular signal regulated kinase 1 and 2 (ERK-1/2) were phosphorylated by rhEPO treatment. Knockdown of EPOR expression in INA-6 cells reduced rhEPO-induced phospo-JAK-2 and phospho-ERK-1/2. Co-cultures of primary myeloma cells with bone marrow-derived stroma cells did not protect the myeloma cells from rhEPO-induced cell death. In four different clinical trials, survival data linked to gene expression analysis indicated that high levels of EPOR mRNA were associated with better survival. Our results demonstrate for the first time active EPOR signaling in malignant plasma cells. EPO

  1. Mast cells and angiogenesis in primary and recurrent pterygia

    Fatma Hüsniye DİLEK


    Full Text Available Pterygium is a common benign lesion of limbus but the pathogenesis are not completely understood. Pterygia have a chronic inflammatory cellular infiltrate and a rich vasculature. Mast cells are a heterogeneous group of multifunctional tissue-resident cells. It has been suggested that mast cells and their products may be responsible for the formation of new blood vessels. We investigated the number and phenotype of mast cells and neovascularization in pterygia specimens and compared with those in normal conjunctival specimensPterygia tissues were obtained during excisional surgery from 32 eyes of 32 consecutive patients. Seventeen of all cases were recurrent pterygia. Superior bulbar conjunctival tissue from the same eye was also sampled as control tissues. The tissue sections were stained with routine hematoxyline-eosin and toluidine blue stain for mast cells. For immunohistochemical studies anti-factor VIII-related antigen, monoclonal anti human mast cell tryptase and chymase were used as an endothelial and mast cell marker.The mean number of mast cells in pterygia was significantly higher than that in the normal conjunctival tissue and microvessel counts was significantly higher than the counts of the controls in both primary and recurrent pterygia. There was no correlation between microvessel numbers and mast cell numbers. There was no phenotypic difference between the mast cells in the ptergyia and those in the normal conjunctival tissues.This study confirms that mast cells are prominent in pterygia and our results suggest that mast cells and angiogenesis are independent factors in the genesis and progress of ptergyium.

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

    Xie, Sijia; Lüttge, Regina


    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

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

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


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

  4. Primary cerebellar extramedullary myeloid cell tumor mimicking oligodendroglioma.

    Ho, D M; Wong, T T; Guo, W Y; Chang, K P; Yen, S H


    Extramedullary myeloid cell tumors (EMCTs) are tumors consisting of immature cells of the myeloid series that occur outside the bone marrow. Most of them are associated with acute myelogenous leukemia or other myeloproliferative disorders, and a small number occur as primary lesions, i.e., are not associated with hematological disorders. Occurrence inside the cranium is rare, and there has been only one case of primary EMCT involving the cerebellum reported in the literature. The case we report here is a blastic EMCT occurring in the cerebellum of a 3-year-old boy who had no signs of leukemia or any hematological disorder throughout the entire course. The cerebellar tumor was at first misdiagnosed as an "oligodendroglioma" because of the uniformity and "fried egg" artifact of the tumor cells. The tumor disappeared during chemotherapy consisting of 12 treatments. However, it recurred and metastasized to the cerebrospinal fluid (CSF) shortly after the therapy was completed. A diagnosis of EMCT was suspected because of the presence of immature myeloid cells in the CSF, and was confirmed by anti-myeloperoxidase and anti-lysozyme immunoreactivity of the cerebellar tumor. The patient succumbed 1 year and 3 months after the first presentation of the disease.

  5. Highly efficient baculovirus-mediated multigene delivery in primary cells

    Mansouri, Maysam; Bellon-Echeverria, Itxaso; Rizk, Aurélien; Ehsaei, Zahra; Cianciolo Cosentino, Chiara; Silva, Catarina S.; Xie, Ye; Boyce, Frederick M.; Davis, M. Wayne; Neuhauss, Stephan C. F.; Taylor, Verdon; Ballmer-Hofer, Kurt; Berger, Imre; Berger, Philipp


    Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells. PMID:27143231

  6. Cell-mediated infection of cervix derived epithelial cells with primary isolates of human immunodeficiency virus.

    Tan, X; Phillips, D M


    We have previously demonstrated that HIV-infected transformed T-cells or monocytes adhere to monolayers of CD4-negative epithelial cells. Adhesion is soon followed by budding of HIV from infected mononuclear cells onto the surface of epithelial cells. Epithelial cells subsequently take up virus and become productively infected. Based on these findings, we proposed that sexual transmission of HIV may involve cell-mediated infection of intact mucosal epithelia of the urogenital tract. However, it has become increasingly clear that primary cells and HIV strains isolated from patients are more appropriate models for HIV infection than established cell lines and lab strains of virus. In the studies described here, we infected cervix-derived epithelial monolayers with primary monocytes infected with patient isolates of non-syncytial inducing (NSI) macrophage-tropic strains of HIV. Under the culture conditions employed, HIV-infected primary monocytes do not remain adherent to the apical surface of the epithelium, as did HIV-infected transformed cells. Instead, following adherence, the primary cells migrate between epithelial cells. Virus is secreted from a pseudopod as HIV-infected primary monocytes pass between cells of the epithelium. Productive infection of the epithelium was detected by p24 ELISA and PCR Southern blot analysis. Infection can be blocked by sera from HIV-seropositive individuals or by certain sulfated polysaccharides. These findings support the supposition that transmission of HIV may occur via cell-mediated infection of intact epithelia. The observations also hint at the possibility that-HIV-infected monocyte/macrophages in semen or cervical-vaginal secretions could cross intact epithelia by passing between epithelial cells. Blocking studies suggest that it may be possible to inhibit sexual transmission of HIV either by antibodies in genital tract secretions or by a topical formulation containing certain sulfated polysaccharides.

  7. Docosahexaenoic acid induces apoptosis in primary chronic lymphocytic leukemia cells

    Romain Guièze


    Full Text Available Chronic lymphocytic leukemia is an indolent disorder with an increased infectious risk remaining one of the main causes of death. Development of therapies with higher safety profile is thus a challenging issue. Docosahexaenoic acid (DHA, 22:6 is an omega-3 fatty acid, a natural compound of normal cells, and has been shown to display antitumor potency in cancer. We evaluated the potential in vitro effect of DHA in primary CLL cells. DHA induces high level of in vitro apoptosis compared to oleic acid in a dose-dependent and time-dependent manner. Estimation of IC50 was only of 4.813 μM, which appears lower than those reported in solid cancers. DHA is highly active on CLL cells in vitro. This observation provides a rationale for further studies aiming to understand its mechanisms of action and its potent in vivo activity.

  8. Primary cutaneous B-cell lymphoma: Role of surgery

    Parbhakar, Sam; Cin, Arianna Dal


    PURPOSE: To evaluate the role of surgery in patients diagnosed with primary cutaneous B-cell lymphoma (PCBCL) – a rare disease entity. The authors offer a rationale for the use of primary surgical excision in the treatment of isolated cutaneous lymphomas. METHODS: A literature review examining the use of primary surgical excision in the treatment of PCBCL was conducted. The lymphoma database at the Juravinski Cancer Centre (Hamilton, Ontario) was searched from January 1995 to July 2008, generating a list of 4924 patients. A simulated computer program was subsequently designed to search for all possible PCBCLs. A retrospective chart review was then conducted on the new list of 1325 patients, identifying 25 patients diagnosed with PCBCL. RESULTS: The mean age of the 25 patients with PCBCL was 59.9 years; nine (36%) were treated with surgery, and sixteen (64%) with radiation. The average follow-up period for patients was 3.6 years. Twenty-four of the 25 patients were completely cured, with only one patient recurring in the radiation subgroup. There were no complications in the surgery subgroup. There were two local complications in the radiation subgroup consisting of chronic ulcerations. CONCLUSIONS: Primary surgical excision is an effective management option in the treatment of PCBCL, particularly the marginal zone and follicle centre subtypes. PMID:22654537

  9. Primary retroperitoneal Merkel cell carcinoma: Case report and literature review

    Quiroz-Sandoval, Osvaldo A.; Cuellar-Hubbe, Mario; Lino-Silva, Leonardo S.; Salcedo-Hernández, Rosa A.; López-Basave, Horacio N.; Padilla-Rosciano, Alejandro E.; León-Takahashi, Alberto M.; Herrera-Gómez, Ángel


    Background Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma that affects elderly patients and typically arises in sun-exposed skin. The disease is very rare and only few cases present with no apparent skin lesion. In the retroperitoneum there are only two cases reported in the literature. Case presentation We report a case of a 54-year-old Mexican male with MCC, which presented as a large retroperitoneal mass. Pathological and immunohistochemical analysis of the transabdominal CT-guided biopsy specimen revealed a MCC. The patient underwent preoperative chemotherapy followed by a laparotomy and the mass was successfully excised. Discussion There are two possible explanations for what occurred in our patient. The most plausible theory is the retroperitoneal mass could be a massively enlarged lymph node where precursor cells became neoplastic. This would be consistent with a presumptive diagnosis of primary nodal disease. Moreover, metastasis to the retroperitoneal lymph nodes has been reported as relatively common when compared to other sites such as liver, bone, brain and skin. The less probable theory is the non-described “regression” phenomena of a cutaneous MCC, but we are not found a primary skin lesion. Conclusion Preoperative chemotherapy and excision of the primary tumor is the surgical treatment of choice for retroperitoneal MCC. We propose that further studies are needed to elucidate the true efficacy of chemotherapy in conventional and unconventional patients with MCC. PMID:26708276

  10. Second Unrelated Donor Hematopoietic Cell Transplantation for Primary Graft Failure

    Schriber, Jeffrey; Agovi, Manza-A.; Ho, Vincent; Ballen, Karen K.; Bacigalupo, Andrea; Lazarus, Hillard M.; Bredeson, Christopher N.; Gupta, Vikas; Maziarz, Richard T.; Hale, Gregory A.; Litzow, Mark R.; Logan, Brent; Bornhauser, Martin; Giller, Roger H.; Isola, Luis; Marks, David I.; Rizzo, J. Douglas; Pasquini, Marcelo C.


    Failure to engraft donor cells is a devastating complication after allogeneic hematopoietic cell transplantation (HCT). We describe the results of 122 patients reported to the National Marrow Donor Program between 1990 and 2005, who received a second unrelated donor HCT after failing to achieve an absolute neutrophil count of ≥ 500/ μL without recurrent disease. Patients were transplanted for leukemia (n=83), myelodysplastic disorders (n=16), severe aplastic anemia (n=20) and other diseases (n=3). The median age was 29 years. Twenty-four patients received second grafts from a different unrelated donor. Among 98 patients who received a second graft from the same donor, 28 received products that were previously collected and cryopreserved for the first transplantation. One-year overall survival after second transplant was 11% with 10 patients alive at last follow up. We observed no differences between patients who received grafts from the same or different donors, or in those who received fresh or cryopreserved product. The outcomes after a second allogeneic HCT for primary graft failure are dismal. Identifying risk factors for primary graft failure can decrease the incidence of this complication. Further studies are needed to test whether early recognition and hastened procurement of alternative grafts can improve transplant outcomes for primary graft failure. PMID:20172038

  11. Oncogenic NRAS Primes Primary Acute Myeloid Leukemia Cells for Differentiation.

    Cornelia Brendel

    Full Text Available RAS mutations are frequently found among acute myeloid leukemia patients (AML, generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1 in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC driven differentiation. Taken together, our findings show that AML with inv(16 and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.

  12. Experiments on Gene Transferring to Primary Hematopoietic Cells by Liposome


    Liposomes have showed many advantages in mediating exogenous gene into many cell types in vitro and in vivo. But few data are available concerning gene transfer into hematopoietic cells. In this report, we described two-marker genes (Neo R and Lac Z) co-transferred into hematopoietic cells of human and mouse by using liposome in vitro. The efficiency of gene transfer was tested by Xgal staining and observation of colony formation. The X-gal blue staining rate of transduced cells was about (13.33±2. 68) % in human and about (16. 28±2.95) % in mouse without G418 selection. After G418 selection, the blue cell rate was (46. 06±3.47)%in human and (43. 45±4. 1) % in mouse, which were markedly higher than those before selection, suggesting that high-efficiency gene transfer and expression could be attained in primary hematopoietic cells using this easy and harmless transduction protocol. At the same time, this protocol provided experimental data for clinicians to investigate the biology of marrow reconstitution and trace the origin of relapse after autologous bone marrow transplantation for the patients with leukemia.

  13. Primary desmoplastic small round cell tumor of the femur

    Yoshida, Akihiko; Garcia, Joaquin [Memorial Sloan-Kettering Cancer Center, Department of Pathology, New York, NY (United States); Edgar, Mark A. [Memorial Sloan-Kettering Cancer Center, Department of Pathology, New York, NY (United States); Weill Medical College of Cornell University, New York, NY (United States); Meyers, Paul A. [Weill Medical College of Cornell University, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Department of Pediatrics, New York, NY (United States); Morris, Carol D. [Weill Medical College of Cornell University, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Department of Surgery, Orthopaedic Service, New York, NY (United States); Panicek, David M. [Weill Medical College of Cornell University, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States)


    Desmoplastic small round cell tumor (DSRCT) is a rare malignant neoplasm typically involving the abdominal cavity of a young male. Extra-abdominal occurrence of this tumor is very rare. We report a 10-year-old girl with primary DSRCT arising within the left femur. The patient presented with knee pain, and radiological findings were strongly suggestive of osteogenic sarcoma. In addition to the typical microscopic appearance and immunophenotype, RT-PCR demonstrated the chimeric transcript of EWS-WT1, which is diagnostic of DSRCT. Pulmonary metastases were present at initial staging studies, but no abdominal or pelvic lesion was present. Despite chemotherapy and complete tumor excision, the patient developed progressive lung and bone metastases and died 3 years after initial presentation. This is the second reported case of primary DSRCT of bone with genetic confirmation. (orig.)

  14. Stem Cell Transplantation for Treatment of Primary Immunodeficiency Disorders

    Susanna M. Müller Wilhelm Friedrich


    Full Text Available Primary Immunodeficiencies constitute a group of highly complex congenital disorders most of which are characterized by a very poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT has become an established curative treatment approach in many of these disorders, which may be permanently corrected. In this presentation basic and practical aspects of HSCT are presented, with an emphasis on its application in lymphocyte disorders such as severe combined immunodeficiency (SCID. Optimal results and outcome of HSCT are highly dependant on early and correct diagnosis of these rare disorders, and HSCT should usually be applied early in the course of the disease in order to prevent irreversible complications from infections. Clinical results will be summarized based on recent analysis performed in large patient cohorts, which have shown steady improvements and have led to a marked change in the prognosis of patients with primary immunodeficiencies.

  15. Cell longevity and sustained primary growth in palm stems.

    Tomlinson, P Barry; Huggett, Brett A


    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.

  16. Attachment of human primary osteoblast cells to modified polyethylene surfaces.

    Poulsson, Alexandra H C; Mitchell, Stephen A; Davidson, Marcus R; Johnstone, Alan J; Emmison, Neil; Bradley, Robert H


    Ultra-high-molecular-weight polyethylene (UHMWPE) has a long history of use in medical devices, primarily for articulating surfaces due to its inherent low surface energy which limits tissue integration. To widen the applications of UHMWPE, the surface energy can be increased. The increase in surface energy would improve the adsorption of proteins and attachment of cells to allow tissue integration, thereby allowing UHMWPE to potentially be used for a wider range of implants. The attachment and function of human primary osteoblast-like (HOB) cells to surfaces of UHMWPE with various levels of incorporated surface oxygen have been investigated. The surface modification of the UHMWPE was produced by exposure to a UV/ozone treatment. The resulting surface chemistry was studied using X-ray photoelectron spectroscopy (XPS), and the topography and surface structure were probed by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which showed an increase in surface oxygen from 11 to 26 atom % with no significant change to the surface topography. The absolute root mean square roughness of both untreated and UV/ozone-treated surfaces was within 350-450 nm, and the water contact angles decreased with increasing oxygen incorporation, i.e., showing an increase in surface hydrophilicity. Cell attachment and functionality were assessed over a 21 day period for each cell-surface combination studied; these were performed using SEM and the alamarBlue assay to study cell attachment and proliferation and energy-dispersive X-ray (EDX) analysis to confirm extracellular mineral deposits, and total protein assay to examine the intra- and extracellular protein expressed by the cells. HOB cells cultured for 21 days on the modified UHMWPE surfaces with 19 and 26 atom % oxygen incorporated showed significantly higher cell densities compared to cells cultured on tissue culture polystyrene (TCPS) from day 3 onward. This indicated that the cells attached and proliferated more

  17. Lacrimal gland primary acinar cell culture: the role of insulin

    Leonardo Tannus Malki


    Full Text Available ABSTRACT Purpose: The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs and to assess the effect of adding insulin to the culture media. Methods: LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL. Results: In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity. Conclusions: The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture.

  18. Primary anaplastic large T cell lymphoma of central nervous system

    ZHANG Yan


    Full Text Available Background Primary anaplastic large T cell lymphoma (ALCL of central nervous system (CNS can occur in people of all ages, and is usually unrelated with immunodeficiency. It is often misdiagnosed as meningitis, especially tuberculous meningitis, on clinical practice and imaging examination. In pathological diagnosis, the morphological changes of primary ALCL of CNS are similar to the systemic ALCL and the anaplastic lymphoma kinase-1 (ALK-1 can be positive or negative. Being misdiagnosed as meningitis, hormone therapy with glucocorticoid before biopsy is always used, and massive necrosis and a lot of histocyte proliferation and phagocytosis can be found under histological findings. Therefore, when the material is not enough, primary ALCL of CNS is often misdiagnosed as cerebral infarction or malignant histocytosis and so on. This paper reports a case of primary ALCL of CNS and makes a review of relevant literature, so as to summarize the clinical manifestations and elevate the recognition of clinicians and pathologists on this disease. Methods and Results A 12-year-old boy was admitted because of fever, worsening headache, numbness and weakness of right limbs. MRI showed local gyri swelling and abnormal enhancement of pia mater in the right parietal lobe, expanding to the right temporal lobe, and pia mater enhancement in the left parietal lobe. The right temporo-parietal lobe lesion biopsy revealed irregularly shaped tumor cells of large size, rich and eosinophilic cytoplasm and horseshoe-shaped or kidney-shaped nuclei. Immunohistochemical examination showed tumor cells positive for CD3, CD45RO, CD30, ALK-1 and epithelial membrane antigen (EMA, and negative for CD20 and CD79a. Conclusion Primary ALCL of CNS is an extremely rare tumor which is usually misdiagnosed as meningitis according to clinical and imaging examinations. Therefore, for those patients who are considered as meningitis but with poor treatment effect and replase of illness, brain

  19. Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors

    Kajimura, Junko; Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Weng, Nan-ping; Young, Lauren F.; Shieh, Jae-Hung; Moore, Malcolm A.; van den Brink, Marcel R.M.; Kusunoki, Yoichiro


    Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34 + Lin−) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure. PMID:27169377

  20. Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors.

    Kajimura, Junko; Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Weng, Nan-Ping; Young, Lauren F; Shieh, Jae-Hung; Moore, Malcolm A; van den Brink, Marcel R M; Kusunoki, Yoichiro


    Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AX (γH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. A proteome map of primary cultured rat Schwann cells

    Shen Mi


    Full Text Available Abstract Background Schwann cells (SCs are the principal glial cells of the peripheral nervous system with a wide range of biological functions. SCs play a key role in peripheral nerve regeneration and are involved in several hereditary peripheral neuropathies. The objective of this study was to gain new insight into the whole protein composition of SCs. Results Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS was performed to identify the protein expressions in primary cultured SCs of rats. We identified a total of 1,232 proteins, which were categorized into 20 functional classes. We also used quantitative real time RT-PCR and Western blot analysis to validate some of proteomics-identified proteins. Conclusion We showed for the first time the proteome map of SCs. Our data could serve as a reference library to provide basic information for understanding SC biology.

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  1. Melleolides induce rapid cell death in human primary monocytes and cancer cells.

    Bohnert, Markus; Scherer, Olga; Wiechmann, Katja; König, Stefanie; Dahse, Hans-Martin; Hoffmeister, Dirk; Werz, Oliver


    The melleolides are structurally unique and bioactive natural products of the basidiomycete genus Armillaria. Here, we report on cytotoxic effects of melleolides from Armillaria mellea towards non-transformed human primary monocytes and human cancer cell lines, respectively. In contrast to staurosporine or pretubulysin that are less cytotoxic for monocytes, the cytotoxic potency of the active melleolides in primary monocytes is comparable to that in cancer cells. The onset of the cytotoxic effects of melleolides was rapid (within 5 h, each). Side-by-side comparison with the detergent triton X-100 and staurosporine in microscopic and flow cytometric analysis studies as well as analysis of the viability of mitochondria exclude cell lysis and apoptosis as relevant or primary mechanisms. Our results rather point to necrotic features of cell death mediated by an as yet elusive but rapid mechanism. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions.

    Hegde, Shrilakshmi; Gabriel, Cordula; Kragl, Martin; Chopra-Dewasthaly, Rohini


    Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae's consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host-pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner.

  3. Cathode catalysts for primary phosphoric acid fuel cells


    Alkylation or carbon Vulcan XC-72, the support carbon, was shown to provide the most stable bond type for linking cobalt dehydrodibenzo tetraazannulene (CoTAA) to the surface of the carbon; this result is based on data obtained by cyclic voltammetry, pulse voltammetry and by release of 14C from bonded CoTAA. Half-cell tests at 100 C in 85% phosphoric acid showed that CoTAA bonded to the surface of carbon (Vulcan XC-72) via an alkylation procedure is a more active catalyst than is platinum based on a factor of two improvement in Tafel slope; dimeric CoTAA had catalytic activity equal to platinum. Half-cell tests also showed that bonded CoTAA catalysts do not suffer a loss in potential when air is used as a fuel rather than oxygen. Commercially available polytetrafluroethylene (PTFE) was shown to be unstable in the fuel cell environment with degradation occurring in 2000 hours or less. The PTFE was stressed at 200 C in concentrated phosphoric acid as well as electrochemically stressed in 150 C concentrated phosphoric acid; the surface chemistry of PTFE was observed to change significantly. Radiolabeled PTFE was prepared and used to verify that such chemical changes also occur in the primary fuel cell environment.

  4. Reference gene for primary culture of prostate cancer cells.

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele


    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  5. Are primary renal cell carcinoma and metastases of renal cell carcinoma the same cancer?

    Semeniuk-Wojtaś, Aleksandra; Stec, Rafał; Szczylik, Cezary


    Metastasis is a process consisting of cells spreading from the primary site of the cancer to distant parts of the body. Our understanding of this spread is limited and molecular mechanisms causing particular characteristics of metastasis are still unknown. There is some evidence that primary renal cell carcinoma (RCC) and metastases of RCC exhibit molecular differences that may effect on the biological characteristics of the tumor. Some authors have detected differences in clear cell and nonclear cell component between these 2 groups of tumors. Investigators have also determined that primary RCC and metastases of RCC diverge in their range of renal-specific markers and other protein expression, gene expression pattern, and microRNA expression. There are also certain proteins that are variously expressed in primary RCCs and their metastases and have effect on clinical outcome, e.g., endothelin receptor type B, phos-S6, and CD44. However, further studies are needed on large cohorts of patients to identify differences representing promising targets for prognostic purposes predicting disease-free survival and the metastatic burden of a patient as well as their suitability as potential therapeutic targets. To sum up, in this review we have attempted to summarize studies connected with differences between primary RCC and its metastases and their influence on the biological characteristics of renal cancer.

  6. Management of primary germ cell tumors of the mediastinum.

    Economou, J S; Trump, D L; Holmes, E C; Eggleston, J E


    Twenty-eight patients with primary malignant germ cell tumors (GCT) of the mediastinum were treated at the University of California at Los Angeles and The Johns Hopkins Hospital in the past 30 years. Of 11 patients with pure seminomas, nine (82%) are free of disease from 6 months to 15 years following therapy. The primary treatment modality in these patients was mediastinal radiation; one patient with metastatic disease had a complete remission and prolonged survival following combination chemotherapy. Seventeen patients had GCT with nonseminomatous elements. Only three (18%) are alive and free of disease. One patient treated only surgically is alive at 15 years and two patients treated with combination chemotherapy and operation are alive and free of disease at 6 months and 3 years. When analyzed by a Kaplan-Meier actuarial survival estimate, patients with nonseminomatous GCT who were treated with cisplatin-bleomycin-based chemotherapy had a median survival of 14.0 months whereas those treated with chemotherapy regimens not employing these agents had a median survival of 4.0 months (generalized Wilcoxon test, p = 0.0495). Patients with pure seminomas are effectively treated with radiation therapy. Patients with nonseminomatous tumors have a much poorer prognosis and deserve aggressive multimodality therapy with cisplatin-bleomycin-based chemotherapy.

  7. Hematopoietic stem cell transplantation for primary immunodeficiency diseases.

    Slatter, Mary A; Cant, Andrew J


    Hematopoietic stem cell transplantation (HSCT) is now highly successfully curing a widening range of primary immunodeficiencies (PIDs). Better tissue typing, matching of donors, less toxic chemotherapy, better virus detection and treatment, improved supportive care, and graft-versus-host disease prophylaxis mean up to a 90% cure for severe combined immunodeficiency patients and a 70-80% cure for other PIDs given a matched unrelated donor, and rising to 95% for young patients with specific PIDs, such as Wiskott-Aldrich syndrome. Precise molecular diagnosis, detailed data on prognosis, and careful pre-HSCT assessment of infective lung and liver damage will ensure an informed benefit analysis of HSCT and the best outcome. It is now recognized that the best treatment option for chronic granulomatous disease is HSCT, which can also be curative for CD40 ligand deficiency and complex immune dysregulation disorders.

  8. Primary orbital precursor T-cell lymphoblastic lymphoma

    Stenman, Lisa; Persson, Marta; Enlund, Fredrik


    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....... There was no involvement of the bone marrow. Based on the clinical and histopathological findings, a diagnosis of T-LBL was made. There was no evidence of NOTCH1 mutation or rearrangements of the ETV6 and MLL genes and high-resolution array-based comparative genomic hybridization (arrayCGH) analysis revealed a normal...... genomic profile. The patient received chemotherapy according to the high-risk NOPHO protocol, followed by myeloablative allogenic bone marrow transplantation. At 35 months after diagnosis, the patient remained in complete first remission, but without light perception on his left eye. To the best of our...

  9. Novel isolation strategy to deliver pure fetal-origin and maternal-origin mesenchymal stem cell (MSC) populations from human term placenta.

    Patel, J; Shafiee, A; Wang, W; Fisk, N M; Khosrotehrani, K


    The placenta is an abundant source of mesenchymal stem/stromal cells (MSC). Although presumed of translationally-advantageous fetal origin, the literature instead suggests a high incidence of either contaminating or pure maternal MSC. Despite definitional criteria that MSC are CD34-, increasing evidence suggests that fetal MSC may be CD34 positive in vivo. We flow sorted term placental digests based on CD34+ expression and exploited differential culture media to isolate separately pure fetal and maternal MSC populations. This method has considerable translational implications, in particular to clinical trials underway with "placental" MSC of uncertain or decidual origin.

  10. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.

    Danto, S I; Zabski, S M; Crandall, E D


    An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken

  11. Evaluation of tumor angiogenesis in patients with non-small cell lung carcinoma

    Suciu B.A.


    Full Text Available The aim of this study is to evaluate tumor angiogenesis in patients with non-small cell lung carcinoma. A total of 20 patients with pulmonary adenocarinoma have been included in the study. In order to evaluate tumor angiogenesis we studied the importance of CD34 expression. Evaluation of vascular density was performed with a semiautomated method using the dedicated software called ImageJ. We introduced in our study 20 patients with lung adenocarcinoma. We were able to identify tumor angiogenesis in 19 cases (95%. Immunolabeling of CD34 positive endothelial cells provided a good overview of tumor vascularization. Immunohistochemical staining of CD34 positive endothelium cells provided a good basis for tumor vascularity assessment, and also an excellent contrast for computer assisted morphometric measurements. Also we studied the intensity of the immunohistochemical staining of CD34 in the tumoral cells. We obtained the following results: a minor expression in 4 cases (20%, a moderate expression in 9 cases (45% and an intense expression in 6 cases (30%. The histological type of adenocarcinomas influences the architecture and branching of the vessels. The density of newly developed vessels is higher in patients with papillary pulmonary adenocarcinomas, which may indicate a possible relationship between the histological type and development of vascular supply.

  12. Enamel matrix derivative promote primary human pulp cell differentiation and mineralization

    Riksen, Elisabeth Aurstad; Landin, Maria A; Reppe, Sjur; Nakamura, Yukio; Lyngstadaas, Ståle Petter; Reseland, Janne E


    ...; however the molecular mechanisms involved are unclear. The effect of EMD (5-50 μg/mL) on primary human pulp cells were compared to untreated cells and cells incubated with 10⁻⁸ M dexamethasone (DEX...

  13. Primary intravascular large B-cell lymphoma of pituitary

    K R Anila


    Full Text Available A 68-year-old retired nurse, who was a known hypertensive on medication, presented with prolonged fever of 2-month duration without any clinical evidence of infection. On examination she had altered mental status. She also had other nonspecific complaints such as sleep disturbances, loss of weight, etc. On investigation, she was found to have anemia, thrombocytopenia, raised erythrocyte sedimentation rate (ESR, C-reactive protein (CRP, and lactate dehydrogenase (LDH values. She also had electrolyte imbalance. Radiological evaluation of brain showed mass lesion in the sella turcica, suggestive of pituitary adenoma. Biochemical evaluation showed hypopituitarism. Trans-sphenoidal biopsy was done. Based on histopathological and immunohistochemical findings a diagnosis of intravascular large B-cell lymphoma (IVLBCL of pituitary was made. Our patient′s condition deteriorated rapidly and she succumbed to her illness before therapy could be initiated. We are reporting this case because of the rare subtype of large B-cell lymphoma presenting at an extremely unusual primary site.

  14. Growth inhibitory activity of Ankaferd hemostat on primary melanoma cells and cell lines

    Turk, Seyhan; Malkan, Umit Yavuz; Ghasemi, Mehdi; Hocaoglu, Helin; Mutlu, Duygu; Gunes, Gursel; Aksu, Salih; Haznedaroglu, Ibrahim Celalettin


    Objective: Ankaferd hemostat is the first topical hemostatic agent about the red blood cell–fibrinogen relations tested in the clinical trials. Ankaferd hemostat consists of standardized plant extracts including Alpinia officinarum, Glycyrrhiza glabra, Thymus vulgaris, Urtica dioica, and Vitis vinifera. The aim of this study was to determine the effect of Ankaferd hemostat on viability of melanoma cell lines. Methods: Dissimilar melanoma cell lines and primary cells were used in this study. These cells were treated with different concentrations of Ankaferd hemostat to assess the impact of different dosages of the drug. All cells treated with different concentrations were incubated for different time intervals. After the data had been obtained, one-tailed T-test was used to determine whether the Ankaferd hemostat would have any significant inhibitory impact on cell growth. Results: We demonstrated in this study that cells treated with Ankaferd hemostat showed a significant decrease in cell viability compared to control groups. The cells showed different resistances against Ankaferd hemostat which depended on the dosage applied and the time treated cells had been incubated. We also demonstrated an inverse relationship between the concentration of the drug and the incubation time on one hand and the viability of the cells on the other hand, that is, increasing the concentration of the drug and the incubation time had a negative impact on cell viability. Conclusion: The findings in our study contribute to our knowledge about the anticancer impact of Ankaferd hemostat on different melanoma cells. PMID:28293423

  15. Mechanisms of Ionizing Radiation-Induced Cell Death in Primary Lung Cells


    Zhai M, et al. 2007. Protein oxidation implicated as the primary determinant of bacterial radioresistance. PLoS Biol 5:e92 43. Demidenko ZN...oxidative stress induces senescence in retinal pigment epithelial cells via TGF-beta release. Investigative ophthalmology & visual science 50:926- 35

  16. Characterization of protocadherin-1 expression in primary bronchial epithelial cells : association with epithelial cell differentiation

    Koning, Henk; Sayers, Ian; Stewart, Ceri E.; de Jong, Debora; ten Hacken, Nick H. T.; Postma, Dirkje S.; van Oosterhout, Antoon J. M.; Nawijn, Martijn C.; Koppelman, Gerard H.


    Protocadherin-1 (PCDH1) is a novel susceptibility gene for asthma that is expressed in airway epithelium. We aimed to characterize PCDH1 mRNA transcripts and protein expression in primary bronchial epithelial cells and to determine regulation of PCDH1 during mucociliary differentiation. Total RNA an

  17. Non-invasive sources of cells with primary cilia from pediatric and adult patients

    Ajzenberg, H.; Slaats, G.G.; Stokman, M.F.; Arts, H.H.; Logister, I.; Kroes, H.Y.; Renkema, K.Y.; Haelst, M.M. van; Terhal, P.A.; Rooij, I.A.L.M. van; Keijzer-Veen, M.G.; Knoers, N.V.; Lilien, M.R.; Jewett, M.A.; Giles, R.H.


    BACKGROUND: Ciliopathies give rise to a multitude of organ-specific pathologies; obtaining relevant primary patient material is useful for both diagnostics and research. However, acquisition of primary ciliated cells from patients, particularly pediatric patients, presents multiple difficulties. Bio

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

    Bhargava, A


    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.

  19. Stem cells decreased neuronal cell death after hypoxic stress in primary fetal rat neurons in vitro.

    Sakai, Tetsuro; Xu, Yan


    To explore stem cell-mediated neuronal protection through extracellular signaling pathways by transplanted stem cells, we sought to identify potential candidate molecules responsible for neuronal protection using an in vitro coculture system. Primary fetal rat hippocampal neurons underwent hypoxia (≤1% oxygen) for 96 h nad then were returned to a normoxic condition. The study group then received rat umbilical cord matrix-derived stem cells, while the control group received fresh media only. The experimental group showed decreased neuronal apoptosis compared to the control group [44.5 ± 1.6% vs. 71.0 ± 4.2% (mean ± SD, p = 0.0005) on day 5] and higher neuronal survival (4.9 ± 1.2 cells/100× field vs. 2.2 ± 0.3, p = 0.02 on day 5). Among 90 proteins evaluated using a protein array, stem cell coculture media showed increased protein secretion of TIMP-1 (5.61-fold), TIMP-2 (4.88), CNTF-Rα (3.42), activin A (2.20), fractalkine (2.04), CCR4 (2.02), and decreased secretion in MIP-2 (0.30-fold), AMPK α1 (0.43), TROY (0.48), and TIMP-3 (0.50). This study demonstrated that coculturing stem cells with primary neurons in vitro decreased neuronal cell death after hypoxia with significantly altered protein secretion. The results suggest that stem cells may offer neuronal protection through extracellular signaling.

  20. Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells.

    Khan, Mohammed I; Czarnecka, Anna M; Lewicki, Sławomir; Helbrecht, Igor; Brodaczewska, Klaudia; Koch, Irena; Zdanowski, Robert; Król, Magdalena; Szczylik, Cezary


    Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs)-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have

  1. Metabolic detoxication pathways for sterigmatocystin in primary tracheal epithelial cells.

    Cabaret, Odile; Puel, Olivier; Botterel, Françoise; Pean, Michel; Khoufache, Khaled; Costa, Jean-Marc; Delaforge, Marcel; Bretagne, Stéphane


    Human health effects of inhaled mycotoxins remain poorly documented, despite the large amounts present in bioaerosols. Among these mycotoxins, sterigmatocystin is one of the most prevalent. Our aim was to study the metabolism and cellular consequences of sterigmatocystin once it is in contact with the airway epithelium. Metabolites were analyzed first in vitro, using recombinant P450 1A1, 1A2, 2A6, 2A13, and 3A4 enzymes, and subsequently in porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. Expressed enzymes and PTECs were exposed to sterigmatocystin, uniformly enriched with (13)C to confirm the relationship between sterigmatocystin and metabolites. Induction of the expression of xenobiotic-metabolizing enzymes upon sterigmatocystin exposure was examined by real-time quantitative real-time polymerase chain reaction. Incubation of 50 μM sterigmatocystin with recombinant P450 1A1 led to the formation of three metabolites: monohydroxy-sterigmatocystin (M1), dihydroxy-sterigmatocystin (M2), and one glutathione adduct (M3), the latter after the formation of a transient epoxide. Recombinant P450 1A2 also led to M1 and M3. P450 3A4 led to only M3. In PTEC, 1 μM sterigmatocystin metabolism resulted in a glucuro conjugate (M4) mainly excreted at the basal side of cells. If PTEC were treated with β-naphthoflavone prior to sterigmatocystin incubation, two other products were detected, i.e., a sulfo conjugate (M5) and a glucoro conjugate (M6) of hydroxy-sterigmatocystin. Exposure of PTEC for 24 h to 1 μM sterigmatocystin induced an 18-fold increase in the mRNA levels of P450 1A1, without significantly induced 7-ethoxyresorufin O-deethylation activity. These data suggest that sterigmatocystin is mainly detoxified and is unable to produce significant amounts of reactive epoxide metabolites in respiratory cells. However, sterigmatocystin increases the P450 1A1 mRNA levels with unknown long-term consequences. These in vitro results obtained in

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

    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

  3. Primary mesenchyme cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan.

    Lane, M C; Solursh, M


    Primary mesenchyme cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous beta-D-xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary mesenchyme cells of a very large, cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary mesenchyme cell migration since treatment of actively migrating cells with chondroitinase ABC reversibly inhibited their migration in vitro.

  4. Primary Mediastinal Large B-Cell Lymphoma during Pregnancy

    Cesar A. Perez


    Full Text Available Non-Hodgkin’s Lymphoma (NHL rarely presents during pregnancy and primary mediastinal large B-cell lymphoma (PMLBCL accounts for approximately 2.5% of patients with NHL. The case of a 22-year-old woman who was diagnosed with Stage IIA PMLBCL during week 13 of her intrauterine pregnancy is described. The staging consisted in computed tomography (CT of the chest and magnetic resonance imaging (MRI of the abdomen and pelvis. She was managed with R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone for a total of six cycles and, because of the early presentation during the second trimester, she received the entire chemotherapy course during the pregnancy. She delivered a healthy baby at 34 weeks of pregnancy and a 18FDG-PET/CT scan demonstrated complete remission after delivery. After 20 months of follow up she remains with no evidence of disease and her 1-year-old son has shown no developmental delays or physical abnormalities. PMLBCL, although an uncommon subgroup of DLBCL, may present during pregnancy and R-CHOP should be considered as one suitable option in this complex scenario.

  5. File list: ALL.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available ALL.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells hg19 All antigens Cardiovascular ...

  6. File list: ALL.CDV.20.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available ALL.CDV.20.AllAg.Primary_umbilical_vein_endothelial_cells hg19 All antigens Cardiovascular ...

  7. File list: ALL.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available ALL.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells hg19 All antigens Cardiovascular ...

  8. File list: ALL.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available ALL.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells hg19 All antigens Cardiovascular ...

  9. Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells

    Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A;


    Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation......-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested...... the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant...

  10. Establishment and characterization of primary lung cancer cell lines from Chinese population

    Chao ZHENG; Yi-hua SUN; Xiao-lei YE; Hai-quan CHEN; Hong-bin JI


    Aim: To establish and characterize primary lung cancer cell lines from Chinese population.Methods: Lung cancer specimens or pleural effusions were collected from Chinese lung cancer patients and cultured in vitro with ACL4 medium (for non-small cell lung carcinomas (NSCLC)) or HITES medium (for small cell lung carcinomas (SCLC)) supplemented with 5%FBS. All cell lines were maintained in culture for more than 25 passages. Most of these cell lines were further analyzed for oncogenic mutations, karyotype, cell growth kinetics, and tumorigenicity in nude mice.Results: Eight primary cell lines from Chinese lung cancer patients were established and characterized, including seven NSCLC cell lines and one SCLC cell line. Five NSCLC cell lines were found to harbor epidermal growth factor receptor (EGFR) kinase domain mutations.Conclusion: These well-characterized primary lung cancer cell lines from Chinese population provide a unique platform for future studies of the ethnic differences in lung cancer biology and drug response.

  11. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

    Hugo Melida; Antonio Encina; Asier Largo-Gosens; Esther Novo-Uzal; Rogelio Santiago; Federico Pomar; Pedro Garca; Penelope Garca-Angulo; Jose Luis Acebes; Jesus Alvarez


    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

  12. Comparative In Vitro Immune Stimulation Analysis of Primary Human B Cells and B Cell Lines

    Van Belle, Kristien; Herman, Jean; Boon, Louis; Waer, Mark


    B cell specific immunomodulatory drugs still remain an unmet medical need. Utilisation of validated simplified in vitro models would allow readily obtaining new insights in the complexity of B cell regulation. For this purpose we investigated which human B lymphocyte stimulation assays may be ideally suited to investigate new B lymphocyte immunosuppressants. Primary polyclonal human B cells underwent in vitro stimulation and their proliferation, production of immunoglobulins (Igs) and of cytokines, and expression of cell surface molecules were analysed using various stimuli. ODN2006, a toll-like receptor 9 (TLR9) agonist, was the most potent general B cell stimulus. Subsequently, we investigated on which human B cell lines ODN2006 evoked the broadest immunostimulatory effects. The Namalwa cell line proved to be the most responsive upon TLR9 stimulation and hence may serve as a relevant, homogeneous, and stable B cell model in an in vitro phenotypic assay for the discovery of new targets and inhibitors of the B cell activation processes. As for the read-out for such screening assay, it is proposed that the expression of activation and costimulatory surface markers reliably reflects B lymphocyte activation. PMID:28116319

  13. EDITORIAL: In vitro experiments with primary mammalian cells: To Pool or not to Pool?

    MJ Stoddart


    Full Text Available There are various strategies that can be adopted when performing in vitro experiments with primary cells such as mesenchymal stem cells. It is generally accepted that multiple donors need to be investigated to take into account donor to donor variability; this is especially critical when investigating primary human cells. However, increasingly it is being seen that studies are pooling the cells from multiple donors prior to performing the experiment. This has obvious advantages but also many disadvantages, the greatest being loss of statistical power. This loss of statistical power is the reason why the pooling of primary cells for experiments is not to be recommended.

  14. Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy.

    Puech, Pierre-Henri; Taubenberger, Anna; Ulrich, Florian; Krieg, Michael; Muller, Daniel J; Heisenberg, Carl-Philipp


    During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

  15. Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes.

    Föllmann, W; Weber, S; Birkner, S


    Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon

  16. Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

    Duressa, Tewodros Firdissa; Huybrechts, Roger


    Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.

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

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


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

  18. Peripheral blood stem cell harvest in patients with limited stage small-cell lung cancer

    Katakami, Nobuyuki; Takakura, Shunji; Fujii, Hiroshi; Nishimura, Takashi; Umeda, Bunichi [Kobe City General Hospital (Japan)


    Chemotherapy plus granulocyte colony-stimulating factor (G-CSF) induced mobilization of peripheral blood stem cells (PBSC) was performed in patients with limited stage small-cell lung cancer. Chemotherapy consisted of cisplatin/etoposide or cisplatin/adriamycin/etoposide. The amounts of CD34 positive cells and granulocyte-macrophage colony forming units (CFU-GM) collected during 2-3 courses of apheresis were 3.1{+-}2.9 x 10{sup 6}/kg (n=10) and 3.1{+-}1.5 x 10{sup 5}/kg (n=8) , respectively. Adequate amounts of PBSC were also harvested even in patients treated with concurrent chemoradiotherapy. Eight patients were successfully treated with high-dose chemotherapy consisting of ifosfamide, carboplatin and etoposide with PBSC transfusion. The patients'-bone marrow reconstruction was rapid and no treatment-related death was observed. (author)

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

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


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

  20. 9 CFR 113.51 - Requirements for primary cells used for production of biologics.


    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for primary cells used for production of biologics. 113.51 Section 113.51 Animals and Animal Products ANIMAL AND PLANT HEALTH... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used...

  1. Application of modified enzyme digestion method in rapid primary culture of human glioma cells

    Wei XIANG


    Full Text Available Objective  To explore the applied value of modified enzyme digestion method in primary culture of human glioma cells. Methods  A traditional enzyme digestion method was modified based on literatures and our work experience. The glioma cells from 32 glioma patients with different grades were primarily cultured by the modified enzyme digestion method. The morphological features of these cells were observed under an inverted phase contrast microscope. The primary cells were purified by differential adhesion during passage. The primary cells were identified by immunofluorescence technique, and the growth curves were drawn by cell proliferation assays (CCK-8 method for investigating the proliferation of the cells cultured in vitro. Results  The primary human glioma cells were successfully cultured and transferred by the new method, with a success rate of 87.5%. The cells cultured successfully in vitro showed good adherent growth, stable morphologies, thus can be passaged. Fluoroimmunoassay showed positive expression of glial fibrillary acidic protein, which confirms the cultured cells were glioma cells. Cell proliferation assays revealed active cell proliferation in vitro, the higher the tumor grade, the higher the proliferative capacity. Conclusion  The modified enzyme digestion method is simpler and more efficient for primary culture of human glioma cells, and the success rate is also higher, thus being able to provide a good guarantee for fundamental research of glioma. DOI: 10.11855/j.issn.0577-7402.2016.06.06

  2. Cell Communication in a Coculture System Consisting of Outgrowth Endothelial Cells and Primary Osteoblasts

    David Paul Eric Herzog


    Full Text Available Bone tissue is a highly vascularized and dynamic system with a complex construction. In order to develop a construct for implant purposes in bone tissue engineering, a proper understanding of the complex dependencies between different cells and cell types would provide further insight into the highly regulated processes during bone repair, namely, angiogenesis and osteogenesis, and might result in sufficiently equipped constructs to be beneficial to patients and thereby accomplish their task. This study is based on an in vitro coculture model consisting of outgrowth endothelial cells and primary osteoblasts and is currently being used in different studies of bone repair processes with special regard to angiogenesis and osteogenesis. Coculture systems of OECs and pOBs positively influence the angiogenic potential of endothelial cells by inducing the formation of angiogenic structures in long-term cultures. Although many studies have focused on cell communication, there are still numerous aspects which remain poorly understood. Therefore, the aim of this study is to investigate certain growth factors and cell communication molecules that are important during bone repair processes. Selected growth factors like VEGF, angiopoietins, BMPs, and IGFs were investigated during angiogenesis and osteogenesis and their expression in the cultures was observed and compared after one and four weeks of cultivation. In addition, to gain a better understanding on the origin of different growth factors, both direct and indirect coculture strategies were employed. Another important focus of this study was to investigate the role of “gap junctions,” small protein pores which connect adjacent cells. With these bridges cells are able to exchange signal molecules, growth factors, and other important mediators. It could be shown that connexins, the gap junction proteins, were located around cell nuclei, where they await their transport to the cell membrane. In

  3. Unusual Presentation of Primary Squamous Cell Carcinoma of Mandible

    Shanmugasundaram, Karpagavalli; Subramanian, Sathasiva; Kumar, Vimal


    Carcinoma arising primarily from the jaw is a locally aggressive lesion with poor prognosis. Primary intraosseous carcinoma (PIOC) lesion develops either de novo remnants of odontogenic epithelium, odontogenic cyst/tumor, epithelium remnants, or/and salivary gland residues. We describe very interesting case of primary intraosseous carcinoma of mandible. This extensive lesion was sent for oncological opinion and further management. Due to the uncertainty of diagnostic criteria of PIOC, only few cases of this lesion with a typical presentation have been reported. This article presents a case of primary intraosseous carcinoma with a unique appearance and detailed review stating its clinicopathological correlation. PMID:28078158

  4. Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells.

    Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A; Mollenkopf, Hans-Joachim; Meyer, Thomas F; Brüggemann, Holger


    Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes.

  5. Innate immune cells in the pathogenesis of primary systemic vasculitis.

    Misra, Durga Prasanna; Agarwal, Vikas


    Innate immune system forms the first line of defense against foreign substances. Neutrophils, eosinophils, erythrocytes, platelets, monocytes, macrophages, dendritic cells, γδ T cells, natural killer and natural killer T cells comprise the innate immune system. Genetic polymorphisms influencing the activation of innate immune cells predispose to development of vasculitis and influence its severity. Abnormally activated innate immune cells cross-talk with other cells of the innate immune system, present antigens more efficiently and activate T and B lymphocytes and cause tissue destruction via cell-mediated cytotoxicity and release of pro-inflammatory cytokines. These secreted cytokines further recruit other cells to the sites of vascular injury. They are involved in both the initiation as well as the perpetuation of vasculitis. Evidences suggest reversal of aberrant activation of immune cells in response to therapy. Understanding the role of innate immune cells in vasculitis helps understand the potential of therapeutic modulation of their activation to treat vasculitis.

  6. CD27-triggering on primary plasma cell leukaemia cells has anti-apoptotic effects involving mitogen activated protein kinases

    Guikema, JEJ; Vellenga, E; Abdulahad, WH; Hovenga, S; Bos, NA


    Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multiple myeloma (MM) and is characterized by a poor prognosis. In a previous study we demonstrated that PCL plasma cells display a high expression of CD27, in contrast to MM plasma cells. The present study was

  7. Primary cell wall composition of bryophytes and charophytes.

    Popper, Zoë A; Fry, Stephen C


    Major differences in primary cell wall (PCW) components between non-vascular plant taxa are reported. (1) Xyloglucan: driselase digestion yielded isoprimeverose (the diagnostic repeat unit of xyloglucan) from PCW-rich material of Anthoceros (a hornwort), mosses and both leafy and thalloid liverworts, as well as numerous vascular plants, showing xyloglucan to be a PCW component in all land plants tested. In contrast, charophycean green algae (Klebsormidium flaccidium, Coleochaete scutata and Chara corallina), thought to be closely related to land plants, did not contain xyloglucan. They did not yield isoprimeverose; additionally, charophyte material was not digestible with xyloglucan-specific endoglucanase or cellulase to give xyloglucan-derived oligosaccharides. (2) Uronic acids: acid hydrolysis of PCW-rich material from the charophytes, the hornwort, thalloid and leafy liverworts and a basal moss yielded higher concentrations of glucuronic acid than that from the remaining land plants including the less basal mosses and all vascular plants tested. Polysaccharides of the hornwort Anthoceros contained an unusual repeat-unit, glucuronic acid-alpha(1-->3)-galactose, not found in appreciable amounts in any other plants tested. Galacturonic acid was consistently the most abundant PCW uronic acid, but was present in higher concentrations in acid hydrolysates of bryophytes and charophytes than in those of any of the vascular plants. Mannuronic acid was not detected in any of the species surveyed. (3) Mannose: acid hydrolysis of charophyte and bryophyte PCW-rich material also yielded appreciably higher concentrations of mannose than are found in vascular plant PCWs. (4) Mixed-linkage glucan (MLG) was absent from all algae and bryophytes tested; however, upon digestion with licheninase, PCW-rich material from the alga Ulva lactuca and the leafy liverwort Lophocolea bidentata yielded penta- to decasaccharides, indicating the presence of MLG-related polysaccharides. Our

  8. Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

    Shaobo Hu; Zifang Song; Qichang Zheng; Jun Nie


    Objective: To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods: Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers,and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion 6me, attachment time and recovery after cryopreservation. Results: Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of singleendothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion: The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin

  9. Expression of tumor antigens on primary ovarian cancer cells compared to established ovarian cancer cell lines

    Kloudová, Kamila; Hromádková, Hana; Partlová, Simona; Brtnický, Tomáš; Rob, Lukáš; Bartůňková, Jiřina; Hensler, Michal; Halaška, Michael J.; Špíšek, Radek; Fialová, Anna


    In order to select a suitable combination of cancer cell lines as an appropriate source of antigens for dendritic cell-based immunotherapy of ovarian cancer, we analyzed the expression level of 21 tumor associated antigens (BIRC5, CA125, CEA, DDX43, EPCAM, FOLR1, Her-2/neu, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MUC-1, NY-ESO-1, PRAME, p53, TPBG, TRT, WT1) in 4 established ovarian cancer cell lines and in primary tumor cells isolated from the high-grade serous epithelial ovarian cancer tissue. More than 90% of tumor samples expressed very high levels of CA125, FOLR1, EPCAM and MUC-1 and elevated levels of Her-2/neu, similarly to OVCAR-3 cell line. The combination of OV-90 and OVCAR-3 cell lines showed the highest overlap with patients' samples in the TAA expression profile. PMID:27323861

  10. Primary Cutaneous Lymphoma-Associated Pseudoepitheliomatous Hyperplasia Masquerading as Squamous Cell Carcinoma in a Young Adult.

    Ansari, Mahsa; Azmoodeh Ardalan, Farid; Najafi, Masoumeh; Goodarzi, Azadeh; Ghanadan, Alireza


    Primary cutaneous anaplastic large cell lymphoma is a T-cell malignancy with atypical CD30 positive lymphocytes. Pseudoepitheliomatous hyperplasia is an uncommon finding in primary cutaneous anaplastic large cell lymphoma, and may mimic squamous cell carcinoma as pseudomalignancy. Careful attention of a pathologist to correct diagnosis of pseudoepitheliomatous hyperplasia and its underlying causes will help physicians to avoid inappropriate management. Here, we present a 22-year-old man referred to our hospital with a solitary nodule persistent on his forearm which was diagnosed as squamous cell carcinoma in the first biopsy. The lesion recurred after two months and histopathologic and immunohistochemistry examination revealed anaplastic large cell lymphoma with florid pseudoepitheliomatous hyperplasia which masquerading as well-differentiated squamous cell carcinoma. Diagnosis of pseudoepitheliomatous hyperplasia must guide the pathologist to search for underlying causes, such as primary cutaneous lymphoma. Pseudoepitheliomatous hyperplasia may mimic squamous cell carcinoma and this can result in inappropriate diagnosis and management.

  11. Reversing effect of exogenous WWOX gene expression on malignant phenotype of primary cultured lung carcinoma cells

    ZHOU Yu-long; LI Yue-chuan; SHOU Feng; LIU Chang-qi; PU Yong; TANG Hua


    Background Whether WW domain containing oxidoreductase (WWOX) gene is a tumor-suppressor is still controversial. Some researchers found that the transcription of the WWOX gene was lacking not only in tumor tissues but also in non-tumorous tissues and sometimes in normal tissues. Hence it is important to explore the role of the expression of the exogenous WWOX gene in the proliferation and apoptosis of primary cultured lung carcinoma cells. Methods Lipofection technique was used to determine primary cultured lung carcinoma cells containing the highly expressed exogenous WWOX gene and primary cultured cells with vectors as controls. An animal model of lung cancer was made by subcutaneous implantation of tumor cells into nude mice. RT-PCR, Western blotting, flow cytometry, and TUN EL were used to detect the transcription, expression of the exogenous gene and the effect of the expression of targeted genes on the proliferation and apoptosis of the primary cultured lung carcinoma cells. Results The growth, clone formation rate (CFR) ((5.33±1.53)%) of the primary lung cancer cells transfected with the WWOX gene, tumor size and weight were significantly lower than those of the non-transfected lung cancer cells (CFR: (14.33±1.53)%) and the primary lung cancer cells transfected with blank plasmids (CFR: (11.00±1.73)%, P<0.05). The apoptosis level of primary lung cancer cells transfected with the WWOX gene ((40.72±5.20)%) was significantly higher than that of the non-transfected lung cancer cells ((2.76±0.02)%) and the primary lung cancer cells transfected with blank plasmids ((2.72±0.15)%, P<0.05). Conclusion The expression of the exogenous WWOX gene can significantly inhibit the proliferation of lung cancer cells and induce their apoptosis, suggesting that the WWOX gene possesses tumor-suppressing effect.

  12. Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells

    Gho, Yong Song; Choi, Dong-Sic; Choi, Do-Young; Hong, Bok Sil; Jang, Su Chul; Kim, Dae-Kyum; Lee, Jaewook; Kim, Yoon-Keun; Kim, Kwang Pyo


    Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastat...

  13. Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus.

    Tanabe, Koji; Kani, Shuichi; Shimizu, Takashi; Bae, Young-Ki; Abe, Takaya; Hibi, Masahiko


    Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

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

    Jan Pavel Kucera


    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.

  15. Primary Intraparenchymal Squamous Cell Carcinoma of the Kidney: A Rare and Unique Entity

    Prithwijit Ghosh


    Full Text Available Primary squamous cell carcinoma (SCC of the renal parenchyma is a very unusual entity which needs to be differentiated from primary SCC of renal pelvis, SCC from another primary site, and urothelial carcinoma with extensive squamous differentiation. We are most probably describing the second case of primary SCC of the renal parenchyma in a 51-year-old male who presented with heaviness of right upper abdomen with intermittent pain in right flank. Contrast-enhanced computed tomography (CECT revealed a mass in the right lower pole of the kidney and histopathology following nephrectomy displayed the features of well-differentiated squamous cell carcinoma without urothelial involvement.

  16. The use of ultrasound in the search for the primary site of unknown primary head and neck squamous cell cancers

    Fakhry, Carole; Agrawal, Nishant; Califano, Joseph; Messing, Barbara; Liu, Jia; Saunders, John; Ha, Patrick; Coquia, Stephanie; Hamper, Ulrike; Gillison, Maura; Blanco, Ray


    Summary Background Although human papillomavirus detection in cervical lymph nodes of head and neck squamous cell cancers (HNSCC) of unknown primary site (UP) is indicative of a primary tumor of the oropharynx (OP), localization can remain elusive. Therefore, we investigated ultrasonography (US) for the identification of the primary tumor. Methods Eligible cases had HNSCC of UP after evaluation by a head and neck surgical oncologist. Controls were healthy volunteers. Transcervical and intraoral ultrasonography was performed by a standard protocol using convex (3.75–6.0 MHz and 5–7.5 MHz) transducers. US findings were compared with operative examination (exam under anesthesia, direct laryngoscopy) and biopsies. The primary outcome of interest was the presence or absence of a lesion on US. Results 10 cases and 20 controls were enrolled. PET/CT scans were negative/nonspecific (9), or suspicious (1) for a primary lesion. On US, predominantly hypoechoic (9 of 10) lesions were visualized consistent with base of tongue (n = 7) or tonsil (n = 3) primary tumors. On operative examination, 5 of 10 were appreciated. Two additional primaries were confirmed with biopsies “directed” by preoperative US. This represents an overall diagnostic rate of 70%, which is 20% higher than our detection rate for 2008–2010. The three cases in which a suspicious lesion was visualized on US, yet remained UP despite further interventions, could represent false positives, misclassification or operator variability. No lesions were suspected among the controls. Conclusion Ultrasound has promise for detection of UPs of the OP and therefore warrants further investigation. PMID:24819862

  17. [Effects of rutaecarpine on inflammatory cytokines in insulin resistant primary skeletal muscle cells].

    Yang, Jian-Wen; Nie, Xu-Qiang; Shi, Hai-Xia; Zhang, Yu-Jin; Zhang, Jian-Yong; Yuan, Ye; Bian, Ka


    It is now well established that inflammation plays an important role in the development of numerous chronic metabolic diseases including insulin resistance (IR) and type 2 diabetes (T2DM). Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Our pre- vious study has shown that rutaecarpine (Rut) can benefit blood lipid profile, mitigate inflammation, and improve kidney, liver, pan- creas pathology status of T2DM rats. However, the effects of Rut on inflammatory cytokines in the development of IR-skeletal muscle cells have not been studied. Thus, our objective was to investigate effects of Rut on inflammatory cytokines interleukiri (IL)-1, IL-6 and tumor necrosis factor (TNF)-α in insulin resistant primary skeletal muscle cells (IR-PSMC). Primary cultures of skeletal muscle cells were prepared from 5 neonate SD rats, and the primary rat skeletal muscle cells were identified by cell morphology, effect of ru- taecarpine on cell proliferation by MTT assay. IR-PSMC cells were induced by palmitic acid (PA), the glucose concentration was measured by glucose oxidase and peroxidase (GOD-POD) method. The effects of Rut on inflammatory cytokines IL-1, IL-6 and TNF-α in IR-PSMC cells were tested by enzyme-linked immunosorbent assay (ELISA) kit. The results show that the primary skeletal muscle cells from neonatal rat cultured for 2-4 days, parallel alignment regularly, and cultured for 7 days, cells fused and myotube formed. It was shown that Rut in concentration 0-180. 0 μmol x L(-1) possessed no cytotoxic effect towards cultured primary skeletal muscle cells. However, after 24 h exposure to 0.6 mmol x L(-1) PA, primary skeletal muscle cells were able to induce a state of insulin resistance. The results obtained indicated significant decrease (P inflammatory cytokines in the IR-PSMC cells.

  18. Campylobacter-induced interleukin-8 responses in human intestinal epithelial cells and primary intestinal chick cells.

    Borrmann, Erika; Berndt, Angela; Hänel, Ingrid; Köhler, Heike


    Campylobacter (C.) jejuni and C. coli can cause gastrointestinal disorders in humans characterized by acute inflammation. Inflammatory signals are initiated during interaction between these pathogens and human intestinal cells, but nothing is known about the stimulation of avian intestinal cells by Campylobacter. Interleukin-8 (IL-8) as a proinflammatory chemokine plays an important role in mobilizing cellular defence mechanism. IL-8 mRNA expression in both human intestinal cells (INT 407) and primary intestinal chick cells (PIC) was determined by quantitative real-time RT-PCR. The secretion of IL-8 protein by INT407 was measured using ELISA. Although C. jejuni and C. coli are considered to be harmless commensals in the gut of birds, the avian Campylobacter isolates investigated were able to induce the proinflammatory IL-8 in PIC as well as in INT407. In an in vitro system, C. jejuni as well as C. coli were able to induce IL-8 mRNA in PIC. Relation between the virulence properties like toxin production, the ability to invade and to survive in Caco-2 cells and the level of IL-8 mRNA produced by INT 407 and PIC after infection with Campylobacter strains was also investigated.

  19. Eye extract improves cell migration out of lymphoid organ explants of L. vannamei and viability of the primary cell cultures.

    Li, Wenfeng; Van Tuan, Vo; Van Thuong, Khuong; Bossier, Peter; Nauwynck, Hans


    Since no cell line from shrimp has been established up till now, an optimization of the primary cell culture protocol is necessary. In this context, the effect of extracts (supernatant of a 1:50 (w/v) suspension) from different shrimp organs (muscle, brain, ganglia, eyestalk, ovary, and eye) on the performance of primary lymphoid cell cultures was evaluated. Ten percent of eye extract and 3% of ovary extract enhanced maximally the migration and survival of cells of the lymphoid organ of Litopenaeus vannamei significantly at 48, 96, and 144 h post seeding. Extracts from the eyestalk (10%), muscle (10%), and brain (1%) significantly promoted the migration and survival of cells at 48 and 96 h post seeding but not anymore at 144 h post seeding. In conclusion, it may be advised to add 10% of eye extract or 3% of ovary extract to cells for the maximal health of primary cell cultures from the lymphoid organ of L. vannamei.

  20. Matrigel improves functional properties of primary human salivary gland cells.

    Maria, Ola M; Zeitouni, Anthony; Gologan, Olga; Tran, Simon D


    Currently, there is no effective treatment available to patients with irreversible loss of functional salivary acini caused by Sjogren's syndrome or after radiotherapy for head and neck cancer. A tissue-engineered artificial salivary gland would help these patients. The graft cells for this device must establish tight junctions in addition to being of fluid-secretory nature. This study analyzed a graft source from human salivary glands (huSG) cultured on Matrigel. Cells were obtained from parotid and submandibular glands, expanded in vitro, and then plated on either Matrigel-coated (2 mg/mL) or uncoated culture dish. Immunohistochemistry, transmission electron microscopy, quantitative real-time-polymerase chain reaction, Western blot, and transepithelial electrical resistance were employed. On Matrigel, huSG cells adopted an acinar phenotype by forming three-dimensional acinar-like units (within 24 h of plating) as well as a monolayer of cells. On uncoated surfaces (plastic), huSG cells only formed monolayers of ductal cells. Both types of culture conditions allowed huSG cells to express tight junction proteins (claudin-1, -2, -3, -4; occludin; JAM-A; and ZO-1) and adequate transepithelial electrical resistance. Importantly, 99% of huSG cells on Matrigel expressed α-amylase and the water channel protein Aquaporin-5, as compared to cells on plastic. Transmission electron microscopy confirmed an acinar phenotype with many secretory granules. Matrigel increased the secretion of α-amylase two to five folds into the media, downregulated certain salivary genes, and regulated the translation of acinar proteins. This three-dimensional in vitro serum-free cell culture method allows the organization and differentiation of huSG cells into salivary cells with an acinar phenotype.

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

    Orcun Celik


    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.

  2. Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells

    Rattner Jerome B


    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

  3. Polystyrene-coated micropallets for culture and separation of primary muscle cells

    Detwiler, David A.; Dobes, Nicholas C.; Sims, Christopher E.; Kornegay, Joseph N.; Allbritton, Nancy L


    Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photo...

  4. Review Article. Technical aspects of oxygen level regulation in primary cell cultures: A review

    Yazdani Mazyar


    Oxygen (O2) is an essential element for aerobic respiration. Atmospheric concentration of O2 is approximately 21%. Mammalian cells, however, are generally adapted to O2 levels much lower than atmospheric conditions. The pericellular levels of O2 must also be maintained within a fairly narrow range to meet the demands of cells. This applies equally to cells in vivo and cells in primary cultures. There has been growing interest in the performance of cell culture experiments under various O2 lev...

  5. Efficient immortalization of primary nasopharyngeal epithelial cells for EBV infection study.

    Yim Ling Yip

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

  6. Cell-surface proteoglycan in sea urchin primary mesenchyme cell migration

    Lane, M.C.


    Early in the development of the sea urchin embryo, the primary mesenchyme cells (PMC) migrate along the basal lamina of the blastocoel. Migration is inhibited in L. pictus embryos cultured in sulfate-free seawater and in S. purpuratus embryos exposed to exogenous {beta}-D-xylosides. An in vitro assay was developed to test the migratory capacity of normal PMC on normal and treated blastocoelic matrix. Sulfate deprivation and exposure to exogenous xyloside render PMC nonmotile on either matrix. Materials removed from the surface of normal PMC by treatment with 1 M urea restored migratory ability to defective cells, whereas a similar preparation isolated from the surface of epithelial cells at the same stage did not. Migration also resumed when cells were removed from the xyloside or returned to normal seawater. The urea extract was partially purified and characterized by radiolabeling, gel electrophoresis, fluorography, ion exchange chromatography, and western blotting. The PMC synthesize a large chondroitin sulfate/dermatan sulfate proteoglycan that is present in an active fraction isolated by chromatography. Chondroitinase ABC digestion of live cells blocked migration reversibly, further supporting the identification of the chondroitin sulfate/dermatan sulfate proteoglycan as the active component in the urea extract. Much of the incorporated sulfate was distributed along the filopodia in {sup 35}SO{sub 4}-labelled PMC by autoradiography. The morphology of normal and treated S. purpuratus PMC was examined by scanning electron microscopy, and differences in spreading, particularly of the extensive filopodia present on the cells, was observed. A model for the role of the chondroitin sulfate/dermatan sulfate proteoglycan in cell detachment during migration is proposed.

  7. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig

    Richter Anne


    Full Text Available Abstract Background Somatic cell nuclear transfer (SCNT is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination. Results PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs and porcine ear fibroblasts (PEFs could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT. Conclusion The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.

  8. Response to microtubule-interacting agents in primary epithelial ovarian cancer cells


    Background Ovarian cancer constitutes nearly 4% of all cancers among women and is the leading cause of death from gynecologic malignancies in the Western world. Standard first line adjuvant chemotherapy treatments include Paclitaxel (Taxol) and platinum-based agents. Taxol, epothilone B (EpoB) and discodermolide belong to a family of anti-neoplastic agents that specifically interferes with microtubules and arrests cells in the G2/M phase of the cell cycle. Despite initial success with chemotherapy treatment, many patients relapse due to chemotherapy resistance. In vitro establishment of primary ovarian cancer cells provides a powerful tool for better understanding the mechanisms of ovarian cancer resistance. We describe the generation and characterization of primary ovarian cancer cells derived from ascites fluids of patients with epithelial ovarian cancer. Methods Chemosensitivity of these cell lines to Taxol, EpoB and discodermolide was tested, and cell cycle analysis was compared to that of immortalized ovarian cancer cell lines SKOV3 and Hey. The relationship between drug resistance and αβ-tubulin and p53 status was also investigated. Results All newly generated primary cancer cells were highly sensitive to the drugs. αβ-tubulin mutation was not found in any primary cell lines tested. However, one cell line that harbors p53 mutation at residue 72 (Arg to Pro) exhibits altered cell cycle profile in response to all drug treatments. Immortalized ovarian cancer cells respond differently to EpoB treatment when compared to primary ovarian cancer cells, and p53 polymorphism suggests clinical significance in the anti-tumor response in patients. Conclusions The isolation and characterization of primary ovarian cancer cells from ovarian cancer patients’ specimens contribute to further understanding the nature of drug resistance to microtubule interacting agents (MIAs) currently used in clinical settings. PMID:23574945

  9. Adherence of Actinobacillus pleuropneumoniae to primary cultures of porcine lung epithelial cells.

    Boekema, B.K.H.L.; Stockhofe, N.; Smith, H.E.; Kamp, E.M.; Putten, van J.P.; Verheijden, J.H.


    To study adherence of Actinobacillus pleuropneumoniae to porcine lower respiratory epithelium, a cell culture model was developed using primary cultures of porcine lung epithelial cells (LEC). Adherence assays were performed and results were compared with data obtained with swine kidney cells (SK6).

  10. Establishing and maintaining primary cell cultures derived from the ctenophore Mnemiopsis leidyi.

    Vandepas, Lauren E; Warren, Kaitlyn J; Amemiya, Chris T; Browne, William E


    We have developed an efficient method for the preparation and maintenance of primary cell cultures isolated from adult Mnemiopsis leidyi, a lobate ctenophore. Our primary cell cultures are derived from tissue explants or enzymatically dissociated cells, and maintained in a complex undefined ctenophore mesogleal serum. These methods can be used to isolate, maintain and visually monitor ctenophore cells to assess proliferation, cellular morphology and cell differentiation in future studies. Exemplar cell types that can be easily isolated from primary cultures include proliferative ectodermal and endodermal cells, motile amebocyte-like cells, and giant smooth muscle cells that exhibit inducible contractile properties. We have also derived 'tissue envelopes' containing sections of endodermal canal surrounded by mesoglea and ectoderm that can be used to monitor targeted cell types in an in vivo context. Access to efficient and reliably generated primary cell cultures will facilitate the analysis of ctenophore development, physiology and morphology from a cell biological perspective. © 2017. Published by The Company of Biologists Ltd.

  11. Real world data on primary treatment for mantle cell lymphoma

    Abrahamsson, Anna; Albertsson-Lindblad, Alexandra; Brown, Peter N


    to prognostic factors and first-line treatment in patients with MCL in a population-based data set. Data were collected from the Swedish and Danish Lymphoma Registries from the period of 2000 to 2011. A total of 1389 patients were diagnosed with MCL. During this period, age-standardized incidence MCL increased...... analysis. Hence, by a population-based approach, we were able to provide novel data on prognostic factors and primary treatment of MCL, applicable to routine clinical practice....

  12. B-cell receptor triggers drug sensitivity of primary CLL cells by controlling glucosylation of ceramides.

    Schwamb, Janine; Feldhaus, Valeska; Baumann, Michael; Patz, Michaela; Brodesser, Susanne; Brinker, Reinhild; Claasen, Julia; Pallasch, Christian P; Hallek, Michael; Wendtner, Clemens-Martin; Frenzel, Lukas P


    Survival of chronic lymphocytic leukemia (CLL) cells is triggered by several stimuli, such as the B-cell receptor (BCR), CD40 ligand (CD40L), or interleukin-4 (IL-4). We identified that these stimuli regulate apoptosis resistance by modulating sphingolipid metabolism. Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of proapoptotic ceramide in BCR/IL-4/CD40L-stimulated primary CLL cells compared with untreated controls. Antiapoptotic glucosylceramide levels were significantly increased after BCR cross-linking. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via UDP-glucose ceramide glucosyltransferase (UGCG). Besides specific UGCG inhibitors, our data demonstrate that IgM-mediated UGCG expression was inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which reverted IgM-induced resistance toward apoptosis of CLL cells. Sphingolipids were recently shown to be crucial for mediation of apoptosis via mitochondria. Our data reveal ABT-737, a mitochondria-targeting drug, as interesting candidate partner for PI3Kδ and BTK inhibition, resulting in synergistic apoptosis, even under protection by the BCR. In summary, we identified the mode of action of novel kinase inhibitors CAL-101 and PCI-32765 by controlling the UGCG-mediated ceramide/glucosylceramide equilibrium as a downstream molecular switch of BCR signaling, also providing novel targeted treatment options beyond current chemotherapy-based regimens.

  13. Thiamine uptake into primary proximal tubule cells and MDCK distal tubule cells; Differential effect of ethanol

    Pochal, M.A.; Taub, M.; Acara, M. (State Univ. of New York, Buffalo (United States))


    Rabbit primary proximal tubule cells (PT) and distal cells from a MDCK cell line (DT) were studied for their ability to accumulate and metabolize {sup 14}C-thiamine and to assess the effect of ethanol on the accumulation. Incubation with 10uM {sup 14}C-thiamine, resulted in a four fold greater accumulation of {sup 14}C in PT compared to DT. Ethanol significantly decreased PT thiamine accumulation to 0.92 {plus minus} 0.09 nmole/mg but had little effect on DT accumulation. Initial thiamine uptake rates were greater in PT than in DT. Ethanol did not produce a significant effect on either initial uptake rate. Ethanol, however, decreased the maximum rate of uptake in PR from 3.20 to 1.75 nmole/mg/min. Although both cell types metabolize {sup 14}C to thiamine phosphates, total amount of metabolite was greater in PT. These data are consistent with cortical slice uptake studies in which thiamine accumulation was associated with its phosphorylation. In these slices both maximal accumulation and metabolism were inhibited by ethanol.

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

    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.

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

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


    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

  16. Polystyrene-coated micropallets for culture and separation of primary muscle cells.

    Detwiler, David A; Dobes, Nicholas C; Sims, Christopher E; Kornegay, Joe N; Allbritton, Nancy L


    Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures' surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells.

  17. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

    Bedayat Babak


    Full Text Available Abstract Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts. With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.

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

    Etienne Caron


    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 primarycells, 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, primarycell 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.

  19. Transoral robotic surgery for the management of head and neck squamous cell carcinoma of unknown primary

    Channir, Hani Ibrahim; Rubek, Niclas; Nielsen, Hans Ulrik


    CONCLUSION: The addition of transoral robotic surgery (TORS) in the diagnostic management of patients classified with head and neck squamous cell carcinoma of unknown primary (SCCUP) is promising and appears to improve detection rates of the primary tumour. The approach presented in this first...

  20. Nanocatalysis for Primary and Secondary High Energy Lithium Oxygen Cells


    included emulsified Teflon (Fuel Cell Earth, 60 wt% in water), and mixtures of styrene butadiene latex ( SBR , Euclid Chemical Company) and sodium...They were, emulsified PTFE (60wt%), 2:1 (by weight) SBR /CMC blend, and lithium acrylate. Figure 43 Comarative perfomence of cahodes containing 6...the oxygen reduction reaction in fuel cell cathodes by nitrogen doped carbon (obtained via pyrolysis of N-containing polymers) has been well

  1. Changes in Natural Killer cell activation and function during primary HIV-1 Infection.

    Vivek Naranbhai

    Full Text Available BACKGROUND: Recent reports suggest that Natural Killer (NK cells may modulate pathogenesis of primary HIV-1 infection. However, HIV dysregulates NK-cell responses. We dissected this bi-directional relationship to understand how HIV impacts NK-cell responses during primary HIV-1 infection. METHODOLOGY/PRINCIPAL FINDINGS: Paired samples from 41 high-risk, initially HIV-uninfected CAPRISA004 participants were analysed prior to HIV acquisition, and during viraemic primary HIV-1 infection. At the time of sampling post-infection five women were seronegative, 11 women were serodiscordant, and 25 women were seropositive by HIV-1 rapid immunoassay. Flow cytometry was used to measure NK and T-cell activation, NK-cell receptor expression, cytotoxic and cytokine-secretory functions, and trafficking marker expression (CCR7, α(4β(7. Non-parametric statistical tests were used. Both NK cells and T-cells were significantly activated following HIV acquisition (p = 0.03 and p<0.0001, respectively, but correlation between NK-cell and T-cell activation was uncoupled following infection (pre-infection r = 0.68;p<0.0001; post-infection, during primary infection r = 0.074;p = 0.09. Nonetheless, during primary infection NK-cell and T-cell activation correlated with HIV viral load (r = 0.32'p = 0.04 and r = 0.35;p = 0.02, respectively. The frequency of Killer Immunoglobulin-like Receptor-expressing (KIR(pos NK cells increased following HIV acquisition (p = 0.006, and KIR(pos NK cells were less activated than KIR(neg NK cells amongst individuals sampled while seronegative or serodiscordant (p = 0.001;p<0.0001 respectively. During HIV-1 infection, cytotoxic NK cell responses evaluated after IL-2 stimulation alone, or after co-culture with 721 cells, were impaired (p = 0.006 and p = 0.002, respectively. However, NK-cell IFN-y secretory function was not significantly altered. The frequency of CCR7+ NK cells was elevated

  2. Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis



    Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli (UPEC) to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium (K-SFM)with epidermal growth factor (EGF) and bovine pituitary extract (BPE). Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678-54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis.The UPEC adherence was performed with observation on the morphological changes of the adhered cells,while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnf1 of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape,unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.

  3. Duck hepatitis B virus replication in primary bile duct epithelial cells.

    Lee, J Y; Culvenor, J G; Angus, P; Smallwood, R; Nicoll, A; Locarnini, S


    Primary cultures of intrahepatic bile duct epithelial (IBDE) cells isolated from duckling livers were successfully grown for studies of duck hepatitis B virus (DHBV). The primary IBDE cells were characterized by immunohistochemistry using CAM 5.2, a cytokeratin marker which was shown to react specifically to IBDE cells in duck liver tissue sections and in primary cultures of total duck liver cells. Immunofluorescence assay using anti-duck albumin, a marker for hepatocytes, revealed that these IBDE cultures did not appear to contain hepatocytes. A striking feature of these cultures was the duct-like structures present within each cell colony of multilayered IBDE cells. Normal duck serum in the growth medium was found to be essential for the development of these cells into duct-like structures. When the primary cultures of duck IBDE cells were acutely infected with DHBV, dual-labeled confocal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 or a combination of anti-pre-S1 proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins. Immunoblot analysis of these cells showed that the DHBV pre-S1 and core proteins were similar to their counterparts in infected primary duck hepatocyte cultures. Southern blot analysis of infected IBDE preparations using a digoxigenin-labeled positive-sense DHBV riboprobe revealed the presence of hepadnavirus covalently closed circular (CCC) DNA, minus-sense single-stranded (SS) DNA, double-stranded linear DNA, and relaxed circular DNA. The presence of minus-sense SS DNA in the acutely infected IBDE cultures is indicative of DHBV reverse transcriptase activity, while the establishment of a pool of viral CCC DNA reveals the ability of these cells to maintain persistent infection. Taken collectively, the results from this study demonstrated that primary duck IBDE cells supported hepadnavirus replication as shown by the de novo synthesis of DHBV proteins and DNA replicative intermediates.

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

    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.

  5. Tenascin-C is required for normal Wnt/β-catenin signaling in the whisker follicle stem cell niche.

    Hendaoui, Ismaïl; Tucker, Richard P; Zingg, Dominik; Bichet, Sandrine; Schittny, Johannes; Chiquet-Ehrismann, Ruth


    Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/β-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed β-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear β-catenin in wild-type mice reflecting active Wnt/β-catenin signaling, whereas β-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/β-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted β-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/β-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/β-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells. Copyright © 2014. Published by Elsevier B.V.

  6. Manipulation of Human Primary Endothelial Cell and Osteoblast Coculture Ratios to Augment Vasculogenesis and Mineralization


    has not been determined for human primary cells. Human umbilical vein ECs were cultured with normal human primary OBs in different EC/OB ratios...MATERIALS AND METHODS Cell Culture Human umbilical vein ECs (Invitrogen, Carlsbad, CA) were cultured in EC media (Lifeline, Walkersville, MD) and used after...into the membrane insert. At each time point, phase contrast microscopy (Nikon, Melville, NY) was used to image the OBs on each well at 10 (n 4

  7. A Rare Case of Primary Insitu Squamous Cell Carcinoma of the Endometrium with Extensive Icthyosis Uteri

    Pailoor K


    Full Text Available Primary squamous cell carcinoma of the endometrium is exceedingly rare. We report a case of 52 years old postmenopausal woman who presented with pelvic pain of four months duration. Gynecologic examination revealed a normal cervix. A possibility of pyometra was considered through pelvic ultrasound. Total abdominal hysterectomy was performed and histopathologically, it was diagnosed as a case of primary in situ squamous cell carcinoma of the endometrium.

  8. Expression of cell cycle-related gene products in different forms of primary versus recurrent PVNS.

    Weckauf, Helgard; Helmchen, Birgit; Hinz, Ulf; Meyer-Scholten, Carola; Aulmann, Sebastian; Otto, Herwart F; Berger, Irina


    Expression patterns of cell cycle regulating gene products and Ki-67 in proliferating synovial cells of primary and recurrent pigmented villonodular synovitis (PVNS) in localized and diffuse lesions were examined by immunohistochemistry. Alterations of cell cycle-related proteins were seen in 98.7% of analyzed lesions. Both RB- and p53 pathways play a role in cell cycle dysregulation in PVNS. The RB pathway was more frequently altered in primary disease, while alterations of the p53 pathway seemed to be more important in recurrent lesions, regardless of the histomorphological type of disease. Ki-67 proliferation rate was elevated in recurrent tumors. Copyright 2004 Elsevier Ireland Ltd.

  9. Microinterferometric characterization of cells isolated from primary hepatomata and from allografts of hepatomata by 4-dimethylaminoazobenzene.

    Tongiani, R; Chieli, E; Malvaldi, G


    Distribution of individual cells according to their solid protoplasmic content has been determined in cell populations isolated from two primary hepatomata by 4-dimethylaminoazobenzene (DAB) and from subcutaneous allografts of two others DAB hepatomata carried for years as transplant lines in rats. In nodular (neoplastic) tissue of the primary hepatomata, the cells maintained the typical distribution of the mammalian hepatocytes in a weight class pattern, but (i) the number of cell clases was reduced due to disappearance of the heavier cells and (ii) the frequency of the light cells was markedly increased with respect to the findings in anodular (non-neoplastic) liver tissue. In the allografts the great majority of the cells had a small content in solids and were normally grouped in one class. It is suggested that a delay in the cell differentiation may be responsible of this change.

  10. Concise Review: Primary Cilia: Control Centers for Stem Cell Lineage Specification and Potential Targets for Cell-Based Therapies.

    Bodle, Josephine C; Loboa, Elizabeth G


    Directing stem cell lineage commitment prevails as the holy grail of translational stem cell research, particularly to those interested in the application of mesenchymal stem cells and adipose-derived stem cells in tissue engineering. However, elucidating the mechanisms underlying their phenotypic specification persists as an active area of research. In recent studies, the primary cilium structure has been intimately associated with defining cell phenotype, maintaining stemness, as well as functioning in a chemo, electro, and mechanosensory capacity in progenitor and committed cell types. Many hypothesize that the primary cilium may indeed be another important player in defining and controlling cell phenotype, concomitant with lineage-dictated cytoskeletal dynamics. Many of the studies on the primary cilium have emerged from disparate areas of biological research, and crosstalk amongst these areas of research is just beginning. To date, there has not been a thorough review of how primary cilia fit into the current paradigm of stem cell differentiation and this review aims to summarize the current cilia work in this context. The goal of this review is to highlight the cilium's function and integrate this knowledge into the working knowledge of stem cell biologists and tissue engineers developing regenerative medicine technologies. Stem Cells 2016;34:1445-1454.

  11. Primary culture and identification of sinoatrial node cells from newborn rat

    宋治远; 钟理; 仝识非; 何国祥


    Objective To establish a reliable approach to primary culture and identification of sinoatrial node (SAN) cells. Methods The SAN cells were cultured from SAN tissue removed from neonatal Wistar rats and purified with differential attachment and 5'-bromodeoxyuridine (BrdU) treatment. The obtained cells were morphologically observed with inverted microscopy and transmission electron microscopy. Its action potential was recorded using electrophysiological methods.Results Three distinctly different cells were observed in the cultured SAN cells: spindle, triangle and irregular. Of these, the spindle cells comprised the greatest proportion, with their shape, structure and electrophysiological characteristics consistent with those of the pacemaker cells of SAN. The triangle cells were similar in features to the similarly shaped myocytes located in the atrial myocardium. Conclusions The culture method of differential attachment combined with BrdU treatment is a reliable approach to growing SAN cells. Of the cells cultured from SAN, the spindle cells appear to function as pacemaker cells.

  12. File list: NoD.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available NoD.CDV.10.AllAg.Primary_umbilical_vein_endothelial_cells hg19 No description Cardiovascular Primary... umbilical vein endothelial cells SRX318770 ...

  13. File list: Pol.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available Pol.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells hg19 RNA polymerase Cardiovascular Primary... umbilical vein endothelial cells ...

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    Full Text Available InP.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells hg19 Input control Cardiovascular Primary... umbilical vein endothelial cells ...

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    Full Text Available Pol.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells hg19 RNA polymerase Cardiovascular Primary... umbilical vein endothelial cells ...

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  1. File list: InP.CDV.10.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

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  2. File list: DNS.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available DNS.CDV.50.AllAg.Primary_umbilical_vein_endothelial_cells hg19 DNase-seq Cardiovascular Primary... umbilical vein endothelial cells ...

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  5. File list: DNS.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

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  6. File list: NoD.CDV.50.AllAg.Primary_endothelial_cells [Chip-atlas[Archive

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  9. File list: DNS.CDV.20.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

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  12. Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia

    Pinkernelle Josephine


    Full Text Available Abstract Background Magnetic nanoparticles (MNPs offer a large range of applications in life sciences. Applications in neurosciences are one focus of interest. Unfortunately, not all groups have access to nanoparticles or the possibility to develop and produce them for their applications. Hence, they have to focus on commercially available particles. Little is known about the uptake of nanoparticles in primary cells. Previously studies mostly reported cellular uptake in cell lines. Here we present a systematic study on the uptake of magnetic nanoparticles (MNPs by primary cells of the nervous system. Results We assessed the internalization in different cell types with confocal and electron microscopy. The analysis confirmed the uptake of MNPs in the cells, probably with endocytotic mechanisms. Furthermore, we compared the uptake in PC12 cells, a rat pheochromocytoma cell line, which is often used as a neuronal cell model, with primary neuronal cells. It was found that the percentage of PC12 cells loaded with MNPs was significantly higher than for neurons. Uptake studies in primary mixed neuronal/glial cultures revealed predominant uptake of MNPs by microglia and an increase in their number. The number of astroglia and oligodendroglia which incorporated MNPs was lower and stable. Primary mixed Schwann cell/fibroblast cultures showed similar MNP uptake of both cell types, but the Schwann cell number decreased after MNP incubation. Organotypic co-cultures of spinal cord slices and peripheral nerve grafts resembled the results of the dispersed primary cell cultures. Conclusions The commercial MNPs used activated microglial phagocytosis in both disperse and organotypic culture systems. It can be assumed that in vivo application would induce immune system reactivity, too. Because of this, their usefulness for in vivo neuroscientific implementations can be questioned. Future studies will need to overcome this issue with the use of cell




    We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or

  14. Organometallic catalysts for primary phosphoric acid fuel cells

    Walsh, Fraser


    A continuing effort by the U.S. Department of Energy to improve the competitiveness of the phosphoric acid fuel cell by improving cell performance and/or reducing cell cost is discussed. Cathode improvement, both in performance and cost, available through the use of a class of organometallic cathode catalysts, the tetraazaannulenes (TAAs), was investigated. A new mixed catalyst was identified which provides improved cathode performance without the need for the use of a noble metal. This mixed catalyst was tested under load for 1000 hr. in full cell at 160 to 200 C in phosphoric acid H3PO4, and was shown to provide stable performance. The mixed catalyst contains an organometallic to catalyze electroreduction of oxygen to hydrogen peroxide and a metal to catalyze further electroreduction of the hydrogen peroxide to water. Cathodes containing an exemplar mixed catalyst (e.g., Co bisphenyl TAA/Mn) operate at approximately 650 mV vs DHE in 160 C, 85% H3PO4 with oxygen as reactant. In developing this mixed catalyst, a broad spectrum of TAAs were prepared, tested in half-cell and in a rotating ring-disk electrode system. TAAs found to facilitate the production of hydrogen peroxide in electroreduction were shown to be preferred TAAs for use in the mixed catalyst. Manganese (Mn) was identified as a preferred metal because it is capable of catalyzing hydrogen peroxide electroreduction, is lower in cost and is of less strategic importance than platinum, the cathode catalyst normally used in the fuel cell.

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

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


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

  16. Preparation and Primary Culture of Liver Cells Isolated from Adult Rats by Dispase Perfusion



    Full Text Available The dispase perfusion technique was used to isolate liver cells from adult rats. The optimum conditions for obtaining many isolated liver cells with high viability were an enzyme concentration of 2000 U/ml, a pH of 7.5 and a perfusion time of 20 min. The population of isolated liver cells prepared with dispase consisted of 43.6% cells with diameters less than 20 micron and 56.4% cells with diameters above 20 micron. The isolated liver cells were cultured in basal culture medium either supplemented with or without dexamethasone (1 X 10(-5M and insulin (10 micrograms/ml. The addition of hormones to the culture medium improved the attachment efficiency of the isolated liver cells and delayed the disappearance of mature hepatocytes. Epithelial-like clear cells proliferated early in primary culture even in the presence of hormones. Therefore, functioning mature hepatocytes and proliferating epithelial-like clear cells coexisted well in the hormone-containing medium. Furthermore, the number of cultured cells reached a maximal level earlier in the presence of hormones than in the absence of hormones. The level of TAT activity in primary cultured cells was higher up to 3 days after inoculation in the presence of hormones than in their absence. No difference between G6Pase activity in primary cultured cells in the presence of hormones and that in the absence of hormones was found.

  17. Primary targets in photochemical inactivation of cells in culture

    Berg, Kristian; Jones, Stuart G.; Prydz, Kristian; Moan, Johan


    The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by tetrasulfonated phenylporphines (TPPS4) are described). Ultracentrifugation studies on postnuclear supernatants indicated that the intracellular distribution of TPPS4 resembles that of (beta) -N-acetyl-D-glucosaminidase ((beta) -AGA), a lysosomal marker enzyme, and that the cytosolic content of TPPS4 is below the detection limit of the ultracentrifugation method. Upon light exposure more than 90% of TPPS4 was lost from the lysosomal fractions, due to lysosomal rupture. The content of TPPS4 in the postnuclear supernatants was reduced by 30 - 40% upon exposure to light. This is most likely due to binding of TPPS4 to the nuclei, which were removed from the cell extracts before ultracentrifugation, after photochemical treatment. The unpolymerized form of tubulin seems to be an important target for the photochemical inactivation of NHIK 3025 cells. Since TPPS4 is mainly localized in lysosomes it was assumed that a dose of light disrupting a substantial number of lysosomes followed by microtubule depolymerization by nocodazole would enhance the sensitivity of the cells to photoinactivation. This was confirmed by using a colony-forming assay. The increased phototoxic effect exerted by such a treatment regime could be explained by an enhanced sensitivity of tubulin to light. Another cytosolic constituent, lactate dehydrogenase, was not photoinactivated by TPPS4 and light.

  18. Senescence of primary amniotic cells via oxidative DNA damage.

    Ramkumar Menon

    Full Text Available OBJECTIVE: Oxidative stress is a postulated etiology of spontaneous preterm birth (PTB and preterm prelabor rupture of the membranes (pPROM; however, the precise mechanistic role of reactive oxygen species (ROS in these complications is unclear. The objective of this study is to examine impact of a water soluble cigarette smoke extract (wsCSE, a predicted cause of pregnancy complications, on human amnion epithelial cells. METHODS: Amnion cells isolated from fetal membranes were exposed to wsCSE prepared in cell culture medium and changes in ROS levels, DNA base and strand damage was determined by using 2'7'-dichlorodihydro-fluorescein and comet assays as well as Fragment Length Analysis using Repair Enzymes (FLARE assays, respectively. Western blot analyses were used to determine the changes in mass and post-translational modification of apoptosis signal-regulating kinase (ASK1, phospho-p38 (P-p38 MAPK, and p19(arf. Expression of senescence-associated β-galectosidase (SAβ-gal was used to confirm cell ageing in situ. RESULTS: ROS levels in wsCSE-exposed amnion cells increased rapidly (within 2 min and significantly (p<0.01 at all-time points, and DNA strand and base damage was evidenced by comet and FLARE assays. Activation of ASK1, P-p38 MAPK and p19(Arf correlated with percentage of SAβ-gal expressing cells after wsCSE treatment. The antioxidant N-acetyl-L-cysteine (NAC prevented ROS-induced DNA damage and phosphorylation of p38 MAPK, whereas activation of ASK1 and increased expression of p19(Arf were not significantly affected by NAC. CONCLUSIONS: The findings support the hypothesis that compounds in wsCSE induces amnion cell senescence via a mechanism involving ROS and DNA damage. Both pathways may contribute to PTB and pPROM. Our results imply that antioxidant interventions that control ROS may interrupt pathways leading to pPROM and other causes of PTB.

  19. Primary cutaneous diffuse large B-cell lymphoma of the upper limb: A fascinating entity

    Manoj Madakshira Gopal


    Full Text Available Primary cutaneous lymphomas are defined as lymphoid neoplasms that present themselves clinically on the skin and do not have extra-cutaneous disease, when the diagnosis is made or even after 6 months of the diagnosis. Primary cutaneous lymphomas of B-cells are less frequent than lymphomas of T-cells. Primary B-cell lymphomas have a better prognosis than secondary B-cell lymphomas. Primary B-cell cutaneous lymphomas are classified into five types according to the World Health Organization and European Organization for Research and Treatment of Cancer classification. The primary diffuse large B-cell cutaneous lymphoma - leg type corresponds to approximately 5-10% of the B-cell cutaneous lymphomas. It is predominantly seen in elderly people and has a female preponderance. Skin lesions can be single, multiple, and even grouped. A 5-year survival rate ranges from 36 to 100% of the cases. The expression of Bcl-2, presence of multiple lesions, and involvement of both the upper limbs lead to a worse prognosis. Very few cases have been described in the literature.

  20. [Primary colorectal lymphoma of diffuse large B-cells: an experience at a general hospital].

    Beltran Gárate, Brady; Morales Luna, Domingo; Quiñones Avila, Pilar; Hurtado de Mendoza, Fernando; Riva Gonzales, Luis; Yabar, Alejandro; Portugal Meza, Karem


    Primary colorectal lymphoma is a very rare disease. Primary colorectal lymphoma of diffuse large B-cells is a more frequent subtype representing 1% of all colon diseases. In a retrospective study, the clinical characteristics and treatment course of primary colorectal lymphoma of diffuse large B-cells between 1997 and 2003 were reviewed. According to Dawson's criteria, fourteen cases were identified. The average age was 65 and the ratio of men to women was 1:3. The most frequent signs and symptoms were abdominal pain (78%), diarrhea (49%) and abdominal tumor (35%). The most frequently involved regions were the cecum (42%), ascending colon (21%) and rectum (21%). Six were in Stage I, four in Stage II and four in Stage III. The 5-year survival per stage was 26, 11 and 5 months, respectively. Primary colorectal lymphoma of diffuse large B-cells usually affects the right part of the colon in an aggressive manner.

  1. Squamous cell carcinoma arising from primary retroperitoneal mature teratoma.

    Joseph, Leena D; Devi, M Kanmani; Sundaram, Sandhya; Rajendiran, S


    A 65 year old postmenopausal female presented with left sided abdominal pain. Sonogram revealed an intra-abdominal 7.4 x 5.7 cm heterogenous mass. On laparotomy, approximately 10 X 10 cm mesenteric mass was seen adherent to the descending colon. Multiple omental tumor deposits were also noted. Gross examination showed solid and cystic tumor with sebaceous material admixed with hair. Histopathology showed mature cystic teratoma with a spectrum of well to poorly differentiated squamous cell carcinoma with omental metastasis.

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

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


    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.

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

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


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

  4. Primary frontal sinus squamous cell carcinoma in a dog treated with surgical excision.

    Grimes, Janet A; Pagano, Candace J; Boudreaux, Bonnie B


    An 8-year-old castrated male mixed breed dog was presented for a squamous cell carcinoma of the left frontal sinus. A partial craniectomy was performed and polytetrafluoroethylene mesh was placed over the craniectomy site. The dog recovered well with a good cosmetic outcome. Histopathology confirmed primary frontal sinus squamous cell carcinoma.

  5. Primary cutaneous marginal zone B-cell lymphoma: clinical and therapeutic features in 50 cases.

    Hoefnagel, J.J.; Vermeer, M.H.; Jansen, P.A.M.; Heule, F.; Voorst Vader, P.C. van; Sanders, C.J.; Gerritsen, M.J.P.; Geerts, M.L.; Meijer, C.J.; Noordijk, E.M.; Willemze, R.


    BACKGROUND: Primary cutaneous marginal zone B-cell lymphoma (PCMZL) is a low-grade B-cell lymphoma that originates in the skin, with no evidence of extracutaneous disease. Studies focusing on the optimal treatment of PCMZL have not been published thus far. We describe 50 patients with PCMZL to

  6. Primary cutaneous marginal zone B-cell lymphoma: Clinical and therapeutic features in 50 cases

    P.P.W. Hoefnagel (Pepijn); P.M. Noordijk (P.); R. Willemze (Roelof); M.H. Vermeer (Maarten); P.M. Jansen (Pieter); F. Heule (Freerk); P.C. Van Voorst Vader (P.); C.J.G. Sanders (C. J G); M.J.P. Gerritsen (M. J P); M.L. Geerts (M.); C.J.L.M. Meijer (Chris)


    textabstractBackground: Primary cutaneous marginal zone B-cell lymphoma (PCMZL) is a low-grade B-cell lymphoma that originates in the skin, with no evidence of extracutaneous disease. Studies focusing on the optimal treatment of PCMZL have not been published thus far. We describe 50 patients with

  7. Primary cutaneous marginal zone B-cell lymphoma - Clinical and therapeutic features in 50 cases

    Hoefnagel, JJ; Vermeer, MH; Jansen, PM; Heule, F; Vader, PCV; Sanders, CJG; Gerritsen, MJP; Geerts, ML; Meijer, CJLM; Noordijk, EM; Willemze, R

    Background: Primary cutaneous marginal zone B-cell lymphoma (PCMZL) is a low-grade B-cell lymphoma that originates in the skin, with no evidence of extracutaneous disease. Studies focusing on the optimal treatment of PCMZL have not been published thus far. We describe 50 patients with PCMZL to

  8. Cellular radiosensitivity of primary and metastatic human uveal melanoma cell lines

    G.J.M.J. van den Aardweg (Gerard J. M.); N.C. Naus (Nicole); A.C. Verhoeven; J.E.M.M. de Klein (Annelies); G.P.M. Luyten (Gré)


    textabstractPURPOSE: To investigate the radiosensitivity of uveal melanoma cell lines by a clonogenic survival assay, to improve the efficiency of the radiation regimen. METHODS: Four primary and four metastatic human uveal melanoma cell lines were cultured in the presence of

  9. Growth hormone-releasing factor induces c-fos expression in cultured primary pituitary cells

    Billestrup, Nils; Mitchell, R L; Vale, W;


    GH-releasing factor (GRF) and somatostatin regulates the secretion and biosynthesis of GH as well as the proliferation of GH-producing cells. In order to further characterize the mitogenic effect of GRF, we studied the expression of the proto-oncogene c-fos in primary pituitary cells. Maximal...

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

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

    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...

  11. Cellular response of mucociliary differentiated primary bronchial epithelial cells to diesel exhaust

    Zarcone, M.C.; Duistermaat, E.; Schadewijk, A. van; Jedynksa, A.D.; Hiemstra, P.S.; Kooter, I.M.


    Cellular response of mucociliary differentiated primary bronchial epithelial cells to diesel exhaust. Am J Physiol Lung Cell Mol Physiol 311: L111–L123, 2016. First published May 17, 2016; doi:10.1152/ajplung.00064.2016.—Diesel emissions are the main source of air pollution in urban areas, and diese

  12. Cellular radiosensitivity of primary and metastatic human uveal melanoma cell lines

    G.J.M.J. van den Aardweg (Gerard J. M.); N.C. Naus (Nicole); A.C. Verhoeven; J.E.M.M. de Klein (Annelies); G.P.M. Luyten (Gré)


    textabstractPURPOSE: To investigate the radiosensitivity of uveal melanoma cell lines by a clonogenic survival assay, to improve the efficiency of the radiation regimen. METHODS: Four primary and four metastatic human uveal melanoma cell lines were cultured in the presence of condi

  13. Cellular response of mucociliary differentiated primary bronchial epithelial cells to diesel exhaust

    Zarcone, M.C.; Duistermaat, E.; Schadewijk, A. van; Jedynksa, A.D.; Hiemstra, P.S.; Kooter, I.M.


    Cellular response of mucociliary differentiated primary bronchial epithelial cells to diesel exhaust. Am J Physiol Lung Cell Mol Physiol 311: L111–L123, 2016. First published May 17, 2016; doi:10.1152/ajplung.00064.2016.—Diesel emissions are the main source of air pollution in urban areas, and

  14. CT findings of primary squamous cell carcinoma of the stomach: a case report

    Kim, Kyoung Min; Lee, Chang Hee; Kim, Kyeong Ah; Park, Cheol Min [Guro Hospital of Korea University, Seoul (Korea, Republic of)


    Primary squamous cell carcinoma is a rare tumor of the stomach with an incidence ranging from 0.04% to 0.4% of all diagnosed gastric cancers. We report a case of squamous cell carcinoma in the stomach associated with hypertrophic gastropathy and observed as a huge mass and wall thickening on the greater curvature site by a multidetector CT.

  15. Sonic Hedgehog signaling pathway in primary liver cancer cells

    Lian-Yi Guo; Pei Liu; Ying Wen; Wei Cui; Ying Zhou


    Objective:To investigate clinical significance ofSonicHedgehog(SHH) signaling pathway molecularShh,Smo andGli2 in primary hepatocellular carcinoma(HCC) tissue.Methods:A total of30HCC tissue samples were collected.Protein expression ofSHH signaling pathway moleculesShh,Smo andGli2 inHCC tissues and para - carcinoma tissue were detected by using immunohistochemical method.Cirrhosis and normal liver tissue specimens were observed as control to analyze the expression ofSHH signaling pathway molecularShh,Smo andGli2 mRNA inHCC tissues and corresponding para-carcinoma tissues and its relationship with the onset of HCC.Results:There was no expression ofShh,Smo andGli2 protein in normal liver tissue, while their positive rates were63.3%,76.7% and66.7% inHCC tissues, respectively, with asignificantly higher expression level than that in the para - carcinoma tissue(P0.05);Shh andSmo protein was detected in part of cirrhosis with positive expression, butGli2 protein was not observable in cirrhosis tissues.Conclusions:InHCC tissues, the high expression level ofSHH signaling pathway molecules signal peptide(Shh), membrane protein receiptor(Smo) and nuclear transcription molecular(Gli2) can be indicators of the onset of liver cancer.

  16. Aortic Cell Apoptosis in Rat Primary Aldosteronism Model

    闫永吉; 欧阳金芝; 王超; 吴准; 马鑫; 李宏召; 徐华; 胡争; 李俊; 王保军; 史涛坪; 龚道静; 倪栋; 张旭


    This study aimed to determine whether aldosterone could induce vascular cell apoptosis in vivo.Thirty-two male rats were randomly divided into 4 groups:vehicle(control),aldosterone,aldosterone plus eplerenone or hydralazine.They were then implanted with an osmotic mini-pump that infused either aldosterone or the vehicle.Systolic blood pressure(SBP) was measured weekly by the tail-cuff method.After 8 weeks,plasma aldosterone concentration(PAC) and renin activity(PRA) were determined by radioimmunoassay.Aorti...


    A. B. Villert


    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.

  18. Primary lymphoblastic B-cell lymphoma of the stomach: A case report

    Miao-Xia He; Ming-Hua Zhu; Wei-Qiang Liu; Li-Li Wu; Xiong-Zeng Zhu


    Primary stomach lymphoblastic B-cell lymphoma (B-LBL) is a rare tumor. We describe a primary stomach B-LBL in a 38 years old female who presented with nonspecific complaints of fatigue and vomiting for 2 mo.Gastrofiberscopy revealed a large gastric ulcer, which was successfully resected. Pathology showed a lymphoblastic cell lymphoma arising from the stomach, and there was no evidence of disease at any extrastomach site.Immunohistochemical staining and gene rearrangement studies supported that the stomach tumor was a clonal B-cell lymphoma. Therefore, the diagnosis of B-LBL was made based on the stomach specimen.

  19. An attempt to eliminate fibroblast-like cells from primary cultures of fetal human livers.



    Full Text Available The elimination of fibroblast-like cells from primary cultures of fetal human livers was studied. A fibroblast-like cell line (HuF, which was obtained by subculturing fetal human liver cells 4 or more times, was briefly treated with hydrocortisone (HC or putrescine (PUT. The growth of HuF cells was inhibited by HC at a concentration of 10(-2 M and by PUT at a concentration higher than 10(-3 M. Long-term treatment of HuF cells with 10(-3 M HC inhibited the growth of the cells. Primary cultures of fetal human livers were made in medium containing HC or PUT, and morphological and functional examinations were made. The cultures were predominantly composed of epithelial-like cells, with few fibroblast-like cells, when the HC concentration was 10(-5M to 10(-3 M. A high amount of albumin was secreted at these concentrations of HC. On the other hand, at 10(-3 M PUT, many epithelial-like cells were seen, but albumin was undetectable. The present results indicate that albumin-producing epithelial-like cells can be selectively maintained in medium containing HC, in primary cultures of fetal human livers.

  20. Primary abdominal wall clear cell carcinoma arising from incisional endometriosis

    Burcu Gundogdu; Isin Ureyen; Gunsu Kimyon; Hakan Turan; Nurettin Boran; Gokhan Tulunay; Dilek Bulbul; Taner Turan; M Faruk Kose


    A 49 year-old patient with the complaint of a mass located in the caesarean scar was admitted. There was a fixed mass 30í30 mm in diameter with regular contour located at the right corner of the pfannenstiel incision. Computed tomography revealed a (40í50í50) mm solid mass lesion with margins that cannot be distinguished from the uterus, bladder and small intestines and a heterogeneous mass lesion (50í45í55) mm in diameter, located in the right side of the anterior abdominal wall. Cytoreductive surgery including total abdominal hysterectomy and bilateral salpingo-oophorectomy was performed. Final pathology was clear cell carcinoma. Clear cell carcinoma arising from an extraovarian endometriotic focus was diagnosed and the patient received 6 cycles paclitaxel-carboplatin chemotherapy as adjuvant treatment. The patient who was lost to follow-up applied to our clinic 2 years after surgery with a recurrent mass in the left inguinal region. After 3 cycles of chemotherapy, the patient's tumoral mass in the left inguinal region was excised. The result of the pathology was carcinoma metastasis. It is decided that the following treatment of the patient should be palliative radiation therapy. The patient who underwent palliative radiation therapy died of disease after 4 months of the second operation.

  1. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu


    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  2. Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

    Corrêa, Natássia C R; Kuasne, Hellen; Faria, Jerusa A Q A


    Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1...... and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted....... This shift in expression may be due to the selection of an 'establishment' phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines...

  3. Bortezomib sensitizes primary human esthesioneuroblastoma cells to TRAIL-induced apoptosis.

    Koschny, Ronald; Holland, Heidrun; Sykora, Jaromir; Erdal, Hande; Krupp, Wolfgang; Bauer, Manfred; Bockmuehl, Ulrike; Ahnert, Peter; Meixensberger, Jürgen; Stremmel, Wolfgang; Walczak, Henning; Ganten, Tom M


    TNF-related apoptosis-inducing ligand (TRAIL), a promising novel anti-cancer cytokine of the TNF superfamily, and Bortezomib, the first-in-class clinically used proteasome inhibitor, alone or in combination have been shown to efficiently kill numerous tumor cell lines. However, data concerning primary human tumor cells are very rare. Using primary esthesioneuroblastoma cells we analyzed the anti-tumor potential and the mechanism employed by Bortezomib in combination with TRAIL for the treatment of this rare but aggressive tumor. Expression of components of the TRAIL pathway was analyzed in tumor specimens and isolated primary tumor cells at the protein level. Cells were treated with TRAIL, Bortezomib, and a combination thereof, and apoptosis induction was quantified. Clonogenicity assays were performed to elucidate the long-term effect of this treatment. Despite expressing all components of the TRAIL pathway, freshly isolated primary esthesioneuroblastoma cells were completely resistant to TRAIL-induced apoptosis. They could, however, be very efficiently sensitized by subtoxic doses of Bortezomib. The influence of Bortezomib on the TRAIL pathway was analyzed and showed upregulation of TRAIL death receptor expression, enhancement of the TRAIL death-inducing signaling complex (DISC), and downregulation of anti-apoptotic proteins of the TRAIL pathway. Of clinical relevance, TRAIL-resistant primary tumor cells could be repeatedly sensitized by Bortezomib, providing the basis for repeated clinical application schedules. This is the first report on the highly synergistic induction of apoptosis in primary esthesioneuroblastoma cells by Bortezomib and TRAIL. This combination, therefore, represents a promising novel therapeutic option for esthesioneuroblastoma.

  4. [Isolation and purification of primary Kupffer cells from mouse liver].

    Sun, Chao; Luo, Qingbo; Lu, Xiuxian; Zheng, Daofeng; He, Diao; Wu, Zhongjun


    Objective To isolate and purify Kupffer cells (KCs) from BALB/c mice by an efficient method of low-speed centrifugation and rapid adherence. Methods The mouse liver tissue was perfused in situ and digested with 0.5 g/L collagenase type IV in vitro by water bath. Then, through the low-speed centrifugation, KCs were separated from the mixed hepatocytes, and purified by rapid adherent characteristics. Finally, the production and activity of KCs obtained by this modified method were compared with those isolated by Percoll density gradient centrifugation. We used F4/80 antibody immunofluorescence technique to observe morphological features of KCs, flow cytometry (FCM) to detect the expression of F4/80 antibody and the ink uptake test to observe the phagocytic activity. Moreover, using FCM, we evaluated the expressions of molecules associated with antigen presentation, including major histocompatibility complex class II (MHC II), CD40, CD86 and CD68 on the surface of KCs subjected to hypoxia/reoxygenation (H/R) modeling. And, ELISA was conducted to measure tumor necrosis factor-α (TNF-α) production of the cultured KCs following H/R. Results The yield of KCs was (5.83±0.54)×10(6) per mouse liver and the survival rate of KCs was up to 92% by low-speed centrifugation and rapid adherent method. Compared with Percoll density gradient centrifugation [the yield of KCs was (2.19±0.43)×10(6) per liver], this new method significantly improved the yield of KCs. F4/80 immunofluorescence showed typical morphologic features of KCs such as spindle or polygon shapes and FCM identified nearly 90% F4/80 positive cells. The phagocytic assay showed that lots of ink particles were phagocytosed into the isolated cells. KC H/R models expressed more MHC II, CD40 and CD86 and produced more TNF-α participating in inflammation. Conclusion The efficient method to isolate and purify KCs from BALB /c mice has been successfully established.

  5. Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies

    Gong Min


    Full Text Available Abstract Background Mesenchymal stem cells (MSCs can be induced to differentiate into neuronal cells under appropriate cellular conditions and transplanted in brain injury and neurodegenerative diseases animal models for neuroregeneration studies. In contrast to the embryonic stem cells (ESCs, MSCs are easily subject to aging and senescence because of their finite ability of self-renewal. MSCs senescence seriously affected theirs application prospects as a promising tool for cell-based regenerative medicine and tissue engineering. In the present study, we established a reversible immortalized mesenchymal stem cells (IMSCs line by using SSR#69 retrovirus expressing simian virus 40 large T (SV40T antigen as an alternative to primary MSCs. Methods The retroviral vector SSR#69 expressing simian virus 40 large T (SV40T antigen was used to construct IMSCs. IMSCs were identified by flow cytometry to detect cell surface makers. To investigate proliferation and differentiation potential of IMSCs, cell growth curve determination and mesodermal trilineage differentiation tests were performed. Neuronal differentiation characteristics of IMSCs were detected in vitro. Before IMSCs transplantation, we excluded its tumorigenicity in nude mice firstly. The Morris water maze tests and shuttle box tests were performed five weeks after HIBD models received cells transplantation therapy. Results In this study, reversible IMSCs were constructed successfully and had the similar morphology and cell surface makers as primary MSCs. IMSCs possessed better ability of proliferation and anti-senescence compared with primary MSCs, while maintained multilineage differentiation capacity. Neural-like cells derived from IMSCs had similar expressions of neural-specific genes, protein expression patterns and resting membrane potential (RMP compared with their counterparts derived from primary MSCs. There was no bump formation in nude mice subcutaneously injected with IMSCs. IMSCs

  6. Primary instabilities in convective cells due to nonuniform heating

    Mancho, A. M.; Herrero, H.; Burguete, J.


    We study a convection problem in a container with a surface open to the air and heated by a long wire placed at the bottom. Coupled buoyancy and thermocapillarity effects are taken into account. A basic convective state appears as soon as a temperature gradient with horizontal component different from zero is applied. It consists of two big rolls that fill the convective cell and are parallel to the heater. A numerical solution allows us to determine this basic state. A linear stability analysis on this solution is carried out. For different values of the applied temperature gradient the basic rolls undergo a stationary bifurcation. The thresholds depend on the fluid properties, on the geometry of the heater, and on the heat exchange on the free surface. This confirms the results obtained in recent experiments.

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

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


    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...

  8. TAT-mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression.

    Simon, Melissa J; Gao, Shan; Kang, Woo Hyeun; Banta, Scott; Morrison, Barclay


    Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT-mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)-TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP-TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum-containing medium; (2) monocultures grown in serum-free medium; (3) cocultures with neurons in serum-free medium. The efficiency of GFP-TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP-TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT-mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT-mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell-type and phenotype-dependence of TAT-mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs.

  9. Primary Blast Causes Delayed Effects without Cell Death in Shell-Encased Brain Cell Aggregates.

    Sawyer, Thomas W; Ritzel, David V; Wang, Yushan; Josey, Tyson; Villanueva, Mercy; Nelson, Peggy; Song, Yanfeng; Shei, Yimin; Hennes, Grant; Vair, Cory; Parks, Steve; Fan, Changyang; McLaws, Lori


    Previous work in this laboratory used underwater explosive exposures to isolate the effects of shock-induced principle stress without shear on rat brain aggregate cultures. The current study has utilized simulated air blast to expose aggregates in suspension and enclosed within a spherical shell, enabling the examination of a much more complex biomechanical insult. Culture medium-filled spheres were exposed to single pulse overpressures of 15-30 psi (∼6-7 msec duration) and measurements within the sphere at defined sites showed complex and spatially dependent pressure changes. When brain aggregates were exposed to similar conditions, no cell death was observed and no changes in several commonly used biomarkers of traumatic brain injury (TBI) were noted. However, similarly to underwater blast, immediate and transient increases in the protein kinase B signaling pathway were observed at early time-points (3 days). In contrast, the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase, as well as vascular endothelial growth factor, both displayed markedly delayed (14-28 days) and pressure-dependent responses. The imposition of a spherical shell between the single pulse shock wave and the target brain tissue introduces greatly increased complexity to the insult. This work shows that brain tissue can not only discriminate the nature of the pressure changes it experiences, but that a portion of its response is significantly delayed. These results have mechanistic implications for the study of primary blast-induced TBI and also highlight the importance of rigorously characterizing the actual pressure variations experienced by target tissue in primary blast studies.

  10. Primary gastric T cell lymphoma mimicking marginal zone B cell lymphoma of mucosa-associated lymphoid tissue.

    Holanda, Danniele; Zhao, Merry Y; Rapoport, Aaron P; Garofalo, Michael; Chen, Qing; Zhao, X Frank


    Primary gastric T cell lymphoma is rare and mostly of large cell type. In this paper, we present a case of gastric T cell lymphoma morphologically similar to the gastric marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT). Morphologically, the cells are small with abundant clear cytoplasm. Lymphoepithelial lesions are readily identified with diffuse destruction of gastric glands. Immunohistochemically, the neoplastic cells are CD3+/CD4+/CD8-/Granzyme B-. Molecular studies revealed monoclonal T cell receptor gamma gene rearrangement. Clinically, the patient responded initially to four cycles of R-CHOP, but then progressed. Because peripheral T cell lymphoma is usually associated with a poor prognosis, whereas marginal zone B cell lymphoma is an indolent lymphoproliferative disorder, this morphologic mimicry should be recognized and completely investigated when atypical small lymphoid infiltrates with lymphoepithelial lesions are encountered in the stomach.

  11. Dependence of sea urchin primary mesenchyme cell migration on xyloside- and sulfate-sensitive cell surface-associated components.

    Lane, M C; Solursh, M


    The migration of sea urchin primary mesenchyme cells (PMC) is inhibited in embryos cultured in sulfate-free seawater and in seawater containing exogenous xylosides. In the present study, primary mesenchyme cells and extra-cellular matrix have been isolated from normal and treated Lytechinus pictus and Strongylocentrotus purpuratus embryos and recombined in an in vitro migration assay to determine whether the cells or the matrix are migration defective. Normal cells were found to migrate on either normal or treated matrix, whereas sulfate-deprived and xyloside-treated PMC failed to migrate in vitro on normal and treated substrata. Migratory ability can be restored to defective cells by returning the PMC to normal seawater, or by exposing the defective cells to materials removed from the surface of normal cells with 1 M urea. The similarity of the results obtained with sulfate-deprived and xyloside-treated PMC suggested that a common molecule may be affected by the two treatments. As a first test of this possibility, xyloside-treated S. purpuratus PMC were given the urea extract prepared from sulfate-deprived S. purpuratus PMC, and this extract did not restore migratory ability. These findings indicate that PMC normally synthesize a surface-associated molecule that is involved in cell migration, and the sensitivity to exogenous xylosides and sulfate deprivation suggests that a sulfated proteoglycan may be involved in primary mesenchyme cell migration.

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

    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.

  13. Interactions of virulent and avirulent leptospires with primary cultures of renal epithelial cells

    Ballard, S A; Williamson, M; Adler, B


    A primary culture system for the cells of mouse renal-tubular epithelium was established and used to observe the adhesion of leptospires. Virulent strains of serovars copenhageni and ballum attached themselves to epithelial cells within 3 h of infection whereas an avirulent variant of serovar...... copenhageni did not adhere to epithelial cells at all within the experimental period of 24 h. The saprophytic Leptospira biflexa serovar patoc became attached non-specifically to inert glass surfaces as well as to the cells. The adhesion of leptospires to epithelial cells was not inhibited by homologous...

  14. BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact

    Scholz, Jean L.; Crowley, Jenni E.; Tomayko, Mary M.; Steinel, Natalie; O'Neill, Patrick J.; Quinn, William J; Goenka, Radhika; Miller, Juli P; Cho, Yun Hee; Long, Vatana; Ward, Chris; Migone, Thi-Sau; Shlomchik, Mark J.; Cancro, Michael P.


    We have used an inhibiting antibody to determine whether preimmune versus antigen-experienced B cells differ in their requisites for BLyS, a cytokine that controls differentiation and survival. Whereas in vivo BLyS inhibition profoundly reduced naïve B cell numbers and primary immune responses, it had a markedly smaller effect on memory B cells and long-lived plasma cells, as well as secondary immune responses. There was heterogeneity within the memory pools, because IgM-bearing memory cells ...

  15. High-Throughput Screening for Bioactive Molecules Using Primary Cell Culture of Transgenic Zebrafish Embryos

    Haigen Huang


    Full Text Available Transgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.

  16. Primary bilateral adrenal B-cell lymphoma associated with EBV and JCV infection

    Guzzardo Vincenza


    Full Text Available Abstract Primary lymphoma of the adrenal gland is a rare and highly aggressive disease, with only a few reports in the literature. The pathogenesis is unknown, but detection of Epstein Barr virus (EBV genome sequences and gene expression in some cases of primary adrenal lymphomas suggested the virus might be a causative agent of the malignancy. While investigating the presence of genome sequences of oncogenic viruses in a large series of adrenal tumors, both EBV and JC polyomavirus (JCV DNA sequences were detected in a diffuse large primary bilateral B-cell non-Hodgkin lymphoma of the adrenal gland, which was diagnosed only at postmortem examination in a 77 year-old woman with incidentally discovered adrenal masses and primary adrenal insufficiency. The presence of both EBV and JCV genome sequences suggests the relevance of EBV and JCV coinfection in the pathogenesis of this rare form of B-cell lymphoma.

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

    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.

  18. ATRX dysfunction induces replication defects in primary mouse cells.

    David Clynes

    Full Text Available The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells.

  19. ATRX dysfunction induces replication defects in primary mouse cells.

    Clynes, David; Jelinska, Clare; Xella, Barbara; Ayyub, Helena; Taylor, Stephen; Mitson, Matthew; Bachrati, Csanád Z; Higgs, Douglas R; Gibbons, Richard J


    The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells.

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  8. File list: Unc.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

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  10. File list: His.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells [Chip-atlas[Archive

    Full Text Available His.CDV.05.AllAg.Primary_umbilical_vein_endothelial_cells hg19 Histone Cardiovascular ...

  11. Phenotyping and auto-antibody production by liver-infiltrating B cells in primary sclerosing cholangitis and primary biliary cholangitis.

    Chung, Brian K; Guevel, Bardia T; Reynolds, Gary M; Gupta Udatha, D B R K; Henriksen, Eva Kristine Klemsdal; Stamataki, Zania; Hirschfield, Gideon M; Karlsen, Tom Hemming; Liaskou, Evaggelia


    Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are immune-mediated biliary diseases that demonstrate prominent and restricted genetic association with human leukocyte antigen (HLA) alleles. In PBC, anti-mitochondrial antibodies (AMA) are specific and used as diagnostic biomarkers. PSC-relevant auto-antibodies remain controversial despite a distinct HLA association that mirrors archetypical auto-antigen driven disorders. Herein, we compared antibody-secreting B cells (ASCs) in PSC and PBC liver explants to determine if liver-infiltrating ASCs represent an opportune and novel source of disease-relevant auto-antibodies. Using enzymatic digestion and mechanical disruption, liver mononuclear cells (LIMCs) were isolated from fresh PSC and PBC explants and plasmablast (CD19+CD27+CD38(hi)CD138-) and plasma cell (CD19+CD27+CD38(hi)CD138+) ASCs were enumerated by flow cytometry. We observed 45-fold fewer plasma cells in PSC explants (n = 9) compared to PBC samples (n = 5, p < 0.01) and 10-fold fewer IgA-, IgG- and IgM-positive ASCs (p < 0.05). Liver-infiltrating ASCs from PSC and PBC explants were functional and produced similar concentrations of IgA, IgG and IgM following 2 weeks of culture. Antibody production by PBC ASCs (n = 3) was disease-specific as AMA to pyruvate dehydrogenase complex E2 subunit (PDC-E2) was detected by immunostaining, immunoblotting and ELISA. Antibody profiling of PSC supernatants (n = 9) using full-length recombinant human protein arrays (Cambridge Protein Arrays) revealed reactivities to nucleolar protein 3 (5/9) and hematopoietic cell-specific Lyn substrate 1 (3/9). Array analysis of PBC supernatants (n = 3) detected reactivities to PDC-E2 and hexokinase 1 (3/3). In conclusion, we detected unique frequencies of liver-infiltrating ASCs in PSC and PBC and in so doing, highlight a feasible approach for understanding disease-relevant antibodies in PSC. Copyright © 2016. Published by Elsevier Ltd.

  12. Primary diffuse large B-cell lymphoma of the corpora cavernosa presented as a perineal mass

    Carlos, González-Satué; Ivanna, Valverde Vilamala; Gustavo, Tapia Melendo; Joan, Areal Calama; Javier, Sanchez Macias; Luis, Ibarz Servio


    Primary male genital lymphomas may appear rarely in testis, and exceptionally in the penis and prostate, but there is not previous evidence of a lymphoma arising from the corpora cavernosa. We report the first case in the literature of a primary diffuse cell B lymphoma of the corpora cavernosa presented with low urinary tract symptoms, perineal pain and palpable mass. Diagnosis was based on trucut biopsy, histopathological studies and computed tomographic images. PMID:22919138

  13. Identification of drugs that restore primary cilium expression in cancer cells.

    Khan, Niamat Ali; Willemarck, Nicolas; Talebi, Ali; Marchand, Arnaud; Binda, Maria Mercedes; Dehairs, Jonas; Rueda-Rincon, Natalia; Daniels, Veerle W; Bagadi, Muralidhararao; Thimiri Govinda Raj, Deepak Balaji; Vanderhoydonc, Frank; Munck, Sebastian; Chaltin, Patrick; Swinnen, Johannes V


    The development of cancer is often accompanied by a loss of the primary cilium, a microtubule-based cellular protrusion that functions as a cellular antenna and that puts a break on cell proliferation. Hence, restoration of the primary cilium in cancer cells may represent a novel promising approach to attenuate tumor growth. Using a high content analysis-based approach we screened a library of clinically evaluated compounds and marketed drugs for their ability to restore primary cilium expression in pancreatic ductal cancer cells. A diverse set of 118 compounds stimulating cilium expression was identified. These included glucocorticoids, fibrates and other nuclear receptor modulators, neurotransmitter regulators, ion channel modulators, tyrosine kinase inhibitors, DNA gyrase/topoisomerase inhibitors, antibacterial compounds, protein inhibitors, microtubule modulators, and COX inhibitors. Certain compounds also dramatically affected the length of the cilium. For a selection of compounds (Clofibrate, Gefitinib, Sirolimus, Imexon and Dexamethasone) their ability to restore ciliogenesis was confirmed in a panel of human cancer cell line models representing different cancer types (pancreas, lung, kidney, breast). Most compounds attenuated cell proliferation, at least in part through induction of the primary cilium, as demonstrated by cilium removal using chloral hydrate. These findings reveal that several commonly used drugs restore ciliogenesis in cancer cells, and warrant further investigation of their antineoplastic properties.

  14. Fertilization in Torenia fournieri: actin organization and nuclear behavior in the central cell and primary endosperm


    Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.

  15. The small GTPase Cdc42 is necessary for primary ciliogenesis in renal tubular epithelial cells.

    Zuo, Xiaofeng; Fogelgren, Ben; Lipschutz, Joshua H


    Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, where they participate in flow sensing. Disruption of cilia function has been linked to the pathogenesis of polycystic kidney disease. We demonstrated previously that the exocyst, a highly conserved eight-protein membrane trafficking complex, localizes to primary cilia of renal tubular epithelial cells, is required for ciliogenesis, biochemically and genetically interacts with polycystin-2 (the protein product of the polycystic kidney disease 2 gene), and, when disrupted, results in MAPK pathway activation both in vitro and in vivo. The small GTPase Cdc42 is a candidate for regulation of the exocyst at the primary cilium. Here, we demonstrate that Cdc42 biochemically interacts with Sec10, a crucial component of the exocyst complex, and that Cdc42 colocalizes with Sec10 at the primary cilium. Expression of dominant negative Cdc42 and shRNA-mediated knockdown of both Cdc42 and Tuba, a Cdc42 guanine nucleotide exchange factor, inhibit ciliogenesis in Madin-Darby canine kidney cells. Furthermore, exocyst Sec8 and polycystin-2 no longer localize to primary cilia or the ciliary region following Cdc42 and Tuba knockdown. We also show that Sec10 directly interacts with Par6, a member of the Par complex that itself directly interacts with Cdc42. Finally, we show that Cdc42 knockdown results in activation of the MAPK pathway, something observed in cells with dysfunctional primary cilia. These data support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis.

  16. The Small GTPase Cdc42 Is Necessary for Primary Ciliogenesis in Renal Tubular Epithelial Cells*

    Zuo, Xiaofeng; Fogelgren, Ben; Lipschutz, Joshua H.


    Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, where they participate in flow sensing. Disruption of cilia function has been linked to the pathogenesis of polycystic kidney disease. We demonstrated previously that the exocyst, a highly conserved eight-protein membrane trafficking complex, localizes to primary cilia of renal tubular epithelial cells, is required for ciliogenesis, biochemically and genetically interacts with polycystin-2 (the protein product of the polycystic kidney disease 2 gene), and, when disrupted, results in MAPK pathway activation both in vitro and in vivo. The small GTPase Cdc42 is a candidate for regulation of the exocyst at the primary cilium. Here, we demonstrate that Cdc42 biochemically interacts with Sec10, a crucial component of the exocyst complex, and that Cdc42 colocalizes with Sec10 at the primary cilium. Expression of dominant negative Cdc42 and shRNA-mediated knockdown of both Cdc42 and Tuba, a Cdc42 guanine nucleotide exchange factor, inhibit ciliogenesis in Madin-Darby canine kidney cells. Furthermore, exocyst Sec8 and polycystin-2 no longer localize to primary cilia or the ciliary region following Cdc42 and Tuba knockdown. We also show that Sec10 directly interacts with Par6, a member of the Par complex that itself directly interacts with Cdc42. Finally, we show that Cdc42 knockdown results in activation of the MAPK pathway, something observed in cells with dysfunctional primary cilia. These data support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis. PMID:21543338

  17. Advanced membranes for alkaline primary and rechargeable alkaline cells with zinc anodes

    Lewis, Harlan; Jackson, Patricia; Salkind, Alvin; Danko, Thomas; Bell, Roger

    Several advanced cellulosic and radiation grafted polypropylene membrane materials are currently under evaluation in the laboratories at Navsea Crane and Rutgers University, for application to alkaline primary and rechargeable cell chemistries which employ zinc as the anode material. A portion of these tests involve model cell evaluations of cellulosic membranes for silver migration rates through the membranes as a function of separation layers and changes in the degree of polymerisation (DP), wet tensile strength (WTS) and voltage changes at both electrodes as a function of model rechargeable cell life cycle. Other testing on the actual membranes is generating data for both cellulosic and polypropylene materials on impedance, swelling properties, and silver and zinc penetration rates. The overall goal of these investigations is to obtain candidate separation membranes which will reduce zinc anode shape change and shedding and resist alkaline oxidative degradation to extend the active wet life in primary cells and both wet and life cycle in rechargeable cells.

  18. A flexible Li polymer primary cell with a novel gel electrolyte based on poly(acrylonitrile)

    Akashi, Hiroyuki; Tanaka, Ko-ichi; Sekai, Koji

    The performance of a Li polymer primary cell with fire-retardant poly(acrylonitrile) (PAN)-based gel electrolytes is reported. By optimizing electrodes, electrolytes, the packaging material, and the structural design of the polymer cell, we succeeded in developing a "film-like" Li polymer primary cell with sufficient performance for practical use. The cell is flexible and less than 0.5 mm thick, which makes it suitable for a power source for some smart devices, such as an IC card. Fast cation conduction in the gel electrolyte minimizes the drop of the discharge capacity even at -20 °C. The high chemical stability of the gel electrolytes and the new packaging material allow the self-discharge rate to be limited to under 4.3%, which is equivalent to that of conventional coin-shaped or cylindrical Li-MnO 2 cells.

  19. Primary squamous cell carcinoma of the liver: A successful surgically treated case

    Hsiang-Lin Lee; Yu-Yin Liu; Chun-Nan Yeh; Kun-Chun Chiang; Tse-Ching Chen; Yi-Yin Jan


    Primary squamous cell carcinoma (SCC) of the liver is rare. Totally nine such cases have been reported in the literature. Primary SCC of the liver has been reported to be associated with hepatic teratoma,hepatic cyst, or hepatolithiasis. Complete remission of poorly differentiated SCC of the liver could be achieved by systemic chemotherapy followed by surgery or remarkably respond to hepatic arterial injection of low dose chemotherapeutic drugs. Here we report the first case of primary SCC of the liver presenting as a solid tumor and receiving successful hepatic resection with 9-mo disease free survival.

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

    Yoshiaki Tagawa


    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.

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

    Tagawa, Yoshiaki; Namba, Kenichi; Ogasawara, Reiki; Kanno, Hiromi; Ishida, Susumu


    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. PMID:26668579

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

    Floriana Rotondo


    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



    Objective: To detect the effect of arsenic trioxide or ATRA on APL cells or HL-60 cells and to investigate the mechanism of the hyperleukocytosis and detect the cross resistance between ATRA and arsenic trioxide. Methods: The number of promyelocytes or more matured granulocytes were counted by regular method, MTT test was used to measure the proliferation of HL-60 cells or APL cells, flow cytometry analysis to measure the apoptosis, NBT method to detect the differentiation of HL-60 cells or APL cells. Results: The proliferation of primary APL cells or HL-60 cells could be inhibited in vitro by either arsenic trioxide or ATRA, which could induce obvious apoptosis or obvious differentiation of primary APL cells or HL-60 cells. Inhibition of proliferation or apoptosis of ATRA resistant HL-60 cells were achieved by exposure toarsenic trioxide in vitro. On the other hand, the results of in vivo treatment showed that arsenic trioxide also induce of hyperleukocytosis. Conclusion: The results indicated that the hyperleukocytosis induced by ATRA is not contributed to the mechanism of more differentiation than apoptosis, there was not cross resistance between ATRA and arsenic trioxide.

  4. Primary de novo malignant giant cell tumor of kidney: a case report

    Torkian Bahman


    Full Text Available Abstract Background Osteoclast-like giant cell tumors are usually observed in osseous tissue or as tumors of tendon sheath, characterized by the presence of multinucleated giant cells and mononuclear stromal cells. It has been reported in various extraosseous sites including breast, skin, soft tissue, salivary glands, lung, pancreas, female genital tract, thyroid, larynx and heart. However, extraosseus occurrence of such giant cell tumors in the kidney is extremely rare and is usually found in combination with a conventional malignancy. De-novo primary malignant giant cell tumors of the kidney are unusual lesions and to our knowledge this is the second such case. Case Presentation We report a rare case of extraosseous primary denovo malignant giant cell tumor of the renal parenchyma in a 39-year-old Caucasian female to determine the histogenesis of this neoplasm with a detailed literature review. Conclusion Primary denovo malignant giant cell tumor of the kidney is extremely rare. The cellular origin of this tumor is favored to be a pluripotential mesenchymal stromal cell of the mononuclear/phagocytic cellular lineage. Awareness of this neoplasm is important in the pathological interpretation of unusual findings at either fine needle aspiration or frozen section of solid renal masses.

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

    Chih-Jen Yang


    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.

  6. A primary pure pancreatic-type acinar cell carcinoma of the stomach: a case report.

    Kim, Kyoung Min; Kim, Chan Young; Hong, Seung-Mo; Jang, Kyu Yun


    Acinar cell carcinoma represents only 1-2% of exocrine pancreatic neoplasms. On exceptionally rare occasions, primary acinar cell carcinoma can occur in ectopic locations. Herein, we report a case of pure pancreatic-type acinar cell carcinoma arising in the stomach. A 54-year-old male presented with a gastric submucosal mass detected by endoscopic examination. Laparoscopic wedge resection was performed. Macroscopically, the 2.7 cm yellowish mass was located in the submucosa of the stomach. Microscopically, the tumor was well circumscribed and had a homogeneous acinar architecture. The tumor cells were small and had a minimal amount of cytoplasm. The nuclei of the tumor cells were round to oval with finely dispersed chromatin. The tumor cells were strongly positive for α1-antitrypsin, chymotrypsin, and α1-antichymotrypsin immunostaining, consistent with pancreatic exocrine differentiation. There was no clinical or radiologic evidence of primary pancreatic or head and neck tumors. After surgical resection of the tumor, there was no recurrence or metastasis during 33 months follow-up. In this report, we have presented a rare case of primary pure pancreatic-type acinar cell carcinoma arising in the stomach and suggest that it could be helpful if the pathologist were aware that pancreatic-type acinar cell carcinoma could arise in the stomach as a polypoid submucosal tumor in the routine diagnostic field of gastric endoscopy.

  7. Mesenchymal Stem Cells Support Survival and Proliferation of Primary Human Acute Myeloid Leukemia Cells through Heterogeneous Molecular Mechanisms

    Brenner, Annette K.; Nepstad, Ina; Bruserud, Øystein


    Acute myeloid leukemia (AML) is a bone marrow malignancy, and various bone marrow stromal cells seem to support leukemogenesis, including osteoblasts and endothelial cells. We have investigated how normal bone marrow mesenchymal stem cells (MSCs) support the in vitro proliferation of primary human AML cells. Both MSCs and primary AML cells show constitutive release of several soluble mediators, and the mediator repertoires of the two cell types are partly overlapping. The two cell populations were cocultured on transwell plates, and MSC effects on AML cells mediated through the local cytokine/soluble mediator network could thus be evaluated. The presence of normal MSCs had an antiapoptotic and growth-enhancing effect on primary human AML cells when investigating a group of 51 unselected AML patients; this was associated with increased phosphorylation of mTOR and its downstream targets, and the effect was independent of cytogenetic or molecular-genetic abnormalities. The MSCs also supported the long-term proliferation of the AML cells. A subset of the patients also showed an altered cytokine network with supra-additive levels for several cytokines. The presence of cytokine-neutralizing antibodies or receptor inhibitors demonstrated that AML cells derived from different patients were heterogeneous with regard to effects of various cytokines on AML cell proliferation or regulation of apoptosis. We conclude that even though the effects of single cytokines derived from bone marrow MSCs on human AML cells differ among patients, the final cytokine-mediated effects of the MSCs during coculture is growth enhancement and inhibition of apoptosis.

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


    This study aimed to investigate the effects of arsenic trioxide(As2O3) on the mitochondrial DNA(mtDNA) of acute promyelocytic leukemia(APL) cells.The NB4 cell line was treated with 2.0 μmol/L As2O3 in vitro,and the primary APL cells were treated with 2.0 μmol/L As2O3 in vitro and 0.16 mg kg-1 d-1 As2O3 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 As2O3 use,but the mutation spots were remarkably increased after As2O3 treatment,which was positively correlated to the rates of cellular apoptosis,the correlation coefficient:rNB4-As2O3=0.973818,and rAPL-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 As2O3 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 As2O3 in APL treatment.

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

    Duncan, Henry F., E-mail: [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)


    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.

  10. Evidence of distinct tumour-propagating cell populations with different properties in primary human hepatocellular carcinoma.

    Federico Colombo

    Full Text Available BACKGROUND AND AIMS: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC compartments within human hepatocellular carcinoma (HCC. METHODS: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice. RESULTS: The primary cell populations (hcc-1, -2 and -3 and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8 differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. CONCLUSIONS: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.


    Darvill, J E; McNeil, M; Darvill, A G; Albersheim, P


    The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.

  12. Double primary non-small cell lung cancer with synchronous small cell lung cancer N2 nodes: a case report

    Gogakos, Apostolos S; Paliouras, Dimitrios; Rallis, Thomas; Chatzinikolaou, Fotios; Xirou, Persefoni; Tsirgogianni, Katerina; Tsavlis,Drosos; Sachpekidis,Nikos; Tsakiridis, Kosmas; Mpakas, Andreas; Zarogoulidis, Konstantinos; Zissimopoulos, Athanasios; Zarogoulidis, Paul; Barbetakis, Nikolaos


    Synchronous multiple primary lung cancer (SMPLC) is rare and very hard to distinguish from metastatic disease. Recent studies indicate the presence of this entity in the lung, with no mention to the involvement of the mediastinum. An extremely rare case of a 68-year-old male with double primary non-small cell lung cancer (NSCLC) in the left upper lobe and N2 positive nodes for small cell lung cancer (SCLC) is presented. Modern diagnostic criteria as well as aggressive curative strategies are ...

  13. Double primary non-small cell lung cancer with synchronous small cell lung cancer N2 nodes: a case report.

    Gogakos, Apostolos S; Paliouras, Dimitrios; Rallis, Thomas; Chatzinikolaou, Fotios; Xirou, Persefoni; Tsirgogianni, Katerina; Tsavlis, Drosos; Sachpekidis, Nikos; Tsakiridis, Kosmas; Mpakas, Andreas; Zarogoulidis, Konstantinos; Zissimopoulos, Athanasios; Zarogoulidis, Paul; Barbetakis, Nikolaos


    Synchronous multiple primary lung cancer (SMPLC) is rare and very hard to distinguish from metastatic disease. Recent studies indicate the presence of this entity in the lung, with no mention to the involvement of the mediastinum. An extremely rare case of a 68-year-old male with double primary non-small cell lung cancer (NSCLC) in the left upper lobe and N2 positive nodes for small cell lung cancer (SCLC) is presented. Modern diagnostic criteria as well as aggressive curative strategies are encouraged, in order to achieve better survival rates for such patients.

  14. Primary cardiac B cell lymphoma: Manifestation of Felty's syndrome or TNFα antagonist.

    Benzerdjeb, Nazim; Ameur, Fatima; Ikoli, Jean-Fortune; Sevestre, Henri


    Primary cardiac B cell lymphoma is rare. To date, fewer than 90 cases have been described in the literature. We report a 67-year-old woman with a 30-year history of rheumatoid arthritis, who had received treatment with leflunomide for 10 years and infliximab for 2 years. Secondary Felty's syndrome appeared. She was admitted to the hospital for abdominal pain. Investigations disclosed a 5cm cardiac mass in the right atrium. Histopathologic examination of tissue specimens obtained at surgical myocardial biopsy demonstrated primary cardiac B cell lymphoma. The other iatrogenic lymphoproliferative disorders are reviewed. This lesion might be a manifestation of long term TNFα antagonists treatment.

  15. Transplantation of primary cultured embryonic mesencephalic neural precursor cells for treating Parkinsonian rats

    Li Fei; Chengchuan Jiang; Linyin Feng; Yaodong Ji; Zhongliang Ding


    BACKGROUND: Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy (CRT) for treating Parkinson disease (PD). Embryonic mesencephalic precursor cells (MPCs) can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far.OBJECTIVE: To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats.DESIGN: A randomized and controlled trial taking SD rats as experimental animals.SETTING: Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University.MATERIALS: This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats,with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences.METHODS: Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1out of 20 model rats after execution, and the other 19 rats were randomly divided into control group (n=5),sham transplantation group (n=5)and cell grafted group (n=9). Primary cultured E12 MPC cell suspension (1.2×1011 L-1)were used as donor cells. 4 μL primary cultured E12 MPC cell suspension prepared freshly was injected

  16. Distinct mesenchymal alterations in N-cadherin and E-cadherin positive primary renal epithelial cells.

    Christof Keller

    Full Text Available BACKGROUND: Renal tubular epithelial cells of proximal and distal origin differ markedly in their physiological functions. Therefore, we hypothesized that they also differ in their capacity to undergo epithelial to mesenchymal alterations. RESULTS: We used cultures of freshly isolated primary human tubular cells. To distinguish cells of different tubular origin we took advantage of the fact that human proximal epithelial cells uniquely express N-cadherin instead of E-cadherin as major cell-cell adhesion molecule. To provoke mesenchymal alteration we treated these cocultures with TGF-β for up to 6 days. Within this time period, the morphology of distal tubular cells was barely altered. In contrast to tubular cell lines, E-cadherin was not down-regulated by TGF-β, even though TGF-β signal transduction was initiated as demonstrated by nuclear localization of Smad2/3. Analysis of transcription factors and miRNAs possibly involved in E-cadherin regulation revealed high levels of miRNAs of the miR200-family, which may contribute to the stability of E-cadherin expression in human distal tubular epithelial cells. By contrast, proximal tubular epithelial cells altered their phenotype when treated with TGF-β. They became elongated and formed three-dimensional structures. Rho-kinases were identified as modulators of TGF-β-induced morphological alterations. Non-specific inhibition of Rho-kinases resulted in stabilization of the epithelial phenotype, while partial effects were observed upon downregulation of Rho-kinase isoforms ROCK1 and ROCK2. The distinct reactivity of proximal and distal cells was retained when the cells were cultured as polarized cells. CONCLUSIONS: Interference with Rho-kinase signaling provides a target to counteract TGF-β-mediated mesenchymal alterations of epithelial cells, particularly in proximal tubular epithelial cells. Furthermore, primary distal tubular cells differed from cell lines by their high phenotypic stability

  17. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration.

    Hu, Chenxia; Li, Lanjuan


    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

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

    Weber, Matheus N; Bauermann, Fernando V; Gómez-Romero, Ninnet; Herring, Andy D; Canal, Cláudio W; Neill, John D; Ridpath, Julia F


    The causes of bovine respiratory disease complex (BRDC) are multifactorial and include infection with both viral and bacterial pathogens. Host factors are also involved as different breeds of cattle appear to have different susceptibilities to BRDC. Infection with bovine pestiviruses, including bovine viral diarrhea virus 1 (BVDV1), BVDV2 and 'HoBi'-like viruses, is linked to the development of BRDC. The aim of the present study was to compare the growth of different bovine pestiviruses in primary testicle cell cultures obtained from taurine, indicine and mixed taurine and indicine cattle breeds. Primary cells strains, derived from testicular tissue, were generated from three animals from each breed. Bovine pestivirus strains used were from BVDV-1a, BVDV-1b, BVDV-2a and 'HoBi'-like virus. Growth was compared by determining virus titers after one passage in primary cells. All tests were run in triplicate. Virus titers were determined by endpoint dilution and RT-qPCR. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Tukey's Multiple Comparison Test (P˂0.05). Significant differences in virus growth did not correlate with cattle breed. However, significant differences were observed between cells derived from different individuals regardless of breed. Variation in the replication of virus in primary cell strains may reflect a genetic predisposition that favors virus replication.

  19. Therapeutic effect of bortezomib for primary plasma cell leukemia followed by auto/allo stem cell transplantation

    Ozasa R


    Full Text Available Ryotaro Ozasa, Masaaki Hotta, Hideaki Yoshimura, Takahisa Nakanishi, Takeshi Tamaki, Shinya Fujita, Naoto Nakamichi, Michihiko Miyaji, Kazuyoshi Ishii, Tomoki Ito, Shosaku NomuraFirst Department of Internal Medicine, Kansai Medical University, Osaka, JapanAbstract: Plasma cell leukemia (PCL is a rare disease that represents approximately 4% of plasma cell malignant disorders. PCL consists of two variants: primary PCL presents in patients with no previous history of multiple myeloma, while secondary PCL consists of a leukemic transformation in a previously recognized multiple myeloma. Primary PCL is an extremely resistant, rapidly progressive, fatal disease, with a median overall survival of 6.8 months. There is no standard therapeutic strategy, because no treatment option has been prospectively evaluated. We describe a successful case of newly diagnosed primary PCL, treated with a regimen that included bortezomib, followed by auto stem cell transplantation and nonmyeloablative allogeneic stem cell transplantation. Our patient has maintained remission status for over 12 months since undergoing the allogeneic stem cell transplantation. This strategy is promising for PCL, which, though an extremely resistant disease, may become curable.Keywords: plasma cell leukemia, multiple myeloma, bortezomib, stem cell transplantation

  20. An expression atlas of human primary cells: inference of gene function from coexpression networks.

    Mabbott, Neil A; Baillie, J Kenneth; Brown, Helen; Freeman, Tom C; Hume, David A


    The specialisation of mammalian cells in time and space requires genes associated with specific pathways and functions to be co-ordinately expressed. Here we have combined a large number of publically available microarray datasets derived from human primary cells and analysed large correlation graphs of these data. Using the network analysis tool BioLayout Express3D we identify robust co-associations of genes expressed in a wide variety of cell lineages. We discuss the biological significance of a number of these associations, in particular the coexpression of key transcription factors with the genes that they are likely to control. We consider the regulation of genes in human primary cells and specifically in the human mononuclear phagocyte system. Of particular note is the fact that these data do not support the identity of putative markers of antigen-presenting dendritic cells, nor classification of M1 and M2 activation states, a current subject of debate within immunological field. We have provided this data resource on the BioGPS web site ( and on (

  1. Primary cilia in the basal cells of equine epididymis: a serendipitous finding.

    Arrighi, Silvana


    Occurrence of a solitary cilium was an unexpected discovery while studying the ultrastructure of epididymal epithelium in equidae. Primary cilia were detected in epididymal basal cells of all individuals of the equines studied - horses, donkey and mules - independently from age and tract of the duct, emerging from the basal cell surface and insinuating into the intercellular spaces. More rarely solitary cilia occurred also at the luminal surface of the principal cells. The ciliary apparatus was constituted by a structurally typical basal body continuous with the finger-like ciliary shaft extending from the cell surface, and an adjacent centriole oriented at right angles to the basal body. The cilium was structured as the typical primary, non-motile cilia found in many mammalian cells, having a 9+0 microtubular pattern. The basal diplosome was randomly associated with other cellular organelles including the Golgi complex, the endoplasmic reticulum, the microfilament network, the plasma membrane, vesicles and pits. Primary ciliogenesis is a new and unexpected finding in the epididymal epithelium. A monitoring role of luminal factors and extracellular liquids might be attributed to this organelle, likely acting as chemical receptor of the luminal environment, thus modulating the epithelial function by a cell-to-cell crosstalk involving the entire epithelium.

  2. In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells.

    Takagi, Mutsumi; Kondo, Hiroki; Yoshida, Toshiomi


    A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.

  3. Pure primary small cell carcinoma of urinary bladder: A rare diagnostic entity

    Sonia Gon


    Full Text Available Small cell carcinoma of the bladder is a rare, aggressive, poorly differentiated neuroendocrine neoplasm accounting for only 0.3-0.7% of all bladder tumors. Since the tumor is very rare, pathogenesis is uncertain. Small cell carcinomas of the urinary bladder are mixed with classic urothelial carcinomas or adenocarcinomas of the bladder in 68% cases, making pure primary small cell carcinoma even a rarer entity. The unknown etiology and natural history of small cell carcinoma of the urinary bladder represent a challenge both to the pathologist and urologists for its diagnosis and treatment, respectively.

  4. A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines

    Rognum Torleiv O


    Full Text Available Abstract Background Tumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the in vivo situation is debated. In a series of 11 microsatellite stable (MSS and 9 microsatellite unstable (MSI colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16INK4a (CDKN2A α-transcript, p14ARF (CDKN2A β-transcript, APC, and E-cadherin (CDH1. We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables. Results The cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14ARF were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14ARF, which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14ARF and p16INK4a was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14ARF. E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling. Conclusions The present study shows that colon cancer cell lines are in general relevant in vitro models, comparable with the in vivo situation, as the cell lines display many of the same

  5. Novel primary thymic defect with T lymphocytes expressing gamma delta T cell receptor

    Geisler, C; Pallesen, G; Platz, P


    Flow cytometric analysis of the peripheral blood mononuclear cells in a six year old girl with a primary cellular immune deficiency showed a normal fraction of CD3 positive T cells. Most (70%) of the CD3 positive cells, however, expressed the gamma delta and not the alpha beta T cell receptor....... Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that most of the gamma delta T cell receptors existed as disulphide-linked heterodimers. Proliferative responses to mitogens were severely reduced, but specific antibody responses after vaccination could be detected...... deficiency associated with a high proportion of T cells expressing the gamma delta T cell receptor has been described in nude mice, and it is suggested that the immune deficiency of this patient may represent a human analogue....

  6. A case of primary pulmonary NK/T cell lymphoma presenting as pneumonia.

    Lee, Sangho; Shin, Bongkyung; Yoon, Hyungseok; Lee, Jung Yeon; Chon, Gyu Rak


    Primary pulmonary lymphoma, particularly non-B cell lymphomas involving lung parenchyma, is very rare. A 46-year-old male was admitted to the hospital with fever and cough. Chest X-ray showed left lower lobe consolidation, which was considered pneumonia. However, because the patient showed no response to empirical antibiotic therapy, bronchoscopic biopsy was performed for proper diagnosis. The biopsied specimen showed infiltrated atypical lymphocytes with angiocentric appearance. On immunohistochemical staining, these atypical cells were positive for CD3, CD30, CD56, MUM-1, and granzyme B, and labeled for Epstein-Barr virus encoded RNA in situ hybridization. These findings were consistent with NK/T cell lymphoma. We report on a case of primary pulmonary NK/T cell lymphoma presenting as pneumonic symptoms and review the literature on the subject.

  7. Expression of polycystins and fibrocystin on primary cilia of lung cells.

    Hu, Qiaolin; Wu, Yuliang; Tang, Jingfeng; Zheng, Wang; Wang, Qian; Nahirney, Drew; Duszyk, Marek; Wang, Shaohua; Tu, Jian-Cheng; Chen, Xing-Zhen


    Mutations in polycystin-1, polycystin-2, or fibrocystin account for autosomal dominant or recessive polycystic kidney disease. Renal cystogenesis is linked to abnormal localization and function of these cystoproteins in renal primary cilia. They are also expressed in extrarenal tissues in which their functions are unclear. Here we found that human type-II alveolar epithelial A549, airway submucosal Calu-3 cells, and rat bronchioles contain primary or multiple cilia in which we detected these cystoproteins. At sub-confluency, polycystin-1 was expressed on plasma membrane, while polycystin-2 was localized to the ER of resting cells. Both polycystins were detected on the spindle and mid-body of mitotic cells, while fibrocystin was on centrosome throughout cell cycle. Polycystins and fibrocystin may participate in regulating mucociliary sensing and transport within pulmonary airways.

  8. Atypical presentation of primary renal squamous cell cancer: a case report

    Mrinal Pahwa


    Full Text Available Renal squamous cell cancer is one of the rare primary urothelial tumors with only a handful of cases reported in literature. Because of high grade, advanced and late presentation, they herald a grave prognosis. They are frequently associated with calculus disease, smoking, phenacetin consumption and foci of squamous metaplasia due to chronic irritation. Nephroureterectomy is the treatment of choice for such tumors. We hereby present a case of 59 year old female who presented with squamous cell cancer of renal pelvis. The case presented here is different from what has already been reported in literature, as the patient had no antecedent risk factors for renal squamous cell carcinoma.-------------------------------------------------Cite this article as: Pahwa M, Pahwa AR, Girotra M, Chawla A. Atypical presentation of primary renal squamous cell cancer: a case report. Int J Cancer Ther Oncol 2014; 2(1:02015.DOI:

  9. Patients' views on improving sickle cell disease management in primary care: focus group discussion.

    Aljuburi, Ghida; Phekoo, Karen J; Okoye, Nv Ogo; Anie, Kofie; Green, Stuart A; Nkohkwo, Asaah; Ojeer, Patrick; Ndive, Comfort; Banarsee, Ricky; Oni, Lola; Majeed, Azeem


    To assess sickle cell disease (SCD) patient and carer perspectives on the primary care services related to SCD that they receive from their general practitioner (GP). A focus group discussion was used to elicit the views of patients about the quality of care they receive from their primary health-care providers and what they thought was the role of primary care in SCD management. The focus group discussion was video recorded. The recording was then examined by the project team and recurring themes were identified. A comparison was made with notes made by two scribes also present at the discussion. Sickle Cell Society in Brent, UK. Ten participants with SCD or caring for someone with SCD from Northwest London, UK. Patients' perceptions about the primary care services they received, and a list of key themes and suggestions. Patients and carers often bypassed GPs for acute problems but felt that GPs had an important role to play around repeat prescriptions and general health care. These service users believed SCD is often ignored and deemed unimportant by GPs. Participants wanted the health service to support primary health-care providers to improve their knowledge and understanding of SCD. Key themes and suggestions from this focus group have been used to help develop an educational intervention for general practice services that will be used to improve SCD management in primary care.

  10. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    Miyoshi, Ko, E-mail: [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan); Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan)


    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  11. Modest Interference with Actin Dynamics in Primary T Cell Activation by Antigen Presenting Cells Preferentially Affects Lamellal Signaling.

    Kole T Roybal

    Full Text Available Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation.

  12. IL-17 Enhances Chemotaxis of Primary Human B Cells during Asthma.

    Halwani, Rabih; Al-Kufaidy, Roua; Vazquez-Tello, Alejandro; Pureza, Mary Angeline; BaHammam, Ahmed S; Al-Jahdali, Hamdan; Alnassar, Sami A; Hamid, Qutayba; Al-Muhsen, Saleh


    IL-17 is a pro-inflammatory mediator that is believed to play a critical role in regulating tissue inflammation during asthma, COPD, as well as other inflammatory disorders. The level of expression of IL-17 has been shown to be upregulated in lung bronchial tissue of asthmatic patients. Several reports have provided further evidence that this cytokine could play a key role in enhancing the migration of inflammatory as well as structural cells of the bronchial lung tissue during asthma and COPD. B cell infiltration to sites of inflammation during inflammatory disorders such as bowel disease, asthma and COPD has been reported. Accordingly, in this study we hypothesized that IL-17 may exert a chemotactic effect on primary B cells during asthma. We observed that B cells from asthmatic patients expressed significantly higher levels of IL-17RA and IL-17RC, compared to those of healthy subjects. Using an in-vitro migration assay, B cells were shown to migrate towards both IL-17A and IL-17F. Interestingly, blocking IL-17A and IL-17F signaling using either anti-IL-17R antibodies or MAP kinase inhibitors prevented in vitro migration of B cell towards IL-17. These observations indicate a direct chemotactic effect of IL-17 cytokines on primary peripheral blood B cells with higher effect being on asthmatic B cells. These findings revealed a key role for IL-17 in enhancing the migration of B cells to the lung tissue during asthma or COPD.

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

    Nikolich-Žugich, J; Davies, J S


    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.

  14. Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts.

    Sascha Schäuble

    Full Text Available Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD, fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P via reversibly cell cycle arrested (C to irreversibly arrested senescent cells (S. In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.

  15. Characterization of the interaction between human respiratory syncytial virus and the cell cycle in continuous cell culture and primary human airway epithelial cells.

    Wu, Weining; Munday, Diane C; Howell, Gareth; Platt, Gareth; Barr, John N; Hiscox, Julian A


    Viruses can modify conditions inside cells to make them more favorable for replication and progeny virus production. One way of doing this is through manipulation of the cell cycle, a process that describes the ordered growth and division of cells. Analysis of model cell lines, such as A549 cells and primary airway epithelial cells, infected with human respiratory syncytial virus (HRSV) has shown alteration of the cell cycle during infection, although the signaling events were not clearly understood. In this study, targeted transcriptomic analysis of HRSV-infected primary airway epithelial cells revealed alterations in the abundances of many mRNAs encoding cell cycle-regulatory molecules, including decreases in the D-type cyclins and corresponding cyclin-dependent kinases (CDK4 and CDK6 [CDK4/6]). These alterations were reflected in changes in protein abundance and/or relocalization in HRSV-infected cells; taken together, they were predicted to result in G(0)/G(1) phase arrest. In contrast, there was no change in the abundances of D-type cyclins in A549 cells infected with HRSV. However, the abundance of the G(1)/S phase progression inhibitor p21(WAF1/CIP1) was increased over that in mock-treated cells, and this, again, was predicted to result in G(0)/G(1) phase arrest. The G(0)/G(1) phase arrest in both HRSV-infected primary cells and A549 cells was confirmed using dual-label flow cytometry that accurately measured the different stages of the cell cycle. Comparison of progeny virus production in primary and A549 cells enriched in G(0)/G(1) using a specific CDK4/6 kinase inhibitor with asynchronously replicating cells indicated that this phase of the cell cycle was more efficient for virus production.

  16. Generation of iPSC lines from primary human chorionic villi cells

    Björn Lichtner


    Full Text Available Primary human chorionic villi (CV cells were used to generate the iPSC line by retroviral transduction of the four Yamanaka-factors OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed both in vivo and in vitro. The transcriptomes of the CV-derived iPSC lines and the human embryonic stem cell lines—H1 and H9 have a Pearson correlation of 0.929 and 0.943 respectively.

  17. Primary Testicular NK/T-Cell Lymphoma: A Study of Two Cases and Review of Literature

    Xiao Lin; Dan Li; Peng Xie; Can Mi; Qing Lin


    Primary testicular NK/T-cell lymphoma is an extremely rare entity progressed rapidly.The aim of this study was to examine clinical and pathological features of primary testicular NK/T-cell lymphoma and to investigate the effective diagnosis and prognosis.In this paper,the two cases of primary testicular NK/T-cell lymphoma were observed by light microscopy,immunohistochemistry and examined by in situ hybridization for Epstein-Barr Virus(EBV)DNA and the literatures were reviewed.The two patients respectively present with bilateral and right-side painless testicular enlargement.The morphology of neoplastic cells of case 1 were small to medium,tumor cells of case 2 were small,medium and large mixed.The tumor cells grew diffusely with irregular and distort nuclear,destructed the organizational structure of the normal testis,and damaged blood vessels,meanwhile,coagulation necrosis was exist.Immunohistochemical staining of neoplastic cells showed positive for CD45,CD2,CD56,CD3ε(cytoplasm staining pattern),CD45RO and Granzyme B,and negative for CD57,CD20,CD79α,CD30,CK,MPO,TdT,Bcl-2 and PLAP were negative.In addition,the EBV DNA was detected in the lymphoma by In situ hybridization.In conclusion,the expression of CD56,CD3ε,and Granzyme B associated proteins and EBV examination by in situ hybridization play a vital role in diagnosis and differential diagnosis of primary testicular NK/T-cell lymphoma.

  18. Primary Testicular NK/T-Cell Lymphoma: A Study of Two Cases and Review of Literature

    Xiao Lin; Dan Li; Peng Xie; Can Mi; Qing Lin


    Primary testicular NK/T-cell lymphoma is an extremely rare entity progressed rapidly.The aim of this study was to examine clinical and pathological features of primary testicular NK/T-cell lymphoma and to investigate the effective diagnosis and prognosis.In this paper,the two cases of primary testicular NK/T-cell lymphoma were observed by light microscopy,immunohistochemistry and examined by in situ hybridization for Epstein-Barr Virus (EBV) DNA and the literatures were reviewed.The two patients respectively present with bilateral and right-side painless testicular enlargement.The morphology of neoplastic cells of case 1 were small to medium,tumor cells of case 2 were small,medium and large mixed.The tumor cells grew diffusely with irregular and distort nuclear,destructed the organizational structure of the normal testis,and damaged blood vessels,meanwhile,coagulation necrosis was exist.Immunohistochemical staining of neoplastic cells showed positive for CD45,CD2,CD56,CD3s (cytoplasm staining pattern),CD45RO and Granzyme B,and negative for CD57,CD20,CD79a,CD30,CK,MPO,TdT,Bcl-2 and PLAP were negative.In addition,the EBV DNA was detected in the lymphoma by In situ hybridization.In conclusion,the expression of CD56,CD3e,and Granzyme B associated proteins and EBV examination by in situ hybridization play a vital role in diagnosis and differential diagnosis of primary testicularNK/T-cell lymphoma.

  19. Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

    Heidegger, Simon; Jarosch, Alexander; Schmickl, Martina; Endres, Stefan; Bourquin, Carole; Hotz, Christian


    Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

  20. Effects of iron on rainbow trout gill cells in primary culture.

    Leguen, Isabelle; Peron, Sandrine; Prunet, Patrick


    This study investigated the effects of iron in the form of iron sulphate (FeSO(4)·7H(2)O), over the range 0.01-1 mM on rainbow trout primary gill cells cultured on semi-permeable membranes. The endpoints measured were cell proliferation, mucous cell numbers, area of mucus in mucous cells, ultrastructural analysis and transepithelial resistance. Regardless of the concentration, FeSO(4) did not modify the apical surface of pavement cells (microridge) and mucous cells. However, at 1 mM, this metal reduced cell numbers, by inhibiting cell proliferation and causing cell death, and induced a decrease in transepithelial resistance. It is interesting to note that cell numbers were also reduced in the presence of 0.5 mM iron salt, although this reduction did not modify transepithelial resistance. FeSO(4) reduced mucous cell number but did not change mucus area in mucous cells suggesting that this metal could induce a discharge of mucous cells, but mucus secretion would be total and not partial. In conclusion, our in vitro model has allowed to study some toxic effect but also resistance of gill epithelium in presence of iron. © Springer Science+Business Media B.V. 2011

  1. Primary renal carcinoid tumor mimicking non-clear cell renal cell carcinoma: A case report

    Joo, Lee Hi; Kim, See Hyung; Kim, Mi Jeong; Choe, Mi Sun [Keimyung University School of Medicine, Dongsan Medical Center, Daegu (Korea, Republic of)


    Carcinoid tumors are neoplasms with neuroendocrine differentiation, and they are most commonly found in the gastrointestinal and respiratory systems. Primary renal carcinoid tumor has rarely been reported. Here, we present a case of primary renal carcinoid tumor manifesting as a small but a gradually enhancing mass with calcification and a cystic component.

  2. Primary macrophages and J774 cells respond differently to infection with Mycobacterium tuberculosis

    Andreu, Nuria; Phelan, Jody; de Sessions, Paola F.; Cliff, Jacqueline M.; Clark, Taane G.; Hibberd, Martin L.


    Macrophages play an essential role in the early immune response to Mycobacterium tuberculosis and are the cell type preferentially infected in vivo. Primary macrophages and macrophage-like cell lines are commonly used as infection models, although the physiological relevance of cell lines, particularly for host-pathogen interaction studies, is debatable. Here we use high-throughput RNA-sequencing to analyse transcriptome dynamics of two macrophage models in response to M. tuberculosis infection. Specifically, we study the early response of bone marrow-derived mouse macrophages and cell line J774 to infection with live and γ-irradiated (killed) M. tuberculosis. We show that infection with live bacilli specifically alters the expression of host genes such as Rsad2, Ifit1/2/3 and Rig-I, whose potential roles in resistance to M. tuberculosis infection have not yet been investigated. In addition, the response of primary macrophages is faster and more intense than that of J774 cells in terms of number of differentially expressed genes and magnitude of induction/repression. Our results point to potentially novel processes leading to immune containment early during M. tuberculosis infection, and support the idea that important differences exist between primary macrophages and cell lines, which should be taken into account when choosing a macrophage model to study host-pathogen interactions. PMID:28176867

  3. Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues.

    van der Merwe, Mathilde; Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay


    The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.

  4. Animal nutrition and breeding conditions modify the physiology of isolated primary cells.

    Milisav, Irina; Banič, Blaž; Šuput, Dušan


    Animal primary cell cultures are widely used in biomedical research to investigate cell metabolism, diseases and to devise novel treatments. Modern animal breeding techniques are developed to unify, control and reduce the amount of microorganisms that the animals are being exposed to. Furthermore, health monitoring and strict caging and handling protocols allow animals to be exposed only to a selected spectrum of microbes. We are starting to appreciate that nutrition can influence composition of gut microbiota that can impact hosting organism's physiology and can even result in development of pathological changes. Evidence is also emerging that acute as well as chronic stresses can profoundly influence the physiology of certain organs, especially heart and liver. Our preliminary data imply that changes in animal nutrition and stress levels initiated up to minutes before the cell isolation could alter the cell stress response of cultured primary hepatocytes after isolation, leading to differences in sensitivity of apoptosis triggering. Therefore, we propose the hypothesis that conditions of animal breeding, especially diet and stress levels, are reflected in the physiology of the isolated primary cells. Variations in animal breeding conditions may influence experimental results on isolated cells and their applicability for studying human disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Primary Intravascular large B-cell lymphoma of lung: a report of one case and review

    Yu Hui


    Full Text Available Abstract Objective To investigate the clinicopathological features of primary intravascular large B-cell lymphoma of lung. Methods A case of primary pulmonary intravascular large B-cell lymphoma was analysed in histopathology and immunophenotype. Results The patient is a 42-year-old female who had cough for one year. Computed tomography showed ground-glass opacities and small nodules in bilateral lung fields. Histopathology demonstrated accumulation of similar sized neoplastic cells within alveolar capillaries, widening the alveolar septae. The alveolar structure sustained in part of districtions. Immunohistologically, the tumor cells were positive for CD20 and negative for CD3,CK, which were similar to the diffuse large B-cell lymphoma. Conclusions Intravascular large B-cell lymphoma is an uncommon type of non-Hodgkin’s lymphoma. Primary pulmonary presentation is even more rare. The diagnosis is based on the histopathology and immunohistochemistry. Virtual slides The virtual slide(s for this article can be found here:

  6. Primary corneal papilloma and squamous cell carcinoma associated with pigmentary keratitis in four dogs.

    Bernays, M E; Flemming, D; Peiffer, R L


    Squamous cell carcinoma (SCC) and squamous papilloma are rarely reported as primary lesions of the cornea in dogs. One case of corneal papilloma and 3 cases of SCC, each arising as a primary central corneal neoplasm rather than spreading from adjacent limbal conjunctiva, were reviewed. The most common cause of SCC in animals is chronic exposure of lightly pigmented epithelium to UV light; however, all dogs in this study had a history of chronic pigmentary keratitis. Three of the 4 dogs were of brachycephalic breeds with naturally proptotic eyes and oversized palpebral fissures that may have exposed the cornea to greater excessive solar radiation. Alternatively, mechanical factors that caused chronic changes in the cornea may have been causative factors for induction of primary dysplastic or neoplastic changes. Primary corneal neoplasia should be considered in the differential diagnosis of corneal masses.

  7. Multiple modes of action potential initiation and propagation in mitral cell primary dendrite

    Chen, Wei R; Shen, Gongyu Y; Shepherd, Gordon M


    The mitral cell primary dendrite plays an important role in transmitting distal olfactory nerve input from olfactory glomerulus to the soma-axon initial segment. To understand how dendritic active properties are involved in this transmission, we have combined dual soma and dendritic patch...... recordings with computational modeling to analyze action-potential initiation and propagation in the primary dendrite. In response to depolarizing current injection or distal olfactory nerve input, fast Na(+) action potentials were recorded along the entire length of the primary dendritic trunk. With weak......-initiation site reflected an independent thresholding mechanism in the distal dendrite. When strong olfactory nerve excitation was paired with strong inhibition to the mitral cell basal secondary dendrites, a small fast prepotential was recorded at the soma, which indicated that an action potential was initiated...

  8. Primary Giant Cell Malignant Fibrous Histiocytoma of the Kidney with Staghorn Calculi

    Chen C


    Full Text Available Malignant fibrous histiocytomas (MFH as primary renal tumours are rare, with less than 50 cases described in the literature. We report a case of primary renal MFH of giant cell type in a 56-year-old man, who presented with bilateral dull flank pain, intermittent gross haematuria and body weight loss (6 kg in 3 months. Intravenous urography, computerized tomography (CT and magnetic resonance image (MRI showed right ureteral stones with mild hydronephrosis, and a solid mass at the lower pole of the left kidney associated with staghorn calculi, as well as tumour thrombi in the left renal vein and inferior vena cava. Left radical nephrectomy and evacuation of tumour thrombi from the left renal vein and inferior vena cava were performed. Histopathologic examination revealed malignant fibrous histiocytoma (MFH of giant cell type. To the best of our knowledge, this is the first report of primary renal MFH associated with staghorn calculi.

  9. Primary intraosseous squamous cell carcinoma in odontogenic keratocyst: A rare entity.

    Saxena, Chitrapriya; Aggarwal, Pooja; Wadhwan, Vijay; Bansal, Vishal


    Squamous cell carcinoma (SCC) arising from the wall of an odontogenic cyst (also known as primary intraosseous carcinoma) is a rare tumor which occurs only in jaw bones. This tumor was first described by Loos in 1913 as a central epidermoid carcinoma of the jaw. Primary intraosseous carcinomas (PIOC) may theoretically arise from the lining of an odontogenic cyst or de novo from presumed odontogenic cell rests. According to the new histological classification of tumors of the World Health Organization, odontogenic keratocyst is nowadays considered a specific odontogenic tumor and the PIOC derived from it is considered as a specific entity which is different from other PIOCs derived from the odontogenic cysts. The following report describes a case of such extremely rare entity that is primary intraosseous SCC of the mandible derived from an OKC in a 60-year-old male patient with brief review of literature.

  10. Cancer Stem Cells in Primary Liver Cancers: Pathological Concepts and Imaging Findings

    Joo, Ijin [Department of Radiology, Seoul National University Hospital, Seoul 110-744 (Korea, Republic of); Kim, Haeryoung [Department of Pathology, Seoul National University Bundang Hospital, Seongnam 463-707 (Korea, Republic of); Lee, Jeong Min [Department of Radiology, Seoul National University Hospital, Seoul 110-744 (Korea, Republic of)


    There is accumulating evidence that cancer stem cells (CSCs) play an integral role in the initiation of hepatocarcinogenesis and the maintaining of tumor growth. Liver CSCs derived from hepatic stem/progenitor cells have the potential to differentiate into either hepatocytes or cholangiocytes. Primary liver cancers originating from CSCs constitute a heterogeneous histopathologic spectrum, including hepatocellular carcinoma, combined hepatocellular-cholangiocarcinoma, and intrahepatic cholangiocarcinoma with various radiologic manifestations. In this article, we reviewed the recent concepts of CSCs in the development of primary liver cancers, focusing on their pathological and radiological findings. Awareness of the pathological concepts and imaging findings of primary liver cancers with features of CSCs is critical for accurate diagnosis, prediction of outcome, and appropriate treatment options for patients.

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

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


    application. In the current study, we selected two synthetic transfection reagents, a cationic lipid-based commercial reagent Lipofectamine RNAiMAX and polyethylenimine (PEI), and two naturally-derived transfection reagents, namely the polysaccharides chitosan (98% deacetylation) and hyaluronic acid (20......% 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...

  12. Characterization of rat parotid and submandibular acinar cell apoptosis in primary culture.

    Limesand, Kirsten H; Barzen, Katherine A; Sanders, Linda A; Sclafani, Robert A; Raynolds, Mary V; Reyland, Mary E; Anderson, Steven M; Quissell, David O


    Apoptosis is a highly organized cellular process that is critical for maintaining glandular homeostasis. We have used primary rat salivary acinar cells from the parotid and submandibular glands to investigate the critical regulatory events involved in apoptosis. Caspase-3 activity, cleavage of caspase substrates, and deoxyribonucleic acid (DNA) fragmentation were assayed in cells treated with etoposide, a DNA-damaging agent, or brefeldin A (BFA), a Golgi toxin. Dose-response studies showed that the sensitivity of both cell types to etoposide and BFA was similar, with 150 microM etoposide or 1.5 microM BFA inducing maximal caspase activation. However, BFA induced a more robust activation of caspase and DNA fragmentation in both cell types. Similar results were observed when the caspase cleavage of poly(adenosine 5'-diphosphate ribose) polymerase and protein kinase C delta were analyzed by Western blot. Analysis of the kinetics of apoptosis showed that caspase-3 activation was maximal at 8 h of etoposide or BFA treatment in the parotid cells and at 8-18 h in the submandibular cells. A similar time course was observed when DNA fragmentation was assayed, although maximal DNA fragmentation in BFA-treated cells was two- to threefold higher than that observed in etoposide-treated cells. Despite slight kinetic differences, it would appear that the apoptotic cascade is very similar in both primary parotid and submandibular acinar cells. Although limited in their long-term stability in culture, the use of primary, nonimmortalized salivary acinar cultures will also permit the use of specific transgenic animals to further characterize the molecular events involved in the regulation of salivary gland acinar cell apoptosis.

  13. Primary cutaneous peripheral T-cell lymphoma, unspecified with an indolent clinical course: a distinct peripheral T-cell lymphoma?

    Ryan, A J A


    Primary cutaneous peripheral T-cell lymphomas (PTL), unspecified, are rare lymphomas, with a poor prognosis. They grow and disseminate rapidly, leading to widespread disease. We report a case of PTL, unspecified occurring on the nose. Despite its aggressive histology, this tumour behaved indolently. It is remarkably similar, clinically and histologically, to four recently described cases that occurred on the ear.

  14. T-Cell Tropism of Simian Varicella Virus during Primary Infection

    W.J.D. Ouwendijk (Werner ); R. Mahalingam (Ravi); R.L. de Swart (Rik); B.L. Haagmans (Bart); G. van Amerongen (Geert); S. Getu (Sarah); D. Gilden (Don); A.D.M.E. Osterhaus (Albert); G.M.G.M. Verjans (George)


    textabstractVaricella-zoster virus (VZV) causes varicella, establishes a life-long latent infection of ganglia and reactivates to cause herpes zoster. The cell types that transport VZV from the respiratory tract to skin and ganglia during primary infection are unknown. Clinical, pathological,

  15. Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

    Kiprilov, Enko N; Awan, Aashir; Desprat, Romain


    Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human h...

  16. Primary pulmonary amyloidosis due to low-grade B cell lymphoma.

    Georghiou, Georgios P; Boikov, Olga; Vidne, Bernardo A; Saute, Milton


    Pulmonary involvement is not an infrequent complication of systemic amyloidosis, although affected patients rarely have significant pulmonary symptoms. In contrast, localized (primary) pulmonary amyloidosis is rare. We report a case of pulmonary low-grade B cell lymphoma with amyloid production, causing localized pulmonary amyloidosis.

  17. Primary rectal signet ring cell carcinoma with peritoneal dissemination and gastric secondaries

    Hsien-Lin Sim; Kok-Yang Tan; Pak-Leng Poon; Anton Cheng


    Disseminated signet ring cell carcinomas frequently arise from the stomach. However, primaries in the colon and rectum have also been reported. We present a 68 year old lady who presented with a change in her bowel habit. Colonoscopy showed a stenosing rectal tumour at 7 cm to 8 cm from the anal verge. Multiple scattered ulcers were also noted along the entire length of the colon. Biopsy of the lesions revealed signet ring cell adenocarcinoma. Gastroscopy showed multiple nodules with ulceration over several areas of the stomach which were similar in appearance to the colonic lesions. However, no primary tumour of the stomach was seen. Biopsy of the gastric lesions also showed signet ring cell adenocarcinoma. Computed tomography scan of the abdomen and pelvis revealed circumferential tumour at the rectosigmoid junction with possible invasion into the left ischiorectal fossa. The overall picture was that of a primary rectal signet ring cell carcinoma with peritoneal dissemination. The patient was referred for palliative chemotherapy in view of the disseminated disease. In the present report, we discuss this interesting pathological entity and review the role of various histolological techniques in helping to identify the primary tumor.

  18. Increased power generation from primary sludge by a submersible microbial fuel cell and optimum operational conditions

    Vologni, Valentina; Kakarla, Ramesh; Angelidaki, Irini;


    Microbial fuel cells (MFCs) have received attention as a promising renewable energy technology for waste treatment and energy recovery. We tested a submersible MFC with an innovative design capable of generating a stable voltage of 0.250 ± 0.008 V (with a fixed 470 Ω resistor) directly from primary...

  19. Intraosseous carcinoma of the jaws: A clinicopathologic review. Part III: Primary intraosseous squamous cell carcinoma

    Woolgar, J.A.; Triantafyllou, A.; Ferlito, A.; Devaney, K.O.; Lewis Jr., J.S.; Rinaldo, A.; Slootweg, P.J.; Barnes, L.


    This is the third part of a review of the clinicopathologic features of intraosseous carcinoma of the jaws (IOCJ). In parts 1 and 2, we discussed metastatic and salivary-type and odontogenic carcinomas, respectively. This part deals with primary intraosseous squamous cell carcinoma. Again, based on

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

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


    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 alcohol

  1. Favorable response to aggressive chemotherapy in a patient with primary plasma cell leukemia.

    Lishner, M; Lang, R; Jutrin, I; Ravid, M


    Primary plasma cell leukemia was diagnosed in a previously healthy 58-year-old man. The unusual presentation with concomitant multiple osteolytic lesions and hepatosplenomegaly, the favorable response to aggressive chemotherapy with COAP, and the relatively long survival of 22 months prompted this report. This and several other cases recently reported should encourage an aggressive therapeutic approach to this disease.

  2. Primary Thyroid-Like Follicular Renal Cell Carcinoma: An Emerging Entity

    S. Malde


    Full Text Available Primary thyroid-like follicular carcinoma of the kidney is a rare but newly emerging histological variant of renal cell carcinoma RCC, with only nine cases reported in the literature to date. We present a further case of this unique condition, discuss the workup and typical histological findings, and review the literature regarding this rare histological variant.

  3. Recycling Spent Primary Cells for the Synthesis of Spinel ZnMn 2 O ...

    Recycling Spent Primary Cells for the Synthesis of Spinel ZnMn 2 O 4 using Waste Polypropylene as Reductant in a Microwave Oven. ... The residual casing was dismantled and scrap iron, plastic and paper separated. The removed mixture ...

  4. Second primary germ cell tumors in patients with seminoma of the testis.

    Cockburn, A G; Vugrin, D; Batata, M; Hajdu, S; Whitmore, W F


    In a review of our experience with seminoma 9 cases of bilateral primary testis germ cell tumors were encountered, including 2 simultaneous and 7 successive. Of the 9 cases 6 were bilateral seminomas and 7 were stage I, contributing to the good survival experience. Treatment policy is specified and discussed.

  5. A fatal case of primary basaloid squamous cell carcinoma in the intrahepatic bile ducts

    Kirkegaard, Johan; Grunnet, Mie; Hasselby, Jane Preuss


    of diagnosis but expired 20 months after surgery with epidural, lung, and spine metastasis. In addition to the unusual clinical presentation, the diagnosis of the liver tumor was that of a primary basaloid squamous cell carcinoma of the intrahepatic bile ducts, an entity with only one previous report...

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

    Sabyasachi Halder

    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.

  7. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren


    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

  8. Selection of mesenchymal-like metastatic cells in primary tumors – an in silico investigation

    Vipin eNarang


    Full Text Available In order to metastasize, cancer cells must undergo phenotypic transition from an anchorage-dependent form to a motile form via a process referred to as epithelial to mesenchymal transition (EMT. It is currently unclear whether metastatic cells emerge late during tumor progression by successive accumulation of mutations, or whether they derive from distinct cell populations already present during the early stages of tumorigenesis. Similarly, the selective pressures that drive metastasis are poorly understood. Selection of cancer cells with increased proliferative capacity and enhanced survival characteristics may explain how some transformations promote a metastatic phenotype. However, it is difficult to explain how disseminated mesenchymal-like cancer cells can be subjected to such selective pressure, since these cells usually remain dormant for prolonged periods of time. In the current study, we have used in silico modeling and simulation to investigate the hypothesis that mesenchymal-like cancer cells evolve during the early stages of primary tumor development, and that these cells exhibit survival and proliferative advantages within the tumor microenvironment. In an agent-based tumor microenvironment model, cancer cell agents with distinct sets of attributes governing nutrient consumption, proliferation, apoptosis, random motility and cell adhesion were allowed to compete for space and nutrients. These simulation data indicated that mesenchymal-like cancer cells displaying high motility and low adhesion proliferate more rapidly and display a survival advantage over epithelial-like cancer cells. Furthermore, the presence of mesenchymal-like cells within the primary tumor influences the macroscopic properties, emergent morphology and growth rate of tumors.

  9. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M


    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

  10. CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

    Ito, Daisuke; Frantz, Aric M.; Williams, Christina; Thomas, Rachael; Burnett, Robert C.; Avery, Anne C.; Breen, Matthew; Mason, Nicola J.; O’Brien, Timothy D.; Modiano, Jaime F.


    Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development; however, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude this system has broad applications for in vitro preclinical development for B-cell malignancies. PMID:22229753

  11. Improved Activation toward Primary Colorectal Cancer Cells by Antigen-Specific Targeting Autologous Cytokine-Induced Killer Cells

    Claudia Schlimper


    Full Text Available Adoptive therapy of malignant diseases with cytokine-induced killer (CIK cells showed promise in a number of trials; the activation of CIK cells from cancer patients towards their autologous cancer cells still needs to be improved. Here, we generated CIK cells ex vivo from blood lymphocytes of colorectal cancer patients and engineered those cells with a chimeric antigen receptor (CAR with an antibody-defined specificity for carcinoembryonic antigen (CEA. CIK cells thereby gained a new specificity as defined by the CAR and showed increase in activation towards CEA+ colon carcinoma cells, but less in presence of CEA− cells, indicated by increased secretion of proinflammatory cytokines. Redirected CIK activation was superior by CAR-mediated CD28-CD3ζ than CD3ζ signaling only. CAR-engineered CIK cells from colon carcinoma patients showed improved activation against their autologous, primary carcinoma cells from biopsies resulting in more efficient tumour cell lysis. We assume that adoptive therapy with CAR-modified CIK cells shows improved selectivity in targeting autologous tumour lesions.

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

    Redouan Mahou


    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.

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

    Mahou, Redouan; Meier, Raphael P H; Bühler, Léo H; Wandrey, Christine


    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.

  14. Primary B-cell deficiencies reveal a link between human IL-17-producing CD4 T-cell homeostasis and B-cell differentiation.

    Rita R Barbosa

    Full Text Available IL-17 is a pro-inflammatory cytokine implicated in autoimmune and inflammatory conditions. The development/survival of IL-17-producing CD4 T cells (Th17 share critical cues with B-cell differentiation and the circulating follicular T helper subset was recently shown to be enriched in Th17 cells able to help B-cell differentiation. We investigated a putative link between Th17-cell homeostasis and B cells by studying the Th17-cell compartment in primary B-cell immunodeficiencies. Common Variable Immunodeficiency Disorders (CVID, defined by defects in B-cell differentiation into plasma and memory B cells, are frequently associated with autoimmune and inflammatory manifestations but we found no relationship between these and Th17-cell frequency. In fact, CVID patients showed a decrease in Th17-cell frequency in parallel with the expansion of activated non-differentiated B cells (CD21(lowCD38(low. Moreover, Congenital Agammaglobulinemia patients, lacking B cells due to impaired early B-cell development, had a severe reduction of circulating Th17 cells. Finally, we found a direct correlation in healthy individuals between circulating Th17-cell frequency and both switched-memory B cells and serum BAFF levels, a crucial cytokine for B-cell survival. Overall, our data support a relationship between Th17-cell homeostasis and B-cell maturation, with implications for the understanding of the pathogenesis of inflammatory/autoimmune diseases and the physiology of B-cell depleting therapies.

  15. Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures

    Aaberg-Jessen, Charlotte; Nørregaard, Annette; Christensen, Karina


    preserving the invasive features and stem cell features of glioma cells. Fluorescently labelled primary glioma spheroids and U87MG cell line-derived spheroids were implanted into organotypic rat corticostriatal slice cultures and the invasion was followed over time by confocal microscopy. The invasion......Gliomas are highly invasive tumors and the pronounced invasive features of gliomas prevent radical surgical resection. In the search for new therapeutics targeting invasive glioma cells, in vivo-like in vitro models are of great interest. We developed and evaluated an in vivo-like in vitro model...... was validated immunohistochemically with paraffin sections using a human-specific vimentin antibody. Moreover, the preservation of immature stem cell features was evaluated immunohistochemically using the stem cell markers CD133, Sox2, Bmi-1 and nestin. The confocal and immunohistochemical results showed...

  16. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression.

    Theerakitthanakul, Korkiat; Khrueathong, Jeerasak; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak


    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression.

  17. Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow.

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


    We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation

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

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


    The application of RNA interference (RNAi) has great therapeutic potential for degenerative diseases of cartilaginous tissues by means of fine tuning the phenotype of cells used for regeneration. However, possible non-specific effects of transfection per se might be relevant for future clinical......% 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...... 3 and day 6 post-transfection. In addition to silencing efficiency, non-specific effects such as cytotoxicity, change in DNA content and differentiation potential of cells were evaluated. Among the four transfection reagents, the commercial liposome-based agent was the most efficient reagent for si...

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

    Higashi Richard M


    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

  20. CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells

    Karolina Skvarova Kramarzova


    Full Text Available Fanconi anemia (FA is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1 primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase. FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

  1. Primary CD56+ NK/T-cell lymphoma of the rectum accompanied with refractory ulcerative colitis.

    Kakimoto, Kazuki; Inoue, Takuya; Nishikawa, Takashi; Ishida, Kumi; Kawakami, Ken; Kuramoto, Takanori; Abe, Yosuke; Morita, Eijiro; Murano, Naoko; Toshina, Ken; Murano, Mitsuyuki; Umegaki, Eiji; Egashira, Yutaro; Okuda, Junji; Tanigawa, Nobuhiko; Hirata, Ichiro; Katsu, Ken-Ichi; Higuchi, Kazuhide


    A case of primary NK/T-cell lymphoma of the rectum accompanied with ulcerative colitis (UC) in a 73-year-old man is reported. He had a 6-year history of repeated admission to our hospital for UC. Total colonoscopy performed 4 months after resolution of refractory UC complicated by cytomegalovirus colitis showed a markedly submucosal tumor in the rectum, which was histologically diagnosed as malignant lymphoma. The findings of computed tomography of the chest and abdomen, gallium scintigraphy, abdominal ultrasonography, and upper gastrointestinal endoscopy showed no abnormal lesions. Therefore, based on a diagnosis of localized rectal lymphoma with UC, proctocolectomy was performed. The resected specimen showed three submucosal tumors in the rectum with local nodal involvement. Histologically, the tumors were characterized by diffusely infiltrating sheets of large atypical lymphoid cells, which were negative for CD4, CD8, and CD20 but were positive for CD56, CD3, and granzyme B. The presence of Epstein-Barr virus (EBV) infection in neoplastic cells was shown by in situ hybridization for EBV-encoded early small RNA1 (EBER-1). Based on these findings, the patient was diagnosed with primary CD56+ NK/T-cell lymphoma of the rectum (stage IIE). This is the first case report of primary rectal NK/T-cell lymphoma accompanied with UC.

  2. Differential expression of alkaline phosphatase gene in proliferating primary lymphocytes and malignant lymphoid cell lines.

    Latheef, S A A; Devanabanda, Mallaiah; Sankati, Swetha; Madduri, Ramanadham


    Alkaline Phosphatase (APase) activity has been shown to be enhanced specifically in mitogen stimulated B lymphocytes committed to proliferation, but not in T lymphocytes. APase gene expression was analyzed in proliferating murine and human primary lymphocytes and human malignant cell lines using reverse transcriptase and real time PCR. In mitogen stimulated murine splenic lymphocytes, enhancement of APase activity correlated well with an increase in APase gene expression. However, in mitogen stimulated murine T lymphocytes and human PBL despite a vigorous proliferative response, no increase in APase enzyme activity or gene expression was observed. A constitutive expression of APase activity concomitant with APase gene expression was observed inhuman myeloma cell line, U266 B1. However, neither enzyme activity nor gene expression of APase were observed in human T cell lymphoma, SUPT-1. The results suggest a differential expression of APase activity and its gene in proliferating primary lymphocytes of mice and humans. The specific expression of APase activity and its gene only in human myeloma cells, but not in proliferating primary B cells can be exploited as a sensitive disease marker.

  3. Breast Carcinoma Cells in Primary Tumors and Effusions Have Different Gene Array Profiles

    Sophya Konstantinovsky


    Full Text Available The detection of breast carcinoma cells in effusions is associated with rapidly fatal outcome, but these cells are poorly characterized at the molecular level. This study compared the gene array signatures of breast carcinoma cells in primary carcinomas and effusions. The genetic signature of 10 primary tumors and 10 effusions was analyzed using the Array-Ready Oligo set for the Human Genome platform. Results for selected genes were validated using PCR, Western blotting, and immunohistochemistry. Array analysis identified 255 significantly downregulated and 96 upregulated genes in the effusion samples. The majority of differentially expressed genes were part of pathways involved in focal adhesion, extracellular matrix-cell interaction, and the regulation of the actin cytoskeleton. Genes that were upregulated in effusions included KRT8, BCAR1, CLDN4, VIL2, while DCN, CLDN19, ITGA7, and ITGA5 were downregulated at this anatomic site. PCR, Western blotting, and immunohistochemistry confirmed the array findings for BCAR1, CLDN4, VIL2, and DCN. Our data show that breast carcinoma cells in primary carcinomas and effusions have different gene expression signatures, and differentially express a large number of molecules related to adhesion, motility, and metastasis. These differences may have a critical role in designing therapy and in prognostication for patients with metastatic disease localized to the serosal cavities.

  4. IL-8 secretion in primary cultures of prostate cells is associated with prostate cancer aggressiveness

    Neveu B


    Full Text Available Bertrand Neveu*, Xavier Moreel*, Marie-Pier Deschênes-Rompré, Alain Bergeron, Hélène LaRue, Cherifa Ayari, Yves Fradet, Vincent FradetDepartment of Surgery, Laval University Cancer Research Centre, CHU de Quebec Research Centre, Quebec, QC, Canada *These authors contributed equally to this workBackground: Chronic inflammation is believed to be a major factor in prostate cancer initiation and promotion and has been studied using prostate cancer cells and immortalized cell lines. However, little is known about the contribution of normal cells to the prostatic microenvironment and inflammation. We aim to study the contribution of normal prostate epithelial cells to prostate inflammation and to link the inflammatory status of normal cells to prostate cancer aggressiveness.Materials and methods: Short-term primary cell cultures of normal epithelial prostate cells were derived from prostate biopsies from 25 men undergoing radical prostatectomy, cystoprostatectomy, or organ donation. Cells were treated with polyinosinic:polycytidylic acid, a mimic of double-stranded viral RNA and a potent inducer of the inflammatory response. Secretion of interleukin (IL-8 in the cell culture medium by untreated and treated cells was measured and we determined the association between IL-8 levels in these primary cell cultures and prostate cancer characteristics. The Fligner–Policello test was used to compare the groups.Results: Baseline and induced IL-8 secretion were highly variable between cultured cells from different patients. This variation was not related to drug use, past medical history, age, or preoperative prostate-specific antigen value. Nonetheless, an elevated secretion of IL-8 from normal cultured epithelial cells was associated with prostate cancer aggressiveness (P=0.0005.Conclusion: The baseline secretion of IL-8 from normal prostate epithelial cells in culture is strongly correlated with cancer aggressiveness and may drive prostate cancer

  5. Primary clear cell carcinoma of parotid gland: Case report and review of literature.

    Rodríguez, Marta Saldaña; Reija, Maria Fe García; Rodilla, Irene González


    Clear cell carcinoma (CCC) is a rare low-grade carcinoma that represents only 1% to 2% of all salivary glands tumors. The finding of a clear cell tumor in a parotid gland involves the necessity of differential diagnosis between primary clear cell parotid tumors and metastases, mainly from kidney. The biological behavior is not very aggressive and development, which is very slow, is usually asymptomatic and indeed, the tumor often reaches considerable dimensions before being diagnosed. The treatment of choice is the surgical excision. There are rare cases of local recurrence and distant metastases. The aim of this article is to report a primary CCC in the parotid gland that microscopically closely resembled a metastatic CCC of renal origin, making microscopic differentiation difficult.

  6. Proliferation rates of bovine primary muscle cells relate to liveweight and carcase weight in cattle.

    Coles, Chantal A; Wadeson, Jenny; Leyton, Carolina P; Siddell, Jason P; Greenwood, Paul L; White, Jason D; McDonagh, Matthew B


    Muscling in cattle is largely influenced by genetic background, ultimately affecting beef yield and is of major interest to the beef industry. This investigation aimed to determine whether primary skeletal muscle cells isolated from different breeds of cattle with a varying genetic potential for muscling differ in their myogenic proliferative capacity. Primary skeletal muscle cells were isolated and cultured from the Longissimus muscle (LM) of 6 month old Angus, Hereford and Wagyu X Angus cattle. Cells were assessed for rate of proliferation and gene expression of PAX7, MYOD, MYF5, and MYOG. Proliferation rates were found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5-20 hours in culture) in vitro (P cattle (P cattle (P cattle.

  7. Primary candidiasis and squamous cell carcinoma of the larynx: report of a case.

    Lee, Dong Hoon; Cho, Hyong Ho


    Primary candidiasis is rare and often confused with a pre-cancerous lesion, squamous cell carcinoma, or verrucous carcinoma. We report an extremely rare case of squamous cell carcinoma of the vocal cord following primary candidiasis. A 62-year-old man presented to our department reporting a 1-month history of hoarseness. He underwent laryngeal microscopic surgery for a presumptive diagnosis of glottic carcinoma. Histopathologic examination revealed candidiasis and scattered moderate dysplasia. He was treated with itraconazole for 4 weeks, and followed up without any recurrence of candidiasis. However, the 42-month follow-up examination revealed a focal whitish lesion on the right true vocal cord, and a repeat biopsy of this area revealed squamous cell carcinoma without evidence of candidiasis. The patient was treated with radiotherapy and remains well with no signs of tumor recurrence or candidiasis.

  8. Primary central nervous system peripheral T-cell lymphoma in a child.

    Gualco, Gabriela; Wludarski, Sheila; Hayashi-Silva, Luciana; Medeiros Filho, Plinio; Veras, Geni; Bacchi, Carlos Eduardo


    A 10-year-old Caucasian boy was admitted to the hospital with a 3-month history of headache, vomiting, ataxia, and right amaurosis. A magnetic resonance imaging (MRI) showed a solid, expansive, parasagittal mass in the right parietal hemisphere that extended sagitally to include the optical chiasm. The lesion was considered unresectable. Histology and immunophenotyping of biopsy tissue revealed characteristics of peripheral T-cell lymphoma. No other anatomical region, including bone marrow, was compromised. Primary T-cell lymphomas of the central nervous system are rare, especially in childhood. Here, we describe the rapidly deteriorating and fatal clinical course of a boy with a primary T-cell lymphoma in the central nervous system.

  9. Spleen histology in children with sickle cell disease and hereditary spherocytosis: hints on the disease pathophysiology.

    Pizzi, Marco; Fuligni, Fabio; Santoro, Luisa; Sabattini, Elena; Ichino, Martina; De Vito, Rita; Zucchetta, Pietro; Colombatti, Raffaella; Sainati, Laura; Gamba, Piergiorgio; Alaggio, Rita


    Hereditary spherocytosis (HS) and sickle cell disease (SCD) are associated with splenomegaly and spleen dysfunction in pediatric patients. Scant data exist on possible correlations between spleen morphology and function in HS and SCD. This study aimed to assess the histologic and morphometric features of HS and SCD spleens, to get possible correlations with disease pathophysiology. In a large series of spleens from SCD, HS, and control patients, the following parameters were considered: (i) macroscopic features, (ii) lymphoid follicle (LF) density, (iii) presence of perifollicular marginal zones, (iv) presence of Gamna-Gandy bodies, (v) density of CD8-positive sinusoids, (vi) density of CD34-positive microvessels, (vii) presence/distribution of fibrosis and smooth muscle actin (SMA)-positive myoid cells, and (viii) density of CD68-positive macrophages. SCD and HS spleens had similar macroscopic features. SCD spleens had lower LF density and fewer marginal zones than did HS spleens and controls. SCD also showed lower CD8-positive sinusoid density, increased CD34-positive microvessel density and SMA-positive myoid cells, and higher prevalence of fibrosis and Gamna-Gandy bodies. HS had lower LF and CD8-positive sinusoid density than did controls. No significant differences were noted in red pulp macrophages. By multivariate analysis, most HS spleens clustered with controls, whereas SCD grouped separately. A multiparametric score could predict the degree of spleen changes irrespective of the underlying disease. In conclusion, SCD spleens display greater histologic effacement than HS, and SCD-related changes suggest impaired function due to vascular damage. These observations may contribute to guide the clinical management of patients.

  10. MET Expression in Primary and Metastatic Clear Cell Renal Cell Carcinoma: Implications of Correlative Biomarker Assessment to MET Pathway Inhibitors

    Brian Shuch


    Full Text Available Aims. Inhibitors of the MET pathway hold promise in the treatment for metastatic kidney cancer. Assessment of predictive biomarkers may be necessary for appropriate patient selection. Understanding MET expression in metastases and the correlation to the primary site is important, as distant tissue is not always available. Methods and Results. MET immunofluorescence was performed using automated quantitative analysis and a tissue microarray containing matched nephrectomy and distant metastatic sites from 34 patients with clear cell renal cell carcinoma. Correlations between MET expressions in matched primary and metastatic sites and the extent of heterogeneity were calculated. The mean expression of MET was not significantly different between primary tumors when compared to metastases (P=0.1. MET expression weakly correlated between primary and matched metastatic sites (R=0.5 and a number of cases exhibited very high levels of discordance between these tumors. Heterogeneity within nephrectomy specimens compared to the paired metastatic tissues was not significantly different (P=0.39. Conclusions. We found that MET expression is not significantly different in primary tumors than metastatic sites and only weakly correlates between matched sites. Moderate concordance of MET expression and significant expression heterogeneity may be a barrier to the development of predictive biomarkers using MET targeting agents.

  11. Structural characterization and primary in vitro cell culture of locust male germline stem cells and their niche.

    Dorn, David C; Dorn, August


    The establishment of in vitro culture systems to expand stem cells and to elucidate the niche/stem cell interaction is among the most sought-after culture systems of our time. To further investigate niche/stem cell interactions, we evaluated in vitro cultures of isolated intact male germline-niche complexes (i.e., apical complexes), complexes with empty niche spaces, and completely empty niches (i.e., isolated apical cells) from the testes of Locusta migratoria and the interaction of these complexes with isolated germline stem cells, spermatogonia (of transit-amplifying stages), cyst progenitor cells, cyst progenitor cell-like cells, cyst cells, and follicle envelope cells. The structural characteristics of these cell types allow the identification of the different cell types in primary cultures, which we studied in detail by light and electron microscopy. In intact testes germline stem cells strongly adhere to their niche (the apical cell), but emigrate from their niche and form filopodia if the apical complex is put into culture with "standard media." The lively movements of the long filopodia of isolated germline stem cells and spermatogonia may be indicative of their search for specific signals to home to their niche. All other incubated cell types (except for follicle envelope cells) expressed rhizopodia and lobopodia. Nevertheless isolated germline stem cells in culture do not migrate to empty niche spaces of nearby apical cells. This could indicate that apical cells lose their germline stem cell attracting ability in vitro, although apical cells devoid of germline stem cells either by emigration of germline stem cells or by mechanical removal of germline stem cells are capable of surviving in vitro up to 56 days, forming many small lobopodia and performing amoeboid movements. We hypothesize that the breakdown of the apical complex in vitro with standard media interrupts the signaling between the germline stem cells and the niche (and conceivably the cyst

  12. Sphere-forming tumor cells possess stem-like properties in human fibrosarcoma primary tumors and cell lines



    Fibrosarcoma is a malignant soft tissue tumor of mesenchymal origin. Despite advances in medical and surgical treatment, patient survival rates have remained poor. According to the cancer stem cell hypothesis, tumors are comprised of heterogeneous cell populations that have different roles in tumor formation and growth. Cancer stem cells are a small cell subpopulation that exhibits stem-like properties to gain aggressiveness and recurrence. These cells have been identified in a variety of cancerous tumors, but not in human fibrosarcoma. In this study, we observed that HT1080 cells and primary fibrosarcoma cells formed spheres and showed higher self-renewal capacity, invasiveness and drug resistance compared with their adherent counterparts. Moreover, we demonstrated that the cells showed higher expression of the embryonic stem cell-related genes Nanog, Oct3/4, Sox2, Sox10 and their encoding proteins, as well as greater tumorigenic capacity in nude mice. In conclusion, our data suggest the presence of a stem-like cell population in human fibrosarcoma tumors, which provides more evidence for the cancer stem cell hypothesis and assistance in designing new therapeutic strategies against human fibrosarcoma. PMID:23205129

  13. BAFF enhances chemotaxis of primary human B cells: a particular synergy between BAFF and CXCL13 on memory B cells.

    Badr, Gamal; Borhis, Gwenoline; Lefevre, Eric A; Chaoul, Nada; Deshayes, Frederique; Dessirier, Valérie; Lapree, Genevieve; Tsapis, Andreas; Richard, Yolande


    B-cell-activating factor of the TNF family, (BAFF), and a proliferation-inducing ligand (APRIL) regulate B-lymphocyte survival and activation. We report that BAFF, but not APRIL, increased the chemotactic response of primary human B cells to CCL21, CXCL12, and CXCL13. The BAFF-induced increase in B-cell chemotaxis was totally abolished by blockade of BAFF-R and was strongly dependent on the activation of PI3K/AKT, NF-kappaB, and p38MAPK pathways. BAFF had similar effects on the chemotaxis of naive and memory B cells in response to CCL21 but increased more strongly that of memory B cells to CXCL13 than that of naive B cells. Our findings indicate a previously unreported role for the BAFF/BAFF-R pair in mature B-cell chemotaxis. The synergy between CXCL13 and BAFF produced by stromal cells and follicular dendritic cells may have important implications for B-cell homeostasis, the development of normal B-cell areas, and for the formation of germinal center-like follicles that may be observed in various autoimmune diseases.

  14. BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact.

    Scholz, Jean L; Crowley, Jenni E; Tomayko, Mary M; Steinel, Natalie; O'Neill, Patrick J; Quinn, William J; Goenka, Radhika; Miller, Juli P; Cho, Yun Hee; Long, Vatana; Ward, Chris; Migone, Thi-Sau; Shlomchik, Mark J; Cancro, Michael P


    We have used an inhibiting antibody to determine whether preimmune versus antigen-experienced B cells differ in their requisites for BLyS, a cytokine that controls differentiation and survival. Whereas in vivo BLyS inhibition profoundly reduced naïve B cell numbers and primary immune responses, it had a markedly smaller effect on memory B cells and long-lived plasma cells, as well as secondary immune responses. There was heterogeneity within the memory pools, because IgM-bearing memory cells were sensitive to BLyS depletion whereas IgG-bearing memory cells were not, although both were more resistant than naïve cells. There was also heterogeneity within B1 pools, as splenic but not peritoneal B1 cells were diminished by anti-BLyS treatment, yet the number of natural antibody-secreting cells remained constant. Together, these findings show that memory B cells and natural antibody-secreting cells are BLyS-independent and suggest that these pools can be separately manipulated.

  15. BAFF promotes regulatory T-cell apoptosis and blocks cytokine production by activating B cells in primary biliary cirrhosis

    Zhang, Bo; Hu, Mintao [Department of Hepatology, Wuxi Infectious Diseases Hospital, Wuxi, Jiangsu (China); Zhang, Peng [Nanjing Medical University, Nanjing, Jiangsu (China); Cao, Hong [Department of Hepatology, Wuxi Infectious Diseases Hospital, Wuxi, Jiangsu (China); Wang, Yongzhen [The Second Hospital of Nanjing, Nanjing, Jiangsu (China); Wang, Zheng; Su, Tingting [Department of Hepatology, Wuxi Infectious Diseases Hospital, Wuxi, Jiangsu (China)


    Primary biliary cirrhosis (PBC) is a chronic and slowly progressive cholestatic liver disease of autoimmune etiology. A number of questions regarding its etiology are unclear. CD4+CD25+ regulatory T cells (Tregs) play a critical role in self-tolerance and, for unknown reasons, their relative number is reduced in PBC patients. B-cell-activating factor (BAFF) is a key survival factor during B-cell maturation and its concentration is increased in peripheral blood of PBC patients. It has been reported that activated B cells inhibit Treg cell proliferation and there are no BAFF receptors on Tregs. Therefore, we speculated that excessive BAFF may result in Treg reduction via B cells. To prove our hypothesis, we isolated Tregs and B cells from PBC and healthy donors. BAFF and IgM concentrations were then analyzed by ELISA and CD40, CD80, CD86, IL-10, and TGF-β expression in B cells and Tregs were measured by flow cytometry. BAFF up-regulated CD40, CD80, CD86, and IgM expression in B cells. However, BAFF had no direct effect on Treg cell apoptosis and cytokine secretion. Nonetheless, we observed that BAFF-activated B cells could induce Treg cell apoptosis and reduce IL-10 and TGF-β expression. We also showed that BAFF-activated CD4+ T cells had no effect on Treg apoptosis. Furthermore, we verified that bezafibrate, a hypolipidemic drug, can inhibit BAFF-induced Treg cell apoptosis. In conclusion, BAFF promotes Treg cell apoptosis and inhibits cytokine production by activating B cells in PBC patients. The results of this study suggest that inhibition of BAFF activation is a strategy for PBC treatment.

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

    Ricci, Claudio; Mota, Carlos; Moscato, Stefania; D'Alessandro, Delfo; Ugel, Stefano; Sartoris, Silvia; Bronte, Vincenzo; Boggi, Ugo; Campani, Daniela; Funel, Niccola; Moroni, Lorenzo; Danti, Serena


    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 alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.

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

    Enas R Yassin

    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.

  18. Sangivamycin induces apoptosis by suppressing Erk signaling in primary effusion lymphoma cells

    Wakao, Kazufumi [Department of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Kofu-shi 400-8511 (Japan); Watanabe, Tadashi [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan); Takadama, Tadatoshi; Ui, Sadaharu [Department of Biotechnology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Kofu-shi 400-8511 (Japan); Shigemi, Zenpei; Kagawa, Hiroki [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan); Higashi, Chizuka; Ohga, Rie; Taira, Takahiro [Department of Molecular Cell Biology, Faculty of Medicine, University of Yamanashi, Chuoh-shi 409-3898 (Japan); Fujimuro, Masahiro, E-mail: [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)


    Highlights: • Sangivamycin induces the apoptosis of B cell lymphoma PEL cells. • Sangivamycin suppresses Erk signaling by inhibiting Erk phosphorylation in PEL cells. • The activation of Erk signaling is essential for PEL cell survival. • Sangivamycin induces the apoptosis of PEL cells without production of progeny virus. • Sangivamycin may serve as a novel drug for the treatment of PEL. - Abstract: Sangivamycin, a structural analog of adenosine and antibiotic exhibiting antitumor and antivirus activities, inhibits protein kinase C and the synthesis of both DNA and RNA. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients and HIV-infected homosexual males. PEL cells are derived from post-germinal center B cells, and are infected with KSHV. Herein, we asked if sangivamycin might be useful to treat PEL. We found that sangivamycin killed PEL cells, and we explored the underlying mechanism. Sangivamycin treatment drastically decreased the viability of PEL cell lines compared to KSHV-uninfected B lymphoma cell lines. Sangivamycin induced the apoptosis of PEL cells by activating caspase-7 and -9. Further, sangivamycin suppressed the phosphorylation of Erk1/2 and Akt, thus inhibiting activation of the proteins. Inhibitors of Akt and MEK suppressed the proliferation of PEL cells compared to KSHV-uninfected cells. It is known that activation of Erk and Akt signaling inhibits apoptosis and promotes proliferation in PEL cells. Our data therefore suggest that sangivamycin induces apoptosis by inhibiting Erk and Akt signaling in such cells. We next investigated whether sangivamycin, in combination with an HSP90 inhibitor geldanamycin (GA) or valproate (valproic acid), potentiated the cytotoxic effects of the latter drugs on PEL cells. Compared to treatment with GA or valproate alone, the addition of sangivamycin enhanced cytotoxic activity. Our data thus indicate that

  19. Ultrastructural identification of Ricinus communis agglutinin-1 positive cells in primary dissociated cell cultures of human embryonic brain.

    Bobryshev, Y; Ashwell, K


    While Ricinus communis agglutinin 1 (RCA-1) can be used as a specific marker to study the development and differentiation of microglial cells in human embryogenesis, little is known about the structural heterogeneity and nature of RCA-1+ cells. To analyse the structural peculiarities of RCA-1+ cells, we have used primary dissociated cultures of human embryonic brain. These have been used as models for investigating many of the aspects of central nervous system (CNS) HIV infection. We have shown that primary dissociated cultures from human embryos as young as 10 weeks gestation contain RCA-1+ cells. The RCA-1+ cells exist in two forms, those without (type I) and those with (type II) processes. The former have a poorly developed ultrastructure, while the latter have well developed ultrastructural features, such as rough endoplasmic reticulum with short cisternae, abundant ribosomes, mitochondria, lysosomes and vacuoles. Furthermore, some of these cells with processes have well developed cytoskeletal features. In this paper, the classification of RCA-1+ cells of embryonic human brain is considered and their morphology compared to microglia identified in rodent CNS.


    Lila Gardenia


    Full Text Available Primary cell culture from tail epidermal tissue of koi carp (Cyprinus carpio koi was developed. Cells were grown in Leibovits-15 medium supplemented with 20% fetal bovine serum and antibiotics (Penicillin/Streptomycin and Kanamycin. Cell growth was observed in a range of incubation temperature (17oC±2oC, 22oC±2oC, 27oC±2oC, and 32oC±2oC in order to determine the optimum temperature. The cells were able to grow at a range of temperature between 17oC to 32oC with optimal growth at 22oC. Primary cells infected with koi herpes virus produced typical cytopathic effects characterized by severe vacuolation and deformation of nuclei, which is consistent with those of previous reports. Artificial injection experiment by using supernatant koi herpes virus SKBM-1 isolate revealed that it could cause 90% mortality in infected fish within two weeks. PCR test with Sph I-5 specific primers carried out with DNA template from supernatant virus, pellet cell, and gills of infected fish showed positive results in all samples (molecular weight of DNA target 290 bp. The cells were found to be susceptible to koi herpes virus and can be used for virus propagation.

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

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


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

  2. Review Article. Technical aspects of oxygen level regulation in primary cell cultures: A review

    Yazdani Mazyar


    Full Text Available Oxygen (O2 is an essential element for aerobic respiration. Atmospheric concentration of O2 is approximately 21%. Mammalian cells, however, are generally adapted to O2 levels much lower than atmospheric conditions. The pericellular levels of O2 must also be maintained within a fairly narrow range to meet the demands of cells. This applies equally to cells in vivo and cells in primary cultures. There has been growing interest in the performance of cell culture experiments under various O2 levels to study molecular and cellular responses. To this end, a range of technologies (e.g. gas-permeable technology and instruments (e.g. gas-tight boxes and gas-controlled incubators have been developed. It should be noted, however, that some of these have limitations and they are still undergoing refinement. Nevertheless, better results should be possible when technical concerns are taken into account. This paper aims to review various aspects of O2 level adjustment in primary cell cultures, regulation of pericellular O2 gradients and possible effects of the cell culture medium.

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

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


    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.

  4. High-throughput gene expression profiling of memory differentiation in primary human T cells

    Russell Kate


    Full Text Available Abstract Background The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1 the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2 a suitable cell-line representative of naive T cells. Results Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput. Conclusion This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes.

  5. Insulinoma Presenting with Long-Standing Depression, Primary Hypogonadism, and Sertoli Cell Only Syndrome

    Usman H. Malabu


    Full Text Available The aim was to report an unusual case of insulinoma presenting with long-standing depression and primary testicular failure. We describe a 34-year-old male with clinical, laboratory, and radiologic data consistent with islet cell tumor and seminiferous tubule failure primary hypogonadism. The literature is reviewed relative to the component of this syndrome, and a possible association is discussed. The subject was investigated for a long-standing history of depression requiring medical attention because of mental confusion and slurred speech and was found to have an insulinoma. He was diagnosed with primary gonadal failure and physical examination showed no evidence of dysmorphic features. Chromosomal analysis revealed normal 46 XY and testicular biopsy showed Sertoli cell only syndrome (SCOS. Biochemistry revealed endogenous hyperinsulinism and histology confirmed an islet cell tumor. He remained euglycemic postoperatively and on followup. From this report, we emphasize drawing clinicians' attention to the possibility of an association between insulinoma and primary testicular failure and suggest consideration of this diagnosis in patients with hypergonadotropic hypogonadism who may present with infertility.

  6. Expression and regulation of Schlafen (SLFN family members in primary human monocytes, monocyte-derived dendritic cells and T cells

    Alexander Puck


    Full Text Available Schlafen (SLFN/Slfn family members have been investigated for their involvement in fundamental cellular processes including growth regulation, differentiation and control of viral replication. However, most research has been focused on the characterization of Slfns within the murine system or in human cell lines. Since little is known about SLFNs in primary human immune cells, we set out to analyze the expression and regulation of the six human SLFN genes in monocytes, monocyte-derived dendritic cells (moDCs and T cells. Comparison of SLFN gene expression across these three cell types showed high mRNA expression of SLFN11 in monocytes and moDCs and high SLFN5 expression in T cells, indicating functional importance within these cell types. Differentiation of monocytes to moDCs leads to the gradual upregulation of SLFN12L and SLFN13 while SLFN12 levels were decreased by differentiation stimuli. Stimulation of moDCs via human rhinovirus, lipopolysaccharide, or IFN-α lead to strong upregulation of SLFN gene expression, while peptidoglycan poorly stimulated regulation of both SLFNs and the classical interferon-stimulated gene MxA. T cell activation was found to downregulate the expression of SLFN5, SLFN12 and SLFN12L, which was reversible upon addition of exogenous IFN-α. In conclusion, we demonstrate, that SLFN gene upregulation is mainly dependent on autocrine type I interferon signaling in primary human immune cells. Rapid decrease of SLFN expression levels following T cell receptor stimulation indicates a role of SLFNs in the regulation of human T cell quiescence.

  7. HCMV Displays a Unique Transcriptome of Immunomodulatory Genes in Primary Monocyte-Derived Cell Types

    Van Damme, Ellen; Thys, Kim; Tuefferd, Marianne; Van Hove, Carl; Aerssens, Jeroen; Van Loock, Marnix


    Human cytomegalovirus (HCMV) is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naïve neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs). This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34+ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread in vivo. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby underlining the potential

  8. Cytotoxic effect of ZnS nanoparticles on primary mouse retinal pigment epithelial cells.

    Bose, Karthikeyan; Lakshminarasimhan, Harini; Sundar, Krishnan; Kathiresan, Thandavarayan


    The multiple properties of zinc sulphide nanoparticles (ZnS-NPs) are attracting great attention in the field of chemical and biological research. ZnS-NPs also find their application in biosensor and photocatalysis. Zinc is an important metal ion in retina and its deficiency leads to age-related macular degeneration. As of now, not much research is available on bio-interaction of ZnS as nanoform with retinal pigment epithelial (RPE) cells. RPE cells in the retina help in maintaining normal photoreceptor function and vision. To begin with, ZnS-NPs were synthesized and characterized using UV-visible spectra, X-ray diffraction, Fourier transform infrared spectrum, transmission electron microscopy and dynamic light scattering. Followed by the confirmation of nanoparticles, our study extended to investigate the impact of ZnS-NPs in primary mouse RPE (MRPE) cells at different concentrations. ZnS-NPs showed dose-dependent cytotoxicity in MRPE cells and no changes were observed in cells' tight intactness at minimal concentration. In addition, exposure to ZnS-NPs increased cellular permeability in dose- and time-dependent manner in MRPE cells. The findings from DCFH-DA analysis revealed that ZnS-NPs-treated cells had elevated level of reactive oxygen species and partial activation of cell apoptosis was identified after exposure to ZnS-NPs at higher concentration. Furthermore, pre-treatment of the primary MRPE cells with ZnS-NPs led to phosphorylation of Akt (Ser 473), which indicates the crucial role of ZnS-NPs in regulating cell survival at minimal concentration. Altogether, this study enumerates requisite dose of using ZnS-NPs to maintain healthy RPE cells and contributes to future studies in development of therapeutic drug and drug carrier for ocular-related disorders.

  9. (18)F-FDG PET/CT in bilateral primary adrenal T-cell lymphoma.

    Santhosh, Sampath; Mittal, Bhagwant Rai; Shankar, Praveen; Kashyap, Raghava; Bhattacharya, Anish; Singh, Baljinder; Das, Ashim; Bhansali, Anil


    Primary adrenal lymphoma is extremely rare. We report a young patient who presented with non- specific symptoms of fever and abdominal pain. Conventional imaging modalities demonstrated bilateral bulky adrenal masses, and whole-body fluorine-18-fluorodesoxyglucose ((18)F-FDG) positron emission tomography/computed tomography showed intense (18)F-FDG-avid bilateral adrenal masses with no evidence of extra-adrenal spread. A pathological diagnosis of non-Hodgkin lymphoma of peripheral T-cell type was made. The present case indicates that primary adrenal lymphoma should be included in the differential diagnosis of bilateral adrenal masses.

  10. Primary cardiac B-cell lymphoma with atrioventricular block and paroxysmal ventricular tachycardia

    Chen Ke-Wei


    Full Text Available Abstract Primary cardiac lymphoma (PCL is very rare, and is extremely challenging to diagnose due to nonspecific symptoms. When discovered, the right atrium and ventricle are most commonly affected, while diffuse cardiac involvement is uncommon. PCL is fatal unless promptly diagnosed and treated. Herein, we present the case of a 36-year-old immunocompetent male who presented with a 5-year history of non-specific chest symptoms and was diagnosed with primary diffuse cardiac large B-cell lymphoma involving the entire heart.

  11. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    Helms, Hans C; Brodin, Birger


    In vitro models of the blood-brain barrier are useful tools to study blood-brain barrier function as well as drug permeation from the systemic circulation to the brain parenchyma. However, a large number of the available in vitro models fail to reflect the tightness of the in vivo blood-brain...... barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  12. Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture.

    Baeza-Squiban, A; Boisvieux-Ulrich, E; Guilianelli, C; Houcine, O; Geraud, G; Guennou, C; Marano, F


    The differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis. The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.

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

    Crystal N. Marconett


    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.

  14. Apoptosis in Primary Oral Squamous Cell Carcinomas without Lymph Node Metastases

    YAN Zhi; WANG Guomin; XIE Zhiyong; ZHANG Handong


    The apoptosis in primary oral squamous cell carcinomas (OSCCs) without lymph node (LN) metastases and its relation with clinical stages and pathological grades was investigated. The terminal deoxynucleotidyl trasferase (TdT)-mediated dUTP nick end labeling (TUNEL) was used to detect the apoptotic cells in 15 cases of OSCCs. The percentage of apoptotic cells among tumor cells were calculated as apoptotic index (AI). The results showed that in all 15 cases of OSCCs,apoptotic cells could be visualized by TUNEL with AI ranging from 0.03 to 0.92 (average 0.32).AI was significantly negatively correlated with pathological grades (P<0. 05). It was concluded that the apoptotic rate was related to the malignant degree of OSCCs without LN metastases.

  15. Lectin from Agaricus Bisporus Suppresses Akt Phosphorylation and Arrests Cell Cycle Progression in Primary Human Retinal Pigment Epithelial Cells

    Y. H. Cheung


    Full Text Available Anomalous retinal pigment epithelial (RPE cells have been implicated in the development of retinal diseases. Lectin from the edible mushroom Agaricus bisporus (ABL was found to inhibit growth of RPE cells. To elucidate the mechanism through which ABL inhibits RPE cell proliferation, we investigated the changes in cell proliferation-related signaling pathways and cell cycle distribution patterns. Primary human RPE cells were grown with or without the lectin (ABL supplement (20ug or 90ug/ml for three days. Phosphorylation statuses of Akt, Jnk and p38 as well as p53 expression level were investigated by Western blotting. Cellular distributions in various cell cycle phases were investigated using flow cytometry. After ABL treatment (90ug/ml, Akt was found to be hypo-phosphorylated while the expression levels of p53, phosphorylated-Jnk and phosphorylated-p38 were not altered. The amount of cells present at S phase was reduced. Our results showed that ABL hypo-phosphorylated Akt and this observation is in line with the finding that ABL could attenuate cell proliferation. As the level of p53 was not significantly altered by ABL, this suggested that the mechanism in which ABL arrested cell proliferation was independent of Akt-mediated MDM2 activation but was possibly mediated by altering G1 to S phase transition.

  16. Human Airway Primary Epithelial Cells Show Distinct Architectures on Membrane Supports Under Different Culture Conditions.

    Min, Kyoung Ah; Rosania, Gus R; Shin, Meong Cheol


    To facilitate drug development for lung delivery, it is highly demanding to establish appropriate airway epithelial cell models as transport barriers to evaluate pharmacokinetic profiles of drug molecules. Besides the cancer-derived cell lines, as the primary cell model, normal human bronchial epithelial (NHBE) cells have been used for drug screenings because of physiological relevance to in vivo. Therefore, to accurately interpret drug transport data in NHBE measured by different laboratories, it is important to know biophysical characteristics of NHBE grown on membranes in different culture conditions. In this study, NHBE was grown on the polyester membrane in a different medium and its transport barrier properties as well as cell architectures were fully characterized by functional assays and confocal imaging throughout the days of cultures. Moreover, NHBE cells on inserts in a different medium were subject to either of air-interfaced culture (AIC) or liquid-covered culture (LCC) condition. Cells in the AIC condition were cultivated on the membrane with medium in the basolateral side only, whereas cells with medium in apical and basolateral sides under the LCC condition. Quantitative microscopic imaging with biophysical examination revealed distinct multilayered architectures of differentiated NHBE cells, suggesting NHBE as functional cell barriers for the lung-targeting drug transport.

  17. Protein Kinase Inhibitors CK59 and CID755673 Alter Primary Human NK Cell Effector Functions

    Scheiter, Maxi; Bulitta, Björn; van Ham, Marco; Klawonn, Frank; König, Sebastian; Jänsch, Lothar


    Natural killer (NK) cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory therapies. In this study, we tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against calmodulin kinase II (CaMKII; CK59) and PKD family kinases (CID755673) that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Treatment with CK59 and CID755673 indeed resulted in a significant dose-dependent reduction of NK cell degranulation markers and cytokine release in freshly isolated Peripheral blood mononuclear cell populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest protein kinase D2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations. PMID:23508354

  18. Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death.

    Grüllich, Carsten; Friske, Viktoria; Finke, Jürgen


    Cytotoxic T lymphocytes and natural killer cells (CTL/NK) induce cell death in leukemia cells by the granzyme B (grB)-dependent granule cytotoxin (GC) pathway. Resistance to GC may be involved in immune evasion of leukemia cells. The delivery of active grB into the cytoplasma is dependent on the presence of perforin (PFN) and grB complexes. We developed a rapid method for the isolation of GC to investigate GC-mediated cell death in primary leukemia cells. We isolated GC containing grB, grB complexes and PFN by detergent free hypotonic lysis of the human NK cell leukemia line YT. The GC induce grB-mediated, caspase-dependent apoptosis in live cells. The human leukemia cell lines KG-1, U937, K562 (myeloid leukemia), Jurkat, Daudi, and BV173 (lymphoblastic leukemia) treated with GC internalized grB and underwent cell death. In primary leukemia cells analyzed ex vivo, we found GC-resistant leukemia cells in three out of seven patients with acute myeloid leukemia and one out of six patients with acute lymphoblastic leukemia. We conclude that our method is fast (approximately 1 h) and yields active GC that induce grB-dependent cell death. Furthermore, resistance to GC can be observed in acute leukemias and may be an important mechanism contributing to leukemia cell immune evasion.

  19. Erythropoietin (EPO)-receptor signaling induces cell death of primary myeloma cells in vitro

    Thea Kristin Våtsveen; Anne-Marit Sponaas; Erming Tian; Qing Zhang; Kristine Misund; Anders Sundan; Magne Børset; Anders Waage; Gaute Brede


    Background: Multiple myeloma is an incurable complex disease characterized by clonal proliferation of malignant plasma cells in a hypoxic bone marrow environment. Hypoxia-dependent erythropoietin (EPO)-receptor (EPOR) signaling is central in various cancers, but the relevance of EPOR signaling in multiple myeloma cells has not yet been thoroughly investigated. Methods: Myeloma cell lines and malignant plasma cells isolated from bone marrow of myeloma patients were used in this st...

  20. Human primary brain tumor cell growth inhibition in serum-free medium optimized for neuron survival.

    Brewer, Gregory J; LeRoux, Peter D


    Glioblastoma is the most common primary brain tumor in adults from which about 15,000 patients die each year in the United States. Despite aggressive surgery, radiotherapy and chemotherapy, median survival remains only 1 year. Here we evaluate growth of primary human brain tumor cells in a defined nutrient culture medium (Neuregen) that was optimized for neuron regeneration. We hypothesized that Neuregen would inhibit tumor cell growth because of its ability to inhibit gliosis in rat brain. Tumor tissue was collected from 18 patients including 10 males and 8 females (mean age 60+/-12 years) who underwent craniotomy for newly diagnosed, histologically confirmed brain tumors. The tissue was shipped overnight in Hibernate transport medium. Tumor cells were isolated and plated in Neurobasal/serum or Neuregen on culture plastic. After 1 week, growth in Neuregen was significantly less in 9/10 glioblastoma multiforme cases, 5/5 meningioma cases and 3/3 cases of brain metastasis. Analysis of deficient formulations of Neuregen and formulations to which selected components were added back implicate no single active component. However, individual cases were sensitive to corticosterone, selenium, ethanolamine, fatty acids and/or antioxidants. Therefore, a defined culture medium that promotes neuron regeneration inhibits the growth of human primary glioblastoma, meningioma and metastatic tumor cells in culture. The possible in vivo efficacy of Neuregen for treatment of brain tumor resections remains to be determined.

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

    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.


    Development of systems that reconstitute hallmark features of human pancreatic intraepithelial neoplasia (PanINs), the precursor to pancreatic ductal adenocarcinoma, could generate new strategies for early diagnosis and intervention. However, human cell-based PanIN models with defined mutations are unavailable. Here, we report that genetic modification of primary human pancreatic cells leads to development of lesions resembling native human PanINs. Primary human pancreas duct cells harbouring oncogenic KRAS and induced mutations in CDKN2A, SMAD4 and TP53 expand in vitro as epithelial spheres. After pancreatic transplantation, mutant clones form lesions histologically similar to native PanINs, including prominent stromal responses. Gene expression profiling reveals molecular similarities of mutant clones with native PanINs, and identifies potential PanIN biomarker candidates including Neuromedin U, a circulating peptide hormone. Prospective reconstitution of human PanIN development from primary cells provides experimental opportunities to investigate pancreas cancer development, progression and early-stage detection. PMID:28272465

  2. Primary outgrowth cultures are a reliable source of human pancreatic stellate cells.

    Han, Song; Delitto, Daniel; Zhang, Dongyu; Sorenson, Heather L; Sarosi, George A; Thomas, Ryan M; Behrns, Kevin E; Wallet, Shannon M; Trevino, Jose G; Hughes, Steven J


    Recent advances demonstrate a critical yet poorly understood role for the pancreatic stellate cell (PSC) in the pathogenesis of chronic pancreatitis (CP) and pancreatic cancer (PC). Progress in this area has been hampered by the availability, fidelity, and/or reliability of in vitro models of PSCs. We examined whether outgrowth cultures from human surgical specimens exhibited reproducible phenotypic and functional characteristics of PSCs. PSCs were cultured from surgical specimens of healthy pancreas, CP and PC. Growth dynamics, phenotypic characteristics, soluble mediator secretion profiles and co-culture with PC cells both in vitro and in vivo were assessed. Forty-seven primary cultures were established from 52 attempts, demonstrating universal α-smooth muscle actin and glial fibrillary acidic protein but negligible epithelial surface antigen expression. Modification of culture conditions consistently led to cytoplasmic lipid accumulation, suggesting induction of a quiescent phenotype. Secretion of growth factors, chemokines and cytokines did not significantly differ between donor pathologies, but did evolve over time in culture. Co-culture of PSCs with established PC cell lines resulted in significant changes in levels of multiple secreted mediators. Primary PSCs co-inoculated with PC cells in a xenograft model led to augmented tumor growth and metastasis. Therefore, regardless of donor pathology, outgrowth cultures produce PSCs that demonstrate consistent growth and protein secretion properties. Primary cultures from pancreatic surgical specimens, including malignancies, may represent a reliable source of human PSCs.

  3. New findings about iron oxide nanoparticles and their different effects on murine primary brain cells.

    Neubert, Jenni; Wagner, Susanne; Kiwit, Jürgen; Bräuer, Anja U; Glumm, Jana


    The physicochemical properties of superparamagnetic iron oxide nanoparticles (SPIOs) enable their application in the diagnostics and therapy of central nervous system diseases. However, since crucial information regarding side effects of particle-cell interactions within the central nervous system is still lacking, we investigated the influence of novel very small iron oxide particles or the clinically approved ferucarbotran or ferumoxytol on the vitality and morphology of brain cells. We exposed primary cell cultures of microglia and hippocampal neurons, as well as neuron-glia cocultures to varying concentrations of SPIOs for 6 and/or 24 hours, respectively. Here, we show that SPIO accumulation by microglia and subsequent morphological alterations strongly depend on the respective nanoparticle type. Microglial viability was severely compromised by high SPIO concentrations, except in the case of ferumoxytol. While ferumoxytol did not cause immediate microglial death, it induced severe morphological alterations and increased degeneration of primary neurons. Additionally, primary neurons clearly degenerated after very small iron oxide particle and ferucarbotran exposure. In neuron-glia cocultures, SPIOs rather stimulated the outgrowth of neuronal processes in a concentration- and particle-dependent manner. We conclude that the influence of SPIOs on brain cells not only depends on the particle type but also on the physiological system they are applied to.

  4. Mouse newborn cells allow highly productive mouse cytomegalovirus replication, constituting a novel convenient primary cell culture system.

    Le-Trilling, Vu Thuy Khanh; Trilling, Mirko


    Mammalian cell culture is indispensable for most aspects of current biomedical research. Immortalized cell lines are very convenient, but transforming principles (e.g. oncogenic viruses or their oncogenes) can heavily influence the experimental outcome. Primary cells do not share this apparent disadvantage but are more laborious to generate. Certain viruses (e.g. mouse cytomegalovirus) do not replicate efficiently in most transformed cell lines. In the past, such viruses have been routinely propagated on primary mouse embryonic fibroblasts (MEF) established around day 17 (d17) of gestation. According to new regulations of the European Union, experiments using gravid mammals and/or their embryos in the last trimester (>d14 in the case of mice) of gestation do require explicit permission of the local authorities responsible for animal care and use. Applying for such permission is time-consuming and often inflexible. Embryonic fibroblasts could also be produced at earlier time points of pregnancy from younger and smaller embryos. Obviously, this approach consumes more pregnant mice and embryos. Newborn mice are larger thus yielding more cells per sacrificed animal and the new Directive (2010/63/EU) excludes the killing of animals solely for the use of their organs or tissues. We established a convenient protocol to generate adherent mouse newborn cells (MNC). A direct comparison of MNC with MEF revealed that MNC fully recapitulate all tested aspects of a broad panel of virological parameters (plaque size, final titers, viral replication kinetics, viral gene expression, drug and interferon susceptibility as well as species specificity). The herein described approach allows researchers the legal use of primary cells and contributes to the 3R (replace, reduce, refine) guiding principles-especially the 'reduce' aspect-for the use of animals in scientific research. Additionally, it offers the option to directly compare in vitro and in vivo experiments when MNC are

  5. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: [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)


    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.

  6. Expression and functional activity of bitter taste receptors in primary renal tubular epithelial cells and M-1 cells.

    Liang, Jie; Chen, Fuxue; Gu, Fu; Liu, Xin; Li, Feng; Du, Dongshu


    The kidney is essential in the maintenance of in vivo homeostasis by body fluid and electrolyte conservation and metabolic waste removal. Previously, we reported the expression of a novel G protein family (Tas2rs), which includes bitter taste receptors, in the kidney tubule system, including the nephrons and the collecting duct system. Bitter taste receptors could affect kidney function via Ca(2+) intake. Alkaloids such as phenylthiocarbamide stimulate these receptors and cause an increase in Ca(2+) intake. In this study, we determined the expression of bitter taste receptors in the immature kidney and small intestine and in primary renal epithelial cells and M-1 (collecting tubule cell line) cells, by using QPCR and immunostaining. We found no expression of bitter taste receptors in the immature kidney and small intestine several days after birth; the relative abundance of Tas2rs transcripts varied depending on the developmental stage. Tas2rs were expressed in primary renal epithelial cells and M-1 cells. The traditional Chinese medicinal plant extracts phellodendrine and coptisine caused a rapid rise in intracellular Ca(2+) concentration, which was inhibited by the phospholipase C (PLC) inhibitor U-73122. Thus, phellodendrine and coptisine could change the physiological status of renal cells in vitro by mediation of bitter taste receptors in a PLC-dependent manner. Our results provide new insights on the expression and role of bitter taste receptors in renal development and function.

  7. Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis.

    Domart-Coulon, I J; Elbert, D C; Scully, E P; Calimlim, P S; Ostrander, G K


    The foundation of marine coral reef ecosystems is calcium carbonate accumulated primarily by the action of hard corals (Coelenterata: Anthozoa: Scleractinia). Colonial hard coral polyps cover the surface of the reef and deposit calcium carbonate as the aragonite polymorph, stabilized into a continuous calcareous skeleton. Scleractinian coral skeleton composition and architecture are well documented; however, the cellular mechanisms of calcification are poorly understood. There is little information on the nature of the coral cell types involved or their cooperation in biocalcification. We report aragonite crystallization in primary cell cultures of a hard coral, Pocillopora damicornis. Cells of apical coral colony fragments were isolated by spontaneous in vitro dissociation. Single dissociated cell types were separated by density in a discontinuous Percoll gradient. Primary cell cultures displayed a transient increase in alkaline phosphatase (ALP) activity, to the level observed in intact corals. In adherent multicellular isolate cultures, enzyme activation was followed by precipitation of aragonite. Modification of the ionic formulation of the medium prolonged maintenance of isolates, delayed ALP activation, and delayed aragonite precipitation. These results demonstrate that in vitro crystallization of aragonite in coral cell cultures is possible, and provides an innovative approach to investigate reef-building coral calcification at the cellular level.

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

    Yashpal Modi


    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.

  9. Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

    Létourneau Isabelle J


    Full Text Available Abstract Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133 were derived from solid tumor (TOV and ascites (OV, at specific time points at diagnosis and relapse (R. Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133, or cisplatin/topotecan (2295. Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2, the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R and OV2295(R2 cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian

  10. Development and validation of primary human myometrial cell culture models to study pregnancy and labour

    Mosher Andrea A


    Full Text Available Abstract Background The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated. Methods Myometrial cells isolated from paired upper segment (US and lower segment (LS biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2, oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8, were measured. ANOVA followed by Bonferroni’s multiple comparisons test was performed. Results We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10. Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1 through to p10. Conclusions Isolated primary myometrial cells maintain expression of

  11. A case report of primary small cell carcinoma of the breast and review of the literature

    Qi-Dong Ge; Wei-Dong Wei; Ning Lv; Yun Cao; Xi Wang; Jun Tang; Ze-Ming Xie; Xiang-Sheng Xiao; Peng Liu; Xiao-Ming Xie


    Primary small cell carcinoma (SCC) of the breast,an exceedingly rare and aggressive tumor,is often characterized by rapid progression and poor prognosis.We report a case of primary SCC of the breast that was diagnosed through pathologic and immunohistochemical examinations.Computed tomography (CT) scans failed to reveal a non-mammary primary site.Due to the scant number of relevant case summaries,this type of tumor is proved to be a diagnostic and therapeutic challenge.Therefore,we also reviewed relevant literature to share expertise in diagnosis,clinicopathologic characteristics,treatment,and prognosis of this type of tumor.Future studies with more cases are required to define more appropriate treatment indications for this disease.

  12. Transmission of HIV-1 by primary human uterine epithelial cells and stromal fibroblasts.

    Asin, Susana N; Fanger, Michael W; Wildt-Perinic, Dunja; Ware, Patricia L; Wira, Charles R; Howell, Alexandra L


    Women can become infected with human immunodeficiency virus type 1 (HIV-1) after the heterosexual transmission of virus from an infected male partner. To understand the events that result in transmission of HIV-1 across the female reproductive tract, we characterized the life-cycle events of HIV-1 in primary cultures of human uterine epithelial cells and stromal fibroblasts. Epithelial cells and stromal fibroblasts released virus particles after exposure to either X4- or R5-tropic strains of HIV-1. Virus released by these cells was able to infect CD4(+) T cells. When exposed to an X4-tropic strain of HIV-1, these cells supported HIV-1 reverse transcription, integration, and viral DNA transcription. When exposed to an R5-tropic strain, however, these cells released unmodified virus. These data suggest that uterine cells are targets for productive infection with X4-tropic strains and release unmodified R5-tropic viruses that would then be able to infect submucosal target cells, including T cells and macrophages.

  13. Protein kinase inhibitors CK59 and CID755673 alter primary human NK cell effector functions

    Maxi eScheiter


    Full Text Available Natural killer (NK cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory theraphies. In this study, we have tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against CaMKII (CK59 and PKD family kinases (CID755673 that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Dose dependent treatment with CK59 and CID755673 indeed results in a significant reduction of NK cell degranulation markers and cytokine release in freshly isolated PBMC populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest PKD2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.

  14. Gonadotropin regulation of the rat proopiomelanocortin promoter: characterization by transfection of primary ovarian granulosa cells.

    Young, S L; Nielsen, C P; Lundblad, J R; Roberts, J L; Melner, M H


    To characterize the transcriptional effects of human (h)FSH and hCG on the POMC gene, primary rat granulosa cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid under the control of the POMC promoter and 5' region. POMC-CAT contains a fragment of the rat POMC gene, extending from nucleotide -704 to nucleotide +63, fused to the CAT gene. Treatment of POMC-CAT-transfected cells with either hFSH (20 ng/ml) or hCG (10 ng/ml) significantly increased CAT enzyme activity; however, neither hCG nor hFSH increased CAT enzyme activity in cells transfected with pSV2-CAT, a reporter plasmid under the control of the SV40 virus promoter and 5' region. The phosphodiesterase inhibitor isobutylmethylxanthine or the nonhydrolyzable cAMP analog cAMP-chlorothiophenyl significantly increased CAT activity in POMC-CAT-transfected granulosa cells. Human FSH stimulated transcription 10, 20, and 40 h after treatment, but FSH stimulation at the two earlier time points was 2.5- to 5.5-fold greater than that at 40 h. Gonadotropin-stimulated steroidogenesis was equivalent in POMC-CAT-transfected granulosa cells, untransfected, and mock-transfected cells. This indicates that transfection left the physiological hormone response intact. These data demonstrate the following. 1) 767 basepairs of the rat POMC gene are enough to confer gonadotropin stimulation on the CAT marker gene in granulosa cells. 2) Although the POMC promotor lacks a well conserved cAMP response element, either of two different pharmacological manipulations of granulosa cells that raise intracellular cAMP can also stimulate POMC-driven CAT expression. 3) Transfected primary cultures of granulosa cells provide a nontransformed, physiologically relevant model with which to study hormone-regulated gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. In situ identification of CD44+/CD24- cancer cells in primary human breast carcinomas.

    Giuseppe Perrone

    Full Text Available Breast cancer cells with the CD44+/CD24- phenotype have been reported to be tumourigenic due to their enhanced capacity for cancer development and their self-renewal potential. The identification of human tumourigenic breast cancer cells in surgical samples has recently received increased attention due to the implications for prognosis and treatment, although limitations exist in the interpretation of these studies. To better identify the CD44+/CD24- cells in routine surgical specimens, 56 primary breast carcinoma cases were analysed by immunofluorescence and confocal microscopy, and the results were compared using flow cytometry analysis to correlate the amount and distribution of the CD44+/CD24- population with clinicopathological features. Using these methods, we showed that the breast carcinoma cells displayed four distinct sub-populations based on the expression pattern of CD44 and CD24. The CD44+/CD24- cells were found in 91% of breast tumours and constituted an average of 6.12% (range, 0.11%-21.23% of the tumour. A strong correlation was found between the percentage of CD44+/CD24- cells in primary tumours and distant metastasis development (p = 0.0001; in addition, there was an inverse significant association with ER and PGR status (p = 0.002 and p = 0.001, respectively. No relationship was evident with tumour size (T and regional lymph node (N status, differentiation grade, proliferative index or HER2 status. In a multivariate analysis, the percentage of CD44+/CD24- cancer cells was an independent factor related to metastasis development (p = 0.004. Our results indicate that confocal analysis of fluorescence-labelled breast cancer samples obtained at surgery is a reliable method to identify the CD44+/CD24- tumourigenic cell population, allowing for the stratification of breast cancer patients into two groups with substantially different relapse rates on the basis of CD44+/CD24- cell percentage.

  16. Primary Adult Human Retinal Pigment Epithelial Cell Cultures on Human Amniotic Membranes

    Singhal Shweta


    Full Text Available Purpose: Retinal pigment epithelial (RPE cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM as substrate. Materials and Methods: Human cadaver eyeballs (16 were obtained from the eye bank after corneal trephination. RPE cells were harvested by a mechanical dissection of the inner choroid surface (10, group 1 or by b enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2. The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students′ ′t′ test. Results: Eleven samples (68.75% showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days in the mechanical group, significantly earlier ( P Conclusions: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes . Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion

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

    兰雨; 张学敏; 杨平地; 胡美茹; 于鸣; 杨怡; 沈倍奋


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

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

    Laura Blumenthal


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

  19. Circulating follicular helper T cells presented distinctively different responses toward bacterial antigens in primary biliary cholangitis.

    Zhou, Zun-Qiang; Tong, Da-Nian; Guan, Jiao; Li, Mei-Fang; Feng, Qi-Ming; Zhou, Min-Jie; Zhang, Zheng-Yun


    Primary biliary cholangitis (PBC) is a chronic and progressive cholestatic liver disease with unknown causes. The initiation of PBC is associated with bacterial infections and abnormal immune correlates, such as the presence of self-reactive anti-mitochondrial antibodies and shifted balance of T cell subsets. In particular, the CD4(+)CXCR5(+) follicular helper T (Tfh) cells are highly activated in PBC patients and are significantly associated with PBC severity, but the underlying reasons are unknown. In this study, we found that the circulating CD4(+)CXCR5(+) T cells were enriched with the interferon (IFN)-γ-secreting Th1-subtype and the interleukin (IL)-17-secreting Th17-subtype, but not the IL-4-secreting Th2 subtype. We further demonstrated that a host of microbial motifs, including Pam3CSK4, poly(I:C), LPS, imiquimod, and CpG, could significantly stimulate IFN-γ, IL-17, and/or IL-21 from circulating CD4(+)CXCR5(+) T cells in PBC patients, especially in the presence of monocytes and B cells. Whole bacterial cells of Escherichia coli, Novosphingobium aromaticivorans, and Mycobacterium gordonae, could also potently stimulate IFN-γ, IL-17, and/or IL-21 production from circulating CD4(+)CXCR5(+) T cells. But interestingly, while the whole cell could potently stimulate circulating CD4(+)CXCR5(+) T cells from both healthy controls and PBC patients, the cell protein lysate could only potently stimulate circulating CD4(+)CXCR5(+) T cells from PBC patients, but not those from healthy controls, suggesting that circulating CD4(+)CXCR5(+) T cells in PBC patients had distinctive antigen-specificity from those in healthy individuals. Together, these data demonstrated that bacterial antigen stimulation is a potential source of aberrant Tfh cell activation in PBC patients. Copyright © 2017. Published by Elsevier B.V.

  20. Primary angiitis of the central nervous system with diffuse cerebral mass effect and giant cells.

    Kinsella, J A


    Primary angiitis of the central nervous system (PACNS), also called primary CNS vasculitis, is an idiopathic inflammatory condition affecting only intracranial and spinal cord vessels, particularly medium-sized and smaller arteries and arterioles. Angiography and histopathology typically do not reveal evidence of systemic vasculitis.(1,2) Histopathology usually reveals granulomatous inflammation affecting arterioles and small arteries of the parenchyma and\\/or leptomeninges, similar to that seen in Takayasu\\'s or giant cell arteritis.(1-3) We report a patient with biopsy-proven PACNS with giant cells and cerebral mass effect on MRI. Magnetic resonance angiography and cerebral angiography appeared normal and there was no evidence of extracranial vasculitis.

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

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


    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.

  2. Primary pancreatic anaplastic large cell lymphoma, ALK negative:A case report

    Christos G Savopoulos; NE Tsesmeli; GD Kaiafa; AT Zantidis; MT Bobos; AI Hatzitolios; ST Papavramidis; IS Kostopoulos


    We present the fourth case of a primary pancreatic anaplastic large cell lymphoma (ALCL), ALK-. An 80-year-old man was admitted to our clinic for further investigation of a fever of unknown origin. He noted anorexia, weight loss and fatigue. His laboratory tests showed anemia and a great elevation of ESR, LDH, and β2 microglobulin. In CT and MRI scan, a soft tissue mass in the pancreas was observed. A repeated endoscopy after his admission revealed an ulcerated mass-like deformity of the duodenal bulb. Explorative laparotomy confirmed a diffuse spread of an unresectable malignant pancreatic mass extending to the adjacent organs. Duodenal and surgical biopsies identified an ALCL of T-cell lineage, ALK-. The patient died in the Intensive Care Unit due to hemodynamic instability.Our case is the first one indicating that primary pancreatic lymphoma should be suspected in a patient with pancreatic mass and elevated serum LDH and β2 microglobulin.

  3. Primary Clear Cell Adenocarcinoma of a Urethral Diverticulum Treated with Multidisciplinary Robotic Anterior Pelvic Exenteration

    Dane Scantling


    Full Text Available Primary urethral carcinoma is extremely rare and is marked by a variety of clinical symptoms. Primary carcinoma of a urethral diverticulum is still rarer and clear cell adenocarcinoma of the urethra is particularly uncommon (Swartz et al., 2006. Such infrequency has led to inadequate management guidance in the literature for a disease that is often late in presentation and carries substantial morbidity and mortality. This treatable but grave disease deserves definitive curative treatment. We present the first published instance in which it was treated with robotic anterior exenteration. In our case, a 47-year-old female was referred to the urology service for investigation of recurring urinary tract infections. During the workup, the patient was found to have an advanced clear cell urethral adenocarcinoma originating in a urethral diverticulum. We discuss the natural history of this condition, its consequences, and the first instance of its treatment using robotic anterior pelvic exenteration.

  4. Effects of β-Carotene and Its Cleavage Products in Primary Pneumocyte Type II Cells

    Cornelia Haider


    Full Text Available β-Carotene has been shown to increase the risk of developing lung cancer in smokers and asbestos workers in two large scale trails, the Beta-Carotene and Retinol Efficacy Trial (CARET and the Alpha-Tocopherol Beta-carotene Cancer Prevention Trial (ATBC. Based on this observation, it was proposed that genotoxic oxidative breakdown products may cause this effect. In support of this assumption, increased levels of sister chromatid exchanges, micronuclei, and chromosomal aberrations were found in primary hepatocyte cultures treated with a mixture of cleavage products (CPs and the major product apo-8′carotenal. However, because these findings cannot directly be transferred to the lung due to the exceptional biotransformation capacity of the liver, potential genotoxic and cytotoxic effects of β-carotene under oxidative stress and its CPs were investigated in primary pneumocyte type II cells. The results indicate that increased concentrations of β-carotene in the presence of the redox cycling quinone dimethoxynaphthoquinone (DMNQ exhibit a cytotoxic potential, as evidenced by an increase of apoptotic cells and loss of cell density at concentrations > 10 µM. On the other hand, the analysis of micronucleated cells gave no clear picture due to the cytotoxicity related reduction of mitotic cells. Last, although CPs induced significant levels of DNA strand breaks even at concentrations ≥ 1 µM and 5 µM, respectively, β-carotene in the presence of DMNQ did not cause DNA damage. Instead, β-carotene appeared to act as an antioxidant. These findings are in contrast with what was demonstrated for primary hepatocytes and may reflect different sensitivities to and different metabolism of β-carotene in the two cell types.

  5. A cancer stem cell model for studying brain metastases from primary lung cancer.

    Nolte, Sara M; Venugopal, Chitra; McFarlane, Nicole; Morozova, Olena; Hallett, Robin M; O'Farrell, Erin; Manoranjan, Branavan; Murty, Naresh K; Klurfan, Paula; Kachur, Edward; Provias, John P; Farrokhyar, Forough; Hassell, John A; Marra, Marco; Singh, Sheila K


    Brain metastases are most common in adults with lung cancer, predicting uniformly poor patient outcome, with a median survival of only months. Despite their frequency and severity, very little is known about tumorigenesis in brain metastases. We applied previously developed primary solid tumor-initiating cell models to the study of brain metastases from the lung to evaluate the presence of a cancer stem cell population. Patient-derived brain metastases (n = 20) and the NCI-H1915 cell line were cultured as stem-enriching tumorspheres. We used in vitro limiting-dilution and sphere-forming assays, as well as intracranial human-mouse xenograft models. To determine genes overexpressed in brain metastasis tumorspheres, we performed comparative transcriptome analysis. All statistical analyses were two-sided. Patient-derived brain metastasis tumorspheres had a mean sphere-forming capacity of 33 spheres/2000 cells (SD = 33.40) and median stem-cell frequency of 1/60 (range = 0-1/141), comparable to that of primary brain tumorspheres (P = .53 and P = .20, respectively). Brain metastases also expressed CD15 and CD133, markers suggestive of a stemlike population. Through intracranial xenotransplantation, brain metastasis tumorspheres were found to recapitulate the original patient tumor heterogeneity. We also identified several genes overexpressed in brain metastasis tumorspheres as statistically significant predictors of poor survival in primary lung cancer. For the first time, we demonstrate the presence of a stemlike population in brain metastases from the lung. We also show that NCI-H1915 tumorspheres could be useful in studying self-renewal and tumor initiation in brain metastases. Our candidate genes may be essential to metastatic stem cell populations, where pathway interference may be able to transform a uniformly fatal disease into a more localized and treatable one.

  6. Primary Cutaneous Peripheral T-Cell Lymphoma Not Otherwise Specified: A Rapidly Progressive Variant of Cutaneous T-Cell Lymphoma

    Kimberly Aderhold; Lisa Carpenter; Krysta Brown; Anthony Donato


    Primary Cutaneous Peripheral T-Cell Lymphoma NOS (PTL-NOS) is a rare, progressive, fatal dermatologic disease that presents with features similar to many common benign plaque-like skin conditions, making recognition of its distinguishing features critical for early diagnosis and treatment (Bolognia et al., 2008). A 78-year-old woman presented to ambulatory care with a single 5 cm nodule on her shoulder that had developed rapidly over 1-2 weeks. Examination was suspicious for malignancy and a ...

  7. Heat shock protein 70 expression in relation to apoptosis in primary bladder transitional cell carcinoma

    HE Ling-feng; GUAN Kao-peng; YE Hai-yun; REN Liang; YAN Zheng; WANG Shen-wu; HOU Shu-kun


    @@ Bladder carcinoma is the most common tumor in the urinary system. In 1996, a sample investigation showed that bladder carcinoma, in which more than 90% was mainly primary bladder transitional cell carcinoma (BTCC), was one of the ten highest mortality malignant tumors in China. Bladder carcinoma represented 2% of all malignant tumors and has the fifth most common malignancy in men in Europe and North America.1

  8. Primary hyperparathyroidism associated with a giant cell tumor: One case in the distal radius.

    Ouzaa, M R; Bennis, A; Iken, M; Abouzzahir, A; Boussouga, M; Jaafar, A


    Hyperparathyroidism can present itself as brown tumors (or osteolytic expansive lesions) that usually disappear after normalization of calcium and phosphate levels. It rarely occurs simultaneously with a giant cell tumor. The authors report one case of a localized form at the distal radius in a patient being followed for primary hyperparathyroidism. The diagnostic challenges related to the clinical and radiological similarities of these two pathological entities are discussed, as they can lead to delays in therapeutic management.

  9. Primary cerebral non-Langerhans cell histiocytosis: MRI and differential diagnosis

    Ernemann, U.; Skalej, M.; Voigt, K. [Department of Neuroradiology, University Hospital Tuebingen, Hoppe-Seyler-Strasse 3, 72076 Tuebingen (Germany); Hermisson, M.; Platten, M. [Department of Neurology, University Hospital Tuebingen, Hoppe-Seyler-Strasse 3, 72076 Tuebingen (Germany); Jaffe, R. [Pathology Department, Children' s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213 (United States)


    We report a young woman with primary cerebral non-Langerhans cell histiocytosis of the juvenile xanthogranuloma family. The clinical course was complicated by extensive infiltration of cranial nerves and meninges and epi- and intramedullary spinal dissemination. Whereas the cutaneous form of juvenile xanthogranuloma is usually benign and self-limited, central nervous system involvement is associated with high morbidity and mortality and might therefore be considered a separate clinical entity. (orig.)

  10. Chapter 10 the primary cilium coordinates signaling pathways in cell cycle control and migration during development and tissue repair

    Christensen, Søren T; Pedersen, Stine F; Satir, Peter


    Cell cycle control and migration are critical processes during development and maintenance of tissue functions. Recently, primary cilia were shown to take part in coordination of the signaling pathways that control these cellular processes in human health and disease. In this review, we present...... with the extracellular matrix, coordinate Wnt signaling, and modulate cytoskeletal changes that impinge on both cell cycle control and cell migration....... an overview of the function of primary cilia and the centrosome in the signaling pathways that regulate cell cycle control and migration with focus on ciliary signaling via platelet-derived growth factor receptor alpha (PDGFRalpha). We also consider how the primary cilium and the centrosome interact...

  11. Primary central nervous system B cell lymphoma with features intermediate between diffuse large B cell lymphoma and Burkitt lymphoma.

    Jiang, Liuyan; Li, Zhimin; Finn, Laura E; Personnet, David A; Edenfield, Brandy; Foran, James M; Jaeckle, Kurt A; Reimer, Ronald; Menke, David M; Ketterling, Rhett P; Tun, Han W


    B cell lymphoma with features intermediate between diffuse large B cell lymphoma and Burkitt lymphoma (DLBCL/BL) is a new lymphoma entity which is recognized in the current World Health Organization (WHO) classification (2008). We report a case of a primary central nervous system lymphoma (PCNSL) with findings consistent with DLBCL/BL. It is characterized by a very aggressive clinical course, and a widespread multifocal involvement of the CNS. Our case shows that a DLBCL/BL can manifest in the CNS alone without any systemic involvement.

  12. Phenotypic and Functional Alterations in Circulating Memory CD8 T Cells with Time after Primary Infection

    Martin, Matthew D.; Kim, Marie T.; Shan, Qiang; Sompallae, Ramakrishna; Xue, Hai-Hui; Harty, John T.; Badovinac, Vladimir P.


    Memory CD8 T cells confer increased protection to immune hosts upon secondary viral, bacterial, and parasitic infections. The level of protection provided depends on the numbers, quality (functional ability), and location of memory CD8 T cells present at the time of infection. While primary memory CD8 T cells can be maintained for the life of the host, the full extent of phenotypic and functional changes that occur over time after initial antigen encounter remains poorly characterized. Here we show that critical properties of circulating primary memory CD8 T cells, including location, phenotype, cytokine production, maintenance, secondary proliferation, secondary memory generation potential, and mitochondrial function change with time after infection. Interestingly, phenotypic and functional alterations in the memory population are not due solely to shifts in the ratio of effector (CD62Llo) and central memory (CD62Lhi) cells, but also occur within defined CD62Lhi memory CD8 T cell subsets. CD62Lhi memory cells retain the ability to efficiently produce cytokines with time after infection. However, while it is was not formally tested whether changes in CD62Lhi memory CD8 T cells over time occur in a cell intrinsic manner or are due to selective death and/or survival, the gene expression profiles of CD62Lhi memory CD8 T cells change, phenotypic heterogeneity decreases, and mitochondrial function and proliferative capacity in either a lymphopenic environment or in response to antigen re-encounter increase with time. Importantly, and in accordance with their enhanced proliferative and metabolic capabilities, protection provided against chronic LCMV clone-13 infection increases over time for both circulating memory CD8 T cell populations and for CD62Lhi memory cells. Taken together, the data in this study reveal that memory CD8 T cells continue to change with time after infection and suggest that the outcome of vaccination strategies designed to elicit protective memory


    Hoti Lal


    Full Text Available Primary Squamous Cell Carcinoma in the kidney is a rare malignant neoplasm associated with nephrolithiasis, typically monobacterial pyonephrosis and rarely Xanthogranulomatous pyelonephritis. It is an aggressive disease with a poor prognosis mostly due to lack of presenting clinical features like a palpable mass, gross haematuria and pain. We report a case presenting with renal calculus and pyonephrosis managed initially with percutaneous nephrostomy followed by nephrectomy due to complete loss of renal function. Histopathological evaluation revealed poorly differentiated squamous cell carcinoma which is managed by chemotherapy, although initially beneficial, patients later develop disseminated metastatic disease which holds a poor prognosis.

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

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


    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...

  15. Primary undifferentiated spindle-cell sarcoma of sella turcica: successful treatment with adjuvant temozolomide.

    Sareen, Pooja; Chhabra, Lovely; Trivedi, Nitin


    Sellar tumours in adults are most commonly pituitary adenomas. Primary spindle cell sarcoma of the sella turcica without a prior history of cranial radiation is extremely rare. We report a case of a large sellar mass with suprasellar and cavernous sinus extension in a geriatric male patient who presented with complete left oculomotor nerve palsy and panhypopituitarism. The patient underwent partial resection of the sellar mass through transcranial route. The pathology of the mass revealed a poorly differentiated spindle cell neoplasm most consistent with a sarcoma. Postoperatively, the size of the residual sellar mass decreased significantly following six cycles of external beam radiation in conjunction with temozolomide.

  16. Primary squamous cell carcinoma of the renal parenchyma: A rare occurrence

    Rina Mukherjee


    Full Text Available Primary squamous cell carcinoma (SCC of renal parenchyma is a very rare entity although renal pelvic SCC is common. We probably are reporting the third case in a 35 years female presenting with left lumbar pain and weight loss. Lower pole mass was found on contrast enhanced computed tomography (CECT study of abdomen for which left total nephrectomy was done. Histopathological examination revealed a well differentiated squamous cell carcinoma. Her physical condition did not permit for any adjuvant therapy. Two months after surgery she expired.

  17. Primary NK/T cell lymphoma nasal type of the colon

    Ana María Chirife


    Full Text Available Since nasal NK/T-cell lymphoma and NK/T-cell lymphoma nasal type are rare diseases, colonic involvement has seldom been seen. We report a case of a patient with a primary NK/T-cell lymphoma nasal type of the colon. The patient had no history of malignant diseases and was diagnosed after exhaustive study in the context of fever of unknown origin. The first therapeutic approach followed the DAEPOCH-protocol: etoposide, prednisone, doxor-rubicin, vincristine and cyclophosphamide. The persistence of constitutional symptoms after the first treatment course motivated the switch to a second line following the SMILE-protocol: dexamethasone, metotrexate, ifosfamide, E.coli L-asparaginase, and etoposide. Despite intensive chemotherapy, the patient died 2 months after the diagnose of an extranodal NK/T-cell lymphoma of the colon and 4 months after the first symptomatic appearance of disease.

  18. Primary stroke in a woman with sickle cell anemia responsive to hydroxyurea therapy.

    Ballas, Samir K; Martinez, Ubaldo; Savage, Michael


    The most common cause of stroke in children with sickle cell anemia is infarction due to ischemia. In adults, however, stroke is most commonly hemorrhagic in nature. Other causes of stroke in patients with sickle cell disease are very rare. In this short communication, we describe a woman with sickle cell anemia responsive to hydroxyurea (HU) therapy who had primary stroke due to paradoxical embolization caused by a large atrial septal defect. Successful management of the stroke included surgical closure of the defect with trans-esophageal echocardiographic guidance. To the best of our knowledge, this is the first patient with sickle cell anemia and stroke due to congenital heart disease who did not require open heart surgery for successful management.

  19. Primary Graft Failure after Myeloablative Allogeneic Hematopoietic Cell Transplantation for Hematologic Malignancies

    Olsson, Richard F.; Logan, Brent R.; Chaudhury, Sonali; Zhu, Xiaochun; Akpek, Görgün; Bolwell, Brian J.; Bredeson, Christopher N.; Dvorak, Christopher C.; Gupta, Vikas; Ho, Vincent T.; Lazarus, Hillard M.; Marks, David I.; Ringdén, Olle T.H.; Pasquini, Marcelo C.; Schriber, Jeffrey R.; Cooke, Kenneth R.


    Clinical outcomes after primary graft failure (PGF) remain poor. Here we present a large retrospective analysis (n=23,272) which investigates means to prevent PGF and early detection of patients at high risk. In patients with hematologic malignancies, who underwent their first myeloablative allogeneic hematopoietic cell transplantation, PGF was reported in 1,278 (5.5%), and there was a marked difference in PGFs using peripheral blood stem cell compared to bone marrow grafts (2.5 vs. 7.3%; Pmarrow transplants, total nucleated cell doses ≤2.4 × 108/kg were associated with PGF (OR 1.39; Ptransplant may provide the rationale for an early request for additional hematopoietic stem cells. PMID:25772027

  20. Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia.

    Graboń, Wojciech; Otto-Ślusarczyk, Dagmara; Chrzanowska, Alicja; Mielczarek-Puta, Magdalena; Joniec-Maciejak, Ilona; Słabik, Krzysztof; Barańczyk-Kuźma, Anna


    High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L-Ser and/or L-Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1-mRNA and AST2-mRNA by RT-PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L-Asp. AST1-mRNA expression was independent on oxygen and cell line, but AST2-mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L-Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells. Copyright © 2016 John Wiley & Sons, Ltd.

  1. Pancreatic-type Acinar Cell Carcinoma of the Stomach Included in Multiple Primary Carcinomas.

    Yonenaga, Yoshikuni; Kurosawa, Manabu; Mise, Masahiro; Yamagishi, Miki; Higashide, Shunichi


    Pancreatic-type acinar cell carcinoma (ACC) in the stomach is extraordinarily rare. We pathologically examined two cases with multiple primary carcinomas, including gastric tumors. Gastric cancer specimens were examined by immunostaining and electron microscopy. Both cases had cancer cells with acinar patterns, resembling pancreatic ACC. The cancer cells in the first case were positive for exocrine markers, including chymotrypsin, lipase and alpha-1 antichymotrypsin (ACT), as well as neuroendocrine markers, including chromogranin A and synaptophysin. The cancer cells in the second case were positive for chymotrypsin and alpha-1 ACT, while being slightly positive for chromogranin A and synaptophysin. Ultrastructurally, cancer cells contained zymogen granules in both cases. The final diagnosis was pancreatic mixed acinar-neuroendocrine carcinoma and pure pancreatic ACC, respectively. We confirmed two cases with gastric pancreatic-type ACC included in multiple primary carcinomas. This type of double cancer has not been reported previously. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  2. Analysis of changes in the expression pattern of claudins using salivary acinar cells in primary culture.

    Fujita-Yoshigaki, Junko


    Primary saliva is produced from blood plasma in the acini of salivary glands and is modified by ion adsorption and secretion as the saliva passes through the ducts. In rodents, acinar cells of salivary glands express claudin-3 but not claudin-4, whereas duct cells express both claudins-3 and -4. The distinct claudin expression patterns may reflect differences in the permeability of tight junctions between acinar and duct cells. To analyze the role of claudins in salivary glands, we established a system for the primary culture of parotid acinar cells, where the expression patterns of claudins are remarkably changed. Real-time RT-PCR and immunoblot analyses reveal that the expression levels of claudins-4 and -6 increased, whereas claudins-3 and -10 decreased. We found that the signal to induce those changes is triggered during cell isolation and is mediated by Src and p38 MAP kinase. Here, we introduce the methods used to determine the signal pathway that induces the change in claudin expression.

  3. [Primary squamous cell carcinoma of the ascending colon: report of a case and Korean literature review].

    Cho, Dong Keun; Kim, Sang Hun; Cho, Sung Bum; Lee, Wan Sik; Joo, Young Eun


    Primary squamous cell carcinoma of the colon is an extremely rare malignancy. A 48-year-old male visited our hospital for screening colonoscopy. Colonoscopic examination showed a 1 cm sized sessile polyp in the ascending colon. The patient underwent endoscopic mucosal resection (EMR) without any complication. The pathologic findings were compatible with squamous differentiation of tumor cells in inflammatory colonic mucosa. The tumor was confined to the mucosa and the margins of the excised tissue were found to be free of the tumor. There were no other primary sites and no distant metastases in the extensive evaluation using a whole body CT scan and PET-CT. Additional surgical resection was not done. Follow-up colonoscopy performed eight month later showed a whitish scar without evidence of local recurrence and follow-up PET-CT demonstrated no evidence of recurrence. Herein, we report a case of primary squamous cell carcinoma of the ascending colon presenting as a sessile polyp which was removed by EMR.

  4. MCP-1 expression by rat type II alveolar epithelial cells in primary culture.

    Paine, R; Rolfe, M W; Standiford, T J; Burdick, M D; Rollins, B J; Strieter, R M


    Recruitment and activation of mononuclear phagocytes are potentially critical regulatory events for control of pulmonary inflammation. Located at the boundary between the alveolar airspace and the interstitium, alveolar epithelial cells are ideally situated to regulate the recruitment and activation of mononuclear phagocytes through the production of cytokines in response to inflammatory stimulation from the alveolar space. To test this hypothesis, we investigated the production of monocyte chemotactic polypeptide-1 (MCP-1), a protein that is chemotactic for and that activates monocytes, by rat type II alveolar epithelial cells in primary culture. Immunocytochemical staining using anti-murine JE, an antibody recognizing rat MCP-1, demonstrated cell-associated MCP-1 Ag throughout the monolayer. The intensity of staining was increased in response to IL-1 beta. When type II epithelial cells formed a tight monolayer on a filter support, there was polar secretion of MCP-1 Ag into the apical compartment by both control and IL-1-stimulated cells as measured by specific MCP-1 ELISA. Northern blot analysis revealed that IL-1 and TNF-alpha stimulated MCP-1 mRNA expression in a dose-dependent manner, whereas dexamethasone blocked MCP-1 expression by cells stimulated with IL-1. In contrast to previous results using transformed epithelial cell lines, MCP-1 mRNA was induced in these primary cultures directly by stimulation with LPS. These data suggest that alveolar epithelial cells may have an important and previously unrecognized role in the initiation and maintenance of inflammatory processes in the lung by recruiting and activating circulating monocytes through the production of MCP-1.

  5. Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells.

    Stephen Shannon

    Full Text Available Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA, increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex, a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively "glues" cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU

  6. Quantifying Epithelial Early Common Progenitors from Long-Term Primary or Cell Line Sphere Culture.

    Clément, Flora; Zhu, Helen He; Gao, Wei-Qiang; Delay, Emmanuel; Maguer-Satta, Véronique


    Here, a protocol to quantify epithelial early common progenitor/stem cells grown as spheres in non-adherent culture conditions is described. This protocol is based on the combination of two functional tests: the sphere assay to maintain and enrich early progenitor/stem cells, and the epithelial colony-forming cells (E-CFC) assay to identify and quantify further differentiated epithelial progenitors. Primary spheres mainly contain progenitors and rare stem/early common progenitor cells while secondary and tertiary spheres contain progenitor cells derived from the early common progenitor/stem cell population maintained through passages and partially differentiated. Spheres are enzymatically and mechanically dissociated; the derived cells are subsequently plated on irradiated NIH-3T3 fibroblasts for further processing, as in the E-CFC assay. The principle of this assay is to quantify the number of epithelial colonies generated by cells present in the different sequential spheres. This assay has therefore been named the early common progenitor-derived colonies assay (ECP-DC).

  7. p16(INK4a prevents centrosome dysfunction and genomic instability in primary cells.

    Kimberly M McDermott


    Full Text Available Aneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16(INK4a generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16(INK4a cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16(INK4a activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16(INK4a suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis.

  8. Analysis of quantum dot fluorescence stability in primary blood mononuclear cells.

    Summers, Huw D; Holton, Mark D; Rees, Paul; Williams, Paul M; Thornton, Catherine A


    A quantitative assessment of fluorescence signal generation and persistence in blood cells, measured at multiple points over a time course, is presented. Quantum dots (QDs) are inorganic fluorophores that are photostable and nonmetabolized and so can provide quantitative measures of cell biology over multiple cell generations. However, if the potential of these nanoparticles for long-term reporting is to be realized, an understanding of the stability of their fluorescence in living cells is essential. CdTe/ZnS and CdSe/ZnS core/shell dots with peak emission wavelengths of 705 nm and 585 nm, respectively, were loaded, via endocytosis into mononuclear cells extracted from primary blood and flow cytometry used to measure the average fluorescence intensity per cell within populations >10⁴. Time-based study showed a saturation-limited uptake of QDs with a characteristic time of 20 min and a maximum fluorescence signal that is linearly proportional to dot solution concentration. The fluorescence signal decreases after attachment and internalization within cells and is accurately described by a biexponential decay with a rapid initial decay followed by a much slower signal loss with characteristic times of 435 and 7,000 min respectively. Comparison with control samples indicates that interaction with the culture media is a major contributory factor to the initial signal decay. These results provide phenomenological descriptions of the evolving QD fluorescence within live cells with associated analytical equations that allow quantitative assessment of QD-based assays.

  9. Telomerase functions beyond telomere maintenance in primary cutaneous T-cell lymphoma.

    Chevret, Edith; Andrique, Laetitia; Prochazkova-Carlotti, Martina; Ferrer, Jacky; Cappellen, David; Laharanne, Elodie; Idrissi, Yamina; Boettiger, Anna; Sahraoui, Wafa; Ruiz, Florian; Pham-Ledard, Anne; Vergier, Beatrice; Belloc, Francis; Dubus, Pierre; Beylot-Barry, Marie; Merlio, Jean-Philippe


    Telomere erosion may be counteracted by telomerase. Here we explored telomere length (TL) and telomerase activity (TA) in primary cutaneous T-cell lymphoma (CTCL) by using quantitative polymerase chain reaction and interphase quantitative fluorescence in situ hybridization assays. Samples from patients with Sézary syndrome (SS), transformed mycosis fungoides (T-MF), and cutaneous anaplastic large cell lymphoma were studied in parallel with corresponding cell lines to evaluate the relevance of TL and TA as target candidates for diagnostic and therapeutic purposes. Compared with controls, short telomeres were observed in aggressive CTCL subtypes such as SS and T-MF and were restricted to neoplastic cells in SS. While no genomic alteration of the hTERT (human telomerase catalytic subunit) locus was observed in patients' tumor cells, TA was detected. To understand the role of telomerase in CTCL, we manipulated its expression in CTCL cell lines. Telomerase inhibition rapidly impeded in vitro cell proliferation and led to cell death, while telomerase overexpression stimulated in vitro proliferation and clonogenicity properties and favored tumor development in immunodeficient mice. Our data indicate that, besides maintenance of TL, telomerase exerts additional functions in CTCL. Therefore, targeting these functions might represent an attractive therapeutic strategy, especially in aggressive CTCL.

  10. Primary mediastinal large B-cell lymphoma arising from thyroid in a renal recipient with Hashimoto's thyroiditis.

    Wu, Fang; Qu, Lu; Li, Dai-Qiang; Hu, Chun-Hong


    Primary mediastinal large B-cell lymphoma is a subtype of diffuse large B-cell lymphoma, arising in the mediastinum from putative thymic B-cell origin with distinctive clinical and genetic features. Generally, primary mediastinal large B-cell lymphoma is believed as only deriving in the mediastinum. The current study presents a rare case of primary mediastinal large B-cell lymphoma which arising from thyroid in a renal recipient with Hashimoto's thyroiditis. Moreover, we devoted a discussion to the relationship among primary mediastinal large B-cell lymphoma, immunomodulatory therapy and autoimmune diseases. The immunologic derangement induced by long-term immunomodulatory therapy and Hashimoto's thyroiditis may be the possible cause for the ectopic lymphoma.

  11. Interleukin-4 Induces CpG Site-Specific Demethylation of the Pendrin Promoter in Primary Human Bronchial Epithelial Cells

    Giada Scantamburlo


    Full Text Available Pendrin is upregulated in bronchial epithelial cells following IL-4 stimulation via binding of STAT6 to an N4 GAS motif. Basal CpG methylation of the pendrin promoter is cell-specific. We studied if a correlation exists between IL-4 sensitivity and the CpG methylation status of the pendrin promoter in human bronchial epithelial cell models. Methods: Real-time PCR and pyrosequencing were used to respectively quantify pendrin mRNA levels and methylation of pendrin promoter, with and without IL-4 stimulation, in healthy and diseased primary HBE cells, as well as NCI-H292 cells. Results: Increases in pendrin mRNA after IL-4 stimulation was more robust in NCI-H292 cells than in primary cells. The amount of gDNA methylated varied greatly between the cell types. In particular, CpG site 90 located near the N4 GAS motif was highly methylated in the primary cells. An additional CpG site (90bis, created by a SNP, was found only in the primary cells. IL-4 stimulation resulted in dramatic demethylation of CpG sites 90 and 90bis in the primary cells. Conclusions: IL-4 induces demethylation of specific CpG sites within the pendrin promoter. These epigenetic alterations are cell type specific, and may in part dictate pendrin mRNA transcription.

  12. Pigment cell differentiation in sea urchin blastula-derived primary cell cultures.

    Ageenko, Natalya V; Kiselev, Konstantin V; Dmitrenok, Pavel S; Odintsova, Nelly A


    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential.

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

    Fornara, O; Bartek, J; Rahbar, A;


    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......-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype...... was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM...

  14. Subcurative radiation significantly increases cell proliferation, invasion, and migration of primary glioblastoma multiforme in vivo

    Adarsh Shankar; Robert A. Knight; Stephen Brown; Ali S. Arbab; Sanath Kumar; Asm Iskander; Nadimpalli RS Varma; Branislava Janic; Ana deCarvalho; Tom Mikkelsen; Joseph A. Frank; Meser M. Ali


    Tumor cellproliferation, infiltration, migration, and neovascularization are known causes of treatment resistance in glioblastoma multiforme (GBM). The purpose of this study was to determine the effect of radiation on the growth characteristics of primary human GBM developed in a nude rat. Primary GBM cells grown from explanted GBM tissues were implanted orthotopically in nude rats. Tumor growth was confirmed by magnetic resonance imaging on day 77 (baseline) after implantation. The rats underwent irradiation to a dose of 50 Gy delivered subcuratively on day 84 postimplantation (n= 8), or underwent no radiation (n= 8). Brain tissues were obtained on day 112 (nonirradiated) or day 133 (irradiated). Immunohistochemistry was performed to determine tumor cell proliferation (Ki-67) and to assess the expression of infiltration marker (matrix metalloproteinase-2, MMP-2) and cell migration marker (CD44). Tumor neovascularization was assessed by microvessel density using von-Willebrand factor (vWF) staining. Magnetic resonance imaging showed well-developed, infiltrative tumors in 11 weeks postimplantation. The proportion of Ki-67-positive cells in tumors undergoing radiation was (71 ± 15)%compared with (25 ± 12)%in the nonirradiated group (P=0.02). The number of MMP-2-positive areas and proportion of CD44-positive cells were also high in tumors receiving radiation, indicating great invasion and infiltration. Microvessel density analysis did not show a significant difference between nonirradiated and irradiated tumors. Taken together, we found that subcurative radiation significantly increased proliferation, invasion, and migration of primary GBM. Our study provides insights into possible mechanisms of treatment resistance fol owing radiation therapy for GBM.

  15. MicroRNA profiling of primary cutaneous large B-cell lymphomas.

    Lianne Koens

    Full Text Available Aberrant expression of microRNAs is widely accepted to be pathogenetically involved in nodal diffuse large B-cell lymphomas (DLBCLs. However, the microRNAs profiles of primary cutaneous large B-cell lymphomas (PCLBCLs are not yet described. Its two main subtypes, i.e., primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT and primary cutaneous follicle center lymphoma (PCFCL are characterized by an activated B-cell (ABC-genotype and a germinal center B-cell (GCB-genotype, respectively. We performed high-throughput sequencing analysis on frozen tumor biopsies from 19 cases of PCFCL and PCLBCL-LT to establish microRNA profiles. Cluster analysis of the complete microRNome could not distinguish between the two subtypes, but 16 single microRNAs were found to be differentially expressed. Single microRNA RT-qPCR was conducted on formalin-fixed paraffin-embedded tumor biopsies of 20 additional cases, confirming higher expression of miR-9-5p, miR-31-5p, miR-129-2-3p and miR-214-3p in PCFCL as compared to PCLBCL-LT. MicroRNAs previously described to be higher expressed in ABC-type as compared to GCB-type nodal DLBCL were not differentially expressed between PCFCL and PCLBCL-LT. In conclusion, PCFCL and PCLBCL-LT differ in their microRNA profiles. In contrast to their gene expression profile, they only show slight resemblance with the microRNA profiles found in GCB- and ABC-type nodal DLBCL.

  16. Cell type- and isotype-specific expression and regulation of β-tubulins in primary olfactory ensheathing cells and Schwann cells in vitro.

    Omar, Mohamed; Hansmann, Florian; Kreutzer, Robert; Kreutzer, Mihaela; Brandes, Gudrun; Wewetzer, Konstantin


    Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are closely-related cell types with regeneration-promoting properties. Comparative gene expression analysis is particularly relevant since it may explain cell type-specific effects and guide the use of each cell type into special clinical applications. In the present study, we focused on β-tubulin isotype expression in primary adult canine glia as a translational large animal model. β-tubulins so far have been studied mainly in non-neuronal tumors and implied in tumorigenic growth. We show here that primary OECs and SCs expressed βII-V isotype mRNA. Interestingly, βIII-tubulin mRNA and protein expression was high in OECs and low in SCs, while fibroblast growth factor-2 (FGF-2) induced its down-regulation in both cell types to the same extent. This was in contrast to βV-tubulin mRNA which was similarly expressed in both cell types and unaltered by FGF-2. Immunocytochemical analysis revealed that OEC cultures contained a higher percentage of βIII-tubulin-positive cells compared to SC cultures. Addition of FGF-2 reduced the number of βIII-tubulin-positive cells in both cultures and significantly increased the percentage of cells with a multipolar morphology. Taken together, we demonstrate cell type-specific expression (βIII) and isotype-specific regulation (βIII, βV) of β-tubulin isotypes in OECs and SCs. While differential expression of βIII-tubulin in primary glial cell types with identical proliferative behaviour argues for novel functions unrelated to tumorigenic growth, strong βIII-tubulin expression in OECs may help to explain the specific properties of this glial cell type.

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

    Shet Tanuja


    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.

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

    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.

  19. Interleukin-1{beta} regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

    Zitta, Karina; Brandt, Berenice [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany); Wuensch, Annegret [Institute of Molecular Animal Breeding and Biotechnology, Ludwig Maximilians University, Munich (Germany); Meybohm, Patrick; Bein, Berthold; Steinfath, Markus; Scholz, Jens [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany); Albrecht, Martin, E-mail: [Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel (Germany)


    Research highlights: {yields} Levels of IL-1{beta} are increased in the pig myocardium after infarction. {yields} Cultured pig heart cells possess IL-1 receptors. {yields} IL-1{beta} increases cell proliferation of pig heart cells in-vitro. {yields} IL-1{beta} increases MMP-2 and MMP-9 activity in pig heart cells in-vitro. {yields} IL-1{beta} may be important for tissue remodelling events after myocardial infarction. -- Abstract: After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1{beta} is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1{beta} on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1{beta}. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1{beta} resulted in a dose-dependent increase of cell proliferation (P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1{beta} (P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1{beta} plays a major role in the events of tissue remodelling in the heart. Combined

  20. Identification of DNA-PKcs as a primary resistance factor of salinomycin in osteosarcoma cells.

    Zhen, Yun-Fang; Li, Song-Tao; Zhu, Yun-Rong; Wang, Xiao-Dong; Zhou, Xiao-Zhong; Zhu, Lun-Qing


    Malignant osteosarcoma (OS) is still a deadly disease for many affected patients. The search for the novel anti-OS agent is extremely urgent and important. Our previous study has proposed that salinomycin is a novel anti-OS agent. Here we characterized DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a primary salinomycin resistance factor in OS cells. DNA-PKcs inhibitors (NU7026, NU7441 and LY294002) or DNA-PKcs shRNA knockdown dramatically potentiated salinomycin-induced death and apoptosis of OS cells (U2OS and MG-63 lines). Further, forced-expression of microRNA-101 ("miR-101") downregulated DNA-PKcs and augmented salinomycin's cytotoxicity against OS cells. Reversely, over-expression of DNA-PKcs in OS cells inhibited salinomycin's lethality. For the mechanism study, we show that DNA-PKcs is required for salinomycin-induced pro-survival autophagy activation. DNA-PKcs inhibition (by NU7441), shRNA knockdown or miR-101 expression inhibited salinomycin-induced Beclin-1 expression and autophagy induction. Meanwhile, knockdown of Beclin-1 by shRNA significantly sensitized salinomycin-induced OS cell lethality. In vivo, salinomycin administration suppressed U2OS xenograft tumor growth in severe combined immuno-deficient (SCID) mice, and its anti-tumor activity was dramatically potentiated with co-administration of the DNA-PKcs inhibitor NU7026. Together, these results suggest that DNA-PKcs could be a primary resistance factor of salinomycin in OS cells. DNA-PKcs inhibition or silence may thus significantly increase salinomycin's sensitivity in OS cells.

  1. Analysis of HIV-1- and CMV-specific memory CD4 T-cell responses during primary and chronic infection.

    Harari, Alexandre; Rizzardi, G Paolo; Ellefsen, Kim; Ciuffreda, Donatella; Champagne, Patrick; Bart, Pierre-Alexandre; Kaufmann, Daniel; Telenti, Amalio; Sahli, Roland; Tambussi, Giuseppe; Kaiser, Laurent; Lazzarin, Adriano; Perrin, Luc; Pantaleo, Giuseppe


    CD4 T-cell-specific memory antiviral responses to human immunodeficiency virus type 1 (HIV-1) and cytomegalovirus (CMV) were investigated in 16 patients with documented primary HIV-1 infection (4 of the 16 subjects also had primary CMV infection) and compared with those observed in patients with chronic HIV-1 and CMV coinfection. Virus-specific memory CD4 T cells were characterized on the basis of the expression of the chemokine receptor CCR7. HIV-1- and CMV-specific interferon-gamma-secreting CD4 T cells were detected in patients with primary and chronic HIV-1 and CMV coinfection and were mostly contained in the cell population lacking expression of CCR7. The magnitude of the primary CMV-specific CD4 T-cell response was significantly greater than that of chronic CMV infection, whereas there were no differences between primary and chronic HIV-1-specific CD4 T-cell responses. A substantial proportion of CD4(+)CCR7(-) T cells were infected with HIV-1. These results advance the characterization of antiviral memory CD4 T-cell response and the delineation of the potential mechanisms that likely prevent the generation of a robust CD4 T-cell immune response during primary infection.

  2. Immune Modulatory Cell Therapy for Hemophilia B Based on CD20-Targeted Lentiviral Gene Transfer to Primary B Cells

    Xiaomei Wang


    Full Text Available Gene-modified B cells expressing immunoglobulin G (IgG fusion proteins have been shown to induce tolerance in several autoimmune and other disease models. However, lack of a vector suitable for gene transfer to human B cells has been an obstacle for translation of this approach. To overcome this hurdle, we developed an IgG-human factor IX (hFIX lentiviral fusion construct that was targeted to specifically transduce cells expressing human CD20 (hCD20. Receptor-specific retargeting by mutating envelope glycoproteins of measles virus (MV-lentiviral vector (LV and addition of a single-chain variable fragment specific for hCD20 resulted in gene delivery into primary human and transgenic hCD20 mouse B cells with high specificity. Notably, this protocol neither required nor induced activation of the B cells, as confirmed by minimal activation of inflammatory cytokines. Using this strategy, we were able to demonstrate induction of humoral tolerance, resulting in suppression of antibody formation against hFIX in a mouse model of hemophilia B (HB. In conclusion, transduction of receptor-specific retargeted LV into resting B cells is a promising method to develop B cell therapies for antigen-specific tolerance induction in human disease.

  3. Final design of the LSST primary/tertiary mirror cell assembly

    Neill, Douglas R.; Muller, Gary; Hileman, Ed; DeVries, Joe; Araujo, Constanza; Gressler, William J.; Lotz, Paul J.; Mills, Dave; Sebag, Jacques; Thomas, Sandrine; Warner, Mike; Wiecha, Oliver


    The Large Synoptic Survey Telescope (LSST) primary/tertiary (M1M3) mirror cell assembly supports both on-telescope operations and off-telescope mirror coating. This assembly consists of the cast borosilicate M1M3 monolith mirror, the mirror support systems, the thermal control system, a stray light baffle ring, a laser tracker interface and the supporting steel structure. During observing the M1M3 mirror is actively supported by pneumatic figure control actuators and positioned by a hexapod. When the active system is not operating the mirror is supported by a separate passive wire rope isolator system. The center of the mirror cell supports a laser tracker which measures the relative position of the camera and secondary mirror for alignment by their hexapods. The mirror cell structure height of 2 meters provides ample internal clearance for installation and maintenance of mirror support and thermal control systems. The mirror cell also functions as the bottom of the vacuum chamber during coating. The M1M3 mirror has been completed and is in storage. The mirror cell structure is presently under construction by CAID Industries. The figure control actuators, hexapod and thermal control system are under developed and will be integrated into the mirror cell assembly by LSST personnel. The entire integrated M1M3 mirror cell assembly will the tested at the Richard F Caris Mirror Lab in Tucson, AZ (formerly Steward Observatory Mirror Lab).

  4. Primary Culture of Alveolar Epithelial Type Ⅱ Cells and Its Bionomic Study

    SHI Xuemei; NI Wang; ZHANG Huilan; XIONG Shengdao; ZHEN Guohua; XIONG Weining; ZHANG Zhenxiang; XU Yongjian; HU Qiongjie; ZHAO Jianping


    To establish a better method of primary culture for alveolar epithelial type Ⅱ cells (AEC Ⅱ) and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with tryp- sin and collagenase, which were then purified by plated into culture flask coated with rat immu- noglobulin G. The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi-croscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expres-sion and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive pu- rification by using IgG, with a yield of about 2-3×107, and a purity of about 75%-84 %. Cells could be quickly identified with AKP staining. AEC Ⅱ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC Ⅱ, and AKP staining is simple in cell identification. AEC Ⅱ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

  5. Vitamin D3 modulated gene expression patterns in human primary normal and cancer prostate cells.

    Guzey, Meral; Luo, Jianhua; Getzenberg, Robert H


    The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors and has potential tumor-suppressive functions in prostate and other cancer types. Vitamin D3 (VD3) exerts its biological actions by binding within cells to VDR. The VDR then interacts with specific regions of the DNA in cells, and triggers changes in the activity of genes involved in cell division, cell survival, and cellular function. Using human primary cultures and the prostate cancer (PCa) cell line, ALVA-31, we examined the effects of VD3 under different culture conditions. Complete G0/G1 arrest of ALVA-31 cells and approximately 50% inhibition of tumor stromal cell growth was observed. To determine changes in gene expression patterns related to VD3 activity, microarray analysis was performed. More than approximately 20,000 genes were evaluated for twofold relative increases and decreases in expression levels. A number of the gene targets that were up- and down-regulated are related to potential mechanisms of prostatic growth regulation. These include estrogen receptor (ER), heat shock proteins: 70 and 90, Apaf1, Her-2/neu, and paxillin. Utilizing antibodies generated against these targets, we were able to confirm the changes at the protein level. These newly reported gene expression patterns provide novel information not only potential markers, but also on the genes involved in VD3 induced apoptosis in PCa.

  6. Enamel Matrix Derivative Promote Primary Human Pulp Cell Differentiation and Mineralization

    Elisabeth Aurstad Riksen


    Full Text Available Enamel matrix derivative (EMD has been found to induce reactive dentin formation; however the molecular mechanisms involved are unclear. The effect of EMD (5–50 μg/mL on primary human pulp cells were compared to untreated cells and cells incubated with 10−8 M dexamethasone (DEX for 1, 2, 3, 7, and 14 days in culture. Expression analysis using Affymetrix microchips demonstrated that 10 μg/mL EMD regulated several hundred genes and stimulated the gene expression of proteins involved in mesenchymal proliferation and differentiation. Both EMD and DEX enhanced the expression of amelogenin (amel, and the dentinogenic markers dentin sialophosphoprotein (DSSP and dentin matrix acidic phosphoprotein 1 (DMP1, as well as the osteogenic markers osteocalcin (OC, BGLAP and collagen type 1 (COL1A1. Whereas, only EMD had effect on alkaline phosphatase (ALP mRNA expression, the stimulatory effect were verified by enhanced secretion of OC and COL1A from EMD treated cells, and increased ALP activity in cell culture medium after EMD treatment. Increased levels of interleukin-6 (IL-6, interleukin-8 (IL-8, and monocyte chemoattractant proteins (MCP-1 in the cell culture medium were also found. Consequently, the suggested effect of EMD is to promote differentiation of pulp cells and increases the potential for pulpal mineralization to favor reactive dentine formation.

  7. Quantification of Lactate Dehydrogenase for Cell Viability Testing Using Cell Lines and Primary Cultured Astrocytes.

    Kaja, Simon; Payne, Andrew J; Naumchuk, Yuliya; Koulen, Peter


    Drug discovery heavily relies on cell viability studies to assess the potential toxicity of drug candidates. L-Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that catalyzes the concomitant interconversions of pyruvate to L-lactate and NADH to NAD(+) during glycolysis, and the reverse reactions during the Cori cycle. In response to cellular damage, induced by endogenous cellular mechanisms or as a result of exogenously applied insults, LDH is released from the cytoplasm into the extracellular environment. Its stability in cell culture medium makes it a well-suited correlate for the presence of damage and toxicity in tissues and cells. We herein present protocols for a reproducible and validated LDH assay optimized for several cell types. In contrast to commercially available LDH assays, often associated with proprietary formulations and high cost, our protocols provide ample opportunities for experiment-specific optimization with low variability and cost. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  8. Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

    Rimann, Markus; Bono, Epifania; Annaheim, Helene; Bleisch, Matthias; Graf-Hausner, Ursula


    Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.

  9. Human umbilical mesenchymal stem cells conditioned medium promote primary wound healing regeneration

    Dwi Liliek Kusindarta


    Full Text Available Aim: This research was conducted to clarify the capability of human umbilical mesenchymal stem cells conditioned medium (HU-MSCM to promote regenerations of primary wound healing on the incision skin injury. Materials and Methods: In this study, two approaches in vitro and in vivo already done. On in vitro analysis, tube formation was performed using HU vein endothelial cells in the presence of HU-MSCM, in some experiments cells line was incubated prior the presence of lipopolysaccharide and HU-MSCM then apoptosis assay was performed. Furthermore, in vivo experiments 12 female rats (Rattus norvegicus were used after rats anesthetized, 7 mm wound was made by incision on the left side of the body. The wound was treated with HU-MSCM containing cream, povidone iodine was run as a control. Wound healing regenerations on the skin samples were visualized by hematoxylin-eosin staining. Results: In vitro models elucidate HU-MSCM may decreasing inflammation at the beginning of wound healing, promote cell migration and angiogenesis. In addition in vivo models show that the incision length on the skin is decreasing and more smaller, HE staining describe decreasing of inflammation phase, increasing of angiogenesis, accelerate fibroplasia, and maturation phase. Conclusions: Taken together our observation indicates that HU-MSCM could promote the acceleration of skin tissue regenerations in primary wound healing process.

  10. Primary Endometrial Squamous Cell Carcinoma In Situ; Report of a rare disease

    Sujata Jetley


    Full Text Available Squamous cell carcinoma (SCC of the endometrium, whether primary or secondary to cervical cancer, is a rare entity. Primary endometrial squamous cell carcinoma in situ is even more uncommon; it usually occurs in postmenopausal women and has a strong association with pyometra. We report a 60-year-old multiparous postmenopausal woman who presented to the Hakeem Abdul Hameed Centenary Hospital, New Delhi, India, in May 2014 with a lower abdominal swelling corresponding in size to a pregnancy of 26 gestational weeks and vaginal discharge of one year’s duration. A total abdominal hysterectomy with a bilateral salpingooophorectomy was performed, which revealed an enlarged uterus with pyometra. Histopathology showed that the entire endometrial lining had been replaced with malignant squamous cells without invasion of the myometrium. Immunohistochemistry revealed that the tumour cells were positive for p63 with a high Ki-67 labelling index. No adjuvant therapy was required and the patient was disease-free at a seven-month follow-up.

  11. Inhibition of autophagy suppresses sertraline-mediated primary ciliogenesis in retinal pigment epithelium cells.

    Kim, Eun Sung; Shin, Ji Hyun; Park, So Jung; Jo, Yoon Kyung; Kim, Jae-Sung; Kang, Il-Hwan; Nam, Jung-Bum; Chung, Doo-Young; Cho, Yoonchul; Lee, EunJoo H; Chang, Jong Wook; Cho, Dong-Hyung


    Primary cilia are conserved cellular organelles that regulate diverse signaling pathways. Autophagy is a complex process of cellular degradation and recycling of cytoplasmic proteins and organelles, and plays an important role in cellular homeostasis. Despite its potential importance, the role of autophagy in ciliogenesis is largely unknown. In this study, we identified sertraline as a regulator of autophagy and ciliogenesis. Sertraline, a known antidepressant, induced the growth of cilia and blocked the disassembly of cilia in htRPE cells. Following treatment of sertraline, there was an increase in the number of cells with autophagic puncta and LC3 protein conversion. In addition, both a decrease of ATG5 expression and the treatment of an autophagy inhibitor resulted in the suppression of the sertraline-induced activation of autophagy in htRPE cells. Interestingly, we found that genetic and chemical inhibition of autophagy attenuated the growth of primary cilia in htRPE cells. Taken together, our results suggest that the inhibition of autophagy suppresses sertraline-induced ciliogenesis.

  12. Trehalose-mediated autophagy impairs the anti-viral function of human primary airway epithelial cells.

    Qun Wu

    Full Text Available Human rhinovirus (HRV is the most common cause of acute exacerbations of chronic lung diseases including asthma. Impaired anti-viral IFN-λ1 production and increased HRV replication in human asthmatic airway epithelial cells may be one of the underlying mechanisms leading to asthma exacerbations. Increased autophagy has been shown in asthmatic airway epithelium, but the role of autophagy in anti-HRV response remains uncertain. Trehalose, a natural glucose disaccharide, has been recognized as an effective autophagy inducer in mammalian cells. In the current study, we used trehalose to induce autophagy in normal human primary airway epithelial cells in order to determine if autophagy directly regulates the anti-viral response against HRV. We found that trehalose-induced autophagy significantly impaired IFN-λ1 expression and increased HRV-16 load. Inhibition of autophagy via knockdown of autophagy-related gene 5 (ATG5 effectively rescued the impaired IFN-λ1 expression by trehalose and subsequently reduced HRV-16 load. Mechanistically, ATG5 protein interacted with retinoic acid-inducible gene I (RIG-I and IFN-β promoter stimulator 1 (IPS-1, two critical molecules involved in the expression of anti-viral interferons. Our results suggest that induction of autophagy in human primary airway epithelial cells inhibits the anti-viral IFN-λ1 expression and facilitates HRV infection. Intervention of excessive autophagy in chronic lung diseases may provide a novel approach to attenuate viral infections and associated disease exacerbations.

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

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


    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.

  14. Cell context-specific expression of primary cilia in the human testis and ciliary coordination of Hedgehog signalling in mouse Leydig cells

    Berg Nygaard, Marie; Almstrup, Kristian; Lindbæk, Louise;


    cells of mature seminiferous epithelium, but present in Sertoli cell-only tubules in Klinefelter syndrome testis. Peritubular cells in atrophic testis produce overly long cilia. Furthermore cultures of growth-arrested immature mouse Leydig cells express primary cilia that are enriched in components...

  15. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

    Saji, Chiaki [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Higashi, Chizuka; Niinaka, Yasufumi [Faculty of Medicine, University of Yamanashi, Chuoh-shi 409-3898 (Japan); Yamada, Koji [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Noguchi, Kohji [Faculty of Pharmacy, Keio University, 1-5-30 Shiba-koen, Minato-ku, Tokyo 105-8512 (Japan); Fujimuro, Masahiro, E-mail: [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)


    Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome

  16. T-cell leukemia 1 expression in nodal Epstein-Barr virus-negative diffuse large B-cell lymphoma and primary mediastinal B-cell lymphoma.

    Gualco, Gabriela; Weiss, Lawrence M; Barber, Glen N; Bacchi, Carlos E


    The physiologic expression of the product of the proto-oncogene TCL1 (T-cell leukemia 1) is primarily restricted to early embryonic cells. In nonneoplastic B cells, the expression of TCL1 is determined by the differentiation step with silencing at the germinal center stage. TCL1 protein is overexpressed in a wide variety of human diseases. It has been shown that TCL1 is a powerful B-cell oncogene, which has been implicated in the pathogenesis of various types of mature B-cell lymphomas. There is no comparative information in the literature addressing the expression of TCL1 in pediatric and adult nodal diffuse large B-cell lymphoma or primary mediastinal large B-cell lymphoma. We studied 55 cases of adult and pediatric diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma to analyze the phenotypic profile of these lymphomas, including TCL1 expression, and its relationship with clinical outcome in different age groups. The cases were analyzed by immunohistochemistry for the expression of TCL1, CD10, BCL-2, BCL-6, and MUM1. We also evaluated c-MYC translocation by fluorescence in situ hybridization. TCL1 was observed in 11 cases, 5 pediatric and 6 adult cases, all but one diffuse large B-cell lymphoma. Pediatric cases showed a significant association between TCL1 expression, high proliferative index, and presence of c-MYC translocation. TCL1 positivity was predominantly found in germinal center phenotype diffuse large B-cell lymphoma. Overall survival was worse in adult TCL1-positive cases than pediatric ones. Primary mediastinal large B-cell lymphomas infrequently expressed TCL1 in both age groups.

  17. Spatial phase sensitivity of complex cells in primary visual cortex depends on stimulus contrast.

    Meffin, H; Hietanen, M A; Cloherty, S L; Ibbotson, M R


    Neurons in primary visual cortex are classified as simple, which are phase sensitive, or complex, which are significantly less phase sensitive. Previously, we have used drifting gratings to show that the phase sensitivity of complex cells increases at low contrast and after contrast adaptation while that of simple cells remains the same at all contrasts (Cloherty SL, Ibbotson MR. J Neurophysiol 113: 434-444, 2015; Crowder NA, van Kleef J, Dreher B, Ibbotson MR. J Neurophysiol 98: 1155-1166, 2007; van Kleef JP, Cloherty SL, Ibbotson MR. J Physiol 588: 3457-3470, 2010). However, drifting gratings confound the influence of spatial and temporal summation, so here we have stimulated complex cells with gratings that are spatially stationary but continuously reverse the polarity of the contrast over time (contrast-reversing gratings). By varying the spatial phase and contrast of the gratings we aimed to establish whether the contrast-dependent phase sensitivity of complex cells results from changes in spatial or temporal processing or both. We found that most of the increase in phase sensitivity at low contrasts could be attributed to changes in the spatial phase sensitivities of complex cells. However, at low contrasts the complex cells did not develop the spatiotemporal response characteristics of simple cells, in which paired response peaks occur 180° out of phase in time and space. Complex cells that increased their spatial phase sensitivity at low contrasts were significantly overrepresented in the supragranular layers of cortex. We conclude that complex cells in supragranular layers of cat cortex have dynamic spatial summation properties and that the mechanisms underlying complex cell receptive fields differ between cortical layers.

  18. [Possibilities and limits of paraffin-embedded cell markers in diagnosis of primary cutaneous histiocytosis].

    Fartasch, M; Goerdt, S; Hornstein, O P


    To date, the rare primary histiocytoses of the skin are diagnosed definitively on the basis of the clinical symptoms, H&E-stained sections, and demonstration of CD1 positivity in frozen sections and of Birbeck granules on electron microscopy. The improvement and analysis of antibodies with the ability to react in paraffin tissue allow retrospective evaluation and classification of these disorders. The antibodies for S-100-protein, peanut agglutinin (PNA) and PCNA (proliferating cell nuclear antigen) have been advocated for differentiation of the specific cells of Langerhans cell histiocytosis (LCH) from other histiocytic cell systems. To date the non-Langerhans cell histiocytoses (non-LCH) have no common ultrastructural and immunohistochemical characteristics. The infiltrate is made up of multiple cell populations, which are of significance for the cellular pathobiology (subtypes of monocytes/macrophages and dendritic cells). The number and distribution of the different monocyte/macrophages and dendritic cells and their ability to react with immunohistochemical markers in paraffin tissue can be completely different in different clinical entities. The antibodies against factor XIIIa (shown on xanthoma disseminatum) and the monoclonal antibody Ki-M1P (shown on juvenile xanthogranuloma) seem to be valuable in discrimination between LCH and non-LCH. Both markers show a positive staining pattern with the characteristic large macrophages. In juvenile xanthogranuloma, the foam cells and giant cells express Ki-M1P, KP1 and anti-cathepsin B. Other monocyte/macrophage markers with the ability to react in paraffin tissue, such as Mac387, lysozyme, alpha 1-antitrypsin and Leu-M1 (Anti-CD 15), in contrast, did not show a typical staining pattern with the characteristic large macrophages dominating the histological picture.

  19. Impact of polyelectrolytes and their corresponding multilayers to human primary endothelial cells.

    Nolte, Andrea; Hossfeld, Susanne; Schroeppel, Birgit; Mueller, Anne; Stoll, Dieter; Walker, Tobias; Wendel, Hans Peter; Krastev, Rumen


    The layer-by-layer technique, which allows simple preparation of polyelectrolyte multilayers, came into the focus of research for development of functionalized medical devices. Numerous literature exist that concentrate on the film build-up and the behaviour of cells on polyelectrolyte multilayers. However, in case of very soft polyelectrolyte multilayers, studies of the cell behaviour on these films are sometimes misleading with regard to clinical applications because cells do not die due to cytotoxicity but due to apoptosis by missing cell adhesion. It turns out that the adhesion in vitro, and thus, the viability of cells on polyelectrolyte multilayers is mostly influenced by their mechanical properties. In order to decide, which polyelectrolyte multilayers are suitable for implants, we take this problem into account by putting the substrates with soft films on top of pre-cultured human primary endothelial cells ('reverse assay'). Hence, the present work aims giving a more complete and reliable study of typical polyelectrolyte multilayers with regard to clinical applications. In particular, coatings consisting of hyaluronic acid and chitosan as natural polymers and sulfonated polystyrene and polyallylamine hydrochlorite as synthetic polymers were studied. The adsorption of polyelectrolytes was characterized by physico-chemical methods which show regular buildup. Biological examination of the native or modified polyelectrolyte multilayers was based on their effect to cell adhesion and morphology of endothelial cells by viability assays, immunostaining and scanning electron microscopy. Using the standard method, which is typically applied in literature--seeding cells on top of films--shows that the best adhesion and thus, viability can be achieved using sulfonated polystyrene/polyallylamine hydrochlorite. However, putting the films on top of endothelial cells reveals that hyaluronic acid/chitosan may also be suitable for clinical applications: This result is

  20. Primary retroperitoneal transitional cell carcinoma presenting as a dumb-bell tumour.

    Basu, S; Ansari, M; Gupta, S; Kumar, A


    We report a retroperitoneal transitional cell carcinoma arising from the primitive urogenital remnants of a 56-year-old married Indian woman. She presented with a huge cystic mass in the hypogastrium and right iliac fossa, which extended into the right thigh as a massive dumb-bell tumour. On exploration, it was found not to be arising from any known retroperitoneal structure. The mass was excised, and the histopathology confirmed transitional cell carcinoma with positive margins. Though she received postoperative chemotherapy with cyclophosphamide, adriamycin and cisplatin, she developed extensive local recurrence and hepatic secondaries, and succumbed to the disease after ten months of follow-up. We highlight the rarity of the disease, its atypical presentation as a cystic dumb-bell lump, its diagnostic challenges and aggressive behaviour, and review the literature on primary retroperitoneal transitional cell carcinomas.